The role of propeptide-mediated autoinhibition and intermolecular chaperone in the maturation of cognate catalytic domain in leucine aminopeptidase.

Leucyl aminopeptidase A from Aspergillus oryzae RIB40 (AO-LapA) is an exo-acting peptidase, widely utilised in food debittering applications. AO-LapA is secreted as a zymogen by the host and requires enzymatic cleavage of the autoinhibitory propeptide to reveal its full activity. Scarcity of structural data of zymogen aminopeptidases hampers a better understanding of the details of their molecular action of autoinhibition and how this might be utilised to improve the properties of such enzymes by recombinant methods for more effective bioprocessing. To address this gap in the literature, herein we report high-resolution crystal structures of recombinantly expressed AO-LapA precursor (AO-proLapA), mature LapA (AO-mLapA) and AO-mLapA complexed with reaction product l-leucine (AO-mLapA-Leu), all purified from Pichia pastoris culture supernatant. Our structures reveal a plausible molecular mechanism of LapA catalytic domain autoinhibition by propeptide and highlights the role of intramolecular chaperone (IMC). Our data suggest an absolute requirement for IMC in the maturation of cognate catalytic domain of AO-LapA. This observation is reinforced by our expression and refolding data of catalytic domain only (AO-refLapA) from Escherichia coli inclusion bodies, revealing a limited active conformation. Our work supports the notion that known synthetic aminopeptidase inhibitors and substrates mimic key polar contacts between propeptide and corresponding catalytic domain, demonstrated in our AO-proLapA zymogen crystal structure. Furthermore, understanding the atomic details of the autoinhibitory mechanism of cognate catalytic domains by native propeptides has wider reaching implications toward synthetic production of more effective inhibitors of bimetallic aminopeptidases and other dizinc enzymes that share an analogous reaction mechanism.

A survey of staffing levels in paediatric diabetes services throughout the UK.

AIMS: To assess staffing levels of healthcare professionals involved in the care of children and young people with diabetes in the UK. METHODS: A web-based questionnaire was distributed to lead consultant paediatricians from all paediatric diabetes services in the UK between October and December 2014. Data on staffing levels and other aspects of diabetes services were collected and differences between the four nations of the UK and across the 10 English diabetes networks were explored. RESULTS: Some 175 services (93%) caring for 29 711 children and young people aged ≤ 24 years with diabetes participated in the survey. Northern Ireland and Wales had the lowest ratio of total staff to patient population. Nursing caseloads per one whole-time equivalent (WTE) nurse ranged from 71 patients in England to 110 patients in Northern Ireland with only 52% of the UK services meeting the Royal College of Nursing recommended nurse-to-patient ratio of > 1 : 70. Scotland and Northern Ireland had the highest ratio of consultants and fully trained doctors per 1000 patients (3.5 WTE). Overall, 17% of consultants had a Certificate of Completion of Training in Endocrinology and Diabetes. Some 44% of dietitians were able to adjust insulin dose. Only 43% of services provided 24-h access to advice from the diabetes team and 82% of services had access to a psychologist. Staffing levels adjusted for volume were not directly related to glycaemic performance of services in England and Wales. CONCLUSIONS: Wide variations in staffing levels existed across the four nations of the UK and important gaps were present in key areas.

Clinic variation in glycaemic control for children with Type 1 diabetes in England and Wales: a population-based, multilevel analysis.

AIM: To understand the scope for improving children's glycaemic outcomes by reducing variation between clinics and examine the role of insulin regimen and clinic characteristics. METHODS: Cross-sectional analysis of 2012-2013 National Paediatric Diabetes Audit data from 21 773 children aged < 19 years with Type 1 diabetes cared for at 176 clinics organized into 11 regional diabetes networks in England and Wales. Variation in HbA1c was explored by multilevel models with a random effect for clinic. The impact of clinic context was quantified by computing the per cent of total variation in HbA1c which occurs between clinics (intraclass correlation coefficient; ICC). RESULTS: Overall, 69 of the 176 diabetes clinics (39%) had a glycaemic performance that differed significantly from the national average after adjusting for patient case-mix with respect to age, gender, diabetes duration, deprivation and ethnicity. However, differences between clinics accounted for 4.7% of the total variation in HbA1c . Inclusion of within-clinic HbA1c standard deviation led to a substantial reduction in ICC to 2.4%. Insulin regimen, clinic volume and diabetes networks had a small or moderate impact on ICC. CONCLUSIONS: Differences between diabetes clinics accounted for only a small portion of the total variation in glycaemic control because most of the variation was within clinics. This implies that national glycaemic improvements might best be achieved not only by targeting poor centres but also by shifting the whole distribution of clinics to higher levels of quality.

Surface and adhesion properties of lactobacilli.

The surface properties of lactobacilli are of significant technological importance as they determine the interaction of the bacterial cells with the gastrointestinal mucosa, and therefore influence their location in the gut and their functionality. Studying the surface of the bacteria is critical for understanding the adhesion process better. This review compiles the knowledge from studies on the characterization Lactobacillus surfaces and evaluates the potential relationship between the cells' physicochemical characteristics and their adhesive abilities. It also discusses the effect that the production processes, such as fermentation and drying, can exert on the surface properties and adhesion abilities of lactobacilli.

Effect of growth time on the surface and adhesion properties of Lactobacillus rhamnosus GG.

AIMS: To investigate the changes in the surface properties of Lactobacillus rhamnosus GG during growth, and relate them with the ability of the Lactobacillus cells to adhere to Caco-2 cells. METHODS AND RESULTS: Lactobacillus rhamnosus GG was grown in complex medium, and cell samples taken at four time points and freeze dried. Untreated and trypsin treated freeze dried samples were analysed for their composition using SDS-PAGE analysis and Fourier transform infrared spectroscopy (FTIR), hydrophobicity and zeta potential, and for their ability to adhere to Caco-2 cells. The results suggested that in the case of early exponential phase samples (4 and 8 h), the net surface properties, i.e. hydrophobicity and charge, were determined to a large extent by anionic hydrophilic components, whereas in the case of stationary phase samples (13 and 26 h), hydrophobic proteins seemed to play the biggest role. Considerable differences were also observed between the ability of the different samples to adhere to Caco-2 cells; maximum adhesion was observed for the early stationary phase sample (13 h). The results suggested that the adhesion to Caco-2 cells was influenced by both proteins and non-proteinaceous compounds present on the surface of the Lactobacillus cells. CONCLUSION: The surface properties of Lact. rhamnosus GG changed during growth, which in return affected the ability of the Lactobacillus cells to adhere to Caco-2 cells. SIGNIFICANCE AND IMPACT OF THE STUDY: The levels of adhesion of Lactobacillus cells to Caco-2 cells were influenced by the growth time and reflected changes on the bacterial surface. This study provides critical information on the physicochemical factors that influence bacterial adhesion to intestinal cells.

Effect of culture medium and cryoprotectants on the growth and survival of probiotic lactobacilli during freeze drying.

AIMS: To investigate the effects of the medium and cryoprotective agents used on the growth and survival of Lactobacillus plantarum and Lactobacillus rhamnosus GG during freeze drying. METHODS AND RESULTS: A complex medium was developed consisting primarily of glucose, yeast extract and vegetable-derived peptone. Trehalose, sucrose and sorbitol were examined for their ability to protect the cells during freeze drying. Using standardized amount of cells and the optimized freeze drying media, the effect of the growth medium on cell survival during freeze drying was investigated. The results showed that glucose and yeast extract were the most important growth factors, while sucrose offered better protection than trehalose and sorbitol during freeze drying. When the cells were grown under carbon limiting conditions, their survival during freeze drying was significantly decreased. CONCLUSIONS: A clear relationship was observed between cell growth and the ability of the cells to survive during the freeze drying process. SIGNIFICANCE AND IMPACT OF THE STUDY: The survival of probiotic strains during freeze drying was shown to be dependent on the cryoprotectant used and the growth medium.

Improving survival of probiotic bacteria using bacterial poly-γ-glutamic acid.

A major hurdle in producing a useful probiotic food product is bacterial survival during storage and ingestion. The aim of this study was to test the effect of γ-PGA immobilisation on the survival of probiotic bacteria when stored in acidic fruit juice. Fruit juices provide an alternative means of probiotic delivery, especially to lactose intolerant individuals. In addition, the survival of γ-PGA-immobilised cells in simulated gastric juice was also assessed. Bifidobacteria strains (Bifidobacteria longum, Bifidobacteria breve), immobilised on 2.5% γ-PGA, survived significantly better (P<0.05) in orange and pomegranate juice for 39 and 11 days respectively, compared to free cells. However, cells survived significantly better (P<0.05) when stored in orange juice compared to pomegranate juice. Moreover, both strains, when protected with 2.5% γ-PGA, survived in simulated gastric juice (pH2.0) with a marginal reduction (<0.47 log CFU/ml) or no significant reduction in viable cells after 4h, whereas free cells died within 2h. In conclusion, this research indicates that γ-PGA can be used to protect Bifidobacteria cells in fruit juice, and could also help improve the survival of cells as they pass through the harsh conditions of the gastrointestinal tract (GIT). Following our previous report on the use of γ-PGA as a cryoprotectant for probiotic bacteria, this research further suggests that γ-PGA could be used to improve probiotic survival during the various stages of preparation, storage and ingestion of probiotic cells.

Evaluation of the effect of malt, wheat and barley extracts on the viability of potentially probiotic lactic acid bacteria under acidic conditions.

In this work, the effect of cereal extracts, used as delivery vehicles for potentially probiotic lactic acid bacteria (LAB), on the acid tolerance of the cells was evaluated under conditions that simulate the gastric tract. More specifically, the effect of malt, barley and wheat extracts on the viability of Lactobacillus plantarum, Lactobacillus acidophilus and Lactobacillus reuteri during exposure for 4 h in a phosphate buffer acidified at pH 2.5 was investigated. In the absence of cereal extracts all strains demonstrated a significant reduction in their cell population, particularly L. plantarum. The viability of L. plantarum was improved by approximately 4 log(10) cycles in the presence of malt and 3 log(10) cycles in the presence of wheat and barley. The survival of L. acidophilus and L. reuteri was increased by more than 1.5 and 0.7 log(10) cycle, respectively, upon addition of cereal extracts. In order to evaluate the contribution of the cereal constituents on cell survival, the individual effect of glucose, maltose and free amino nitrogen (FAN), which were added at concentrations that correlated to the reducing sugar and FAN content of the cereal extracts, was examined. The viability of L. plantarum was progressively improved as the maltose or glucose concentration increased; an increase by approximately 2 log(10) cycles was observed in the presence of 8.33 g/l sugar. The survival of L. acidophilus increased by more than 1 log(10) cycle, even at very low concentrations of maltose and glucose (e.g., 0.67 g/l), while L. reuteri stability was enhanced in the presence of maltose but no appreciable effect was demonstrated in the presence of glucose. Sugar analysis indicated that glycolysis was inhibited in all cases. Addition of tryptone and yeast extract, used as sources of FAN, enhanced L. acidophilus acid tolerance, but did not affect L. reuteri and L. plantarum. The results presented in this study indicate that malt, wheat and barley extracts exhibit a significant protective effect on the viability of L. plantarum, L. acidophilus and L. reuteri under acidic conditions, which could be mainly attributed to the amount of sugar present in the cereal extracts.

Application of cereals and cereal components in functional foods: a review.

The food industry is directing new product development towards the area of functional foods and functional food ingredients due to consumers' demand for healthier foods. In this respect, probiotic dairy foods containing human-derived Lactobacillus and Bifidobacterium species and prebiotic food formulations containing ingredients that cannot be digested by the human host in the upper gastrointestinal tract and can selectively stimulate the growth of one or a limited number of colonic bacteria have been recently introduced into the market. The aim of these products is to affect beneficially the gut microbial composition and activities. Cereals offer another alternative for the production of functional foods. The multiple beneficial effects of cereals can be exploited in different ways leading to the design of novel cereal foods or cereal ingredients that can target specific populations. Cereals can be used as fermentable substrates for the growth of probiotic microorganisms. The main parameters that have to be considered are the composition and processing of the cereal grains, the substrate formulation, the growth capability and productivity of the starter culture, the stability of the probiotic strain during storage, the organoleptic properties and the nutritional value of the final product. Additionally, cereals can be used as sources of nondigestible carbohydrates that besides promoting several beneficial physiological effects can also selectively stimulate the growth of lactobacilli and bifidobacteria present in the colon and act as prebiotics. Cereals contain water-soluble fibre, such as beta-glucan and arabinoxylan, oilgosaccharides, such as galacto- and fructo-oligosaccharides and resistant starch, which have been suggested to fulfil the prebiotic concept. Separation of specific fractions of fibre from different cereal varieties or cereal by-products, according to the knowledge of fibre distribution in cereal grains, could be achieved through processing technologies, such as milling, sieving, and debranning or pearling. Finally, cereal constituents, such as starch, can be used as encapsulation materials for probiotics in order to improve their stability during storage and enhance their viability during their passage through the adverse conditions of the gastrointestinal tract. It could be concluded that functional foods based on cereals is a challenging perspective, however, the development of new technologies of cereal processing that enhance their health potential and the acceptability of the food product are of primary importance.

Determinants of adherence to gluten-free diet in Greek children with coeliac disease: a cross-sectional study.

BACKGROUND/OBJECTIVE: A strict and lifelong commitment to a gluten-free diet (GFD) remains one of the most challenging issues in children with coeliac disease. The present study aimed to record compliance rates and investigate the connection between dietary compliance and demographics, disease-related factors and parental knowledge. SUBJECTS/METHODS: Parents of 90 Greek children diagnosed with coeliac disease were recruited from the outpatient gastroenterology clinic of a children's hospital in Athens, Greece. Dietary compliance and a range of demographic and clinical data were obtained from parents through a specially constructed questionnaire. Further data included parental perceived and actual knowledge about coeliac disease and GFD. Compliant and noncompliant groups were compared for measured factors and a multivariate approach was followed to elicit independent effects of compliance determinants. RESULTS: Overall, 44.4% of children with coeliac disease were reported to be compliant to a strict GFD. A 1-year increase in the age of the child was associated with 15% lower odds of adhering to a strict diet after adjusting for other variables (odds ratio (OR)=0.85, 95% CI: 0.75-0.96). Parental perceived knowledge was also independently and significantly associated with dietary compliance (OR=3.3, 95% CI=1.1-9.8). No statistically significant correlation emerged between dietary compliance and other clinical or demographic variables. DISCUSSION: Low compliance rates to GFD were observed in children with coeliac disease. Information based on children's age and perceived parental knowledge can be used to develop risk profiles that health care professionals can utilise to identify children likely to be noncompliant and thus adjust their counselling strategy accordingly.

Emulsifying, rheological and physicochemical properties of exopolysaccharide produced by Bifidobacterium longum subsp. infantis CCUG 52486 and Bifidobacterium infantis NCIMB 702205.

The rheological, emulsification and certain physicochemical properties of purified exopolysaccharides (EPS) of Bifidobacterium longum subsp. infantis CCUG 52486 and Bifidobacterium infantis NCIMB 702205 were studied and compared with those of guar gum and xanthan gum. The two strains were grown in skim milk supplemented with 1.5% (w/v) casein hydrolysate at 37 °C for 24h; they both produced heteropolysaccharides with different molecular mass and composition. The carbohydrate content of both polymers was more than 92% and no protein was detected. The EPS of B. longum subsp. infantis CCUG 52486 showed highly branched entangled porous structure under scanning electron microscopy. Higher intrinsic viscosity was observed for the EPS of B. longum subsp. infantis CCUG 52486 compared to the EPS of B. infantis NCIMB 702205 and guar gum. Both polymers showed pseudoplastic non-Newtonian fluid behaviour in an aqueous solution. The EPS of B. infantis NCIMB 702205 and B. longum subsp. infantis CCUG 52486 produced more stable emulsions with orange oil, sunflower seed oil, coconut oil and xylene compared to guar and xanthan gum. The EPS of B. longum subsp. infantis CCUG 52486 is the most promising one for applications in the food industry, as it had higher intrinsic viscosity, higher apparent viscosity in aqueous solution, porous dense entangled structure and good emulsification activity.

Effect of food models and low-temperature storage on the adhesion of Lactobacillus rhamnosus GG to Caco-2 cells.

This study evaluated the effects of fat and sugar levels on the surface properties of Lactobacillus rhamnosus GG during storage in food model systems, simulating yogurt and ice cream, and related them with the ability of the bacterial cells to adhere to Caco-2 cells. Freeze-dried L. rhamnosus GG cells were added to the model food systems and stored for 7 days. The bacterial cells were analyzed for cell viability, hydrophobicity, ζ potential, and their ability to adhere to Caco-2 cells. The results indicated that the food type and its composition affected the surface and adhesion properties of the bacterial cells during storage, with yogurt being a better delivery vehicle than ice cream in terms of bacterial adhesion to Caco-2 cells. The most important factor influencing bacterial adhesion was the storage time rather than the levels of fats and sugars, indicating that conformational changes were taking place on the surface of the bacterial cells during storage.

Growth studies of potentially probiotic lactic acid bacteria in cereal-based substrates.

AIMS: The overall growth kinetics of four potentially probiotic strains (Lactobacillus fermentum, Lact. reuteri, Lact. acidophilus and Lact. plantarum) cultured in malt, barley and wheat media were investigated. The objectives were to identify the main factors influencing the growth and metabolic activity of each strain in association with the cereal substrate. METHODS AND RESULTS: All fermentations were performed without pH control. A logistic-type equation, which included a growth inhibition term, was used to describe the experimental data. In the malt medium, all strains attained high maximum cell populations (8.10-10.11 log10 cfu ml(-1), depending on the strain), probably due to the availability of maltose, sucrose, glucose, fructose (approx. 15 g l(-1) total fermentable sugars) and free amino nitrogen (approx. 80 mg l(-1)). The consumption of sugars during the exponential phase (10-12 h) resulted in the accumulation of lactic acid (1.06-1.99 g l(-1)) and acetic acid (0.29-0.59 g l(-1)), which progressively decreased the pH of the medium. Each strain demonstrated a specific preference for one or more sugars. Since small amounts of sugars were consumed by the end of the exponential phase (17-43%), the decisive growth-limiting factor was probably the pH, which at that time ranged between 3.40 and 3.77 for all of the strains. Analysis of the metabolic products confirmed the heterofermentative or homofermentative nature of the strains used, except in the case of Lact. acidophilus which demonstrated a shift towards the heterofermentative pathway. All strains produced acetic acid during the exponential phase, which could be attributed to the presence of oxygen. Lactobacillus plantarum, Lact. reuteri and Lact. fermentum continued to consume the remaining sugars and accumulate metabolic products in the medium, probably due to energy requirements for cell viability, while Lact. acidophilus entered directly into the decline phase. In the barley and wheat media all strains, especially Lact. acidophilus and Lact. reuteri, attained lower maximum cell populations (7.20-9.43 log10 cfu ml(-1)) than in the malt medium. This could be attributed to the low sugar content (3-4 g l(-1) total fermentable sugar for each medium) and the low free amino nitrogen concentration (15.3-26.6 mg l(-1)). In all fermentations, the microbial growth ceased at pH values (3.73-4.88, depending on the strain) lower than those observed for malt fermentations, which suggests that substrate deficiency in sugars and free amino nitrogen contributed to growth limitation. CONCLUSIONS: The malt medium supported the growth of all strains more than barley and wheat media due to its chemical composition, while Lact. plantarum and Lact. fermentum appeared to be less fastidious and more resistant to acidic conditions than Lact. acidophilus and Lact. reuteri. SIGNIFICANCE AND IMPACT OF THE STUDY: Cereals are suitable substrates for the growth of potentially probiotic lactic acid bacteria.