Enzyme production by solid-state fermentation on soybean meal: A comparative study of conventional and ultrasound-assisted extraction methods.

Protease, cellulase, and α-amylase producing Bacillus subtilis strain was cultivated by solid-state fermentation technique using soybean meal as a substrate. The aim of the present study was to establish a highly efficient enzymes' extraction method as a first stage in downstream processing. The conventional extraction procedure was optimized by determining pH, stirring rate, solid/liquid ratio, and time of extraction on enzymes' recoveries from fermented soybean meal. Yields of leached enzymes were compared to the amounts of enzymes that are achieved with ultrasound-assisted extraction (UAE). UAE was established to be superior method for obtaining higher yields of proteases (up to 330 IU) and α-amylases (825 IU), under significantly shorter extraction time and gaining more concentrated product. However, the obtained model predicts that conventional process led to a product with a higher cellulolytic activity (≥7.5 IU).

Effect of immobilisation materials on viability and fermentation activity of dairy starter culture in whey-based substrate.

BACKGROUND: The main objectives of the paper were to study influence of immobilisation of dairy starter culture 'Lactoferm ABY 6' on fermentation and probiotic potential of fermented whey-based substrate. RESULTS: Fermentation with free cells takes 1.5 h less than fermentation with encapsulated cells, but samples with encapsulated cells have better characteristics after 28 days of storage. Chitosan coating provides additional protection of cells in bile salt solution (95.86% of viable cells compared to the initial number) and simulated gastric juice (37.8% for pH 2.5) compared to the alginate beads (94.54% in bile salt solution and 36.18% in simulated gastric juice for pH 2.5). Free cells had a drastic reduction in the number of viable cells (83.0% in bile salt solution and no viable cells in simulated gastric juice for pH 2.5). CONCLUSION: Samples with alginate beads and chitosan-coated alginate beads have significantly (P < 0.05) higher viable cell count than samples with free cells, during 4 h monitoring survival at pH 2.5, pH 3.0 and 0.3% bovine bile solution. These beads can be used to improve survival of probiotic cells in fermented whey-based beverage during storage and consummation, which improves the quality of the product.

Integrated production of lactic acid and biomass on distillery stillage.

The possibilities of parallel lactic acid and biomass production in batch and fed-batch fermentation on distillery stillage from bioethanol production were studied. The highest lactic acid yield and productivity of 92.3 % and 1.49 g L(-1) h(-1) were achieved in batch fermentation with initial sugar concentration of 55 g L(-1). A significant improvement of the process was achieved in fed-batch fermentation where the concentration of lactic acid was increased to 47.6 % and volumetric productivity for 21 % over the batch process. A high number of Lactobacillus rhamnosus ATCC 7469 viable cells of 10(9) CFU ml(-1) was attained at the end of fed-batch fermentation. The survival of 92.9 % of L. rhamnosus cells after 3 h of incubation at pH 2.5 validated that the fermentation media remained after lactic acid removal could be used as a biomass-enriched animal feed thus making an additional value to the process.

Effects of poly(vinyl alcohol) blending with Ag/alginate solutions to form nanocomposite fibres for potential use as antibacterial wound dressings.

In this work, nanocomposite fibres and microfibres based on alginate and poly(vinyl alcohol) (PVA) with silver nanoparticles (AgNPs) were produced and characterized for potential application as antibacterial wound dressings. PVA/Ag/Na-alginate colloid solution was used for the preparation of the fibres by a simple extrusion technique followed by freezing-thawing cycles. UV-Visible spectroscopy confirmed successful preservation of AgNPs in fibres while Fourier transform infrared spectroscopy has shown a balanced combined effect on the Ca-alginate spatial arrangement with the addition of both AgNPs and PVA. The presence of PVA in fibres induced an increase in the swelling degree as compared with that of Ag/Ca-alginate fibres (approx. 28 versus approx. 14). Still, the initially produced PVA/Ca-alginate fibres were mechanically weaker than Ca-alginate fibres, but after drying and rehydration exhibited better mechanical properties. Also, the obtained fibres released AgNPs and/or silver ions at the concentration of approximately 2.6 µg cm-3 leading to bacteriostatic effects against Staphylococcus aureus and Escherichia coli. These results are relevant for practical utilization of the fibres, which could be stored and applied in the dry form with preserved mechanical stability, sorption capacity and antibacterial activity.

Antibacterial graphene-based hydroxyapatite/chitosan coating with gentamicin for potential applications in bone tissue engineering.

Electrophoretic deposition process (EPD) was successfully used for obtaining graphene (Gr)-reinforced composite coating based on hydroxyapatite (HAP), chitosan (CS), and antibiotic gentamicin (Gent), from aqueous suspension. The deposition process was performed as a single step process at a constant voltage (5 V, deposition time 12 min) on pure titanium foils. The influence of graphene was examined through detailed physicochemical and biological characterization. Fourier transform infrared spectroscopy, field emission scanning electron microscopy, thermogravimetric analysis, X-ray diffraction, Raman, and X-ray photoelectron analyses confirmed the formation of composite HAP/CS/Gr and HAP/CS/Gr/Gent coatings on Ti. Obtained coatings had porous, uniform, fracture-free surfaces, suggesting strong interfacial interaction between HAP, CS, and Gr. Large specific area of graphene enabled strong bonding with chitosan, acting as nanofiller throughout the polymer matrix. Gentamicin addition strongly improved the antibacterial activity of HAP/CS/Gr/Gent coating that was confirmed by antibacterial activity kinetics in suspension and agar diffusion testing, while results indicated more pronounced antibacterial effect against Staphylococcus aureus (bactericidal, viable cells number reduction >3 logarithmic units) compared to Escherichia coli (bacteriostatic, <3 logarithmic units). MTT assay indicated low cytotoxicity (75% cell viability) against MRC-5 and L929 (70% cell viability) tested cell lines, indicating good biocompatibility of HAP/CS/Gr/Gent coating. Therefore, electrodeposited HAP/CS/Gr/Gent coating on Ti can be considered as a prospective material for bone tissue engineering as a hard tissue implant.

Multifunctional ternary composite films based on PLA and Ag/alginate microbeads: Physical characterization and silver release kinetics.

Novel multifunctional composite poly(lactic acid) (PLA) films with alginate microbeads containing silver nanoparticles (AgNPs) were developed for potential antimicrobial food packaging applications. AgNPs, 10-20 nm in size, were synthesized in a Na-alginate solution by a hydrothermal method yielding a sterile, pH neutral colloid solution of low viscosity that was electrostatically extruded to produce Ag/alginate microbeads (190 µm in size) with retained AgNPs. Dried microbeads were uniformly dispersed in PLA films with retained AgNPs as confirmed by UV-Vis spectroscopy and scanning electron microscopy. The films were characterized regarding thermal and mechanical properties as well as silver release in different food simulants. Results show that PLA matrix served as a diffusion barrier so that the released silver concentration in water after 10 days was within the prescribed limit of 0.05 mg kg-1 while the films induced inhibitory effects against Staphylococcus aureus in the agar diffusion test.

Evaluation of ß-galactosidase from Lactobacillus acidophilus as biocatalyst for galacto-oligosaccharides synthesis: Product structural characterization and enzyme immobilization.

ß-Galactosidase is an important industrial enzyme that catalyzes reaction of lactose hydrolysis and recently more interesting reaction of transgalactosylation, yielding a highly valuable group of prebiotic compounds named galacto-oligosaccharides (GOS). In this paper, parameters for achieving high yields of tailor-made GOS using crude ß-galactosidase obtained from Lactobacillus acidophilus ATCC 4356, probiotic bacteria regarded as safe for human consumption, were optimized. At the same time, detailed structural elucidation of obtained GOS was conducted, and it was concluded that ß-galactosidase from L. acidophilus shows a particular specificity towards the formation of ß-(1→6) glycosidic bonds. In order to develop more stable and economically cost-effective preparation, crude enzyme was successfully immobilized on a methacrylic polymer carrier Lifetech ECR8409, leading to its simultaneous 2-fold purification. This immobilized preparation showed unchanged specificity towards the transgalactosylation reaction, thus yielding 86 g/l GOS under the previously optimized conditions (lactose concentration 400 g/l in 0.1 M sodium phosphate buffer, pH 6.8 and temperature 50°C).

Gentamicin-Loaded Bioactive Hydroxyapatite/Chitosan Composite Coating Electrodeposited on Titanium.

Composite coating of antibiotic gentamicin (Gent), natural polymer chitosan (CS), and hydroxyapatite (HAP) was successfully assessed by applying the electrophoretic deposition (EPD) technique. EPD was performed under optimized deposition conditions (5 V, 12 min) on pure titanium plates, to obtain HAP/CS and HAP/CS/Gent composite coatings in a single step from three-component aqueous suspension, with favorable antibacterial properties. Composite coatings were characterized by X-ray diffraction (XRD), field emission scanning electron microscopy, Fourier transform infrared spectroscopy, thermogravimetric analysis, and X-ray photoelectron analysis, confirming the formation of composite HAP/CS and HAP/CS/Gent coatings on the titanium surface, which is due to intermolecular hydrogen bonds. Employing the XRD technique, HAP was detected by obtaining the characteristic diffraction maximums. Good antibacterial activity of the composite coating loaded with antibiotic (HAP/CS/Gent) was confirmed against Staphylococcus aureus and Escherichia coli, pointing to the high potential for bioapplication. Introduction of gentamicin in HAP/CS/Gent coating caused very mild cytotoxicity in the tested cell lines MRC-5 and L929. MTT testing was used to evaluate cell viability, and HAP/CS was classified as noncytotoxic.

A comprehensive approach to in vitro functional evaluation of Ag/alginate nanocomposite hydrogels.

In this work, we present a comprehensive approach to evaluation of alginate microbeads with included silver nanoparticles (AgNPs) at the concentration range of 0.3-5mM for potential biomedical use by combining cytotoxicity, antibacterial activity, and silver release studies. The microbeads were investigated regarding drying and rehydration showing retention of ∼ 80-85% of the initial nanoparticles as determined by UV-vis and SEM analyses. Both wet and dry microbeads were shown to release AgNPs and/or ions inducing similar growth delays of Staphylococcus aureus and Escherichia coli at the total released silver concentrations of ∼ 10 µg/ml. On the other hand, these concentrations were highly toxic for bovine chondrocytes in conventional monolayer cultures while nontoxic when cultured in alginate microbeads under biomimetic conditions in 3D perfusion bioreactors. The applied approach outlined directions for further optimization studies demonstrating Ag/alginate microbeads as potentially attractive components of soft tissue implants as well as antimicrobial wound dressings.

Effect of different fermentation parameters on L-lactic acid production from liquid distillery stillage.

Expansion of lactic acid applications, predominantly for the preparation of biodegradable polymers increased the research interest for new, economically favourable production processes. Liquid stillage from bioethanol production can be an inexpensive, valuable source of nutrients for growth of lactic acid bacteria. Utilisation of residual biomass with spent fermentation media as a functional animal feed can greatly influence the process value and its ecological aspect. In this paper, the kinetics of lactic acid and biomass production on liquid stillage by Lactobacillus rhamnosus ATCC 7469 was studied. In addition, the impact of temperature, inoculum concentration, shaking and pH control by addition of CaCO(3) was evaluated. Maximal lactic acid yield of 73.4%, as well as high biomass production (3×10(8) CFU ml(-1)) were achieved under selected conditions (41°C, 5% (v/v) of inoculum, 1% (w/v) of CaCO(3), initial pH of 6.5 and shaking rate of 90 rpm). These results were achieved without supplementation of the stillage with nitrogen or mineral sources.