Evaluation of microbial adjuncts and their effect on the ripening of cheddar cheese

A bacteriocin-producing strain of Lactobacillus paracasei DPC 4715 was used as an adjunct culture in Cheddar cheese in order to control the growth of “wild” nonstarter lactic acid bacteria. No suppression of growth of the indicator strain was observed in the experimental cheese. The bacteriocin produced by Lactobacillus paracasei DPC 4715 was sensitive to chymosin and cathepsin D and it may have been cleaved by the rennet used for the cheese manufactured or by indigenous milk proteases. A series of studies were performed using various microbial adjuncts to influence cheese ripening. Microbacterium casei DPC 5281, Corynebacterium casei DPC 5293 and Corynebacterium variabile DPC 5305 were added to the cheesemilk at level of 109 cfu/ml resulting in a final concentration of 108 cfu/g in Cheddar cheese. The strains significantly increased the level of pH 4.6-soluble nitrogen, total free amino acids after 60 and 180 d of ripening and some individual free amino acids after 180 d. Yarrowia lipolytica DPC 6266, Yarrowia lipolytica DPC 6268 and Candida intermedia DPC 6271 were used to accelerate the ripening of Cheddar cheese. Strains were grown in YG broth to a final concentration of 107 cfu/ml, microfluidized, freeze-dried and added to the curd during salting at level of 2% w/w. The yeasts positively affected the primary, secondary proteolysis and lipolysis of cheeses and had aminopeptidase, dipeptidase, esterase and 5’ phosphodiestere activities that contributed to accelerate the ripening and improve the flavor of cheese. Hafia alvei was added to Cheddar cheesemilk at levels of 107 cfu/ml and 108 cfu/ml and its contribution during ripening was evaluated. The strain significantly increased the level of pH 4.6-soluble nitrogen, total free amino-acids, and some individual free amino-acids of Cheddar cheese, whereas no differences in the urea-polyacrylamide gel electrophoresis (urea-PAGE) electrophoretograms of the cheeses were detected. Hafia alvei also significantly increased the level of some biogenic amines. A low-fat Cheddar cheese was made with Bifidobacterium animalis subsp. lactis, strain BB-12® at level of 108 cfu/ml, as a probiotic adjunct culture and Hi-Maize® 260 (resistant high amylose maize starch) at level of 2% and 4% w/v, as a prebiotic fiber which also played the role of fat replacer. Bifidobacterium BB-12 decreased by 1 log cycle after 60 d of ripening and remained steady at level of ~107 cfu/g during ripening. The Young’s modulus also increased proportionally with increasing levels of Hi-maize. Hencky strain at fracture decreased over ripening and increased with increasing in fat replacer. A cheese based medium (CBM) was developed with the purpose of mimicking the cheese environment at an early ripening stage. The strains grown in CBM showed aminopeptidase activity against Gly-, Arg-, Pro- and Phe-p-nitroanalide, whereas, when grown in MRS they were active against all the substrates tested. Both Lb. danicus strains grown in MRS and in CBM had aminotransferase activity towards aromatic amino acids (Phe and Trp) and also branched-chain amino acids (Leu and Val). Esterase activity was expressed against p-nitrophenyl-acetate (C2), pnitrophenyl- butyrate (C4) and p-nitrophenyl-palmitate (C16) and was significantly higher in CBM than in MRS.

The role played by angiotensin (1-7) in the regulation of renal haemodynamics and excretory function in anesthetized rats

Initial studies have demonstrated that intra- renal infusion of Ang (1-7) caused a diuresis and natriuresis that was proportional to the degree of activation of the Renin Angiotensin Aldosterone System (RAAS). This raised the question as why the magnitude of this diuresis and natriuresis was compromised in rats receiving a high sodium diet (suppressed RAAS) and enhanced in low sodium fed rats (activated RAAS)? Could the answer lie with changes in intra-renal AT1 or Mas receptor expression? Interestingly, the observed Ang (1-7) induced increases in sodium and water excretion in rats receiving either a low or normal sodium diet were and blocked in the presence of the AT 1 receptor antagonist (Losartan) in the presence of the, 'Mas' receptor antagonist (A-779). These data suggest that both AT1 and 'Mas' receptors need to be functional in order to fully mediate the renal responses to intra-renal Ang (1-7) infusion. Importantly, further experimentation also revealed that there is a proportional relationship between AT 1 receptor expression in the rat renal cortex and the magnitude of the excretory actions of intra renal Ang (1-7) infusion, which is only partially dependent on the level of 'Mas' receptor expression. These observations suggest that although Ang (1-7) induced increases in sodium and water excretion are mediated by the Mas receptor, the magnitude of these excretory responses appear to be dependent upon the level of AT 1 receptor expression and more specifically Ang II/ AT 1 receptor signalling. Thus in rats receiving a low sodium diet, Ang (1-7) acts via the Mas receptor to inhibit Ang II/ AT 1 receptor signalling. In rats receiving a high sodium diet the down regulated AT 1 receptor expression implies a reduction in Ang II/ AT 1 receptor signalling which renders the counter-regulatory effects of intra-renal Ang (1-7) infusion redundant.

The impact of whey protein isolate on energy balance regulation

Using C57BL/6J mice fed whey protein isolate (WPI) enriched high fat (HFD) or low-fat diets (LFD), this study tested the hypothesis that WPI directly impacts on adiposity by influencing lipid metabolism. WPI suppressed HFD-induced body fat and increased lean mass at 8 weeks of dietary challenge despite elevated plasma triacylglycerol (TAG) levels, suggesting reduced TAG storage. WPI reduced HFD-associated hypothalamic leptin and insulin receptor (IR) mRNA expression, and prevented HFD-associated reductions in adipose tissue IR and glucose transporter 4 expression. These effects were largely absent at 21 weeks of HFD feeding, however WPI increased lean mass and cause a trend towards decreased fat mass, with notable increased Lactobacillus and decreased Clostridium gut bacterial species. Increasing the protein to carbohydrate ratio enhanced the above effects, and shifted the gut microbiota composition away from the HFD group. Seven weeks of WPI intake with a LFD decreased insulin signalling gene expression in the adipose tissue in association with an increased fat accumulation. WPI reduced intestinal weight and length, suggesting a potential functional relationship between WPI, gastro-intestinal morphology and insulin related signalling in the adipose. Extending the study to 15 weeks, did not affect adipose fat weight, but decreased energy intake, weight gain and intestinal length. The functionality of protein sensing lysophosphatidic acid receptor 5 (LPA5) in 3T3-L1 pre-adipocytes was assessed. Over-expression of the receptor in 3T3-L1 pre-adipocytes provided a growth advantage to the cells and suppressed cellular differentiation into mature fat cells. In conclusion, the data demonstrates WPI impacts on adiposity by influencing lipid metabolism in a temporal manner, resulting possibly due to changes in lean mass, hypothalamic and adipose gene expression, gut microbiota and gastrointestinal morphology. The data also showed LPA5 is a novel candidate in regulating of preadipocyte growth and differentiation, and may mediate dietary protein effects on adipose tissue.

The impact of whey protein consumption and exercise on the composition and diversity of the gut microbiota: a high through-put DNA sequencing approach

Advances in culture independent technologies over the last decade have highlighted the pivotal role which the gut microbiota plays in maintaining human health. Conversely, perturbations to the composition or actions of the ‘normal/functioning’ microbiota have been frequently associated with the pathogenesis of several disease states. Therefore the selective modulation of enteric microbial communities represents a viable target for the development of novel treatments for such diseases. Notably, while bovine whey proteins and exercise have been shown to positively influence several physiological processes, such as energy balance, their effect on the composition or functionality of the gut microbiota remains largely unknown. In this thesis, a variety of ex vivo, murine and human models are used in conjunction with high-throughput DNA sequencing-based analysis to provide valuable and novel insights into the impact of both whey proteins and exercise on enteric microbial communities. Overall the results presented in this thesis highlight that the consumption both whey protein isolate (WPI), and individual component proteins of whey such as bovine serum albumin (BSA) and lactoferrin, reduce high fat diet associated body weight gain and are associated with beneficial alterations within the murine gut microbiota. Although the impact of exercise on enteric microbial communities remains less clear, it may be that longer term investigations are required for the true effect of exercise on the gut microbiota to be fully elucidated.

Longitudinal study of changes in body mass index, anthropometric measures, dietary intake and physical activity in cohorts of school-going Irish adolescents

The prevalence of obesity worldwide has increased dramatically over the last few decades. Poor dietary habits and low levels of exercise in adolescence are often maintained into adulthood where they can impact on the incidence of obesity and chronic diseases. A 3-year longitudinal study of anthropometric, dietary and exercise parameters was carried out annually (2005 - 2007) in 3 Irish secondary schools. Anthropometric measurements were taken in each year and analysed longitudinally. Overweight and obesity were at relatively low levels in these adolescents. Height, weight, BMI, waist and hip circumferences and TST increased significantly over the 3 years. Waist-to-hip ratio (WHR) decreased significantly over time. Boys were significantly taller than girls across the 3 years. A 3-day weighed food diary was used to assess food intake by the adolescents. Analysis of dietary intake data was determined using WISP©. Mean daily energy and nutrient intakes were reported. Mean daily energy and macronutrient intakes were analysed longitudinally. The adolescents’ diet was characterised by relatively high saturated fat intakes and insufficient fruit and vegetable consumption. The dietary pattern did not change significantly over the 3 years. Boys consumed more energy than girls over the study period. A validated questionnaire was used to assess physical activity and sedentary activity levels. Boys were substantially more active and had higher energy expenditure estimates than girls throughout the study. A significant longitudinal decrease in physical activity levels among the adolescents was observed. Both genders spent more than the recommended amount of time (hrs/day) pursing sedentary activities. The dietary pattern in these Irish adolescents is relatively poor. Of additional concern is the overall longitudinal decrease in physical activity levels. Promoting consumption of a balanced diet and increased exercise levels among adolescents will help to reduce future public health care costs due to weight-related diseases.

The formulation, testing and stability of 16% fat cream liqueurs

Cream liqueurs manufactured by a one-step process, where alcohol was added before homogenisation, were more stable than those processed by a two -step process which involved addition of alcohol after homogenisation. Using the one-step process, it was possible to produce creaming-stable liqueurs by using one pass through a homogeniser (27.6 MPa) equipped with "liquid whirl" valves. Test procedures to characterise cream liqueurs and to predict shelf life were studied in detail. A turbidity test proved simple, rapid and sensitive for characterising particle size and homogenisation efficiency. Prediction of age thickening/gelation in cream liqueurs during incubation at 45 °C depended on the age of the sample when incubated. Samples that gelled at 45 °C may not do so at ambient temperature. Commercial cream liqueurs were similar in gross chemical composition, and unlike experimentally produced liqueurs, these did not exhibit either age-gelation at ambient or elevated temperatures. Solutions of commercial sodium caseinates from different sources varied in their calcium sensitivity. When incorporated into cream liqueurs, caseinates influenced the rate of viscosity increase, coalescence and, possibly, gelation during incubated storage. Mild heat and alcohol treatment modified the properties of caseinate used to stabilise non-alcoholic emulsions, while the presence of alcohol in emulsions was important in preventing clustering of globules. The response to added trisodium citrate varied. In many cases, addition of the recommended level (0.18%) did not prevent gelation. Addition of small amounts of NaOH with 0.18 % trisodium citrate before homogenisation was beneficial. The stage at which citrate was added during processing was critical to the degree of viscosity increase (as opposed to gelation) in the product during 45 °C incubation. The component responsible for age-gelation was present in the milk-solids non fat portion of the cream and variations in the creams used were important in the age-gelation phenomenon Results indicated that, in addition to possibly Ca++, the micellar casein portion of serum may play a role in gelation. The role of the low molecular weight surfactants, sodium stearoyl lactylate and monodiglycerides in preventing gelation, was influenced by the presence of trisodium citrate. Clustering of fat globules and age-gelation were inhibited when 0.18 % citrate was included. Inclusion of sodium stearoyl lactylate, but not monodiglycerides, reduced the extent of viscosity increase at 45 °C in citrate containing liqueurs.

A study of diet and health in the elderly; the gut microbiota as a source of bioactive agents

The global proportion of older persons is increasing rapidly. Diet and the intestinal microbiota independently and jointly contribute to health in the elderly. The habitual dietary patterns and functional microbiota components of elderly subjects were investigated in order to identify specific effector mechanisms. A study of the dietary intake of Irish community-dwelling elderly subjects showed that the consumption of foods high in fat and/or sugar was excessive, while consumption of dairy foods was inadequate. Elderly females typically had a more nutrient- dense diet than males and a considerable proportion of subjects, particularly males, had inadequate intakes of calcium, magnesium, vitamin D, folate, zinc and vitamin C. The association between dietary patterns, glycaemic index and cognitive function was also investigated. Elderly subjects consuming ‘prudent’ dietary patterns had better cognitive function compared to those consuming ‘Western’ dietary patterns. Furthermore, fully-adjusted regression models revealed that a high glycaemic diet was associated with poor cognitive function, demonstrating a new link between nutrition and cognition. An extensive screening study of the elderly faecal-derived microbiota was also undertaken to examine the prevalence of antimicrobial production by intestinal bacteria. A number of previously characterised bacteriocins were isolated (gassericin T, ABP-118, mutacin II, enterocin L-50 and enterocin P) in this study. Interestingly, a Lactobacillus crispatus strain was found to produce a potentially novel antimicrobial compound. Full genome sequencing of this strain revealed the presence of three loci which exhibited varying degrees of homology with the genes responsible for helveticin J production in Lb. helveticus. An additional study comparing the immunomodulatory capacity of ‘viable’ and ‘non-viable’ Bifidobacterium strains found that Bifidobacterium-fermented milks (BFMs) containing ‘non-viable’ cells could stimulate levels of IL-10 and TNF-α in a manner similar to those stimulated by BFMs containing ‘viable’ cells in vitro.