Lactobacillus plantarum and subspecies of Lactobacillus casei were isolated from good quality mature Cheddar cheese and characterized with respect to metabolic functions that would allow their use in cheesemaking. In this way microbiological control of the maturation process with particular emphasis on protein catabolism was achieved. The lactobacilli isolated were selected for low growth rates (and acid production) in milk, and low proteinase activity to allow for their addition in high numbers to cheesemilk together with the normal starter flora (group N streptococci). The growth and acid production of the starter bacteria were unaffected by the presence of the lactobacilli during cheese manufacture and it was found that the added lactobacilli were able to grow and function under the conditions prevalent in Cheddar cheese during maturation. It was also demonstrated that the lactobacilli could be grown in an artificial medium to high numbers under controlled conditions and could be harvested for the preparation of cell concentrates, a necessary characteristic for commercialization. The lactobacilli also metabolized citrate, a potential problem in cheese maturation associated with C02 production but this did not adversely affect the maturation process under the conditions used. Compared to the group N streptococci the non-starter lactobacilli possessed a proteinase system that had a higher temperature optimum and was less affected by heat and sodium chloride. They also possessed a more active peptidase system although both the lactobacilli and the starter organisms possessed a similar range of peptidases. Non-starter lactobacilli were added to normal cheese and cheese made with proteinase negative starter. The added organisms did not adversely affect manufacturing parameters and did not metabolize citrate or lead to the formation of biogenic amines. However protein catabolism rates, particularly with respect to peptide degradation, were increased, as was flavour development and intensity. It was observed that the body and texture of the cheeses was unaffected by the treatment. By controlling both the starter and non-starter microflora in the cheeses a practical system for favourably influencing cheese maturation was possible. The investigation has demonstrated that carefully selected and characterized non-starter lactobacilli can be incorporated into Cheddar cheese manufacture in order to influence flavour development during maturation. Moreover the organisms can be added to the vat stage of manufacture without causing problems to the manufacturing process. This approach is a simple cost effective means of improving the cost of Cheddar cheese production and provides an unique opportunity to improve and control quality of all Cheddar cheese produced.

Studies were conducted using cultured immortalized wild type (non-CF) and mutant (CF) DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) tracheal epithelial cells on the anti-inflammatory impact of agents that may alter ceramide and glutathione (GSH) metabolism. The CF cells demonstrated abnormally high levels of GSH and glutathione disulfide (GSSG), which could diminish intracellular production of ceramide, a key modulator of inflammation and apoptosis. Hence, additional cell culture studies were carried out with a known inducer of in situ ceramide synthesis, N-4(4-hydroxyphenyl) retinamide (fenretinide) on interleukin (IL)-8 release, intracellular ceramide content, and cellular proliferation in both the basal state and following the inflammatory stimuli of tumor necrosis factor (TNF) -alpha. Fenretinide treatment was associated with a dose-dependent increase in the cellular content of ceramide in both CF and non CF cells. Also, an inhibition of IL-8 release in the inflammatory condition of TNF-alpha treatment was observed following fenretinide treatment in the CF cells. As hyperbaric treatment of whey proteins was previously associated with improved survivability and higher GSH content in a Pseudomonas aeruginosa murine model of cystic fibrosis (CF), the anti-inflammatory role of low molecular weight peptides (< 1kDa) generated from enzymatic hydrolysis of native and pressurized whey protein isolates (WPI) was examined. Pressure treatment of WPI was associated with an enhanced protein digestibility and an altered peptide profile following in vitro digestion. The whey peptides were tested CF and non-CF lung epithelial cells to identify for their effects on GSH metabolism. The impact of the combined treatment of fenretinide and WPH was also tested in terms of apoptosis and cytokine release in cell culture. As opposed to non-CF cells, CF cells showed a strong downtrend in release of IL-8 following the combined fenretinide and whey peptide treatment. In addition, whey peptides protected wild type epithelial cells from the apoptotic effect of fenretinide. Our results suggest the usefulness of these agents as a pharmacological treatment in CF.

The growth hormone receptor gene (GHR) has been previously documented to be a good candidate gene for detection of a quantitative trait locus (QTL) which influences milk production in Holstein cattle. In this study, the promoter region of the GHR gene and microsatellite markers AGAL29 and BM5004 were studied. Their effects on milk yield (MY), fat yield (FY), protein yield (PY), fat percentage (FP) and protein percentage (PP) were examined. DNA was isolated from 1746 used by the artificial insemination (AI) industry representing 26 half-sibling families. Three polymorphisms in the GHR gene were genotyped (GHRAlu, GHRAcc and GHR Stu) along with both microsatellites. The markers were analyzed in a cross-family analysis. The model included a population mean, a fixed grandsire effect, a fixed allele effect and a random residual error. The data was also analyzed using a nested model in a granddaughter design to investigate a possible consistency in the allelic effect in individual families. Lastly, the data was analyzed using the haplotypes of GHRAlu and GHR Acc, using the same model as the cross-family analysis. It included an analysis of a fixed haplotype effect instead of a fixed allele effect. (Abstract shortened by UMI.)

Laboratory scale Cheddar type cheese were made from 411 milk samples originating from Ayrshire, Brown Swiss, Canadienne and Jersey with different phenotypes of kappa-casein (kappa-CN) and beta-lactoglobulin (beta-LG). From the milk input, cheese and whey outputs, the cheese yield, cheese composition and cheesemaking efficiency and other parameters were determined. The overall 37% moisture adjusted cheese yield and cheese yield efficiency were 11.19g, 10.19g, 11.97g, 11.46g and 98.91%, 90.10%, 90.76% and 90.92% for Ayrshire, Brown Swiss, Canadienne and Jersey respectively. The milk of the four respective breeds contained 12.91, 12.69, 13.50, 14.57% total solids; 3.97, 3.58, 4.40, 4.61% fat; 3.61, 3.73, 3.81, 4.00% protein. In Ayrshire, the combination BB/BB and BB/AA (kappa-CN/beta-LG) were associated with higher 37% moisture adjusted cheese yield with the values of 12.51 and 12.83 g/100 g milk respectively. The cheese composition for these two types of milk were 62.28 and 63.96% total solids, 24.22 and 20.40% protein; 32.75 and 37.92% fat. For Brown Swiss, type AA/BB was associated with higher cheese yield (11.18g) with composition of 62.15% total solids, 22.54% protein, 33.23% fat. The phenotype with the highest cheese yield for Canadienne is BB/AB (12.45g) with cheese composition of 63.61% total solids, 23.27% protein and 63.61% fat. In Jersey, the phenotype combination with higher cheese yield (14.59g) is BB/BB. The cheese composition corresponding to this phenotype was 58.64% total solids, 22.96% protein and 29.59% fat. Phenotypes associated with better coagulating properties for Ayrshire, Brown Swiss, Canadienne and Jersey were BB/AB, BB/BB, BB/BB and BB/BB for kappa-CN/beta-LG respectively.

This study was based on the 462 milk samples collected from approximately 2000 cows registered in Dairy Herd Analysis Service (DHAS). Milk samples from fresh milks were phenotyped by gel electrophoresis. Milk samples were selected according to the nine possibilities of phenotype combination of $ kappa$-casein AA, AB, BB and $ beta$-lactoglobulin AA, AB and BB. Selected milk samples from fresh milks were heated at 25$ sp circ$C, 60$ sp circ$C, 70$ sp circ$C, 80$ sp circ$C and 90$ sp circ$C, respectively. Whole casein and whey protein were separated by adjusting the pH to 4.6. Quantitative determination of milk protein were performed by reverse-phase HPLC. Whole casein was separated to $ kappa$-casein ($ kappa$-Cn), $ beta$-casein ($ beta$-Cn) and $ rm alpha sb{s}$-casein ($ rm alpha sb{s}$-Cn). Whey protein was separated as immunoglobulin (Ig), bovine serum albumin (BSA), $ alpha$-lactalbumin ($ alpha$-La) and $ beta$-lactoglobulin ($ beta$-Lg). Individual milk protein fraction was quantitatively determined by relative peak area and their ratios to whey protein or casein. The denaturation of individual milk protein at different heating temperature was investigated. (Abstract shortened by UMI.)

No description available.

Hydrostatic pressure has been demonstrated to induce major changes in secondary structure of whey proteins resulting in an increased digestibility in vitro, and possibly an improvement of the glutathione (GSH) inducing effect of whey proteins in vivo. Micro filtration and ion-exchange, two commonly used processing techniques in whey protein manufacture, generate whey proteins with different compositions. Two animal studies were designed to compare the digestibility and GSH inducing effects of whey protein isolates (WPIs) treated with three repeated pulse cycling of pressure (3-cycle) or single pulse of high pressure (1-cycle) and pressurized microfiltrated and ion-exchange WPIs. The results indicate that special hydrostatic pressure treatment on the proteins improves its growth stimulating effect, but does not enhance the GSH-inducing effect of WPI in the healthy growing rats. Difference among commercial whey protein products is also an important factor that affects the biological properties of the pressurized whey proteins. In conclusion, both proper pressure treatment and product composition should be considered in order to find the most bio-effective whey protein preparation.

Recent studies have demonstrated that applied hydrostatic pressure can affect the functional properties of whey protein isolate (WPI). In this work, the effects of applied hydrostatic pressure on the tertiary and secondary structure of whey proteins were investigated by spectroscopic and rheological techniques to elucidate the molecular basis of such pressure-induced changes in protein functionality. The individual protein components of WPI and various samples of WPI obtained from different sources were subjected to different single-cycle pressure treatments of up to 400 MPa in 100 MPa increments with 30-min holding time as well as to pressures ranging from 450 to 650 MPa without a holding time. Electrospray ionization-mass spectrometry, circular dichroism, and Fourier transform Raman spectroscopic studies of pressure-treated samples of beta-lactoglobulin, the major protein component of WPI, revealed significant changes in tertiary structure. Fourier transform infrared spectroscopic studies revealed that the secondary structure of beta-lactoglobulin was also sensitive to applied pressure and holding time. The secondary and tertiary structure of alpha-lactalbumin, the second most prevalent protein in WPI, was unaffected by applied hydrostatic pressure. The spectroscopic behaviour of the various samples of WPI subjected to pressure treatment was variable and indicated that the response of WPI to applied hydrostatic pressure is dependent on the method used to isolate the WPI from whey. The rheological profiles of beta-lactoglobulin, alpha-lactalbumin, and WPI samples after various pressure treatments were also recorded. Both beta-lactoglobulin and WPI exhibited marked increases in viscosity with increasing pressure, whereas alpha-lactalbumin remained solutions exhibited no significant change in viscosity. These studies have furthered the understanding of the effects of applied hydrostatic pressure on the molecular structure and rheological pr

DNA methylation has been shown to be involved in switching a number of genes on or off in particular cells. The relationship between DNA methylation and $ beta$-casein gene expression in the mammary tissue of lactating cows and mammary epithelial cells was examined. A positive correlation existed between hypomethylation of two MspI/HpaII sites in the body and one MspI/HpaII site in the 3$ sp prime$ end of the $ beta$-casein gene and its expression. In addition to these sites, hypomethylation of a distal MspI/HpaII site and HindIII sensitivity at a HindIII site also correlated with gene expression. Five DNase I hypersensitive sites were located within a 8 kb fragment. These sites designated as H1 to H5 were mapped approximately $-5, -1.3, -0.2,$ 1.7 and 2.5 kb with respect to the start site of transcription, respectively. The H2 and H3 sites were within a 1790 bp sequence that has been reported to contain a responsive element for prolactin and extracellular matrix dependent regulation and the binding site for mammary gland specific factor. / To study the dynamic changes in hypomenthylation at the MspI/HpaII sites and HindIII sensitivity, mammary tissues from pregnant heifers were evaluated. Site specific demethylation was observed depending on the stage of gestation. Demethylation of two MspI/HpaII sites (denoted M2 and M4) occurred during the early gestation, progressed slowly until mid-pregnancy, and rapidly during the last part of pregnancy. During the early stages of gestation, changes in the HindIII sensitivity in the coding domain of the $ beta$-casein gene also took place. Despite changes in HindIII sensitivity, the second HindIII site remained resistant to HindIII. By the fifth stage of gestation, the third MspI/HpaII site (M3) became less methylated and during this time the H2 site became more sensitive to HindIII. Northern analysis confirmed that demethylation of the M3 site and the acquisition of HindIII sensitivity at the H2 site was correlated with $ beta$-casein transcription. / Although $ alpha$-lactalbumin and $ beta$-casein genes are structurally and evolutionarily unrelated, they likely share common regulatory features, since both are expressed in the mammary gland during lactation. To investigate this possibility, methylation of the $ alpha$-lactalbumin gene was examined. In vivo studies revealed hypomethylation of the bovine $ alpha$-lactalbumin gene at two MspI sites and a cluster of two HhaI sites during the first and second stage of gestation, respectively. Furthermore, hypomethylation events occured only in the functional gene and not in pseudogenes, and the hypomethylation pattern was established prior to gene expression. / Taken together, the present finding suggest that DNA hypomethylation is necessary for the expression of two mammary-specific milk protein genes, $ beta$-casein and $ alpha$-lactalbumin. Hypermethylation within the body of these genes may silence these genes in non-expressing tissues and in non-epithelial cells within the mammary gland during lactation.

Thesis (M.S.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on January 16, 2008) Includes bibliographical references.