Yeasts are essential for mucilage degradation of coffee beans during wet fermentation.

During wet fermentation, mucilage layers in coffee cherries must be removed completely. To explain mucilage degradation, several controversial hypotheses have been proposed. The aim of this work was to improve our understanding of the kinetics of mucilage breakdown. Pulped coffee beans were wet fermented with seven different treatments for 36 h. Endogenous bacteria and yeasts are selectively suppressed, and pectinases or lactic acid are added. They also involve maintaining the beans at pH 7 throughout fermentation and using spontaneous fermentation without additives as a control. During spontaneous fermentation, yeast and lactic acid bacteria were detected and significantly increased to 5.5 log colony-forming units (CFU)/mL and 5.2 log CFU/mL, respectively. In the first 12 h of fermentation, there was a significant degree of endogenous pectinolytic activity, which resulted in partly destroyed beans in the absence of microorganisms. By adding pectinase and lactic acid to the fermentation mass, the breakdown process was accelerated in less than 8 h. When yeast was present throughout the fermentation, complete degradation was achieved. Bacteria played no critical role in the degradation. Klebsiella pneumoniae and Erwinia soli were found in a lower population and showed weaker pectinolytic activities compared to Hanseniaspora uvarum and Pichia kudriavzevii. During wet fermentation, mucilage degradation appears to be mediated by endogenous enzymes at the early stage, whereas microbial contributions, mainly yeasts, occur subsequently. H. uvarum and P. kudriavzevii may be promising candidates to be tested in future studies as coffee starter cultures to better control the mucilage degradation process.

Amino acid sequence of two new milk-clotting proteases from the macroalga Gracilaria edulis.

This study is aimed at identifying and characterising the proteases we previously extracted from the red seaweed Gracilaria edulis with the potential as milk-clotting enzymes. The protease extract was first analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and zymography. Two protease bands with a molecular weight of 44 and 108 kDa were identified, and analysed using in-gel digestion and liquid chromatography-tandem mass spectrometry/mass spectrometry (LC-MS/MS). Eight peptides from the LC-MS/MS analysis matched those in existing protein databases but they were not related to any protease of the genera Gracilaria and Hydropuntia. Further analysis revealed that more than 80% of the peptide sequence of the algal proteases matched with those from members of the bacteria kingdom, including Gallaecimonas and Alteromonas. Among these, twelve matching homolog proteases were identified as metalloprotease and serine protease. The results indicated that the algal proteases have a close relationship with both algae and bacteria, and suggest that the proteases might have resulted from past bacterial colonisation of the algae and subsequent horizontal gene transfer between bacteria and algae.

Health economic analysis for the 'CURE Project' pilot: a hospital-based tobacco dependency treatment service in Greater Manchester.

INTRODUCTION: Treating tobacco dependency in patients admitted to acute care National Health Service (NHS) trusts is a key priority in the NHS 10-year plan. This paper sets out the results of a health economic analysis for 'The CURE Project' pilot; a new hospital-based tobacco dependency service. METHODS: A health economic analysis to understand the costs of the intervention (both for the inpatient service and postdischarge costs), the return on investment (ROI) and the cost per quality-adjusted life year (QALY) of the CURE Project pilot in Greater Manchester. ROI and cost per QALY were calculated using the European Study on Quantifying Utility of Investment in Protection from Tobacco and Greater Manchester Cost Benefit Analysis Tools. RESULTS: The total intervention costs for the inpatient service in the 6-month CURE pilot were £96 224 with a cost per patient who smokes of £40.21. The estimated average cost per patient who was discharged on pharmacotherapy was £97.40. The cost per quit (22% quit rate for smokers at 12 weeks post discharge) was £475. The gross financial ROI ratio was £2.12 return per £1 invested with a payback period of 4 years. The cashable financial ROI ratio was £1.06 return per £1 invested with a payback period of 10 years. The public value ROI ratio was £30.49 per £1 invested. The cost per QALY for this programme was £487. DISCUSSION: The CURE Project pilot has been shown to be exceptionally cost-effective with highly significant ROI in this health economic analysis. This supports the NHS priority to embed high-quality tobacco addiction treatment services in acute NHS trusts, and the CURE Project provides a blueprint and framework to achieve this.

Microbiological and Chemical Characteristics of Wet Coffee Fermentation Inoculated With Hansinaspora uvarum and Pichia kudriavzevii and Their Impact on Coffee Sensory Quality.

Hansinaspora uvarum and Pichia kudriavzevii were used as starter cultures to conduct inoculated wet fermentations of coffee beans, and their growth, metabolic activities and impact on the flavor, aroma and overall sensory quality of coffee were compared with spontaneous fermentation (control). H. uvarum and P. kudriavzevii dominated the fermentations, growing to maximum populations of about 10.0 log CFU/ml compared with 8.0 log CFU/ml in the spontaneous fermentation. The dominance of the inoculated yeasts led to faster and more complete utilization of sugars in the mucilage, with resultant production of 2-3 fold higher concentrations of metabolites such as glycerol, alcohols, aldehydes, esters, and organic acids in the fermented green beans. Cup tests showed coffee produced from the inoculated fermentations, especially with P. kudriavzevii, received higher scores for flavor, aroma and acidity than the control. The findings of this study confirmed the crucial role of yeasts in the wet fermentation of coffee beans and their contribution to high quality coffee, and demonstrated the potential H. uvarum and P. kudriavzevii as starter cultures in the process.

The crucial role of yeasts in the wet fermentation of coffee beans and quality.

The objective of this study was to investigate the role of yeasts in the wet fermentation of coffee beans and their contribution to coffee quality using a novel approach. Natamycin (300 ppm) was added to the fermentation mass to suppress yeast growth and their metabolic activities, and the resultant microbial ecology, bean chemistry and sensory quality were analyzed and compared to non-treated spontaneous fermentation we reported previously. The yeast community was dominated by Hanseniaspora uvarum and Pichia kudriavzevii and grew to a maximum population of about 5.5 log CFU/g in the absence of Natamycin, while when Natamycin was added yeasts were suppressed. The major bacterial species in both the spontaneous and yeast-suppressed fermentations included the lactic acid bacteria Leuconostoc mesenteroides and Lactococcus lactis, the acetic acid bacteria Gluconobacter cerinus and Acetobacter persici and the Enterobacteriaceae Enterobacter, Citrobacter and Erwinia. For both fermentations, the mucilage layers were completely degraded by the end of the process and the absence of yeast activities had no significant impact on mucilage degradation. During fermentation, reducing sugars were consumed while lactic acid was accumulated inside the beans, and its concentration was significantly higher in the spontaneous fermentation (3 times) than that where yeasts were suppressed by Natamycin. Glycerol was detected with a concentration of 0.08% in the absence of Natamycin and was not identified when Natamycin was added. Green beans fermented with yeast growth contained a higher amount of isoamyl alcohol (21 times), ethanol (3.7 times), acetaldehyde (8 times), and ethyl acetate (25 times) compared to beans fermented in the absence of yeast activities, which remained higher in the former after roasting. Beans fermented without yeast activities had a mild fruity aroma, and lower sensory scores of fragrances (7.0), flavor (6.5), acidity (6.3), body (7.0) and overall score (6.5) compared to the former. These findings demonstrated the crucial roles of yeasts in wet fermentation of coffee beans and for producing high quality coffee.

Extraction, partial purification and characterization of proteases from the red seaweed Gracilaria edulis with similar cleavage sites on κ-casein as calf rennet.

Enzymes currently used in cheesemaking have various drawbacks, and there is a continual need to find new coagulants. This study describes the extraction and biochemical characterization of two proteases from the red alga Gracilaria edulis. The proteases were extracted with phosphate buffer and partially purified by ammonium sulphate precipitation and dialysis. The enzymes exhibited optimum caseinolytic activity at 60 °C and a pH range of 6-8. They showed a high ratio of milk-clotting over caseinolytic activity, indicating they had an excellent milk-clotting ability. The proteases were confirmed to be serine protease and metalloprotease with molecular weight (MW) of 44 and 108 kDa. They exhibited high hydrolytic activity on κ-caseins, cleaving κ-casein at four main sites, one of which being the same as that of calf rennet, which is the first reported for an algal protease. The findings demonstrated that the proteases could potentially be used as a milk coagulant in cheesemaking.

Ecological diversity, evolution and metabolism of microbial communities in the wet fermentation of Australian coffee beans.

The microbial ecology in the fermentation of Australian coffee beans was investigated in this study. Pulped coffee beans were kept underwater for 36 h before air dried. Samples were collected periodically, and the microbial communities were analyzed by culture-dependent and independent methods. Changes in sugars, organic acids and microbial metabolites in the mucilage and endosperm of the coffee beans during fermentation were monitored by HPLC. Culture-dependent methods identified 6 yeast and 17 bacterial species, while the culture-independent methods, multiple-step total direct DNA extraction and high throughput sequencing, identified 212 fungal and 40 bacterial species. Most of the microbial species in the community have been reported for wet fermentation of coffee beans in other parts of the world, but the yeast Pichia kudriavzevii was isolated for the first time in wet coffee bean fermentation. The bacterial community was dominated by aerobic mesophilic bacteria (AMB) with Citrobacter being the predominant genus. Hanseniaspora uvarum and Pichia kudriavzevii were the predominant yeasts while Leuconostoc mesenteroides and Lactococcus lactis were the predominant LAB. The yeasts and bacteria grew significantly during fermentation, utilizing sugars in the mucilage and produced mannitol, glycerol, and lactic acid, leading to a significant decrease in pH. The results of this study provided a preliminary understanding of the microbial ecology of wet coffee fermentation under Australian conditions. Further studies are needed to explore the impact of microbial growth and metabolism on coffee quality, especially flavour.

The SIPHER Consortium: Introducing the new UK hub for systems science in public health and health economic research.

The conditions in which we are born, grow, live, work and age are key drivers of health and inequalities in life chances. To maximise health and wellbeing across the whole population, we need well-coordinated action across government sectors, in areas including economic, education, welfare, labour market and housing policy. Current research struggles to offer effective decision support on the cross-sector strategic alignment of policies, and to generate evidence that gives budget holders the confidence to change the way major investment decisions are made. This open letter introduces a new research initiative in this space. The SIPHER ( Systems Science in Public Health and Health Economics Research) Consortium brings together a multi-disciplinary group of scientists from across six universities, three government partners at local, regional and national level, and ten practice partner organisations. The Consortium's vision is a shift from health policy to healthy public policy, where the wellbeing impacts of policies are a core consideration across government sectors. Researchers and policy makers will jointly tackle fundamental questions about: a) the complex causal relationships between upstream policies and wellbeing, economic and equality outcomes; b) the multi-sectoral appraisal of costs and benefits of alternative investment options; c) public values and preferences for different outcomes, and how necessary trade-offs can be negotiated; and d) creating the conditions for intelligence-led adaptive policy design that maximises progress against economic, social and health goals. Whilst our methods will be adaptable across policy topics and jurisdictions, we will initially focus on four policy areas: Inclusive Economic Growth, Adverse Childhood Experiences, Mental Wellbeing and Housing.

Systematic Population Screening, Using Biomarkers and Genetic Testing, Identifies 2.5% of the U.K. Pediatric Diabetes Population With Monogenic Diabetes.

OBJECTIVE: Monogenic diabetes is rare but is an important diagnosis in pediatric diabetes clinics. These patients are often not identified as this relies on the recognition of key clinical features by an alert clinician. Biomarkers (islet autoantibodies and C-peptide) can assist in the exclusion of patients with type 1 diabetes and allow systematic testing that does not rely on clinical recognition. Our study aimed to establish the prevalence of monogenic diabetes in U.K. pediatric clinics using a systematic approach of biomarker screening and targeted genetic testing. RESEARCH DESIGN AND METHODS: We studied 808 patients (79.5% of the eligible population) <20 years of age with diabetes who were attending six pediatric clinics in South West England and Tayside, Scotland. Endogenous insulin production was measured using the urinary C-peptide creatinine ratio (UCPCR). C-peptide-positive patients (UCPCR ≥0.2 nmol/mmol) underwent islet autoantibody (GAD and IA2) testing, with patients who were autoantibody negative undergoing genetic testing for all 29 identified causes of monogenic diabetes. RESULTS: A total of 2.5% of patients (20 of 808 patients) (95% CI 1.6-3.9%) had monogenic diabetes (8 GCK, 5 HNF1A, 4 HNF4A, 1 HNF1B, 1 ABCC8, 1 INSR). The majority (17 of 20 patients) were managed without insulin treatment. A similar proportion of the population had type 2 diabetes (3.3%, 27 of 808 patients). CONCLUSIONS: This large systematic study confirms a prevalence of 2.5% of patients with monogenic diabetes who were <20 years of age in six U.K. clinics. This figure suggests that ∼50% of the estimated 875 U.K. pediatric patients with monogenic diabetes have still not received a genetic diagnosis. This biomarker screening pathway is a practical approach that can be used to identify pediatric patients who are most appropriate for genetic testing.

Response of layer and broiler strain chickens to parenteral administration of a live Salmonella Typhimurium vaccine.

Responses to the parenteral administration of a live aroA deletion Salmonella serovar Typhimurium vaccine given to three brown egg layer strains and two broiler strains were studied. Twenty-five birds of each strain were reared together in floor pens to 6 weeks of age and then moved as individual strains to new floor pens and injected with 10(8) colony forming units (CFU) per bird of the vaccine bacteria intramuscularly or subcutaneously, 10(6) CFU per bird subcutaneously, or phosphate buffered saline (PBS) subcutaneously as a vaccination control. Three birds of one layer strain were injected intramuscularly with 0.5mg/ bird S. Typhimurium lipopolysaccharide (LPS) to evaluate whether response was similar for vaccine and endotoxin. Birds were weighed, and rectal temperatures recorded at the time of injection, then observed over 24 hours. Rectal temperatures were measured and blood samples collected for serum IL-6 assay at 3 hours post injection (PI). At 12 hours PI blood samples were drawn for analyses for plasma phosphorus (P), glucose (Glu), cholesterol (Cho), aspartate transaminase (AST), total protein (Ptn) and creatinine kinase (CK). Blood was sampled 14 days PI and tested for serum antibody to S. Typhimurium. Vaccination resulted in significant seroconversion by 14 days PI in all strains compared to the controls. The three layer strains exhibited a clinical malaise, evident within 90 minutes of injection, lasting for 12 hours, with complete recovery by 24 hours PI. Only the 10(8) CFU dose given subcutaneously produced an increase in rectal temperature 3 hours PI. Vaccination had no effect on IL-6 or Ptn. All vaccine doses increased P and the higher dose by either route decreased Cho in all bird strains. The 10(8) vaccine dose increased Glu and intramuscular injection markedly elevated CK only in the layer strains. The response was not completely congruous with that to LPS alone. The results highlight the need for consideration of differences in response of bird strain when consideration is given to the parenteral administration of live Salmonella vaccines.

The interplay between Campylobacter and Helicobacter species and other gastrointestinal microbiota of commercial broiler chickens.

BACKGROUND: Poultry represent an important source of foodborne enteropathogens, in particular thermophilic Campylobacter species. Many of these organisms colonize the intestinal tract of broiler chickens as harmless commensals, and therefore, often remain undetected prior to slaughter. The exact reasons for the lack of clinical disease are unknown, but analysis of the gastrointestinal microbiota of broiler chickens may improve our understanding of the microbial interactions with the host. METHODS: In this study, the fecal microbiota of 31 market-age (56-day old) broiler chickens, from two different farms, was analyzed using high throughput sequencing. The samples were then screened for two emerging human pathogens, Campylobacter concisus and Helicobacter pullorum, using species-specific PCR. RESULTS: The gastrointestinal microbiota of chickens was classified into four potential enterotypes, similar to that of humans, where three enterotypes have been identified. The results indicated that variations between farms may have contributed to differences in the microbiota, though each of the four enterotypes were found in both farms suggesting that these groupings did not occur by chance. In addition to the identification of Campylobacter jejuni subspecies doylei and the emerging species, C. concisus, C. upsaliensis and H. pullorum, several differences in the prevalence of human pathogens within these enterotypes were observed. Further analysis revealed microbial taxa with the potential to increase the likelihood of colonization by a number of these pathogens, including C. jejuni. CONCLUSION: Depletion of these taxa and the addition of taxa that compete with these pathogens, may form the basis of competitive exclusion strategies to eliminate them from the gastrointestinal tract of chickens.

A novel miniaturized most probable number method for the enumeration of Campylobacter spp. from poultry-associated matrices.

A novel miniaturized most probable number (mMPN) method was developed for the enumeration of thermophilic Campylobacter spp. using a modification of blood-free Bolton broth (supplemented with 25mg/l of sulfamethoxazole) and CampyFood ID agar. The mMPN was evaluated by comparison with direct plating (modified ISO/TS, 10272-2:2006) for the analysis of samples (n=149) representing various poultry matrices (carcases, broiler ceca and feces, scald tank water and feed). A sensitivity of 95%, specificity of 90% and Cohen KAPPA agreement of 0.84 was determined for the mMPN method compared to direct plating. Quantitative comparison found 83% of enumerations to be less than ±1log10 different (Student's t-test P<0.001). Financial analysis showed that the mMPN required 51% less media and 60% less labor than the direct plating protocol. The mMPN provides a method that can be used for complete through-chain analysis that has a single enrichment step and multiple dilutions to extinction for a variety of samples (containing low, medium and high populations).

Diversity and toxigenicity among members of the Bacillus cereus group.

Members of the Bacillus cereus group were isolated from rice products by centrifugation-plating and conventional spread-plating methods. Random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) results showed broad diversity among the strains and revealed some associations among isolates from raw and cooked rice samples, at the genotypic level. A comparatively greater diversity among strains was observed in isolates from raw rice than those from cooked rice and, generally, the RAPD profiles of isolates from raw and cooked rice were different, with only a few of them common to both types of rice. The toxigenic potential of the isolates was also determined by molecular and immunoassay analyses. The results revealed that most isolates from the B. cereus group were potentially or actually toxigenic, and some isolates could produce both diarrhoeal and emetic toxins. Generally, isolates belonging to the B. cereus group with the same RAPD pattern were shown to have a similar profile of enterotoxigenicity.

Development and validation of a drag swab method using tampons and different diluents for the detection of members of Salmonella in broiler houses.

Members of the genus Salmonella represent a significant public health concern and also a colonizer of commercial poultry. Therefore, the early detection and management of colonized broiler breeders and their progeny is essential. There have been numerous methods for farm-based detection, with gauze-based drag swabs being the most commonly used. In the present study, the wet (boiled water, buffered peptone water and double-strength skin milk) tampon was compared with the gauze to determine the recovery rate (10(2) colony-forming units/swab) of five common poultry serovars of Salmonella and after cold (4°C/48 h) storage. The recovery was found to be equivalent when tested using the ISO6572:2002 method, for all diluents (Cohen's κ =1.0; sensitivity = 1.0; specificity = 1.0). The subsequent field trial (n = 15 farms) compared the tampon drag swab (TDS) with a statistically appropriate (90% confidence, detect 10% prevalence) number of faecal swabs (n = 22), which also showed high agreement between the TDS and faecal sampling (κ = 0.86; McNemar's χ(2) = 1.0; sensitivity = 0.9; specificity = 1.0). However, direct faecal sampling showed a wider diversity of serovars of Salmonella than the corresponding TDS. The TDS is a very sensitive, readily available and cost-effective screening method for salmonellas in broiler breeder houses. This TDS technique may be used for routinely screening of broiler houses, and faecal sampling would only be used to confirm colonization or contamination, and to measure flock serovar variance.

Application of a dense gas technique for sterilizing soft biomaterials.

Sterilization of soft biomaterials such as hydrogels is challenging because existing methods such as gamma irradiation, steam sterilization, or ethylene oxide sterilization, while effective at achieving high sterility assurance levels (SAL), may compromise their physicochemical properties and biocompatibility. New methods that effectively sterilize soft biomaterials without compromising their properties are therefore required. In this report, a dense-carbon dioxide (CO(2) )-based technique was used to sterilize soft polyethylene glycol (PEG)-based hydrogels while retaining their structure and physicochemical properties. Conventional sterilization methods such as gamma irradiation and steam sterilization severely compromised the structure of the hydrogels. PEG hydrogels with high water content and low elastic shear modulus (a measure of stiffness) were deliberately inoculated with bacteria and spores and then subjected to dense CO(2) . The dense CO(2) -based methods effectively sterilized the hydrogels achieving a SAL of 10(-7) without compromising the viscoelastic properties, pH, water-content, and structure of the gels. Furthermore, dense CO(2) -treated gels were biocompatible and non-toxic when implanted subcutaneously in ferrets. The application of novel dense CO(2) -based methods to sterilize soft biomaterials has implications in developing safe sterilization methods for soft biomedical implants such as dermal fillers and viscosupplements.

Utilization of a novel autologous killed tri-vaccine (serogroups B [Typhimurium], C [Mbandaka] and E [Orion]) for Salmonella control in commercial poultry breeders.

An autologous killed trivalent vaccine (3x10(8) colony-forming units [CFU]), based on three Salmonella serovars (Typhimurium - serogroup B, Mbandaka - serogroup C, and Orion - serogroup E) prevalent in the flocks of Australian poultry companies, was developed using Salenvac techniques. At 20 weeks, hens vaccinated at 12 and 17 weeks as well as non-vaccinated hens were challenged (250 microl of 10(7) CFU) with autologous and heterologous serovars belonging to serogroup B (Typhimurium and Agona), serogroup C (Mbandaka and Infantis) and serogroup E (Orion and Zanzibar). Overall, vaccination resulted in a significant difference in carriage of Salmonella between non-vaccinated and vaccinated commercial Cobb hens (P <0.05) for serogroups B and C. However, due to low colonization rates in the non-vaccinated birds, no significant difference (P>0.05) could be determined for serogroup E. All vaccinated flocks produced a significant antibody response (P<0.001) to the S. Typhimurium vaccine strain, measured using a S. Typhimurium enzyme-linked immunosorbent assay (Guildhay), which peaked at 20 weeks of age, with 39% of the hens positive. Maternal antibodies were detected in 16% of the yolks from eggs produced by these flocks. There was a significant difference after challenge with Salmonella (P <0.05) among 1-day-old chicks from vaccinated versus non-vaccinated parents, when challenged using 10(4) CFU but not when challenged with 10(8) CFU. The success of this trial resulted in the incorporation of this vaccine into a Salmonella control system in commercial broiler breeder production.

Yeast ecology of Kombucha fermentation.

Kombucha is a traditional fermentation of sweetened tea, involving a symbiosis of yeast species and acetic acid bacteria. Despite reports of different yeast species being associated with the fermentation, little is known of the quantitative ecology of yeasts in Kombucha. Using oxytetracycline-supplemented malt extract agar, yeasts were isolated from four commercially available Kombucha products and identified using conventional biochemical and physiological tests. During the fermentation of each of the four products, yeasts were enumerated from both the cellulosic pellicle and liquor of the Kombucha. The number and diversity of species varied between products, but included Brettanomyces bruxellensis, Candida stellata, Schizosaccharomyces pombe, Torulaspora delbrueckii and Zygosaccharomyces bailii. While these yeast species are known to occur in Kombucha, the enumeration of each species present throughout fermentation of each of the four Kombucha cultures demonstrated for the first time the dynamic nature of the yeast ecology. Kombucha fermentation is, in general, initiated by osmotolerant species, succeeded and ultimately dominated by acid-tolerant species.