Encapsulation of Bifidobacterium longum in alginate-dairy matrices and survival in simulated gastrointestinal conditions, refrigeration, cow milk and goat milk

The aim of this study was to microencapsulate Bifidobacterium longum subsp. infantis CCUG 52486 using the extrusion method in a variety of matrices, namely sodium alginate (SA), sodium alginate-cow milk (SACM), sodium alginate-goat milk (SAGM) and sodium alginate-casein hydrolysate (SACH), and to evaluate the survival of free and encapsulated bacterial cells under different conditions. The encapsulation yield, size and surface morphology of the microcapsules were evaluated. The survival of microencapsulated bacterial cells and free bacterial cells were evaluated under simulated gastrointestinal conditions as well as in refrigeration, cow milk and goat milk during storage at 4 oC for 28 days. The average size of SACM capsules and SAGM capsules was 2.8±0.3 mm and 3.1±0.2 mm respectively. Goat milk and cow milk based matrices resulted in dense microcapsules which led to better performances in simulated gastrointestinal conditions than SA and SACH microcapsules. The bacterial cells encapsulated in SAGM showed the highest survival rate in cow milk (7.61 log cfu g-1) and goat milk (8.10 log cfu g-1) after the storage of 28 d. The cells encapsulated in SA and SACH and the free cells performed poorly under the simulated gastrointestinal conditions and in all different storage conditions. This study showed that SACM and SAGM are suitable to encapsulate B. longum subsp. infantis CCUG 52486 using the extrusion technique and more specifically, SAGM has a potential to be used as a new encapsulation material for encapsulating probiotic bacteria, resulting milk and goat milk-based products with higher probiotic cell concentrations during refrigerated storage

Poly-(γ-glutamic acid) Production and Optimization from Agro-Industrial Bioresources as Renewable Substrates by Bacillus sp. FBL-2 through Response Surface Methodology

We optimized culture conditions using Bacillus sp. FBL-2 as a poly-(γ-glutamic acid) (PGA) producing strain isolated from cheonggukjang. All experiments were performed under aerobic conditions using a laboratory scale 2.5 L fermentor. We investigated the effects of fermentation parameters (temperature, pH, agitation, and aeration) and medium components (glutamic acid, citric acid, and yeast extract) on poly-(γ-glutamic acid) production, viscosity, and dry cell mass. A non-optimized fermentation method (1.5 vvm, 350 rpm, and 37 °C) yielded PGA, viscosity, and dry cell mass at levels of 100.7 g/L, 483.2 cP, and 3.4 g/L, respectively. L-glutamic acid, citric acid, and yeast extract supplementation enhanced poly-(γ-glutamic acid) production to 175.9 g/L. Additionally, the production of poly-(γ-glutamic acid) from rice bran and wheat bran was assessed using response surface methodology (central composite rotatable design). Agricultural by-products (rice bran and wheat bran) and H2SO4 were selected as factors, and experiments were performed by combining various component concentrations to determine optimal component concentrations. Our experimentally-derived optimal parameters included 38.6 g/L of rice bran, 0.42% of H2SO4, 28.0 g/L of wheat bran, and 0.32% of H2SO4. Under optimum conditions, rice bran medium facilitated poly-(γ-glutamic acid) production of up to 22.64 g/L, and the use of wheat bran medium yielded up to 14.6 g/L. Based on a validity test using the optimized culture conditions, poly-(γ-glutamic acid) was produced at 47.6 g/L and 36.4 g/L from these respective mediums, and both results were higher than statistically predicted. This study suggests that rice bran can be used as a potential alternative substrate for poly-(γ-glutamic acid) production

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

Probiotic microcarrier: a continuous folate producer

The recommended daily intake of folate (B-complex vitamin) for an adult varies between 200-400 µg, being the intake of folate inefficient due its extremely unstable chemical forms. The aim of this work is the creation of model to folate in situ production using probiotics. However, three main issues need to be overcome: (a) probiotic bacteria should be protected towards the gastric medium (encapsulation); (b) microcarriers size should be smaller than 100 µm, to avoid modifying food texture; and (c) microcarriers should adhere to gut epithelium in order to increase bacteria residence time. Lactococcus lactis cremoris was grown in milk (30ºC). Alginate-based microcarriers were produced and three layers were built using the layer-by-layer technique in that worder: poly-L-lysine; sodium alginate; chitosan. Confocal microscopy was used to confirm the consequent adhesion of the layers (poly-L-lysine/FITC; chitosan/rhodamine). After production the microcarriers where put into a 10 mL solution of KCl-HCl (pH 2 - 1 hour), at 100 rpm and then into a PBS solution (pH 7.2 - 3 hours) in order to mimic the passage through the gastrointestinal tract. The utilization of free bacteria (LLC) in milk showed an increase of folate content in 4.73 µg/L after 6 h. The average size of the microcarriers from 21.01 ± 0.49 µm to 39.84 ± 0.79 µm when the pH increased from 2 to 7.2. The size averages obtained were smaller than 100 µm and showed a swelling capacity (particles duplicate their size upon passing from pH 2 to pH 7.2), being confirmed by confocal microscopy images the correct adhesion of the different layers after this experiment and the stability of the microcarriers. Microcarriers produced through LbL showed great potential for encapsulation of probiotics, allowing their protection against harsh gastrointestinal conditions, predicting their use as a microcarrier for in situ folate production

Combining creative writing and narrative analysis to deliver new insights into the impact of pulmonary hypertension

Introduction Pulmonary hypertension is life-limiting. Delays in diagnosis are common, and even after treatment has been initiated, pulmonary hypertension has marked effects on many aspects of social and physical function. We believed that a new approach to examining disease impact could be achieved through a combination of narrative research and creative writing. Methods Detailed unstructured narrative interviews with people with pulmonary hypertension were analysed thematically. Individual moments were also summarised and studied using creative writing, in which the interviewer created microstories from narrative and interview data. Stories were shared with their subjects, and with other patients, clinicians, researchers and the wider public. The study was carried out in hospital and in patients’ homes. Results Narrative analysis generated a rich data set which highlighted profound effects of pulmonary hypertension on identity, and demonstrated how the disease results in very marked personal change with ongoing and unpredictable requirement for adaptation. The novel methodology of microstory development proved to be an effective tool to summarise, communicate, and explore the consequences of pulmonary hypertension and the clinical challenges of caring for patients with this illness. Conclusions A holistic approach to treatment of chronic respiratory diseases such as pulmonary hypertension requires and benefits from explicit exploration of the full impacts of the illness. Narrative analysis and the novel approach of targeted microstory development can form a valuable component of the repertoire of approaches to effectively comprehend chronic disease and can also facilitate patient-focused discussion and interventions

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 (B. longum, B. 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 (pH 2.0) with a marginal reduction (<0.47 log CFU/ml) or no significant reduction in viable cells after four hours, whereas free cells died within two hours. 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

Lipoprotein-Associated Phospholipase A2 Bound on High-Density Lipoprotein Is Associated With Lower Risk for Cardiac Death in Stable Coronary Artery Disease Patients A 3-Year Follow-Up

ObjectivesThe aim of this study was to examine the prognostic value of lipoprotein-associated phospholipase A2 (Lp-PLA2) associated with high-density lipoprotein (HDL) (HDL-Lp-PLA2) in patients with stable coronary artery disease (CAD).BackgroundLp-PLA2 is a novel risk factor for cardiovascular disease. It has been postulated that the role of Lp-PLA2 in atherosclerosis may depend on the type of lipoprotein with which it is associated.MethodsTotal plasma Lp-PLA2 and HDL-Lp-PLA2 mass and activity, lipids, and C-reactive protein were measured in 524 consecutive patients with stable CAD who were followed for a median of 34 months. The primary endpoint was cardiac death, and the secondary endpoint was hospitalization for acute coronary syndromes, myocardial revascularization, arrhythmic event, or stroke.ResultsFollow-up data were obtained from 477 patients. One hundred twenty-three patients (25.8%) presented with cardiovascular events (24 cardiac deaths, 47 acute coronary syndromes, 28 revascularizations, 22 arrhythmic events, and 2 strokes). Total plasma Lp-PLA2 mass and activity were predictors of cardiac death (hazard ratio [HR]: 1.013; 95% confidence interval [CI]: 1.005 to 1.021; p = 0.002; and HR: 1.040; 95% CI: 1.005 to 1.076; p = 0.025, respectively) after adjustment for traditional risk factors for CAD. In contrast, HDL-Lp-PLA2 mass and activity were associated with lower risk for cardiac death (HR: 0.972; 95% CI: 0.952 to 0.993; p = 0.010; and HR: 0.689; 95% CI: 0.496 to 0.957; p = 0.026, respectively) after adjustment for traditional risk factors for CAD.ConclusionsTotal plasma Lp-PLA2 is a predictor of cardiac death, while HDL-Lp-PLA2 is associated with lower risk for cardiac death in patients with stable CAD, independently of other traditional cardiovascular risk factors

A laminated polymer film formulation for enteric delivery of live vaccine and probiotic bacteria

Live bacterial cells (LBC) are administered orally as attenuated vaccines, to deliver biopharmaceutical agents, and as probiotics to improve gastrointestinal health. However, LBC present unique formulation challenges and must survive gastrointestinal antimicrobial defenses including gastric acid after administration. We present a simple new formulation concept, termed Polymer Film Laminate (PFL). LBC are ambient dried onto cast acid-resistant enteric polymer films that are then laminated together to produce a solid oral dosage form. LBC of a model live bacterial vaccine and a probiotic were dried directly onto a cast film of enteric polymer. The effectiveness at protecting dried cells in a simulated gastric fluid (pH 2.0) depended on the composition of enteric polymer film used, with a blend of ethylcellulose plus Eudragit L100 55 providing greater protection from acid than Eudragit alone. However, although PFL made from blended polymers films completely released low molecular weight dye into intestinal conditions (pH 7.0), they failed to release LBC. In contrast, PFL made from Eudragit alone successfully protected dried probiotic or vaccine LBC from simulated gastric fluid for 2h, and subsequently released all viable cells within 60min of transfer into simulated intestinal fluid. Release kinetics could be controlled by modifying the lamination method

Antioxidant activity, total phenolics and flavonoids contents: should we ban in vitro screening methods?

As many studies are exploring the association between ingestion of bioactive compounds and decreased risk of non-communicable diseases, the scientific community continues to show considerable interest in these compounds. In addition, as many non-nutrients with putative health benefits are reducing agents, hydrogen donors, singlet oxygen quenchers or metal chelators, measurement of antioxidant activity using in vitro assays has become very popular over recent decades. Measuring concentrations of total phenolics, flavonoids, and other compound (sub)classes using UV/Vis spectrophotometry offers a rapid chemical index, but chromatographic techniques are necessary to establish structure-activity. For bioactive purposes, in vivo models are required or, at the very least, methods that employ distinct mechanisms of action (i.e., single electron transfer, transition metal chelating ability, and hydrogen atom transfer). In this regard, better understanding and application of in vitro screening methods should help design of future research studies on ‘bioactive compounds’

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