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Site-specific peptide conjugation to carrier moleculesKozakowska, K. A. January 2015 (has links)
Clinically used peptides and antibodies can be modified to improve their therapeutic potential. As peptides are relatively small molecules, they display rapid renal clearance. Modifying a peptide by conjugation to poly(ethylene glycol) (PEG) or a protein carrier, such as an antibody are clinically valid examples to make peptides therapeutically more efficacious. Peptide modification using an efficient site-selective conjugation strategy is required to prepare homogenous conjugates that if efficacious, then have the potential for development. Mono-thiol specific conjugation can be accomplished using maleimide reagents. However, maleimide based conjugates are prone to de-conjugation or exchange reactions with plasma proteins. To address this limitation a mono-sulfone PEG reagent 1 that can undergo efficient conjugation to a single cysteine thiol in a therapeutic peptide that had been developed has been evaluated with peptides. In contrast to maleimide derived conjugates, any resulting conjugate from reagent 1 can be further stabilised with sodium borohydride to prevent PEG de-conjugation by a retro-Michael reaction. A dual-agonist peptide, PEP5 underwent conjugation with reagent 1 derived from a 40 kDa PEG with 90% conversion to give the PEG40-PEP5 conjugate. The pure conjugate was obtained in 50% yield after purification by ion exchange chromatography (IEX). An in vitro receptor binding assay showed that the PEG40-PEP5 conjugate displayed 0.11-4% and 0.08-2.2% binding affinities against two relevant receptors. A mouse PK study showed the PEG40-PEP5 conjugate had a 60-fold improved circulation time (15.9 h) compared to the non-modified PEP5 (0.26 h) while also reducing blood glucose levels in obese mice. Conjugation by bis-alkylation to the two thiols that are derived from a disulfide bond is another efficient way to prepare stable and homogenous PEG-peptide conjugates. The thiol bis-alkylating PEG reagent 3 can selectively alkylate both sulfurs derived from a naturally occurring disulfide bond to form a conjugate with a three-carbon bridge between the two cysteine thiols. Octreotide (OCT) is a cyclic octapeptide with a disulfide that underwent reaction at the two cysteine thiols with the 10 kDa PEG variant of reagent 3 at 78% conversion to give a PEG10-OCT conjugate. The PEG10-OCT conjugate maintained its selectivity towards somatostatin receptor 2 (sst2) and 5 (sst5) in an in vitro receptor-binding assay. A molecular dynamics (MD) study showed that insertion of the three-carbon bridge between the cysteine thiols maintained the active structure of OCT responsible for the interactions with the somatostatin receptors. PEG10-OCT also displayed a significant inhibitory effect on IGF-1 at 24 and 48 h in an in vivo study while displaying a longer in vivo circulation time than the non-modified OCT at the same 0.3 mg/kg dose. The PEG10-OCT was detected at average 43 ng/mL and 55 ng/mL at 4 h and 8 h respectively, while OCT was below the detection limit (<10 ng/mL) at both time points. Since peptides are much smaller relative to the PEG molecule, a bis-functional PEG reagent 5 was examined to conjugate two copies of OCT to prepare OCT-PEG10-OCT conjugate. OCT-PEG10-OCT inhibited IMR-32 cell growth in vitro and was more efficient than PEG10-OCT by 29% at 0.1 µg/mL. In the MD study less PEG shielding effect was observed in OCT-PEG10-OCT than in PEG10-OCT resulting in 13% greater solvent accessible surface area (SASA) of OCT. Conjugation of a peptide to an antibody to make an antibody drug conjugate (ADC), rather than to a large molecular weight PEG, is a clinically proven strategy for increasing the efficacy of cytotoxic peptides. Colleagues used the bis-alkylating PEG reagent 3 as a basis to design two new reagents 7 and 8 with the cytotoxic peptide, monomethyl auristatin E (MMAE). These reagents are being used for the selective conjugation of MMAE to the thiols derived from the four interchain disulfide bonds in an IgG1 to produce more homogenous and stable ADCs. The linker between the MMAE and the antibody must be stable in circulation to avoid systemic toxicity upon premature release of the payload. Strategies were developed to purify a model ADC, prepared by bis-alkylation to evaluate the antibody-drug ratio (DAR) and its in vivo stability. A protein precipitation method was developed that with LC-MS analysis enabled the detection of unconjugated MMAE reagent 7 species below 1% (wt) in the purified ADC at <1 mg scale. An affinity capture method was also developed to purify trastuzumab-based ADCs from animal serum from an in vivo study. Hydrophobic interaction chromatography (HIC) enabled analysis of the DAR profile of these ADCs on <2 µg scale. Finally HER2 which is a trastuzumab-specific antigen was efficiently biotinylated and coated onto magnetic beads with >99% efficiency. The functionalised beads allowed a 80% recovery of trastuzumab-PEG(24u)-val-cit-PAB-MMAE conjugate from mouse serum. The DAR profile of the purified ADC conjugate was assessed by HIC and showed there was drug loss (~30%) possibly being due to instability of the val-cit-PAB cleavable linker in blood circulation but not de-conjugation as shown by mass spectrometry analysis.
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Evaluation of the in vitro biological activities and phytochemical profiling of eight Ficus species collected in ZambiaBwalya, A. G. January 2015 (has links)
Infectious diseases are responsible for an overwhelming number of deaths and morbidity worldwide. In tropical regions of the world, in particular, developing countries like Zambia, poor health is prevalent and diseases such as malaria, meningitis, pneumonia, tuberculosis and gastrointestinal infections strongly persist. Folkloric medicines are still actively used against some of these infections as primary care before seeking conventional treatment at hospitals. Members of the genus Ficus (Moraceae) are traditionally used in Zambia against many diseases caused by bacterial, fungal and protozoal infections. Thus according to the plant parts used traditionally for herbal preparations, aerial and root parts of eight Ficus species namely; F. ingens, F. lutea, F. natalensis, F. ovata, F. sansibarica subsp. macrosperma, F. sycomorus subsp. gnaphalocarpa, F. sycomorus subsp. sycomorus and F. wakefieldii were collected from different parts of Zambia. The main aim of this thesis was to evaluate the medicinal potential of members of the genus Ficus. This was achieved by three objectives, which involved the phytochemical profiling of the crude extracts and subextracts of the Ficus for their constituents using chromatographic methods such as thin layer chromatography (TLC), proton Nuclear Magnetic Resonance (1H NMR) and high performance liquid chromatography (HPLC). Secondly, the extracts were screened for various biological activities after which they were evaluated against recombinant FAS-II elongation enzymes, FabG, FabI and FabZ as potential targets in liver stage malaria parasites. In this case, finely ground dried plant material was extracted with methanol (MeOH) to yield the crude methanol extracts (CR-MeOH) which, were further partitioned to provide a coarse separation of the crude extracts according to polarity. The three subextracts obtained included n-hexane, chloroform (CHCl3) and aqueous methanol (aq-MeOH). The obtained extracts and subextracts were screened for biological activities such as: antifungal and antibacterial activities using the broth dilution and agar disc diffusion assays, antitubercular activity using the MTT assay, antischistosomal activity using the microscopic in vitro assay. In addition, antiprotozoal activitites which included antileshmanial activity using an assay against amastigotes of L. donovani strain MHOM/ET/67/L82, trypanocidal activity against T. cruzi and T. brucei rhodesiense STIB 900 strain, and antiplasmodial activity by modified [3H]-hypoxanthine incorporation assay, using the chloroquine/pyrimethamine resistant K1 strain were performed. Cytotoxity activity was also performed using rat skeletal myoblasts L6-cells. The chemical profiling was done by TLC, NMR and RP-HPLC. Meanwhile the chemical compound isolation for F. sansibarica was attempted by different chromatographic techniques and characterization by spectroscopic methods. The phytochemical profiling revealed the presence of closely related polyphenolic compounds to which some of the biological activities were attributed to. For instance, the antibacterial and the FAS-II enzyme inhibition activities were mostly retained in the aq-MeOH subextracts, which were composed of very polar metabolites including flavonoids. Antiplasmodial activity was observed mostly in the less polar metabolites which were retained in the hexane and CHCl3 subextracts of the stem barks. This pattern was similar with antitrypanosomal and antileishmanial activities, though with lesser sensitivity. The same subextracts including those of the root barks showed the most activity against M. tuberculosis with MIC values of 256 and 128 μg/ml, and against Schistosoma, for both larval and adult worms. The extracts did not exert any antifungal activity by the agar disc diffusion method we used. Detailed phytochemical investigation of the leaves of F. sansibarica was performed, and led to the isolation of two compounds; epicatechin and apigenin-6-C-glucoside from the chloroform and aq. MeOH subextracts respectively. The predominant constituent of the CR-MeOH extract of F. sansibarica was identified as having a molecular weight of 432 g/mol by LC-MS analysis which could be set as an identification chemical marker for F. sansibarica. The results highlight the potential that Ficus species could have as a valuable source for potent compounds which can be identified as scaffolds for the development of novel liver stage antimalarial drugs. Our results support previous research on the antimicrobial activity of Ficus species and they also provide an in vitro scientific basis supporting the use of Ficus species in traditional herbal preparations against some bacterial and parasitic infections.
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Engineering excipient-free particles for inhalationMueannoom, W. January 2014 (has links)
The size of inhalable particles should be approximately between 1-5 µm to be delivered to the lower respiratory tract. However, there are some disadvantages of the powders having such a small particle size, such as the difficulty of aerosolisation due to their intrinsic cohesiveness. Generating porous particles is a way of increasing the physical size of particles to enable effective aerosolisation but remain sufficiently small to reach the lower airways. A modified Hewlett-Packard thermal inkjet printer (TIJ) and airbrush (Model 200-3, USA) were used to engineer drug solutions of the inhalable drugs salbutamol sulphate (SS) and combination particles of beclometasone dipropionate (BDP) and SS (drug-drug particles). Subsequently, L-leucine was added with the aim of improving dispersibility and aerosol performance of SS and BDP:SS particles. The results demonstrated that the spray freeze dried (SFD) particles produced from both techniques had a low density of less than 0.1 g/cm3, and were spherical and porous. The SFD SS was amorphous; whereas, the inclusion of either L-leucine or BDP in formulations produced particles exhibiting partial crystallinity. This led to an enhancement of the BDP release from the SFD BDP:SS particles in the dissolution study due to: a) the largely amorphous nature of the particles b) SS acting as a solubility enhancer and c) the high surface area of the porous structure, compared to Easyhaler® Beclometasone. The SFD particles produced by the airbrush had a smaller physical size (ca: 4-10 µm) than those from the TIJ (ca: 5-30 µm). Next generation impactor (NGI) analysis indicated that the particles sprayed from the airbrush had a smaller aerodynamic size (MMAD; 0.5-5 µm) and higher fine particle fraction (FPF) (47.8-70.8 % FPF) than those particles jetted by the TIJ. In addition, engineering the combined particles of BDP:SS using the TIJ and airbrush proved that homogeneity of the drugs was achieved due to the equality of the drugs’ deposition in each NGI stage (p > 0.05). The presence of L-leucine enhanced the dispersibility of the SFD particles as the L-leucine content was increased, due to enrichment with L-leucine on the particle’s surface, and this led to a high percentage FPF being obtained. Spray freeze-drying using the TIJ and airbrush requires only a small volume of the drug solution (approximately < 5 mL for TIJ and 5-20 mL for the airbrush), which means the potential for pulmonary formulation can be assessed early in the preformulation stage.
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The transformation of traditional Asian medical knowledge into international commodities : the link between traditional medicines and the international marketBooker, A. J. January 2014 (has links)
Aims and Objectives Medicinal plant value chains have been overlooked compared with food commodities. Revenue generation tends to be weighted towards the retail end of the chain and consequently the farm labourers, farmers and processors (primary producers) are the lowest beneficiaries. This project aims to investigate medicinal plant value chains and interpret the impact different value chains have on the livelihoods of primary producers in developing countries and for the first time analytically assess the quality implications for the manufacturers and end users in Europe. Methodological Approach Case studies were undertaken on three separate sites in India. Data was gathered on medicinal plant value chains by means of semi-structured interviews and non-participant observations. Samples were collected from locations in India, China, Europe and the USA and analysed using nuclear magnetic resonance spectroscopy and high performance thin layer chromatography. Results There were benefits for primary producers that belonged to a vertically integrated value chain and resulting products were subject to a higher standard of processing and storage. The analysis demonstrated that there was variation in the chemical composition of the samples tested and that products obtained from a vertically integrated value chain were more similar chemically to fresh turmeric rhizomes than other samples tested. Conclusions Using analytical methods, it was possible to correlate important variations in product composition for selected samples and identify strengths and weaknesses of some key value chains. Through establishing direct contracts with farmers in India, the vertically integrated value chain investigated was able to exert greater control over cultivation and manufacturing processes than found in other chains. Consequently the vertically integrated value chain is able to produce a higher quality product than generally found on the market. This results in a value addition that was passed back down the chain for the benefit of the primary producers.
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Toxicological risk asessment of Aristolochia speciesMichl, J. January 2014 (has links)
Aristolochia species are toxic plants used as herbal medicines worldwide. They are known to cause aristolochic acid nephropathy (AAN), a disease associated with kidney failure and kidney cancer. Aristolochic acid (AA) I and AA II are considered to be responsible for AAN. However, a wide range of aristolochic acid analogues exists and their implication in AAN may have been overlooked. The aims of this project are to assess the health risks associated with different Aristolochia species and to elucidate the principles behind their nephrotoxic effects. 44 medicinally used Aristolochia species were analyzed using LC-MS and 1H- NMR. The cytotoxicity and genotoxicity of 28 Aristolochia extracts was measured in human kidney (HK-2) cells. Several AAAs were isolated from natural sources. Their molecular mechanisms were analysed in hepatoma G2 (Hep G2) cells. Furthermore, we studied ethnobotanical uses of Aristolochia indica L. in Bangladesh. AA I and AA II are the most common AAAs. However, aristolactam (AL) I, AA IV and AL BI are widespread as well. Several of the extracts caused cytotoxic effects and micronuclei induction in HK-2 cells. No correlation was found between the amounts of AA I or AA II and the extractsʼ toxicity. While both AA I and AA II formed DNA adducts in Hep G2 cells, only AA I was cytotoxic and caused oxidative stress. AL I and AA IIIa caused oxidative stress to a lesser extent. Other components exhibited no toxic effects. Furthermore we demonstrated that Aristolocha indica is widely available in Bangladesh and more awareness needs to be raised about the health risks associated its use. By linking a metabolomic analysis with in vitro studies, we were able to show that the assumption that the toxicity of Aristolochia species is caused by AA I and AA II alone is incorrect.
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Synthesis and discovery of the putative cognitive enhancer BRS-015 : effect on glutamatergic transmission and synaptic plasticitySzulc, B. R. January 2015 (has links)
This thesis is concerned with the discovery of a novel heterocyclic compound – BRS-015, its synthesis and an analysis of its effects on excitatory synaptic transmission at a major pathway in the brain. BRS-015 is related to the natural product clausenamide, which has been shown to facilitate synaptic transmission. As such, clausenamide and related analogues may possess therapeutic potential as memory enhancing drugs, which are in urgent need of development due to the increasing numbers of patients diagnosed with memory disorders and for which there is no current effective therapy. BRS-015 was synthesized using a novel approach to the core structure of clausenamide involving an intramolecular acylal cyclisation reaction, which has not previously been reported. The first section of the thesis opens with a description of the discovery, structure and biological activity of clausenamide and discussion of previous synthetic strategies adopted by a number of research groups and attempts to classify these into the varying approaches towards the central core of clausenamide. The second section describes the structure of the rat brain and the types of processes involved in memory formation, as well as the neurophysiological assays used to investigate synaptic transmission and plasticity. The second group of chapters describes our own approach to the core of clausenamide and the synthesis of BRS-015, with a detailed discussion of the structural analysis and investigation of the intramolecular acylal cyclisation reaction used during the synthetic process. The third chapter describes the neurophysiological assays used in our investigations into the effects of BRS-015, which was tested against glutamatergic synaptic transmission and plasticity in acute rat hippocampal slices. BRS-015 was shown to reversibly enhance the amplitude of AMPA receptor mediated EPSCs recorded from CA3 pyramidal neurones and evoked by dentate stimulation. When tested in the presence of selective glutamate receptor antagonists, BRS-015 did not have this powerful enhancing effect on kainate or NMDA receptor mediated EPSCs. In addition, BRS-015 increased the amplitude of glutamate-evoked currents in CA3 pyramidal neurones and did not alter short-term synaptic plasticity but facilitated the induction of mossy fibre LTP, with little effect at associational/commissural synapses. BRS-015 has striking enhancing properties on AMPA receptor mediated synaptic transmission at mossy fibre synapses either by directly interacting with AMPA receptors or via indirect modulation, the mechanisms of which could lead to synapse strengthening.
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A direct interaction between the Parkinson's disease protein leucine-rich repeat kinase 2 and specific β-tubulin isoforms regulates tubulin acetylationSpain, V. January 2015 (has links)
Mutations in LRRK2 are a common cause of Parkinson’s disease (PD). LRRK2 encodes leucine-rich repeat kinase 2 (LRRK2), a ROCO protein. It has an enzymatic core consisting of a Ras of complex proteins (Roc) GTPase domain and kinase domain, surrounded by protein-protein interaction regions. Pathogenic LRRK2 mutations modify activity in these enzymatic domains, but how this leads to neurodegeneration is still to be elucidated. One of the few confirmed LRRK2 interactors is tubulin, the main constituent of microtubules (MTs) and part of the cytoskeletal network. Disease-causing mutations in LRRK2 alter this network, reducing neurite outgrowth and leading to accumulation of hyperphosphorylated MT-associated protein (MAP) tau. Meanwhile changes in post-translational modifications of tubulin and MAPs alter the dynamic instability of MTs, leading to aberrant axonal transport, synaptic dysfunction and axonal degeneration. I investigated the LRRK2-tubulin interaction. Using yeast two-hybrid I demonstrated that the interaction is conferred by the LRRK2 Roc domain and the C-terminus of the β-tubulin isoforms TUBB, TUBB4 and TUBB6. The interaction requires Lys362 and Ala364 and is blocked in isoforms expressing a serine at these positions. This site is on the luminal face of MT protofibrils, close to the paclitaxel binding site and α-tubulin Lys40 acetylation site, both of which are involved in MT stability. This location is poorly accessible within mature, stabilised MTs but exposed in dynamic MT populations. Consistent with this finding, endogenous LRRK2 located to dynamic growth cone MTs in SH-SY5Y cells. Overexpression and knock-out studies in HEK cells and mouse embryonic fibroblasts showed that LRRK2 is associated with reduced α-tubulin acetylation. These results demonstrate the specificity of the LRRK2-tubulin interaction, suggesting LRRK2 distribution at the cytoskeleton is determined by the tubulin composition and may vary between cell types. Changes in MT acetylation in the presence of disease-causing LRRK2 mutations could contribute to pathogenic mechanisms, with altered MT stability implicated in PD neurodegeneration. As mutations affecting the β-tubulin C-terminal residues could disrupt the LRRK2 interaction without compromising MT integration, a cohort of late-onset familial PD cases was also screened for mutations within the cytoskeleton.
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The rational design of topical formulationsDuszynska-Krupa, A. M. January 2015 (has links)
This thesis addresses the development of topical formulations designed to treat atopic dermatitis (AD) using nicotinamide (NA). A rational approach to the development of topical formulations based on the physical and chemical properties of the drug and vehicle components is studied. This approach is an alternative to the model of formulation development where the drug is added into an existing vehicle without optimisation of the formulation in terms of the active delivery to its site of action. The work encompasses preliminary pre-formulation studies, in vitro uptake and permeation studies using a model silicone membrane and pig ear skin. Moreover, the influence of topical formulations containing the model drug on the parameters indicative of skin health is tested in the in vivo studies. The primary objective is to optimise the skin delivery of NA with the use of appropriate excipients. The solvents are chosen on the basis of their physicochemical parameters, namely solubility parameter (δ), mutual miscibility and ability to dissolve the model drug. The performance of rationally developed simple formulations is tested in vitro and compared with prototype formulations containing more complex vehicles. In vitro uptake and permeation studies using silicone are performed to determine the influence of chosen solvents on NA permeation in a membrane which is less complex than skin. In addition NA skin delivery is evaluated with in vitro and in vivo techniques and the relationship between the physicochemical parameters of the solvents used and the drug percutaneous absorption is examined. Finally, the efficacy of prototype NA formulations in improving the skin state in vivo is investigated. The performance of prototype formulations in terms of NA percutaneous absorption is determined with reference to their influence on the skin condition.
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Impact of epicatechin gallate on the structural integrity of the PBP2/PBP2a complex in methicillin resistant Staphylococcus aureusPaulin, S. January 2014 (has links)
Introduction: The selective pressure imposed by the misuse and overuse of antibiotics has led to the emergence and dissemination of methicillin resistant Staphylococcus aureus (MRSA), forcing a re-evaluation of therapeutic approaches to the treatment of MRSA infections. Epicatechin gallate (ECg), a constituent of the tea plant Camellia sinensis, has the capacity to abrogate the resistance of MRSA to β-lactam antibiotics and may be useful as an adjunct to conventional chemotherapy. Current evidence suggests that ECg sensitises resistant strains to β-lactam agents by disruption of the penicillin binding protein (PBP) complex PBP2/PBP2a at the septal site of cell division following its intercalation into the cytoplasmic membrane (CM) bilayer. Methods: Styrene maleic acid lipid co-polymer (SMALP) was used to solubilise and extract PBP2/PBP2a membrane complexes from the CM of EMRSA-16 and ECg-exposed cells. Cell walls were partially digested and membrane proteins excised and solubilised with hydrolysed styrene maleic acid (SMA). SMALP particles were visualised by TEM and size distribution determined by dynamic light scattering. Membrane protein complexes were cross-linked within SMALPs, protein complexes recovered by co-immunoprecipitation and the constituents determined by Western blotting and flow cytometry. Results: PBP2/PBP2a complexes were identified in both ECg-exposed and control EMRSA-16 cells when SMALPs were pulled down with anti-PBP2 and anti-PBP2a antibodies. Fewer complexes were recovered from ECg exposed cells. Co-immunoprecipitation of SMALPs with antibody against the division scaffold protein FtsZ led to the identification of FtsZ/PBP2/PBP2a complexes. ECg displaced PBP2a from this complex. The PBP2/PBP4 complex was also identified, however there was no difference observed following ECg exposure. Conclusion: Intercalation of ECg into the MRSA phospholipid palisade led to partial disruption of PBP2a from PBP2/PBP2a and FtsZ/PBP2/PBP2a complexes. The data suggest that ECg-mediated conversion of MRSA to β-lactam susceptibility may in part be related to loss of functional integrity of the cellular replication machinery. The therapeutic approach with the use of antibiotic resistance modifying agent, such as ECg, in combination with a previously ineffective β-lactam antibiotic, presents a novel therapy to combat antibiotic resistance in MRSA.
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The application of isothermal microcalorimetry for studying mixed probiotic culturesFredua-Agyeman, M. January 2015 (has links)
The main aim of this research was to explore the potential of the isothermal microcalorimeter to detect bacteria in mixed cultures; applied to investigate the antagonistic effect of commercial probiotics against pathogens and each other; and also the prebiotic potential of a substrate. Gastric tolerance of commercial probiotic products was also investigated with an improvement on current methods. An initial mixed culture study with Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli in the microcalorimeter showed that the microcalorimeter could detect their growth in mixed cultures; S. aureus was always outcompeted in growth. Antagonistic activity of probiotic strains, Lactobacillus acidophilus, Bifidobacterium lactis, Bifidobacterium bifidum or commercial probiotic products against P. aeruginosa, E. coli, S. aureus and the clinically important gut pathogen, Clostridium difficile was demonstrated in the microcalorimeter and was shown to be pH-dependent using neutralized and unmodified cell free culture supernatant (CFS) produced by the probiotic strains. But concentrated CFS of the probiotics also inhibited the pathogenic species in a non pH-dependent manner, likely due to specific antimicrobial substances or bacteriocins. The result also demonstrated that probiotic strains could compete with each other in growth when put together. The prebiotic potential of inulin was demonstrated with the microcalorimeter using faecal slurry and pure probiotic strains. Gastric tolerance assay of commercial probiotic products in porcine gastric fluid, SGF (acidified NaCl solution) and FaSSGF (acidified NaCl solution with biorelevant amounts of bile salt, pepsin and lecithin) mimicking the fed and fasted states showed significant differences between the products and fluids. In conclusion, the research showed that the microcalorimeter is a useful in vitro tool for detecting bacterial growth in mixed cultures and studying functional characteristics of probiotics and prebiotics; overcoming some of the limitations of the conventional methods.
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