Efficacy of Sakacin on Selected Food Pathogenic Microorganisms Isolated from Fermented Milk Products

The efficacy of sakacin on selected food pathogenic microorganisms isolated from fermented milk products was investigated. The L. sake was isolated using the pour plate technique and was characterized based on it colony, cell morphology and some biochemical tests. This isolate was identified using standard scheme. The L. sake FCF 33 was propagated in De Man Rogosa Sharpe (MRS) broth for bacteriocin (sakacin) production. The sakacin had inhibitory effects on all test microorganisms (ranging from +5mm to +6mm) except Shigella dysenteriae N11, Salmonella typhimurium N8, Klebsiella ozaenae W24 and Proteus mirabilis N16a). Bacteriocins are antimicrobial substances of lactic acid bacteria (LAB) have gained tremendous attention as potential bio preservatives in the food and dairy industries. The LAB can serve as probiotics, which are products aimed at delivering living, potentially beneficial bacterial cells to the gut ecosystem of humans and other animals.

Síntese do Poli (ácido láctico) por policondensação direta utilizando um planejamento fatorial

The environmental impact caused by the disposal of non-biodegradable polymer packaging on the environment, as well as the high price and scarcity of oil, caused increase of searches in the area of biodegradable polymers from renewable resources were developed. The poly (lactic acid) (PLA) is a promising polymer in the market, with a large availability of raw material for the production of its monomer, as well as good processability. The aimed of this study was synthesis PLA by direct polycondesation of lactic acid, using the tool of experimental design (DOE) (central composite rotatable design (CCRD)) to optimize the conditions of synthesis. The polymer obtained was characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), viscosimetric analysis, differential scanning calorimeter (DSC) and size exclusion chromatography (SEC). The results confirmed the formation of a poly (lactic acid) semicrystalline in the syntheses performed. Through the central composite rotatable design was possible to optimize the crystallization temperature (Tc) and crystallinity degree (Xc). The crystallization temperature maximum was found for percentage of catalyst around the central point (0,3 (%W)) and values of time ranging from the central point (6h) to the upper level (+1) (8h). The crystallization temperature maximum was found for the total synthesis time of 4h (-1) and percentage of catalyst 0,1(W%) (-1). The results of size exclusion chromatography (SEC) showed higher molecular weights to 0,3 (W%) percent of catalyst and total time synthesis of 3,2h

Survival and fermentation activity of probiotic bacteria and oxidative stability of omega-3 oil in co-microcapsules during storage

Tuna oil (O) and probiotic bacteria Lactobacillus casei (P) were co-microencapsulated in whey protein isolate (WPI)-gum Arabic (GA) complex coacervate. The co-microcapsules (WPI-P-O-GA), L. casei microcapsules (WPI-P-GA) and tuna oil microcapsules (WPI-O-GA) were converted into powder using spray and freeze drying. The interaction between probiotic bacteria and omega-3 oil in co-microcapsules, particularly in terms of oxidative stability of omega-3 oil and vitality/viability of probiotic bacteria and any synergistic outcome, was studied. The effect of storage temperature (5 and 25 °C) and time (90 days) on the survival and fermentation activity of L. casei and oxidative stability of tuna oil in the microcapsules/co-microcapsules was determined. A synergism between oxidative stability of omega-3 oil and vitality of probiotic bacteria was observed, when they were co-microencapsulated and spray dried. These co-microcapsules will likely have utility in functional food formulations due to simple and cost effective stabilisation and delivery of two important functional ingredients.

A BACKING DEVICE BASED ON AN EMBEDDED STIFFENER AND RETRACTABLE INSERTION TOOL FOR THIN-FILM COCHLEAR ARRAYS

Intracochlear trauma from surgical insertion of bulky electrode arrays and inadequate pitch perception are areas of concern with current hand-assembled commercial cochlear implants. Parylene thin-film arrays with higher electrode densities and lower profiles are a potential solution, but lack rigidity and hence depend on manually fabricated permanently attached polyethylene terephthalate (PET) tubing based bulky backing devices. As a solution, we investigated a new backing device with two sub-systems. The first sub-system is a thin poly(lactic acid) (PLA) stiffener that will be embedded in the parylene array. The second sub-system is an attaching and detaching mechanism, utilizing a poly(N-vinylpyrrolidone)-block-poly(d,l-lactide) (PVP-b-PDLLA) copolymer-based biodegradable and water soluble adhesive, that will help to retract the PET insertion tool after implantation. As a proof-of-concept of sub-system one, a microfabrication process for patterning PLA stiffeners embedded in parylene has been developed. Conventional hotembossing, mechanical micromachining, and standard cleanroom processes were integrated for patterning fully released and discrete stiffeners coated with parylene. The released embedded stiffeners were thermoformed to demonstrate that imparting perimodiolar shapes to stiffener-embedded arrays will be possible. The developed process when integrated with the array fabrication process will allow fabrication of stiffener-embedded arrays in a single process. As a proof-of-concept of sub-system two, the feasibility of the attaching and detaching mechanism was demonstrated by adhering 1x and 1.5x scale PET tube-based insertion tools and PLA stiffeners embedded in parylene using the copolymer adhesive. The attached devices survived qualitative adhesion tests, thermoforming, and flexing. The viability of the detaching mechanism was tested by aging the assemblies in-vitro in phosphate buffer solution. The average detachment times, 2.6 minutes and 10 minutes for 1x and 1.5x scale devices respectively, were found to be clinically relevant with respect to the reported array insertion times during surgical implantation. Eventually, the stiffener-embedded arrays would not need to be permanently attached to current insertion tools which are left behind after implantation and congest the cochlear scala tympani chamber. Finally, a simulation-based approach for accelerated failure analysis of PLA stiffeners and characterization of PVP-b-PDLLA copolymer adhesive has been explored. The residual functional life of embedded PLA stiffeners exposed to body-fluid and thereby subjected to degradation and erosion has been estimated by simulating PLA stiffeners with different parylene coating failure types and different PLA types for a given parylene coating failure type. For characterizing the PVP-b-PDLLA copolymer adhesive, several formulations of the copolymer adhesive were simulated and compared based on the insertion tool detachment times that were predicted from the dissolution, degradation, and erosion behavior of the simulated adhesive formulations. Results indicate that the simulation-based approaches could be used to reduce the total number of time consuming and expensive in-vitro tests that must be conducted.

Characterization of plasmids in a human clinical strain of Lactococcus garvieae

The present work describes the molecular characterization of five circular plasmids found in the human clinical strain Lactococcus garvieae 21881. The plasmids were designated pGL1-pGL5, with molecular sizes of 4,536 bp, 4,572 bp, 12,948 bp, 14,006 bp and 68,798 bp, respectively. Based on detailed sequence analysis, some of these plasmids appear to be mosaics composed of DNA obtained by modular exchange between different species of lactic acid bacteria. Based on sequence data and the derived presence of certain genes and proteins, the plasmid pGL2 appears to replicate via a rolling-circle mechanism, while the other four plasmids appear to belong to the group of lactococcal theta-type replicons. The plasmids pGL1, pGL2 and pGL5 encode putative proteins related with bacteriocin synthesis and bacteriocin secretion and immunity. The plasmid pGL5 harbors genes (txn, orf5 and orf25) encoding proteins that could be considered putative virulence factors. The gene txn encodes a protein with an enzymatic domain corresponding to the family actin-ADP-ribosyltransferases toxins, which are known to play a key role in pathogenesis of a variety of bacterial pathogens. The genes orf5 and orf25 encode two putative surface proteins containing the cell wall-sorting motif LPXTG, with mucin-binding and collagen-binding protein domains, respectively. These proteins could be involved in the adherence of L. garvieae to mucus from the intestine, facilitating further interaction with intestinal epithelial cells and to collagenous tissues such as the collagen-rich heart valves. To our knowledge, this is the first report on the characterization of plasmids in a human clinical strain of this pathogen.

Microbially derived bioactive peptides to improve human health

This thesis describes a study of various methods to produce bioactive peptides. Initially, the generation of anti-Cronobacter spp. peptides by fermentation of milk protein is described. Lactobacillus johnsonii DPC6026 was used to generate two previously described antimicrobial peptides. Phenotypic analysis indicated unsatisfactory casein hydrolysis. The genome of the strain was sequenced and annotated. Results showed a number of unique features present, most notably a large symmetrical inversion of approximately 750kb in comparison with the human isolate L. johnsonii NCC 533. The data suggest significant genetic diversity and intra-species genomic rearrangements within the L. johnsonii spp.. Cronobacter spp. have emerged as pathogens of concern to the powdered infant formula industry. Chapters 3 and 4 of this thesis describe novel methods to generate two antimicrobial peptides, Caseicin A and B. In Chapter 3 a bank of Bacillus strains was generated and investigated for caseicin production. Following casein hydrolysis by specific B. cereus and B. thuringiensis strains the peptides of interest were generated. Chapter 4 describes a sterile enzymatic method to generate peptides from casein. Bioinformatic tools were used to predict enzymes capable of liberating caseicin peptides from casein. Hydrolysates were generated using suitable enzymes, examined and some were found to produce peptides with activity against Cronobacter spp.. This study establishes a potential industrial-grade method to generate antimicrobial peptides. Administration of GLP-1 leads to improved glycaemic control in diabetes patients. Generation of a recombinant lactic acid bacteria capable of producing a GLP-1 analogue is described in Chapter 5. In-vivo analysis confirmed insulinotropic activity. The results illustrate a method using bacteriocin producing cellular machinery to generate bioactive peptides. This thesis describes the generation of bioactive peptides by bacterial fermentation, tailored enzymatic hydrolysis and recombinant bacterial methods. The techniques described contribute to bioactive peptide research with regards novel methods of production and industrial scale-up.

Lactobacillus rossiae, a vitamin B12 producer, represents a metabolically versatile species within the genus Lactobacillus

Lactobacillus rossiae is an obligately hetero-fermentative lactic acid bacterium, which can be isolated from a broad range of environments including sourdoughs, vegetables, fermented meat and flour, as well as the gastrointestinal tract of both humans and animals. In order to unravel distinctive genomic features of this particular species and investigate the phylogenetic positioning within the genus Lactobacillus, comparative genomics and phylogenomic approaches, followed by functional analyses were performed on L. rossiae DSM 15814(T), showing how this type strain not only occupies an independent phylogenetic branch, but also possesses genomic features underscoring its biotechnological potential. This strain in fact represents one of a small number of bacteria known to encode a complete de novo biosynthetic pathway of vitamin B-12 (in addition to other B vitamins such as folate and riboflavin). In addition, it possesses the capacity to utilize an extensive set of carbon sources, a characteristic that may contribute to environmental adaptation, perhaps enabling the strain's ability to populate different niches.

Microbiological quality and safety of minimally processed fruits in the marketplace of southern Portugal

The availability of fresh-cut fruit (FCF) in the marketplace has been increasing in Portugal, although reports of its microbial quality are not known. Due to the growing concerns of these commodities over their microbial safety, the objectives of this work were to study the microbiological quality and prevalence of Salmonella and Listeria monocytogenes on fresh-cut fruits sold in southern Portugal. A study to examine the changes in pH and microbial counts, before and after the expiration dates, was also made. A total of 160 samples was purchased in the local grocery stores between September 2011 and August 2014, before their sell-by date. These samples were assayed for aerobic mesophilic (AM) and psychrotrophic (AP) microorganisms, yeasts and molds (YM), lactic-acid bacteria (LAB), coliforms (TC), Escherichia coli and coagulase positive staphylococci as well as L. monocytogenes and Salmonella. The microbiological counts ranged from 3.0-9.2 lg cfu/g (AM); 2.2–10.7 lg cfu/g (AP); 2.3–10.4 lg cfu/g (YM); 1.9–9.0 lg cfu/g (LAB) and less than 1–9.1 lg cfu/g (TC). The melons and watermelon presented the highest levels of the microbial quality parameters studied. However, no E. coli, staphylococci, Salmonella and L. monocytogenes were detected in any of the samples. After the sell-by date, an increase of the AM, AP, LAB and YM values was observed in all fruits. Conversely, the differences found in TC counts before and after the best-before date had no statistical significance. A decrease in pH was observed in all fruits except pineapple whose pH slightly increased after 14 days of storage. The results highlight the importance of preventing contamination and cross contamination, selecting adequate decontamination technologies and maintaining a strict temperature control during processing, distribution and selling of FCF.

Physicochemical and microbial analysis of feces from horses fed diets containing citrus pulp.

This study aimed to evaluate the e ect of diets containing increasing levels of citrus pulp on the physic-chemical and microbiological characteristics of horses feces. Five mares, at an average age of 3.5 years old and body weight of 492 ± 44.5 kg were arranged in a 5 x 5 Latin Square. The experimental diet consisted of 60% coast-cross hay and 40 % of concentrate with increasing levels of citrus pulp (0, 7, 14, 21, and 28 %). To determine the fecal pH, samples were collected directly from the oor, immediately after defecation, in the rst feces the day at 07:00 a.m., and color and fecal consistency were evaluated. For microbiological analysis, an aliquot was reserved in plastic bags, frozen, and sent to the microbiological laboratory for further analysis. Lactic acid bacteria were counted for Lactobacillus spp. and Streptococcus spp. from fecal samples under anaerobic conditions. The diet produced di erences (P<0.05) in feces consistency 98% had normal and rm stools, while 2% had loose ruminant-type feces. We observed no di erence (P<0.05) for color, verifying 100% of greenish feces, normal for equines. There was no e ect (P>0.05) on pH and on the number of Lactobacillus spp. and Streptococcus spp. The inclusion of up to 28% citrus pulp concentrates for horses did not promote change in the physio-chemical characteristics and on the population of lactic acid-producing bacteria in feces.

Effect of autochthonous starter cultures in the production of "Paio", a traditional Portuguese dry- cured sausage

Traditional dry-cured sausages are highly appreciated in Mediterranean countries. The aim of the present study was to evaluate the effect of different starter cultures in the sausages Alentejano pig meat was used to prepare drycured sausages in a local factory. Staphylococcus xylosus, Lactobacillus sakei and a yeast strain were inoculated at a concentration of 106 cfu/g meat batter both in separate and in mixed culture. Three independent batches with two replicates per treatment were produced. Samples were collected throughout the ripening process. pH and aw were determined according to the ISO standards. Microbiological counts of total mesophiles, total psycrotrophs, anaerobes, coagulase-negative staphylococci (CNS), lactic acid bacteria (LAB), enterobacteria, yeasts and moulds and Listeria monocytogenes were done according to the respective ISO standards, as well as detection of Salmonella spp. Biogenic amines quantification was performed by HPLC as described by Roseiro et al. (1). The treatment with L. sakei alone was the most effective in reducing the contamination level both with Salmonella spp. and L. monocytogenes, however this effect seems to be lost in the mixed cultures. The presence of the yeast strain seems to increase the levels of phenylethylamine and histamine. The contents in cadaverine, putrescine and tyramine were generally lower in the inoculated sausages. Regarding tyramine, the treatments with L. sakei showed significantly lower values. No significant differences between treatments were observed for both spermine and spermidine.

Firmicutes dominate the bacterial taxa within sugar-cane processing plants

Sugar cane processing sites are characterised by high sugar/hemicellulose levels, available moisture and warm conditions, and are relatively unexplored unique microbial environments. The PhyloChip microarray was used to investigate bacterial diversity and community composition in three Australian sugar cane processing plants. These ecosystems were highly complex and dominated by four main Phyla, Firmicutes (the most dominant), followed by Proteobacteria, Bacteroidetes, and Chloroflexi. Significant variation (p , 0.05) in community structure occurred between samples collected from ‘floor dump sediment’, ‘cooling tower water’, and ‘bagasse leachate’. Many bacterial Classes contributed to these differences, however most were of low numerical abundance. Separation in community composition was also linked to Classes of Firmicutes, particularly Bacillales, Lactobacillales and Clostridiales, whose dominance is likely to be linked to their physiology as ‘lactic acid bacteria’, capable of fermenting the sugars present. This process may help displace other bacterial taxa, providing a competitive advantage for Firmicutes bacteria.

Microparticle-mediated gene delivery for the enhanced expression of a 19-KDa fragment of merozoite surface protein 1 of Plasmodium falciparum

The 19 kDa carboxyl-terminal fragment of merozoite surface protein 1 (MSP119) is a major component of the invasion-inhibitory response in individual immunity to malaria. A novel ultrasonic atomization approach for the formulation of biodegradable poly(lactic-co-glycolic acid) (PLGA) microparticles of malaria DNA vaccines encoding MSP119 is presented here. After condensing the plasmid DNA (pDNA) molecules with a cationic polymer polyethylenimine (PEI), a 40 kHz ultrasonic atomization frequency was used to formulate PLGA microparticles at a flow rate of 18 mL h1. High levels of gene expression and moderate cytotoxicity in COS-7 cells were achieved with the condensed pDNA at a nitrogen to phosphate (N/P) ratio of 20, thus demonstrating enhanced cellular uptake and expression of the transgene. The ability of the microparticles to convey pDNA was examined by characterizing the formulated microparticles. The microparticles displayed Z-average hydrodynamic diameters of 1.50-2.10 lm and zeta potentials of 17.8-23.2 mV. The encapsulation efficiencies were between 78 and 83%, and 76 and 85% of the embedded malaria pDNA molecules were released under physiological conditions in vitro. These results indicate that PLGA-mediated microparticles can be employed as potential gene delivery systems to antigen-presenting cells in the prevention of malaria.

Evaluation of Inoculated Lablab Silage for Growing Dairy Heifers

A summer grown forage legume crop – Lablab (Lablab purpureus) harvested in autumn, was ensiled as plastic wrapped, large round bales. Of the 30 bales produced, 13 were inoculated with a bacterial inoculant containing Lactobacillus plantarum and Enterococcus faecium. Inoculant was premixed at 30 g/litre water, cultured overnight (18 hours) then sprayed onto cut forage during the baling and wrapping procedure at 1 litre per tonne of silage. A replicated feeding experiment was conducted in July - August 1998 (5 weeks), using 24 eight month old Holstein Friesian heifers group fed non-inoculated or inoculated silage to appetite plus 2 kg rolled sorghum grain/heifer.day. Chemical composition and nutritive value of well preserved bales of control and inoculated silages were similar (P>0.05) with 50% DM and 26 g N and 6.8 MJ ME per kg DM. Lactic acid and acetic acid concentrations were 11.4 v. 11.4 and 4.90 v. 3.75 g/kg DM for control and inoculated silages respectively (P>0.05). Heifers preferentially selected leaf from the silage offered and maintained liveweight gains of 0.70 and 0.61 kg/day respectively (P>0.05) during the silage feeding period. High DM and low WSC content of the parent forage may have reduced the opportunity for the bacterial inoculant to have effect. Animal production for a consuming world : proceedings of 9th Congress of the Asian-Australasian Association of Animal Production Societies [AAAP] and 23rd Biennial Conference of the Australian Society of Animal Production [ASAP] and 17th Annual Symposium of the University of Sydney, Dairy Research Foundation, [DRF]. 2-7 July 2000, Sydney, Australia.

Spontaneous fermentation of traditional sago starch in Papua New Guinea

Sago starch is an important dietary carbohydrate in lowland Papua New Guinea (PNG). An investigation was conducted to determine whether microbes play a role in its preservation using traditional methods. In 12 stored sago samples collected from PNG villages, lactic acid bacteria (LAB) were present (>= 3.6 x 10(4) cfu/g) and pH ranged from 6.8 to 4.2. Acetic and propionic acids were detected in all samples, while butyric, lactic and valeric acids were present in six or more. In freshly prepared sago, held in sealed containers in the laboratory at 30 degrees C, spontaneous fermentation by endogenous microflora of sago starch was observed. This was evident by increasing concentrations of acetic, butyric and lactic acids over 4 weeks, and pH reducing from 4.9 to 3.1: both LAB and yeasts were involved. Survival of potential bacterial pathogens was monitored by seeding sago starch with similar to 10(4)/g of selected organisms. Numbers of Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus fell to <30/g within 7 days. Salmonella sp. was present only in low numbers after 7 days (<36/g), but Escherichia coli was still detectable after three weeks (>10(2)/g). Fermentation appeared to increase the storability and safety of the product.