Enteric coated spheres produced by extrusion/spheronization provide effective gastric protection and efficient release of live therapeutic bacteria

We present a novel but simple enteric coated sphere formulation containing probiotic bacteria (Lactobacillus casei). Oral delivery of live bacterial cells (LBC) requires live cells to survive firstly manufacturing processes and secondly GI microbicidal defenses including gastric acid. We incorporated live L. casei directly in the granulation liquid, followed by granulation, extrusion, spheronization, drying and spray coating to produce dried live probiotic spheres. A blend of MCC, calcium-crosslinked alginate, and lactose was developed that gave improved live cell survival during manufacturing, and gave excellent protection from gastric acid plus rapid release in intestinal conditions. No significant loss of viability was observed in all steps except drying, which resulted in approximately 1 log loss of viable cells. Eudragit coating was used to protect dried live cells from acid, and microcrystalline cellulose (MCC) was combined with sodium alginate to achieve efficient sphere disintegration leading to rapid and complete bacterial cell release in intestinal conditions. Viability and release of L. casei was evaluated in vitro in simulated GI conditions. Uncoated spheres gave partial acid protection, but enteric coated spheres effectively protected dried probiotic LBC from acid for 2 h, and subsequently released all viable cells within 1h of transfer into simulated intestinal fluid.

Stability of probiotic Lactobacillus plantarum in dry microcapsules under accelerated storage conditions

This study investigated the stability of freeze dried and fluid bed dried alginate microcapsules coated with chitosan containing model probiotic bacteria, Lactobacillus plantarum, during storage for up to 45 days at different water activities (0.11, 0.23, 0.40 and 0.70) and temperatures (4, 30 and 37 °C). The loss in cell viability was around 0.8 log in the case of fluid bed drying and around 1.3 in the case of freeze drying, with the former method resulting in dried capsules of smaller size (~ 1 mm vs 1.3 mm), more irregular shape, and with a rougher surface. In both cases, the water activity and water content were less than 0.25 and 10% w/w, respectively, which favours high storage stability. The storage stability studies demonstrated that as the water activity and temperature decreased the survival of the dried encapsulated cells increased. Considerably better survival was observed for fluid bed dried encapsulated cells compared to freeze dried encapsulated cells and freeze dried free cells with 10% sucrose (control), and in some cases, e.g. at 4 and 30 °C at water activities of 0.11, 0.23 and 0.40, there was more than 1 log difference after 45 days, with concentrations higher than 108 CFU/g after 45 days of storage. The results indicate that fluid bed drying is an effective and efficient manufacturing method to produce probiotic containing capsules with enhanced storage stability.

In vitro study on the cell adhesion ability of immobilized lactobacilli on natural supports

The aim of the present study was to investigate the effect of probiotic immobilization onto wheat grains, both wet and freeze dried, on the adhesion properties of the probiotic cells and make comparisons with wet and freeze dried free cells. Lactobacillus casei ATCC 393 and Lactobacillus plantarum NCIMB 8826 were used as model probiotic strains. The results showed satisfactory adhesion ability of free cells to a monolayer of Caco-2 cells (> 1000 CFU/100 Caco-2 cells for wet cells). Cell immobilization resulted in a significant decrease in adhesion, for both wet and freeze dried formulations, most likely because immobilized cells did not have direct access to the Caco-2 cells, but it still remained in adequate levels (> 100 CFU/100 Caco-2 cells for wet cells). No clear correlation could be observed between cell adhesion and the hydrophobicity of the bacterial cells, measured by the hexadecane adhesion assay. Most notably, immobilization enhanced the monolayer integrity of Caco-2 cells, demonstrated by a more than 2-fold increase in transepithelial electrical resistance (TEER) compared to free cells. SEM micrographs ascertained the adhesion of both immobilized and free cells to the brush border microvilli. Finally, the impact of the food matrix on the adhesion properties of probiotic bacteria and on the design of novel functional products is discussed.

Effect of air-drying pre-treatment on the characterization of forest soil carbon pools

Archived soils could represent a valuable resource for the spatio-temporal inventory of soil carbon stability. However, archived soils are usually air-dried before storage and the impact of a drying pretreatment on physically and chemically-defined C fractions has not yet been fully assessed. Through the comparison of field-moist and corresponding air-dried (at 25oC for 2 weeks) forest soil samples, we examined the effect of air-drying on: a) the quantity and the quality of cold- (CWEC) and hot-water (HWEC) extractable C and b) the concentration of C in physically isolated fractions (free- and intra-aggregate light and organo-mineral). Soil samples were collected from the organic (O) and mineral (A and B) horizons of three different forest soils from southeastern England: (i) Cambisol under Pine (Pinus nigra); (ii) Cambisol under Beech (Fagus sylvatica) and (iii) Gleysol under oak (Quercus robur). CWEC concentrations for dry samples were up to 2 times greater than for corresponding field moist samples and had significantly (p < 0.001) higher phenolic content. However, the effect of drying pretreatment on HWEC, its phenolic content was not significant (p > 0.05) for most samples. Dried soils had significantly (p < 0.001) higher concentrations of free light-C while having lower concentrations of intra-aggregate-C when compared to moist samples (p < 0.001). However, fine silt and clay fractions were not significantly affected by the drying pretreatment (p=0.789). Therefore, based on the results obtained from gleysol and cambisol forest soils studied here, C contents in hot-water extractions and fine particle size physical fractions (< 25µm) seem to be robust measurements for evaluating C fractions in dried stored forest soils. Further soil types should be tested to evaluate the wider generality of these findings.

Extractability and characteristics of proteins deriving from wheat DDGS

Wheat Distillers’ Dried Grains with Solubles (DDGS) and in-process samples were used for protein extraction. Prolamins were the predominant protein components in the samples. The absence of extractable α- and γ-gliadins in DDGS indicated protein aggregation during the drum drying processing stage. Prolamin extraction was performed using 70% (v/v) ethanol or alkaline-ethanol solution in the presence of reducing agent. DDGS extracts had relatively low protein contents (14-44.9%, w/w), regardless of the condition applied. The wet solids were the most suitable raw material for protein extraction, with recovery yields of ~ 55% (w/w) and protein content of ~58% (w/w) in 70% (v/v) ethanol. Protein extracts from wet solids were significantly rich in glutamic acid and proline. Mass balance calculations demonstrated the high carbohydrate content (~ 50%, w/w) of solid residues. Overall, the feasibility of utilising in-process samples of DDGS for protein extraction with commercial potential was demonstrated.

Cognitive effects following acute wild blueberry supplementation in 7 – 10 year old children

Purpose: Previously, anthocyanin-rich blueberry treatments have shown positive effects on cognition in both animals and human adults. However, little research has considered whether these benefits transfer to children. Here we describe an acute time-course and dose–response investigation considering whether these cognitive benefits extend to children. Methods: Using a double-blind cross-over design, on three occasions children (n = 21; 7–10 years) consumed placebo (vehicle) or blueberry drinks containing 15 or 30 g freeze-dried wild blueberry (WBB) powder. A cognitive battery including tests of verbal memory, word recognition, response interference, response inhibition and levels of processing was performed at baseline, and 1.15, 3 and 6 h following treatment. Results: Significant WBB-related improvements included final immediate recall at 1.15 h, delayed word recognition sustained over each period, and accuracy on cognitively demanding incongruent trials in the interference task at 3h. Importantly, across all measures, cognitive performance improved, consistent with a dose–response model, with the best performance following 30 g WBB and the worst following vehicle. Conclusion: Findings demonstrate WBB-related cognitive improvements in 7- to 10-year-old children. These effects would seem to be particularly sensitive to the cognitive demand of task.

Short communication: estimation of the financial benefit of using Jersey milk at different inclusion rates for Cheddar cheese production using partial budgeting

Partial budgeting was used to estimate the net benefit of blending Jersey milk in Holstein-Friesian milk for Cheddar cheese production. Jersey milk increases Cheddar cheese yield. However, the cost of Jersey milk is also higher; thus, determining the balance of profitability is necessary, including consideration of seasonal effects. Input variables were based on a pilot plant experiment run from 2012 to 2013 and industry milk and cheese prices during this period. When Jersey milk was used at an increasing rate with Holstein-Friesian milk (25, 50, 75, and 100% Jersey milk), it resulted in an increase of average net profit of 3.41, 6.44, 8.57, and 11.18 pence per kilogram of milk, respectively, and this additional profit was constant throughout the year. Sensitivity analysis showed that the most influential input on additional profit was cheese yield, whereas cheese price and milk price had a small effect. The minimum increase in yield, which was necessary for the use of Jersey milk to be profitable, was 2.63, 7.28, 9.95, and 12.37% at 25, 50, 75, and 100% Jersey milk, respectively. Including Jersey milk did not affect the quantity of whey butter and powder produced. Althoug further research is needed to ascertain the amount of additional profit that would be found on a commercial scale, the results indicate that using Jersey milk for Cheddar cheese making would lead to an improvement in profit for the cheese makers, especially at higher inclusion rates.

Self-assembled arginine-capped peptide bolaamphiphile nanosheets for cell culture and controlled wettability surfaces

The spontaneous assembly of a peptide bolaamphiphile in water, namely, RFL4FR (R, arginine; F, phenylalanine; L, leucine) is investigated, along with its novel properties in surface modification and usage as substrates for cell culture. RFL4FR self-assembles into nanosheets through lateral association of the peptide backbone. The L4 sequence is located within the core of the nanosheets, whereas the R moieties are exposed to the water at the surface of the nanosheets. Kinetic assays indicate that the self-assembly is driven by a remarkable two-step process, where a nucleation phase is followed by fast growth of nanosheets with an autocatalysis process. The internal structure of the nanosheets is formed from ultrathin bolaamphiphile monolayers with a crystalline orthorhombic symmetry with cross-β organization. We show that human corneal stromal fibroblast (hCSF) cells can grow on polystyrene films coated with films dried from RFL4FR solutions. For the first time, this type of amphiphilic peptide is used as a substrate to modulate the wettability of solid surfaces for cell culture applications.

Design and techno-economic evaluation of microbial oil production as a renewable resource for biodiesel and oleochemical production

Experimental results from the open literature have been employed for the design and techno-economic evaluation of four process flowsheets for the production of microbial oil or biodiesel. The fermentation of glucose-based media using the yeast strain Rhodosporidium toruloides has been considered. Biodiesel production was based on the exploitation of either direct transesterification (without extraction of lipids from microbial biomass) or indirect transesterifaction of extracted microbial oil. When glucose-based renewable resources are used as carbon source for an annual production capacity of 10,000 t microbial oil and zero cost of glucose (assuming development of integrated biorefineries in existing industries utilising waste or by-product streams) the estimated unitary cost of purified microbial oil is $3.4/kg. Biodiesel production via indirect transesterification of extracted microbial oil proved more cost-competitive process compared to the direct conversion of dried yeast cells. For a price of glucose of $400/t oil production cost and biodiesel production cost are estimated to be $5.5/kg oil and $5.9/kg biodiesel, correspondingly. Industrial implementation of microbial oil production from oleaginous yeast is strongly dependent on the feedstock used and on the fermentation stage where significantly higher productivities and final microbial oil concentrations should be achieved.

Evaluating and optimizing oral formulations of live bacterial vaccines using a gastro-small intestine model

Gastrointestinal (GI) models that mimic physiological conditions in vitro are important tools for developing and optimizing biopharmaceutical formulations. Oral administration of live attenuated bacterial vaccines (LBV) can safely and effectively promote mucosal immunity but new formulations are required that provide controlled release of optimal numbers of viable bacterial cells, which must survive gastrointestinal transit overcoming various antimicrobial barriers. Here, we use a gastro-small intestine gut model of human GI conditions to study the survival and release kinetics of two oral LBV formulations: the licensed typhoid fever vaccine Vivotif comprising enteric coated capsules; and an experimental formulation of the model vaccine Salmonella Typhimurium SL3261 dried directly onto cast enteric polymer films and laminated to form a polymer film laminate (PFL). Neither formulation released significant numbers of viable cells when tested in the complete gastro-small intestine model. The poor performance in delivering viable cells could be attributed to a combination of acid and bile toxicity plus incomplete release of cells for Vivotif capsules, and to bile toxicity alone for PFL. To achieve effective protection from intestinal bile in addition to effective acid resistance, bile adsorbent resins were incorporated into the PFL to produce a new formulation, termed BR-PFL. Efficient and complete release of 4.4x107 live cells per dose was achieved from BR-PFL at distal intestinal pH, with release kinetics controlled by the composition of the enteric polymer film, and no loss in viability observed in any stage of the GI model. Use of this in vitro GI model thereby allowed rational design of an oral LBV formulation to maximize viable cell release.

Can milk proteins be a useful tool in the management of cardiometabolic health? An updated review of human intervention trials

The prevalence of cardiometabolic diseases is a significant public health burden worldwide. Emerging evidence supports the inverse association between greater dairy consumption and reduced risk of cardiometabolic diseases. Dairy proteins may have in important role in the favourable impact of dairy on human health such as blood pressure (BP) control, blood lipid and glucose control. The purpose of this review is to update and critically evaluate the evidence on the impacts of casein and whey protein in relation to metabolic function. Evidence from acute clinical studies assessing postprandial responses to milk protein ingestion suggests benefits on vascular function independent of BP, as well as improvement in glycaemic homeostasis. Chronic interventions have been less conclusive, with some showing benefits and others indicating a lack of improvement in vascular function. During chronic consumption BP appears to be lowered and both dyslipidaemia and hyperglacaemia seems to be controlled. Limited number of trials investigated the effects of dairy proteins on oxidative stress and inflammation. The beneficial changes in cardiometabolic homeostasis are likely mediated through improvements in insulin resistance, however to gain more detailed understanding on the underlying mechanism of milk proteins warrants further research. The incorporation of meals enriched with dairy protein in the habitual diet may result in the beneficial effects on cardiometabolic health. Nevertheless, future well-designed, controlled studies are needed to investigate the relative effects of both casein and whey protein on BP, vascular function, glucose homeostasis and inflammation.

Biofilm on the apical region of roots in primary teeth with vital and necrotic pulps with or without radiographically evident apical pathosis

Aim To evaluate, by scanning electron microscopy (SEM), the presence of biofilms on the external surfaces of the apical third of roots of human primary teeth with vital or necrotic pulps with and without radiographically evident periradicular pathosis. Methodology Eighteen teeth were selected: group I - normal pulp (n = 5), group II - pulp necrosis without radiographic evidence of periapical pathosis (n = 7) and group III - pulp necrosis with well-defined radiographic periapical pathosis (n = 6). After extraction, the teeth were washed with saline and immersed in 0.03 g mL(-1) trypsin solution for 20 min. The teeth were then washed in sodium cacodilate buffer and stored in receptacles containing modified Karnovsky solution. The teeth were sectioned, dehydrated in an ethanol series, critical-point dried with CO(2), sputter coated with gold and the external root surface in the apical third examined by SEM. Results In the teeth of groups I and II, the apical root surfaces were covered by collagen fibres, with no evidence of bacteria (100%). In the teeth of group III, the root apices had no collagen fibres but revealed resorptive areas containing microorganisms (cocci, bacilli, filaments and spirochetes) in all cases (100%). Conclusion Microorganisms organized as biofilms on the external root surface (extraradicular infection) were detected in primary teeth with pulp necrosis and radiographically visible periapical pathosis.

Effect of blood contamination on the shear bond strength at resin/dentin interface in primary teeth

Purpose: To assess in vitro the shear bond strength at the resin/dentin interface in primary teeth after contamination with fresh human blood. Methods: 75 crowns of primary molars were embedded in acrylic resin and mechanically ground to expose a flat dentin surface. The specimens were randomly assigned to five groups (n=15), according to the surface treatment. Group I (control) had no blood contamination. The other groups were blood-contaminated and subjected to different post-contamination protocols: in Group 2, the surfaces were rinsed with water; in Group 3, the surfaces were air-dried; in Group 4, the surfaces were rinsed and air-dried; and in Group 5, no post-contamination treatment was done. In all groups, a 3-mm dentin bonding site was demarcated, Single Bond adhesive system was applied and resin composite cylinders were bonded. After 24 hours in distilled water, shear bond strength was tested at a crosshead speed of 0.5 mm/minute. Results: Means (in MPa) were: Group 1: 7.1 (+/- 4.2); Group 2: 4.0 (+/- 1.8); Group 3: 0.9 (+/- 0.7); Group 4: 3.9 (+/- 2.2) and Group 5: 1.3 (+/- 1.5). Data were analyzed statistically by the Kruskal-Wallis test at 5% significance level. Groups 2 and 4 were similar to each other (P > 0.05) and both ware similar to Group 1 (P > 0.05). These groups (2, 3 and 4) had statistically significantly higher bond strengths than Groups 3 and 5 (P < 0.05). Blood contamination negatively affected the shear bond strength to primary tooth dentin. Among the blood-contaminated groups, water-rinsed specimens had higher bond strengths than those that were exclusively air-dried or not submitted to any post-contamination protocol before adhesive application.

3-D volume rendering maxillofacial analysis of angular measurements by multislice CT

Objective. This study was designed to determine the precision and accuracy of angular measurements using three-dimensional computed tomography (3D-CT) volume rendering by computer systems. Study design. The study population consisted of 28 dried skulls that were scanned with a 64-row multislice CT, and 3D-CT images were generated. Angular measurements, (n = 6) based upon conventional craniometric anatomical landmarks (n = 9), were identified independently in 3D-CT images by 2 radiologists, twice each, and were then performed by 3D-CT imaging. Subsequently, physical measurements were made by a third examiner using a Beyond Crysta-C9168 series 900 device. Results. The results demonstrated no statistically significant difference between interexaminer and intraexaminer analysis. The mean difference between the physical and 3-D-based angular measurements was -1.18% and -0.89%, respectively, for both examiners, demonstrating high accuracy. Conclusion. Maxillofacial analysis of angular measurements using 3D-CT volume rendering by 64-row multislice CT is established and can be used for orthodontic and dentofacial orthopedic applications.

Amaranth bars enriched with fructans: acceptability and nutritional value

Amaranth bars enriched with fructans: acceptability and nutritional value. There is an increasing appeal for convenience foods with potential health benefits to the consumer. Raw materials with high nutritional value and functional properties must be used on the development of these food products. Amaranth is a gluten-free grain with high nutrition value. Inulin and oligofructose are prebiotic ingredients presenting effects as the enhancement of calcium absorption. Amaranth bars enriched with inulin and oligofructose were developed in the flavors: banana, Brazilian nuts and dried grape, coconut, peach, strawberry and wall nut. The proximate composition were determined and compared to commercial cereal bars, available in traditional (n=59), light (n=60), diet (n=8), with soy (n=10) and quinoa (n=1) categories. Amaranth bars present mean global acceptance values from 6.3 to 7.6 on a 9-point hedonic scale, nutritional advantages as compared to commercial cereal bars (caloric reduction and higher levels of dietary fiber). Although amaranth is an unknown raw material in Brazil, it shows good potential to be used in the manufacturing of ready-to-eat products. As they are gluten free, these amaranth bars are also an alternative product for celiacs, also contributing to the enhancement of calcium absorption, a problem frequently observed in these patients.