The metabolic pathways required for adenosine triphosphate (ATP) production within the cell are well understood, however recent publications suggest that metabolic pathways are closely linked to immune cell activation and inflammatory diseases. There has been little examination of the metabolic pathways that modulate mast cell activation and the feedback regulator lactic acid. Here we examine metabolic pathways and regulation within mast cells in the context of lipopolysaccharide (LPS) and interleukin (IL-33) activation, for which there has been little to no reported studies. First, we examine the effects of lactic acid, previously considered only a by-product of glycolysis and now understood to act as a negative feedback regulator of inflammation in the context of LPS activation and sepsis. Lactic acid is elevated in septic patients and associated with mortality, potentially due to suppressive effects on LPS signaling and contribution to late phase immunosuppression. By attenuating glycolysis and reducing ATP availability for signaling and cytokine transcription, lactic acid impairs the function of immune cells to fight the initial or subsequent infections. We support this with in vitro and in vivo data. Additionally, our lab has published that lactic acid can suppress IL-33 activation, potentially by metabolic modulation as with LPS activation; however there has been no study of the metabolic requirements for IL-33 activation. We report here that glycolysis is required for ATP and reactive oxygen species (ROS) production to augment signaling and cytokine production downstream of the IL-33 receptor. Together, these studies examine the contribution of metabolism to mast cell activation and may provide potential targets for treatments of diseases that involve LPS- or IL-33-dependent mast cell activation.

In the field of maxillofacial surgery, the treatment for the recovery of the fracture at condyle region of the mandible has been carried out using Bio-metals such as Titanium, Cobalt, Stainless-Steel because they were considered the standard materials for Implant plate devices for fracture fixation. Using these materials have led to undesired disturbances where the patient must undergo secondary surgery after recovery leading to exposure of the fracture site to the surrounding, metal-ion release into the human system, stress-shielding and interruption during imaging i.e. (Computerized tomography scans). The healing of the mandible requires a delicate and stable fixation procedure for the bone structure to heal. Bio-resorbable materials are the renovation for substituting metals for recovery of the fracture. The main dis-advantage using resorbable plates is absence of mechanical strength and stability. Bio-composites are the innovation for the treatment of the fractures the main study for this thesis is comparing the combination bio-ceramic and bio-resorbable materials using Finite Element Analysis software.

A new innovative product, stewsticks, made with beef, pork, spices, and dehydrated vegetables, was developed as a nutritious snack. Lactic or citric acid was added at pH 5.2 or 4.6 to both meatsticks and stewsticks. Meatsticks and stewsticks were prepared by mixing ingredients until a cohesive mass was obtained. This mixture was then extruded into sticks that were cooked to about 50% of original weight. Sticks were then cut to desired length, packaged, and stored. Then meatsticks (beef, pork, and spices) were compared to stewsticks for appearance, texture, flavor, and overall acceptability. The stewsticks had excellent shelf life due to combined hurdles of pH 5.2, water activity of 0.95 or less, salt, and vacuum packaging. Compared to meatsticks, one serving (2 ounces) of stewsticks had less fat (9 vs 11 g respectively), less cholesterol (75 vs 90 mg) and more dietary fiber (4 vs 2 g), carbohydrates (20 vs 4 g), vitamin A (11 vs 2% RDA), and vitamin c {32 vs 1% RDA). The type of acid did not affect panel preference, but the samples at pH 5.2 were preferred over samples at pH 4.6. overall, meatsticks were preferred by the consumer panel over stewsticks although there were 25% of them who rated stewsticks as moderately acceptable or higher.

We explored using de-oiled algal biomass (algal cake) as a low-value substrate for production of lactic acid in fermentations with Lactobacillus casei, and strategies for increasing lactic acid production at low pH. L. casei 12A algal cake (AC) fermentations showed carbohydrate and amino acid availability limit growth and lactic acid production. These nutritional requirements were effectively addressed with enzymatic hydrolysis of the AC using α-amylase, cellulase, and pepsin. Producing 0.075 g lactic acid per g AC from AC digested with all three enzymes. We explored heterologous expression of the cellulase gene (celE) from Clostridium thermocellum and the α-amylase gene (amyA) from Streptococcus bovis in L. casei 12A. Functional activity of CelE was not detected, but low-level activity of AmyA was achieved, and increased > 1.5-fold using a previously designed synthetic promoter. Nonetheless, the improvement was insufficient to significantly increase lactic acid production. Thus, substantial optimization of amyA and celE expression in L. casei 12A would be needed to achieve activities needed to increase lactic acid production from AC. We explored transient inactivation of MutS as a method for inducing hypermutability and increasing adaptability of L. casei 12A and ATCC 334 to lactic acid at low pH. The wild type cells and their ΔmutS derivatives were subject to a 100-day adaptive evolution experiment, followed by repair of the ΔmutS lesion in representative isolates. Growth studies at pH 4.0 revealed that all four adapted strains grew more rapidly, to higher cell densities, and produced significantly more lactic acid than untreated wild-type cells. The greatest increases were observed from the adapted ΔmutS derivatives. Further examination of the 12A adapted ΔmutS derivative identified morphological changes, and increased survival at pH 2.5. Genome sequence analysis confirmed transient MutS inactivation decreased DNA replication fidelity, and identified potential genotypic changes in 12A that might contribute to increased acid lactic acid resistance. Targeted inactivation of three genes identified in the adapted 12A ΔmutS derivative revealed that a NADH dehydrogenase (ndh), phosphate transport ATP-binding protein PstB (pstB), and two-component signal transduction system (TCS) quorum-sensing histidine kinase (hpk) contribute to increased acid resistance in 12A.

Compounds derived from the breakdown of glyceride esters of milk fat, such as free fatty acids and short chain esters, are recognised as playing an important role in the flavour of a range of fermented foods. Esterases, capable of hydrolysing ester bonds, and in some cases, synthesising them via an acyltransferase mechanism, typically enter the fermentation from the starter and adjunct lactic acid bacteria that are used to inoculate milk to initiate the fermentation process. With such an important role in the development of both desirable and undesirable flavours, understanding how these enzymes operate is essential for product control. In this study, the crystal structures of three lactic acid bacterial esterases were solved: EstA from Lactococcus lactis, and AA7 from Lactobacillus rhamnosus which are both capable of hydrolysis of short chain triglycerides as well as synthesising esters via a transferase mechanism, and AZ4, an esterase from L. rhamnosus which appears to be limited to hydrolysis reactions. Whilst all three were found to be members of the hydrolase family, unique features were found for each enzyme, reflecting the large differences in their primary sequences, substrate specificities and activities. EstA and AA7 were both found to have a shallow substrate binding cleft, bisected by the catalytic machinery. The divided binding cleft suggests that during a transferase reaction the transferred group binds in one pocket, with the donor and acceptor groups (dependant on the stage of catalysis) binding in the other. In contrast, AZ4 was found to have a single deep substrate binding cavity, extending into the enzyme interior, with the catalytic residues located near its entrance. The absence of a second binding site for an acceptor is consistent with AZ4 having only one function – that of a hydrolase. The structures presented in this study are the first three dimensional structures of esterases from lactic acid bacteria to be reported. Their analyses, both in native form, and complexed with a varity of ligands mimicking various stages of the reaction cycle have highlighted how this basic fold can be adapted to efficiently catalyse different reactions. More importantly, in the case of AZ4, these structures have suggested that there is a novel mechanism used by the esterases to promote the enzyme reaction to proceed to completion, by preventing a futile catalytic reaction.

A study was undertaken to determine the food safety of the fermented Māori delicacy, Toroi. Ten batches of Toroi were prepared by a commonly used traditional method that consisted of boiling the vegetable component, either watercress or puha, and combining it with chopped mussel flesh. The mixture was cooled and then stored in a refrigerator for up to eight months to allow natural fermentation to take place. All ingredients were sourced from retail outlets. The Toroi was examined at intervals over eight months for a range of pathogens (seven in all) that have been related to incidents of food poisoning in ready-to-eat foods in New Zealand. The survival of a faecal contamination indicator, the laboratory grown strain Escherichia coli NZRM 916, was mapped over eight months. Two strategies to prevent the growth of Listeria monocytogenes in Toroi were also investigated. Only one of the seven pathogens sought was recovered from any sample. This pathogen was Bacillus cereus, a spore-former known to be associated with vegetables. All batches contained B. cereus on the day of preparation but after two weeks refrigerated storage there was no further recovery from any sample. There was a very low incidence of natural E. coli in the Toroi, consistent with levels permitted in mussels sold in retail outlets. The laboratory grown strain, E. coli declined substantially over two months and was not recovered from any samples at eight months. A laboratory grown strain of Listeria monocytogenes, (L70) was added to Toroi and grew well with an increase in concentration of about seven-fold, over 19 days storage in a refrigerator. A bacteriocin producing lactic acid bacterium, Lactobacillus sake Lb706, was added in combined culture with L. monocytogenes to Toroi. It was found that at least 5 x108 L. sake cells were required as an inoculum to ensure elimination of L. monocytogenes from the Toroi. When a purified bacteriocin; nisin, was added, a concentration of 10 mg g-1 in the Toroi was required to eliminate L. monocytogenes. The inhibition study results suggest that unacceptably high inocula or purified bacteriocin would be required to prevent the growth of L. monocytogenes in Toroi. The results of this suggest that Toroi be prepared from mussels either purchased from a retail outlet or harvested from sites known to be free from contamination. Toroi should be safe to eat if prepared carefully, chilled promptly and thoroughly and allowed to ferment for at least two weeks. In addition, care should be taken to maintain Toroi at refrigerated temperatures until it is eaten.

Several lactic acid bacteria contain β-galactosidases. Beta galactosidases catalyze lactose hydrolysis and transfer acceptor sugars onto galactose, producing galactooligosaccharides. The aim of this work was to exploit β-galactosidases of lactic acid bacteria as crude cell extracts to produce novel oligosaccharides with mannose, N-acetylglucosamine, and fucose. Of 17 strains of lactic acid bacteria, transferase activity was the strongest in crude cell extracts of Lactobacillus delbrueckii subsp. bulgaricus, followed by Streptococcus thermophilus, Lactobacillus animalis, and Lactobacillus reuteri in a buffered 19% (w/w) lactose solution. Incorporation of 6 % (w/w) glycerol increased transferase activity and enzyme stability at higher incubation temperatures. Incorporation of 10% (w/w) mannose, N-acetylglucosamine and fucose as acceptor sugars yielded three distinct oligosaccharides with mannose and two with N-acetylglucosamine and fucose, with the composition confirmed using gas chromatography-mass spectrometry. This is the first public report indicating production of oligosaccharides containing N-acetylglucosamine and fucose from β-galactosidases of lactic acid bacteria.

With the increasing demand for biodiesel, the accumulation of byproduct, crude glycerol has become a problem which needs to be solved. Lactic acid is one of the value-added chemical which can be produced from glycerol that has wide uses in food and chemical industry. Although glycerol can be converted to lactic acid with an alkali as the catalyst at high glycerol conversion (100 mol%) and lactic acid yield (around 90 mol%), the high alkalinity would cause severe corrosiveness to a stainless steel reactor. In this study two tasks were performed to convert glycerol to lactic acid with satisfactory conversion and selectivity, and to reduce the corrosiveness of reaction medium. First, CaO was used as solid base catalyst. The highest lactic acid yield achieved was 40.8 mol% with a 97.8 mol% glycerol conversion, when operating at 290°C after 150 min reaction with molar ratio of CaO: glycerol=0.3. Also CaO has advantages such as high lactic acid productivity (3.35 g/(min·L)) and reusability. Meanwhile, CaO can be used as the catalyst for both biodiesel production and the following crude glycerol conversion to lactic acid. Second, for glycerol conversion with NaOH as catalyst, a fed-batch reactor was applied to continuously supply NaOH during reaction process, compensating the OH- neutralized by newly formed lactic acid. The optimal lactic acid yield of 80.5 mol%, with 92.8% glycerol conversion was obtained at 300 °C for 220min, with 1.1 M glycerol initial concentration. A first-order kinetic model for glycerol concentration versus time was developed and verified experimentally under conditions with different initial glycerol concentration and reaction temperature. Although crude glycerol samples contained large amount of impurities, both methods, conversion with solid base catalyst and with fed-batch reactor, were applied successfully to three crude glycerol samples provided by biodiesel manufacturers, and the lactic acid yield reached 52.3 mol% and 72.7 mol% respectively. Finally, the corrosion issue of different methods was compared based on the Fe3+ concentration (analyzed with atomic-absorption spectroscopy) in the products. Both methods of glycerol conversions, with solid base catalyst and fed-batch reactor, can reduce the corrosiveness of glycerol conversion with an alkali as the catalyst.

Broiler flocks become frequently asymptomatically contaminated by the enteric bacteria Salmonella sp. and Campylobacter sp. which are human pathogens. Among the strategies developed at farm level to reduce the incidence of these pathogens, some lactic acid bacteria have been shown to be interesting because of their antimicrobial activity and their stimulatory properties on the immune system of poultry. The aim of this thesis was to select bacteria with antagonistic activity against Salmonella or Campylobacter, and to improve their inhibitory effect by the combination with enzymes of polysaccharidase type. The first step of the thesis was an epidemiological study carried out in the Walloon region in order to determine the contamination way of broilers by Campylobacter in free range production. Results showed that the major way of contamination is the open-air range to which the animals have access during the rearing period. A preventive treatment of the open-air range and the straw litter with an antagonistic strain in combination with an enzyme seems thus to be suitable in this case. The second step of the work aimed at the selection of a xylanase for using as a dietary additive in combination with an antagonistic bacterial strain against Salmonella. Four xylanases were studied in vivo for their effect on growth performances of broiler chickens. Diet supplementation with enzyme led to an increased final body weight and daily weight gain (P < 0.05), without difference according to the bacterial or fungal origin of the xylanase. The Belfeed B1100MP xylanase, which is commercialized in he Walloon region, was selected in order to develop a probiotic-xylanase feed additive. The purpose of the third part was to select a bacterial strain with antagonistic activity against Campylobacter for applying on open-air range and broiler litter. An in vitro screening of 12 lactic acid bacteria was realised using a co-culture assay with a growth medium based on straw and dehydrated poultry excreta, supplemented with different cellulase concentrations. Lactobacillus pentosus and Enterococcus faecium showed inhibitory effect against Campylobacter without enzyme which was intensified by cellulose from 200 ppm. Finally, the effect of dietary supplementation with a L. plantarum strain combined with the Belfeed B1100MP (PE treatment) on growth performance, microflora, and faecal Salmonella Typhimurium concentrations, was studied with experimentally infected broiler chickens. The PE diet allowed to partially overcome the negative effects associated with the infection on growth performance and microflora, and to significantly reduce faecal Salmonella concentration.

According to WHO, the addition of fluoride to milk could be considered as an alternative to water fluoridation for community-based caries prevention in childhood. School-based schemes in developing as well as industrial countries have demonstrated substantial benefits on oral health, but there are limited data available on the local events in the oral cavity after consumption of fluoridated milk. The general aim of the present investigations was to investigate the concentration of fluoride obtained in saliva and dental plaque after ingestion of Fmilk and to explore the possible effects on the oral ecology. A series of controlled studies were performed in vivo in which samples of saliva and dental plaque were collected and analysed with respect to fluoride content, microbial composition and acidogenicity. An in vitro study evaluated the effect on enamel lesion formation. In paper I, significantly increased concentrations of fluoride (p&lt;0.05) were disclosed in saliva 15 minutes after drinking the fluoride-containing water or milk. In the plaque samples however, the F-increase remained significantly elevated still after 2 hours. The availability of fluoride from milk was generally somewhat lower than from water but the differences were not statistically significant in either plaque or saliva. In paper II, the fluoride concentration in plaque was further explored after a single intake or habitual consumption of fluoridated milk together with a regular meal. The results showed that cariesinhibiting levels of fluoride persisted up to 4 hours after intake. There were no significant differences between the single intakes when compared with repeated intakes. In paper III, the influence of fluoridated milk on the salivary microorganisms associated with dental caries was evaluated. No significant alterations of the microflora were found compared with baseline. There was a slight reduction in the proportion of mutans streptococci after 2 and 4 weeks during consumption with fluoridated milk but the difference failed to reach statistical significance. In paper IV it was demonstrated that fluoridated milk significantly (p&lt;0.05) could counteract the lactic acid formation in dental plaque as initiated with sucrose. In paper V, laser fluorescence technique was used to monitor the effect of fluoridated milk on enamel lesion formation in an experimental caries model. The results reinforced previous research and showed a hampering effect of fluoridated milk. No side effects were reported in any of the investigations. The findings of this thesis substantiate that milk is a suitable vehicle for fluoride administration and contribute to the understanding and possible explanations for the anti-caries properties of fluoridated milk. The main conclusions were: a) intake of fluoridated milk resulted in significantly elevated fluoride levels in saliva within the first 15 minutes and up to 4 hours in dental plaque when fluoridate milk was consumed together with meal, b) no significant alteration of the salivary microflora was disclosed after habitual intake of fluoridated milk but a delayed carbohydrate-mediated lactic acid formation in suspensions of dental plaque could be demonstrated, c) the fluoride concentrations in plaque were not negatively influence by the food intake, and d) the in vitro findings advocated that fluoride added to milk reduced enamel lesion formation as assessed by laser fluorescence technique in an experimental caries model.According to WHO, the addition of fluoride to milk could be considered as an alternative to water fluoridation for community-based caries prevention in childhood. School-based schemes in developing as well as industrial countries have demonstrated substantial benefits on oral health, but there are limited data available on the local events in the oral cavity after consumption of fluoridated milk. The general aim of the present investigations was to investigate the concentration of fluoride obtained in saliva and dental plaque after ingestion of Fmilk and to explore the possible effects on the oral ecology. A series of controlled studies were performed in vivo in which samples of saliva and dental plaque were collected and analysed with respect to fluoride content, microbial composition and acidogenicity. An in vitro study evaluated the effect on enamel lesion formation. In paper I, significantly increased concentrations of fluoride (p&lt;0.05) were disclosed in saliva 15 minutes after drinking the fluoride-containing water or milk. In the plaque samples however, the F-increase remained significantly elevated still after 2 hours. The availability of fluoride from milk was generally somewhat lower than from water but the differences were not statistically significant in either plaque or saliva. In paper II, the fluoride concentration in plaque was further explored after a single intake or habitual consumption of fluoridated milk together with a regular meal. The results showed that cariesinhibiting levels of fluoride persisted up to 4 hours after intake. There were no significant differences between the single intakes when compared with repeated intakes. In paper III, the influence of fluoridated milk on the salivary microorganisms associated with dental caries was evaluated. No significant alterations of the microflora were found compared with baseline. There was a slight reduction in the proportion of mutans streptococci after 2 and 4 weeks during consumption with fluoridated milk but the difference failed to reach statistical significance. In paper IV it was demonstrated that fluoridated milk significantly (p&lt;0.05) could counteract the lactic acid formation in dental plaque as initiated with sucrose. In paper V, laser fluorescence technique was used to monitor the effect of fluoridated milk on enamel lesion formation in an experimental caries model. The results reinforced previous research and showed a hampering effect of fluoridated milk. No side effects were reported in any of the investigations. The findings of this thesis substantiate that milk is a suitable vehicle for fluoride administration and contribute to the understanding and possible explanations for the anti-caries properties of fluoridated milk. The main conclusions were: a) intake of fluoridated milk resulted in significantly elevated fluoride levels in saliva within the first 15 minutes and up to 4 hours in dental plaque when fluoridate milk was consumed together with meal, b) no significant alteration of the salivary microflora was disclosed after habitual intake of fluoridated milk but a delayed carbohydrate-mediated lactic acid formation in suspensions of dental plaque could be demonstrated, c) the fluoride concentrations in plaque were not negatively influence by the food intake, and d) the in vitro findings advocated that fluoride added to milk reduced enamel lesion formation as assessed by laser fluorescence technique in an experimental caries model.