Dissertations / Theses on the topic 'Milk proteins'

Various methods for extraction and analysis of high value minor proteins (lactoferrin, lactoperoxidase and immunoglobulins) directly from raw milk were explored. Extraction, purification and analysis of high-value minor proteins directly from milk without pre-treatment are major challenges for dairy industry, largely due to the complexity of milk and the presence of colloidal solids (casein micelles and milk fat globules). To overcome some of these challenges, this work focused on three main objectives: 1) characterization of cryogel monolith chromatography for purification of lactoferrin (LF) and lactoperoxidase (LP) directly from raw milk in single step, 2) identification and characterization of Protein A Mimetic affinity ligands for purification of immunoglobulins (Igs) from milk and 3) development and validation of a surface plasmon resonance method for simultaneous quantification of five whey proteins in multiple samples. Results portrayed the possibility of 40–50 column volumes of various milk samples (whole milk, skim milk and acid whey) to pass through a 5 mL cryogel monolith chromatography column at 525 cm hr⁻¹ without exceeding its pressure limits if the processing temperature is maintained around 35–37°C. Ideally, this should be the milk secretion temperature. The dynamic binding capacity obtained for the cryogel matrix (2.1 mg mL⁻¹) was similar to that of the binding capacity (2.01 mg mL⁻¹) at equilibrium with 0.1 mg mL⁻¹ of lactoferrin in the feed samples. Lactoferrin and lactoperoxidase was selectively bound to the cryogel column with trivial leakage in flowthrough fractions. Lactoferrin was recovered from elution fractions with a yield of 85% and a purity of 90%. These results, together with the ease of manufacture, low cost and versatile surface chemistry of cryogels suggest that they may be a good alternative to packed-bed chromatography for direct capture of proteins from milk, provided that the binding capacity can be increased. A Protein A Mimetic (PAM) hexapeptide (HWRGWV) peptide ligand that binds to the Fc portion of antibody molecules was explored for affinity purification of immunoglobulins from milk. The peptide has the ability to purify IgG from various milk and whey samples with a purity of greater than 85% in single step. More than 90% bound IgG was recovered with 0.2 M acetate buffer at pH 4.0 and total column regeneration was successfully achieved by 2.0 M guanidine-HCl. At 9.0 mg mL⁻¹ of IgG feed concentration, an equilibrium binding capacity of 21.7 mg mL⁻¹ and dynamic binding capacity of approximately 12.0 mg mL⁻¹ of resin was obtained. Recoveries and yields of IgG were significantly influenced by the feed IgG concentration. PAM hexamer ligand also contributed a significant amount of cross-reactivity with casein, glycomacropeptides and β-lactoglobulin proteins, however majority of these proteins were recovered in the regeneration step, except β-lactoglobulin, which co-eluted with IgG. Higher IgG concentration in feed vastly reduced the amount of cross-reactivity whilst increasing the recoveries and purities in the final product. PAM affinity ligands also showed interactions towards other classes of bovine immunoglobulins. These findings established the possibility of using PAM hexamer peptide as an alternative to conventional Protein A/G affinity chromatography for the isolation of Igs from milk in single step process. A surface plasmon resonance (SPR) method was developed for simultaneous, quantitative determination of commercially important whey proteins in raw and processed milk samples, whey fractions and various milk-derived products, with six samples per assay. Immobilized antibody stability and reproducibility of analyses were studied over time for 25 independent runs (n=300), giving a relative standard deviation (RSD) of <4%. Immobilized antibodies showed negligible non-specific interactions (<2–4 SPR response units (RU)) and no cross-reactivity towards other milk components (<1 RU). Regeneration of immobilised antibodies with glycine at pH 1.75 was determined to be optimal for maintaining the SPR response between samples. This method compared and validated well with reversed phase high performance liquid chromatography (RP-HPLC) and standard enzyme-linked immunosorbent assays (ELISA).

The research in this thesis deals with the influence of charge on the adsorption of milk proteins to surfaces. A variety of charged surfaces were used including negatively charged and zwitterionic liposomes prepared from phosphatidylglycerol and phosphatidylcholine respectively and positively and negatively charged polystyrene latices. Adsorption was determined by measuring the increase in the hydrodynamic radius of the particles by photon correlation spectroscopy and also by solution depletion techniques. In some instances, electrophoretic mobility measurements were also used in order to determine changes in the surface charge of the particle as a result of protein adsorption. The ionic strength and pH of the buffer were found to be important in the adsorption of protein to liposomes. In the absence of NaCl, adsorption did not occur. At low pH values, addition of both k-casein and B-Iactoglobulin to negatively charged liposomes caused very large increases in size presumably as a result of aggregation. At pH6.2, protein layer thicknesses on the negatively charged liposomes were significantly greater than on the zwitterionic ones due to charge repulsion between the negatively charged surface and the negatively charged regions of the proteins. Removal of the negatively charged phosphate groups which form a cluster in the hydrophilic region of B-casein resulted in a reduction in the thickness of the adsorbed protein layer on the negatively charged liposome but did not have any effect on the thickness on the zwitterionic surface. The thickness of adsorbed layers of as1-, k-, and B-casein and B-lactoglobulin on the phosphatidylcholine liposomes were all very similar at around 6nm. Addition of as1-casein to the negatively charged liposomes appeared to cause aggregation as a result of protein molecules bridging between liposomes. Attempts to determine the fraction of added protein which bound to the surface of the liposomes were unsuccessful and therefore, the binding of native, dephosphorylated and methyl-esterified l3-casein to small, monodisperse, positively and negatively charged polystyrene latices was studied. As with the liposomes, the thickness of the adsorbed B-casein layer was greater on the negatively charged surface. Removal of the phosphate groups from the protein decreased the layer thickness by about 4nm on the negatively charged surface but had relatively little effect on the thickness on the positively charged surface, once again showing the effect of charge interactions. As with dephosphorylation, methylation also reduces the net negative charge of the protein, but by a different mechanism. This also resulted in a reduction in the thickness of the adsorbed protein layers but only after a significant proportion of the free carboxyl groups had been esterified. Thus methylation of 35 % of these groups had relatively little effect on the thickness of the layer on the positively charged latex and no effect on the negatively charged, but esterification of a further 9% (equivalent to two residues) caused a substantial decrease in thickness on both surfaces. These changes are believed to result from alterations in both the charge and hydrophilicity of particular regions of the B-casein molecule. Bridging was found to occur when low levels of native or modified B-casein were added to the positively charged latex. Protein loading was found to range from 2.5 to 5.5mg m-2 depending on the nature of the protein and the charge on the surface. The thickness of adsorbed native and dephosphorylated B-casein layers on the negatively charged latex was found to be influenced by the presence of calcium and increasing ionic strength. Increasing levels of either calcium ions or NaCI in the medium resulted in a very pronounced decrease in the thickness of pre-adsorbed phosphorylated B-casein layers. The changes in dephosphorylated protein layers were less pronounced. The results are discussed in terms of the proposed loop-and-train configuration of the B-casein at the surface of the latex. The influence of protein phenotype and the extent of glycosylation on the adsorption of k-casein was also determined. The more highly glycosylated protein molecules, which also had a higher net negative charge, formed thicker layers on the negatively charged surface. Again, layer thicknesses were less on the positively charged surface, but for each x-cn phenotype glycosylation increased the thickness, presumably as a result of the increased hydrophilicity of the protein. k-Casein A, which has one more negative charge than the B phenotype, was found to give a slightly thicker layer on the negatively charged latex. Under certain conditions, adsorbed k-casein could be cleaved by the enzyme chymosin as shown by the reduction in the size of the coated latex.

Genetic variants of milk proteins are the result of mutation causing substitutions or deletions of amino acids in the peptides. Certain variants are closely associated with milk production, milk composition and milk qualities such as heat stability and cheesemaking properties. All of the milk protein variants known so far, have been detected by electrophoresis because mutations which have occurred gave rise to changes in net charges. "Silent" variants involve amino acid substitutions which are not accompanied by charge differences and hence would not be identified by electrophoresis. The aim of this project was to search for silent variants of $ alpha sb{ rm s1}$-casein, $ beta$-casein and $ kappa$-casein by application of reversed-phase HPLC for the identification of mutants causing changes in hydrophobicities of peptides.
Whole casein from 281 Holstein and Ayrshire milk samples were fractionated by DEAE-cellulose ion-exchange chromatography, using an FPLC system. The pure forms of major caseins ($ alpha sb{ rm s1}$-casein, $ beta$-casein and $ kappa$-casein) were hydrolysed by trypsin and the digests obtained therefrom were resolved by reversed-phase HPLC. Comparison of peptide profiles within the same electrophoretic variant of homozygous caseins indicated that 24 out of 264 $ alpha sb{ rm s1}$-casein BB, 9 out of 57 $ beta$-casein A$ sp1$A$ sp1$, 5 out of 55 $ beta$-casein A$ sp2$A$ sp2$ and 8 out of 188 $ kappa$-casein AA could be classified as potential silent variants.

Centrifugation of coconut milk resulted in cream, skim milk, and insoluble solids. Proteins were isolated from skim milk by the addition of acid, with or without heating. The separation and isolation gave the following coconut protein preparations: coconut milk, coconut skim milk, insoluble solids, acid precipitate, and acid-heat precipitate.
Trypsin inhibitory activity (TIA) of the coconut protein preparations was relatively low while tryptic digestibility of the isolated proteins was considerably lower than those of the coconut milk and skim milk, the digestibility of coconut protein preparations was lower than that of casein. In general, the emulsifying and farming properties of coconut protein preparations were lower than casein. The insoluble solids showed the highest viscosity when compared with the coconut protein preparations. In contrast to the whey protein concentrate (WPC), the apparent strain of gels from the acid precipitate increased as the pH increased. The gelation properties at pH 3 of the insoluble solids were better than WPC.
The estimated molecular weight by size-exclusion chromatography of coconut protein preparations gave 3 fractions with MW ranging from 6850 Da to 229402 Da. In native PAGE, coconut proteins were separated into at least 3 subunits and under SDS-denatured conditions, the major protein subunits showed MW of 54531 Da and 25008 Da, respectively. RP-HPLC separation of coconut milk, acid precipitate, and acid-heat precipitate gave 3 fractions containing several species of MW ranging between 35574 Da to 51209 Da when analyzed by mass spectometry.

This research investigates the protein interactions that occur when soy protein is added to milk and subjected to renneting or heating. Milk was fortified with 20% soy protein and enzymic coagulation studied at 35°C at various pH's and CaCl2 levels. The first part deals with the interaction between milk and soy proteins during rennet-induced milk coagulation. The first goal was to determine how soy proteins affected milk coagulation. The effects of native versus heat-denatured soy proteins on rennet coagulation time and curd firmness were compared. lmmunogold labeling along with transmission electron microscopy was used to identify and localfze soy proteins in coagulated milk. Partitioning of ß-conglycinin and glycinin, the two main soy protein fractions, between cheese and whey was determined by electrophoresis. Soy proteins affected milk coagulation to the greatest extent at pH 6.6. Both heat-denatured and native soy proteins increased rennet coagulation time. Only heat-denatured soy proteins affected final curd firmness. Most of ß-conglycinin was lost in whey, whereas glycinin was retained in curd. Soy proteins existed in the curd as aggregates that were less electron dense than casein micelles. At pH 6.6, heat-denatured soy proteins were fibrous and adhered to the surfaces of casein micelle, preventing direct micelle-micelle contact. This would delay aggregation rate and decrease curd firmness by decreasing the number and strength of links between casein micelles. Native soy proteins did not bind to the casein micelles but rather were physically trapped within curd. Their effect of delaying aggregation is thought to be a function of their binding of calcium. Adding CaCl2 or lowering the pH to 6.3 or 6.0 helped restore coagulation properties. The second goal was to determine what heat-induced interaction occurs between milk and soy proteins, specifically between κ-casein and glycinin. Both κ-casein and glycinin are heat labile and form insoluble aggregates when heated. When glycinin and κ-casein were heated together, some acidic polypeptides of glycinin crosslinked with κ-casein via disulfide linkages. However, when disulfide linkage was prevented by adding ß-mercaptoethanol , non-covalent interactions between κ-casein and both acidic and basic polypeptides of glycinin occurred that prevented the heat precipitation of glycinin. This non-covalent interaction between glycinin polypeptides and κ-casein may explain why the heat-treated soy proteins became attached to the surfaces of casein micelles during rennet coagulation of milk.

The control of milk protein production was investigated utilising two different approaches. The first model is one of intravenous infusion of atropine. Atropine, which decreases milk protein yield, has been theorised to act either by decreasing blood somatotropin (ST) concentration or by decreasing blood amino acid (AA) concentration. Thus, the first experiment was designed to test which mechanism, or both, is responsible for the effects on milk protein yield. Five lactating dairy cows were assigned to the following treatments which were administered intravenously: Saline (CONT), atropine (ATR), ATR + ST, ATR + AAs, and ATR + ST + AAs. Atropine treatment failed to decrease plasma ST concentration but did decrease plasma alpha-amino nitrogen concentration. Atropine treatment decreased milk protein yield but neither ST, AAs, nor ST + AAs were able to maintain milk protein yield at the CONT level when infused with ATR. It is clear that the treatments tested are not directly responsible for the decrease in milk protein yield due to ATR. Therefore, neither ST, AAs, nor ST + AAs appear to have direct control of milk protein production. Plasma insulin (INS) concentration was decreased and plasma IGF-I concentration was not decreased by ATR treatment. Insulin, therefore, presents itself as a candidate for direct control over milk protein synthesis. The second model is one of monitoring endocrine response to abomasal infusion of AAs mimicking the profile of milk protein with selective deletion of certain AAs. Six lactating dairy cows were subjected to the following treatments: Saline (negative control, NC), AAs (positive control, PC), PC minus methionine (PC-Met), PC minus lysine (PC-Lys), PC minus histidine (PC-His), and PC minus the branched-chain AAs (PC-BCAAs). All endocrine factors studied (ST, INS, glucagon & IGF-I) were affected by treatment. Plasma IGF-I concentration responded similarly, except for the PC-Met treatment, to milk protein yield (Weekes and Cant, 200

The purpose of this study was to develop a short, easy procedure to measure five major proteins in milk and to detect concentrations of added protein to dairy products. Combinations of casein or whey protein with nonfat-dry milk were made with concentration ratios from 0:10 to 10:0. Similar mixtures of defatted goat milk with defatted cow milk were prepared. Samples were hydrolyzed in 6 N HCl at 145°C for 4 h and analyzed for amino acid composition. Multiple regression equations were derived to estimate the relative content of whey protein or casein added to nonfat-dry milk and goat milk added to cow milk employing amino acid profiles of whey protein, casein, nonfat-dry milk, goat milk and cow milk. Correlation coefficient values were all greater than .99. Measuring individual concentrations of milk proteins required separating casein and why proteins by reverse phase high performance liquid chromatography on a C3 column. αs-, β-, and κ-casein were separated after dissociating casein micelles with mercaptoethanol and urea. A 40:60 to 0:100 gradient of .15 M sodium chloride/triethylamine (pH 2.5) and 40% acetonitrile was used. Whey proteins, α-lactalbumin and β-lactoglobulin were separated with a 95:5 to 0:100 gradient of .15 M sodium chloride (pH 2.4) and acetonitrile. Eluted proteins were collected from the column, analyzed for purity by electrophoresis, and hydrolyzed in 6 N HCl at 145°C for 4 h. Purified proteins and mixtures of purified proteins were analyzed for amino acid composition. Estimates of individual protein concentrations in mixtures were made by solving simultaneous equations based on amino acid composition using a tektronix 4052 computer.

Milk expression of recombinant proteins of pharmaceutical value in transgenic dairy animals is an emerging solution for molecules that cannot be made efficiently using the standard bioreactor platform. However, the expression of large quantities of recombinant proteins in the mammary gland can result in phenotypes with compromised lactation physiology. These negative consequences have been vaguely described in the literature and, in most cases; they have not been studied in depth with view to understanding the mechanisms of mammary disruption by transgene expression. Therefore, using a transgenic herd of goats expressing recombinant human butyrylcholinesterase, the main goals of this research were to: a) study the lactation performance and basic milk parameters; b) assess milk composition; c) explore the integrity of epithelial tight junctions, endoplasmic reticulum stress and accelerated cell death; d) investigate delayed lactogenesis; and e) assess the effects of "compensatory treatments" on transgenic lactation performance. Our findings showed that transgenic lactations were characterized by a slow/delayed start of milk production, a relatively normal milk volume at peak and a premature shutdown of milk production compared to controls. These compromised productivities were associated with a disrupted lipid secretion at the level of the secretory epithelium, and a dramatic raise in the presence of phagocytes in milk that was not associated with mammary infection. Milk composition studies indicated that transgenic goats produce lower quantities of caseins and short chain fatty acids. Through determination of serum album presence and Na:K ratio in milk we established the development of permeable tight junctions as an apparent mechanism of lactation disruption in the transgenic animals. Delayed expression of α-lactalbumin was proposed as a major determinant of the delayed lactogenesis observed in the tra
L'expression des protéines recombinantes de valeur pharmaceutique dans la glande mammaire des animaux transgéniques est une solution émergeante pour les molécules qui ne peuvent pas être produites efficacement par les bioréacteurs standards. Toutefois, l'expression de grandes quantités de protéines recombinantes dans la glande mammaire peut entraîner des phénotypes avec une physiologie de la lactation compromise. Ces conséquences négatives ont été vaguement décrites dans la littérature et, dans la plupart des cas ; elles n'ont pas été étudiées en profondeur pour comprendre les mécanismes qui perturbent la glande mammaire suite à l'expression des transgènes. Par conséquent, en utilisant un troupeau de chèvres transgéniques qui expriment la forme recombinée de la butyrylcholinesterase humaine, les principaux objectifs de cette recherche étaient: a) d'étudier la performance de la production laitière et les paramètres de base du lait; b) d'évaluer la composition du lait; c) d'explorer l'intégrité des jonctions serrés épithéliales, le stress du réticulum endoplasmique (RE) et la mort cellulaire accélérée; d) d'enquêter sur la lactogénèse retardée; et e) d'évaluer les effets des "traitements compensatoires" sur la performance de la production laitière des chèvres transgéniques. Nos résultats ont démontré que les lactations transgéniques étaient caractérisées par un départ lent/retardé de la production laitière, un volume de lait relativement normal au pic de la production et un arrêt prématuré de la production laitière par rapport aux contrôles. Ces productivités compromises étaient associées à une perturbation de la sécrétion des lipides au niveau de l'épithélium sécrétoire et une augmentation dramatique des phagocytes dans le lait qui n'était pas associée à une infection mammaire. Les études sur la composition du lait ont indiqués

The properties of genetic variants of $ alpha sb{ rm s1}$-casein (BB, AB), $ beta$-casein (A$ sp1$A$ sp1$, A$ sp2$A$ sp2$, A$ sp1$A$ sp2$, A$ sp1$A$ sp3$, A$ sp2$A$ sp3$, A$ sp1$B, A$ sp2$B, BB), $ kappa$-cn (AA, AB, BB) and $ beta$-lactoglobulin (AA, AB, BB) were compared. The caseins and $ beta$-lactoglobulin were purified by mass ion exchange chomatography. Model systems of $ alpha sb{ rm s1}$- and $ kappa$-caseins were compared with respect to their stability towards calcium ions and other major milk salts precipitation. $ beta$-Casein, $ alpha sb{ rm s1}$-casein BB + $ beta$-caseins and $ kappa$-casein were also compared in their ability to resist calcium ion precipitation. $ kappa$-casein AB was a better stabilizer of $ alpha sb{ rm s1}$-casein than the BB and AA variants. $ alpha sb{ rm s1}$-Casein BB resisted calcium ion precipitation more than the AB variant in presence of $ alpha$-lactose, citrate, chloride and magnesium ions. $ kappa$-Casein BB stabilized $ alpha sb{ rm s1}$-casein AB more than the BB variant in presence of calcium and phosphate ions. Solubility of $ kappa$-casein AA, however, was greater than that of $ kappa$-cn BB and $ kappa$-cn AB variants in calcium and phosphate solutions. As in $ alpha sb{ rm s1}$-casein, significant differences were also found among $ kappa$-casein variants in stabilizing $ beta$-casein against calcium ion precipitation. $ beta$-Caseins A$ sp1$A$ sp1$, A$ sp2$A$ sp2$ and A$ sp1$B produced the most stable micelles while those of A$ sp2$B and B variants the least at 0.03-0.25 $ kappa$-cn/$ beta$-casein ratios and 20 mM Ca$ sp{2+}$. However, stability of micelles formed from $ alpha sb{ rm s1}$-cn + $ beta$-cn A$ sp2$A$ sp3$, $ alpha sb{ rm s1}$-cn + $ beta$-cn A$ sp1$A$ sp3$, and $ alpha sb{ rm s1}$-cn + $ beta$-cn A$ sp2$A$ sp2$ caseins were higher than those of $ alpha sb{ rm s1}$-cn + $ beta$-cn A$ sp1$A$ sp2$, $ alpha sb{ rm s1}$-cn + $ beta$-cn A$ sp1$B and $ alpha sb{ rm s1}$-cn + $ beta$-cn A$ sp1$A$ sp1$.

A total of 596 milk samples with varying fat (3.0 to 4.0%) and protein (3.0 to 4.0%) contents were used to make laboratory-scale cheese and to determine coagulating properties. Higher levels of fat and protein in milk were associated with higher cheese yield. Milk protein has greater effect on cheese yield than milk fat. Adjusted yield increased by 1.91 and 1.29% for every percentage increase in the protein and fat of milk, respectively. Higher levels of fat in milk produced a cheese containing higher fat content and lower protein content. Similarly, higher levels of protein in milk produced higher protein content and lower fat content of cheese. Higher protein to fat ratio (or casein to fat ratio) in milk was associated with better efficiency of fat retention in the cheese. Casein retention in the cheese was not affected by the levels of fat and protein, or protein to fat ratio in milk. Milk adjusted for fat and protein resulted in delayed coagulation and a significant decrease in the curd firmness when compared with unadjusted bulk tank milk having the same levels of the two components.

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.

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.)

Isolated $ alpha sb{ rm s1}$-casein, $ alpha sb{ rm s2}$-casein, $ beta$-casein, $ kappa$-casein of different phenotypes, whole casein of different phenotype combinations of $ alpha sb{ rm s1}$-/$ beta$-/$ kappa$-casein, and $ beta$-lactoglobulin of different phenotypes were prepared and used in the study. Surface properties at the air-water interface, voluminosity and hydration, emulsifying capacity and emulsion stability, foaming and gelling properties were investigated to understand the relationship between the structure and functionalities of milk proteins. Phenotype CC of $ alpha sb{ rm s1}$-casein, A$ sp1$A$ sp1$ and A$ sp1$A$ sp2$ of $ beta$-casein, AB of $ kappa$-casein decreased surface tension at a faster rate than the other, phenotypes within the casein system under consideration. $ beta$-Casein, when compared to $ alpha sb{ rm s1}$-casein, $ alpha sb{ rm s2}$-casein and $ kappa$-casein, was the most surface active protein. The $ alpha sb{ rm s1}$-/$ beta$-/$ kappa$-casein haplotypes of BB/A$ sp2$A$ sp2$/AA and BB/A$ sp1$A$ sp1$/BB were found to be associated with higher surface activity than the other ten combinations of whole casein. $ alpha sb{ rm s-1}$-Casein BB, $ beta$-casein A$ sp2$A$ sp2$ and $ kappa$-casein AB had higher values for voluminosity and hydration than the other phenotypes of the corresponding individual casein. Within the four caseins, $ beta$-casein had the strongest ability to entrap water and the highest value for voluminosity, followed by $ alpha sb{ rm s1}$-casein, $ alpha sb{ rm s2}$-casein and $ kappa$-casein. Whole casein with the combination of BB/A$ sp2$B/BB had the highest value for voluminosity and hydration. Analysis of model emulsions suggested that the emulsifying properties of different sources of protein were dependent on the oil content. $alpha sb{ rm s1}$-Casein BB in 10% oil emulsion, $ beta$-casein A$ sp2$A$ sp2$ in 10% and $ beta$-casein A$ sp2$B in 40% oil emulsions were the best stabilizers. $ kappa$-Casein

With the increased demands of therapeutic proteins, there is going to be a need for new purification technologies which have high throughput, high yield and high resolution. Three purification technologies were explored as potential new technology to isolate recombinant and endogenous milk proteins: Expanded bed adsorption chromatography(EBAC) combined with hydrophobic interaction chromatography(HIC), Recycle continuous flow electrophoresis(RCFE) and Free flow isoelectric focusing(FFIEF). The first process(EBAC/HIC) used with Zn2+ as a selective precipitating agent, purified recombinant human protein C(rhPC) and IgG(contaminated with less than 1% IgA) from swine milk with high resolution and high yield while processing about 10-20 grams in a single operation. The second process(RCFE) was able to isolate the active sub-populations of rhPC from major milk contaminants( - and -pig casein) as wells as from the inactive sub-populations of rhPC. RCFE was able to process 1.5g total protein per hour on a small scale and is currently being researched to process 1kg total protein per hour. The third and final purification process(FFIEF) sub-fractionated 100mg of immuno-purified rhPC into 50 fractions. The FFIEF was able to produce a linear pH gradient over the range of 3-10 using 2% ampholytes. The fractionated rhPC showed differing degrees of activity that resulted from the -carboxylated glutamic acids and the sialic acids.
Ph. D.

Due to its strength, flexibility, and biocompatibility, spider silk is a highly appealing material for applications in the medical field. Unfortunately, natural spider silk is difficult to obtain in large quantities because spiders are territorial and cannibalistic, making them impractical to farm. Synthetic spider silk proteins produced by transgenic hosts such as bacteria and goats have made it possible to obtain the quantities of spider silk needed to study it more fully and to investigate its potential uses. The spider silk proteins produced in our laboratory do not have an optimal purification method to remove all of the non-biocompatible contaminants and have not previously been tested for their biocompatibility. The first focus of this dissertation was to create goat cells that can be used to create new goats. These new goats will produce proteins that can be purified more efficiently and more completely. The second focus of this dissertation was to perform biocompatibility tests on goat-derived spider silk proteins. Prior to performing any biocompatibility tests, a method was established for removing endotoxins – an impurity that causes an immune response in the body – from the proteins. This work has shed light on areas for improvement in the silk protein purification process and laid groundwork for the production of new goat-derived proteins. These steps will help make it possible for synthetic spider silk to progress further toward becoming a viable biomaterial.

A UHT -processed skim milk (85%)/orange juice (15%) drink was developed. Product integrity and stability were maintained by two methods. Proper homogenization of the blend before UHT processing stabilized a drink formulation containing .25% carboxymethyl cellulose and .025% carrageenan. Adjusting the pH of the blend (pH 6.3 and 6.5) resulted in a different stabilization. After 28 days at room temperature, settling of milk solids was 5.2% of volume height in the prehomogenized sample and 86.9% of volume height in the same blend that had not been homogenized prior to UHT processing. After storage, the two treatments were analyzed to verify that there was no perceived textural difference between the pH adjusted and unadjusted blends. A consumer product acceptability evaluation resulted in a split population, and more panelists liked the product than disliked it.

Milk-derived peptides are milk components able to influence specific physiologicalfunctions, finally acting on body health condition. At present, the bioactivitiesdescribed for milk-derived peptides include opiate, antithrombotic, antihypertensive, immunomodulating, antioxidative, antimicrobial, anticancer, mineral-carrying andgrowth-promoting activities. In this thesis, special attention has been given to bioactive peptides with ACE-inhibitory and immunomodulatory activities. In the Experiment 1 Enterococcus faecalis TH563 (E. faecalis TH563) and Lactobacillus delbrueckii subsp. bulgaricus LA2 (L. delb. bulgaricus LA2), two bacterial strains isolated from traditional North Eastern Italy dairy products, have been evaluated for their ability to produce fermented milk rich in ACE-inhibitory and immunomodulatory activities. The preliminary results obtained from this experiment demonstrated that E. faecalis TH563 produced a fermented milk with high ACEinhibitory activity while L. delb. bulgaricus LA2 showed an immunomodulatory activity on bovine lymphocytes. To better understand the mechanisms underlying the immunomodulatory activity of fermented milks, in the Experiment 2 the immunomodulatory effects of the milkderived bioactive tri-peptide YGG have been examined. YGG could be generated during milk fermentation from a–lactalbumin hydrolysis operated by bacterial enzymes, so it could be present in milk fermented by L. delb. bulgaricus LA2. YGG has been administered to purified peripheral blood lymphocytes in different culture conditions (presence/absence of activators of lymphocyte proliferation, different concentration of newborn calf serum) and its effects on lymphocyte proliferation and cytokine RNA expression (IL2 and INFg) have been analyzed. YGG modulated lymphocytes proliferation, in a manner dependent from culture conditions but its effects did not seem mediated by the modulation of IL2 or INFg RNA expression. An important limiting factor of the large-scale diffusion of food carrying potential bioactivities is the bioavailability of the peptides responsible of such bioactivities. The main factors influencing the bioavailability of peptides are the resistance to digestion enzymes of and the absorption by the intestinal epithelium. In the Experiments 3 and 4 the sensitivity to gastrointestinal enzymes and the mechanisms of absorption of the peptide b-CN (193-209) have been evaluated. b-CN (193-209) is a long hydrophobic peptide derived from b-casein that has been already isolated and identified from fermented milks and yogurt and displayed immunomodulatory properties. The pattern of digestion and the mechanisms of absorption have been evaluated in well-known in vitro models for the intestinal epithelium, as the brush border membrane vesicles (BBMV) and the Caco-2 cell line. The results of these studies demonstrated that the b-CN (193-209) peptide is absorbed intact by the Caco-2 monolayer, probably via a vesicles-mediated mechanism. In conclusion, the main contribution of this PhD thesis was to provide new knowledge about milk-derived products with bioactivities. In particular, original contributions are in relation to the mechanisms by which milk-derived bioactive peptides are generated, express their bioactivities, and their fate in the gastrointestinal tract. As a result, new questions have arisen on this area that could constitute the objective of further research programs in the future.
I peptidi bioattivi derivati dal latte costituiscono una parte importante del latte, in grado di influenzare lo stato di salute. Attualmente nel latte e nei suoi derivati sono stati identificati e caratterizzati peptidi ad azione oppioide, anti-trombotica, antiipertensiva, immunomodulatoria, antiossidante, antimicrobica, anticancro, stimolanti l’assorbimento di minerali e la crescita. In questa tesi particolare attenzione è stata rivolta ai peptidi bioattivi ad attività ACE-inibitoria e immunomodulatoria. Nell'Esperimento 1 Enterococcus faecalis TH563 (E. faecalis TH563) e Lactobacillus delbrueckii subsp. bulgaricus LA2 (L. delb. bulgaricus LA2), due ceppi batterici isolati da formaggi tradizionali del Nord Italia, sono stati caratterizzati per la loro capacità di produrre latti fermentati arricchiti in attività ACE-inibitoria e immunomodulatoria. I risultati preliminari hanno dimostrato che il ceppo E. faecalis TH563 è in grado di produrre un latte fermentato con elevata attività ACE-inibitoria mentre il ceppo L. delb. bulgaricus LA2 produce un latte fermentato con attività immunomodulatoria su linfociti bovini. Per meglio comprendere i meccanismi che regolano l’attività immunomodulatoria manifestata dal latte fermentato, nell’Esperimento 2 sono stati riportati i risultati di un esperimento atto a valutare gli effetti immunomodulatori del peptide bioattivo YGG. Tale tripeptide può essere generato durante il processo di fermentazione del latte dalla proteina alfa–lattoalbumina mediante l’azione proteolitica degli enzimi batterici, e quindi anche durante la fermentazione operata dai ceppi E. faecalis TH563 e L. delb. bulgaricus LA2. YGG è stato somministrato a linfociti isolati da sangue bovino e ne è stata studiata la capacità di modulare la proliferazione dei linfociti e l’espressione (RNA) di due citochine (IL2 e INFg) in diverse condizioni di coltura (presenza/assenza di attivatori della proliferazione, diverse concentrazioni di siero bovino). Lo studio ha dimostrato che il peptide YGG è in grado di modulare la proliferazione delle cellule e che tale modulazione è influenzata dalle condizioni di coltura ma non sembra essere mediata dalle citochine oggetto di studio. Un fattore importante che limita l’impiego su larga scala di alimenti con proprietà bioattive è la biodisponibilità dei peptidi portatori di tali bioattività. I fattori che maggiormente influenzano la biodisponibilità dei peptidi sono la resistenza alla digestione operata dagli enzimi gastrointestinali e la possibilità che tali peptidi possano essere assorbiti dall’epitelio intestinale. A questo scopo, negli Esperimenti 3 e 4 sono stati esaminati il profilo di digestione e i meccanismi di assorbimento del peptide b-CN (193-209). b-CN (193-209) è un peptide bioattivo lungo e idrofobico,derivato dalla b-caseina ed è già stato isolato e identificato in diversi prodotti derivati dal latte come yogurt e latte fermentati. Tale peptide possiede inoltre diverse attività immunomodulatorie. Il profilo di digestione di tale peptide e i meccanismi di assorbimento intestinale sono stati studiati in modelli in vitro adatti a rappresentare la mucosa intestinale, come le vescicole della membrana a orletto a spazzola (BBMV) e la linea cellulare Caco-2. Tali esperimenti hanno dimostrato che il peptide viene assorbito intatto dalle cellule Caco-2, probabilmente attraverso un trasporto mediato da vescicole. In conclusione, il contributo principale di questa tesi di dottorato è stato il fornire nuova conoscenza sui prodotti derivati dal latte ad azione bioattiva. Più specificatamente, questa tesi ha permesso di ottenere nuove informazioni sui meccanismi di produzione dei peptidi bioattivi derivati dal latte, sul loro meccanismo d’azione e sulla loro stabilità nel sistema gastrointestinale. Infine, i risultati ottenuti hanno contribuito a generare nuove idee che potranno costituire nuovi spunti per futuri progetti di ricerca.

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.)

Milk coagulation properties (MCP) are a fundamental aspect in cheese production, but un unfavorable trend over year on MCP have been observed in several countries. The cheese yield has decreased, accentuating the necessity to provide dairies with milk well suited for dairy products manufacture. During the past decades the focus of milk production has been kg’s of milk protein, but total milk protein content is a poor indicator of MCP, and the lack of an appropriate high-throughput analysis for routine determination of milk coagulation is currently limiting the opportunity to improve MCP by direct selection. Milk protein composition has long been a subject of interest for worldwide dairy researchers. As a consequence, information on milk protein genotype could be utilized to improve milk protein composition and MCP trough marker assisted selection without having to phenotype large progeny groups. Considering such options, it would be desirable to gain further knowledge about effects of milk protein genetic variants on milk protein composition and on MCP. Aims of the study were to investigate the effects of CSN2-CSN3 haplotypes (β-κ-casein) and BLG (β-Lactoglobulin, β-LG) genotypes on milk production traits, contents of protein fractions and detailed protein composition; to investigate the effects of CSN2-CSN3 haplotypes, BLG genotypes, contents of milk protein fractions and protein composition on MCP; to investigate the effect exerted by the relative ratio of κ-CN A to κ-CN B content on MCP and industrial cheese yield of three Italian cheese varieties. The final aim was to estimate genetic parameters of major milk protein fractions and estimate genetic and phenotypic correlation between milk protein fractions and MCP. A new reversed-HPLC method for the separation and quantification of the most common genetic variants of bovine milk proteins was developed and validated testing linearity, repeatability, reproducibility and accuracy. Contents of major protein fractions were measured by this new method in individual milk samples of 2,167 Simmental cows. Protein composition was measured as weight percentage of each casein (CN) fraction to total casein (TCN) and as weight percentage of β-LG to total whey protein (WH). Genotypes at CSN2, CSN3 and BLG loci were also assessed by HPLC and CSN2-CSN3 haplotype probabilities were estimated for each cow. Rennet coagulation time (RCT) and curd firmness (a30) were measured using a computerized renneting meter. Effects of haplotypes and BLG genotypes on yields were weak or trivial. Haplotypes carrying CSN2 B and CSN3 B exhibited greater TCN and casein number (CI), in comparison with all other haplotypes. Genotype BB at BLG was associated with increased protein, TCN and CI, when compared to genotype AA. Haplotypes including CSN3 B were associated with greater κ-CN content and percentage. Allele CSN2 B was associated with an increase of β-CN content, which occurred at the expense of content of αS1-CN. Haplotypes including allele CSN2 A1 exhibited decreased β-, αS2- and γ-CN concentrations and increased αS1- and κ-CN contents, whereas CSN2 I exerted positive effects on β-CN concentration, without altering other protein fractions content. Effects exerted by haplotypes on CN composition were similar to those exhibited on CN fractions contents. Allele BLG A increased β-LG concentration and altered the β-LG to α-Lactalbumin (α-LA) ratio. When protein fractions contents or protein composition were not included in the statistical model, haplotypes carrying CSN3 B allele exhibited shorter RCT and greater a30, in comparison with those carrying CSN3 A, and haplotypes carrying CSN2 B allele were responsible for a noticeable decrease of RCT and for an increase of a30, when compared to haplotype A2A. When effects of protein fractions contents or of protein composition were added to the model, no difference across haplotypes due to CSN3 and CSN2 alleles was observed for MCP, with the exception of the effect of CSN2 B on RCT, which remained markedly favorable. Also, the favorable effect exerted by CSN2 B on a30 was mediated by the increase of β-CN B in milk. Conversely, β-CN B is likely to exert a molecular effect on RCT, which does not depend upon variation of β-CN content associated to allele B. To test if the lack of effect of κ-CN genetic variant would have been observed also on cheese yield, milks with different κ-CN A to κ-CN B content ratios were separately manufactured to produce Montasio, Asiago and Caciotta cheese. Milk was characterized by having similar composition in terms of protein, TCN, CI, CN composition, β-CN composition and pH. Milk with the higher proportion of κ-CN B (HIGHB) exhibited similar coagulation properties but a higher cheese yield in all the investigated cheese in comparison with milk with a lower proportion of κ-CN B (LOWB). However, the increment of yield observed for HIGHB milk in Montasio cheese was ascribed to a greater fat content of HIGHB milk in comparison with LOWB milk. The probability of HIGHB milk giving a cheese yield 5 % greater than that of LOWB milk ranged from 51 to 67 % for Montasio cheese, but was lower than 21 % for Asiago and Caciotta cheeses. Thus, the ratio of κ-CN A to κ-CN B content did not relevantly affect industrial cheese yield when milks of similar CN composition were processed, and an indirect effect due to the higher κ-CN content of κ-CN B milk on cheese yield is to be suggested. Values of heritability for αS1-CN%, κ-CN% and β-CN% were similar and ranging from 0.61 to 0.70, whereas heritability of αS2-CN%, γ-CN% and β-LG% were 0.28, 0.29 and 0.33, respectively. When CSN2-CSN3 haplotype and BLG genotype were accounted for by the model, heritability estimates of all the protein fractions became similar suggesting that proteins synthesis is regulated by specific genes which control the overall production of milk protein. Genetic correlations among the contents of the five CN fractions and between CN fractions and WH fractions were generally low. Generally, all the CN fractions were also moderately positively correlated with WH. When data where adjusted for CSN2-CSN3 haplotype and BLG genotype, genetic correlations among the contents of protein fractions markedly increased confirming that all the fractions undergone a common regulation. The content and the relative proportion of κ-CN were not genetically correlated with RCT, αS1- and αS2-CN were unfavourately correlated with RCT, but increasing the content of β-CN in milk would result in a shorter RCT. Stronger curds were associated with higher κ-CN and β-CN, and with lower αS1-, αS2-, and γ-CN contents and proportions. Results confirm the lack of favorable associations between TCN and MCP indicating that other traits, i.e. milk protein fractions, should be used for the genetic improvement of cheese-making properties.
Le proprietà di coagulazione del latte (MCP) sono un aspetto fondamentale nella produzione di formaggio, tuttavia, negli ultimi anni, è stato registrato un andamento sfavorevole della coagulazione del latte in diversi Paesi. La resa in formaggio è diminuita, accentuando la necessità di fornire i caseifici con latte più adatto per la trasformazione in formaggio. Nel corso degli ultimi decenni il miglioramento genetico si è focalizzato sui kg di proteina del latte, ma il contenuto totale di proteina non sembra essere un buon indicatore delle MCP, e la mancanza di un metodo di analisi che consenta la determinazione delle MCP su larga scala attualmente limita la possibilità di migliorare le MCP attraverso una selezione diretta. La composizione proteica del latte è stato a lungo oggetto di interesse per i ricercatori di tutto il mondo. Di conseguenza, le informazioni sul genotipo delle proteine del latte potrebbero essere utilizzate per migliorare la composizione della proteina oppure nella selezione assistita da marcatori per migliorare le MCP, senza dover fenotipizzare grandi gruppi di progenie. Alla luce di tali possibilità, sarebbe auspicabile poter acquisire ulteriori conoscenze sugli effetti delle varianti genetiche delle proteine sulla composizione proteica del latte e sulle MCP. Obiettivi di questa tesi sono stati: studiare gli effetti dell’aplotipo CSN2-CSN3 (β-κ-caseina) e del genotipo al locus BLG (β-lattoglobulina, β-LG) su caratteri produttivi, contenuto di frazioni proteiche e composizione proteica; studiare gli effetti dell’aplotipo CSN2-CSN3 e del genotipo al locus BLG, del contenuto di frazioni proteiche e della composizione proteica sulle MCP, studiare l'effetto esercitato dal rapporto relativo tra κ-CN A e B sulle MCP e sulla resa industriale in tre varietà di formaggi italiani. Inoltre, ultimo obiettivo del lavoro è stato la stima dei parametri genetici delle principali frazioni proteiche del latte e delle correlazioni genetiche e fenotipiche tra le frazioni proteiche e le MCP. Un nuovo metodo di analisi HPLC a fase inversa per la separazione e la quantificazione delle più comuni varianti genetiche delle proteine del latte bovino è stato sviluppato e validato attraverso test di linearità, ripetibilità, riproducibilità e accuratezza. Il contenuto delle principali frazioni proteiche è stato misurato con questo nuovo metodo in campioni di latte individuale di 2,167 bovine di razza Simmental. La composizione proteica è stata espressa come percentuale in peso di ogni frazione caseinica rispetto al contenuto totale di caseina (TCN) e come percentuale del peso della β-LG sul totale di proteine del siero (WH). Il genotipo ai loci CSN2, CSN3 e BLG è stato determinato tramite HPLC e le probabilità aplotipiche per gli aplotipi CSN2-CSN3 sono state stimate per ogni animale. Tempo di coagulazione (RCT) e consistenza del coagulo (a30) sono stati misurati utilizzando un lattodinamografo. Gli effetti dell’aplotipo delle caseine e del genotipo al locus BLG sui caratteri produttivi sono stati limitati o trascurabili. Gli aplotipi contenenti gli alleli CSN2 B e CSN3 B hanno mostrato valori più elevati di TCN e un indice caseinico (CI) superiore, rispetto a tutti gli altri aplotipi. Il genotipo BB al locus BLG è stato associato ad un aumento del contenuto proteico e ad un CI superiore rispetto al genotipo AA. Gli aplotipi contenenti l’allele CSN3 B sono stati associati a contenuti e percentuali di κ-CN maggiori. L’allele CSN2 B è risultato associato con un aumento del contenuto di β-CN, che si è verificato a scapito del contenuto di αS1-CN. Gli aplotipi che includevano la variante CSN2 A1 hanno mostrato una diminuzione del contenuto di β-, αS2- e γ-CN e un aumento del contenuto di αS1- e κ-CN, mentre la variante CSN2 I ha esercitato effetti positivi sulla concentrazione di β-CN, senza alterare il contenuto delle altre frazioni proteiche. L’allele A al locus BLG è stato associato ad una maggiore concentrazione di β-LG e ad un più elevato rapporto tra β-LG e α-lattoalbumina (α-LA). Quando il contenuto delle frazioni proteiche o la composizione della proteina non erano inclusi nel modello statistico, gli aplotipi contenenti l’allele CSN3 B erano associati ad RCT più brevi ed a30 maggiori, rispetto a quelli che includevano l’allele CSN3 A, e gli aplotipi contenenti la variante CSN2 B erano responsabili di una notevole diminuzione dei valori di RCT e per valori di a30 maggiori, rispetto agli aplotipi contenente la variante A2. Quando gli effetti del contenuto delle frazioni proteiche o della composizione proteica sono stati inclusi nel modello statistico, nessuna differenza tra aplotipi riconducibile agli alleli ai loci CSN3 e CSN2 è stata osservata per le MCP, con l'eccezione dell’effetto della CSN2 B su RCT, che è rimasto molto favorevole. L'effetto favorevole esercitato dall’allele CSN2 B su a30 è risultato mediato dall’aumento di β-CN B nel latte. Al contrario, la β-CN B esercita probabilmente un effetto diretto su RCT, che non dipende dalla variazione del contenuto di β-CN associato all’allele B. Per verificare se la mancanza di effetto diretto delle varianti genetiche di κ-CN sarebbe stato osservato anche sulla resa in formaggio, latte con differenti rapporti tra κ-CN A e B sono stati lavorati separatamente per la produzione di Montasio, Asiago e Caciotta. Il latte lavorato aveva composizione simile in termini di proteina, TCN, CI, composizione caseinica, composizione della β-CN e pH simile. Il latte con la percentuale maggiore di κ-CN B (HIGHB) ha presentato valori di MCP simili, ma una resa superiore in tutti i tipi di formaggio esaminati, rispetto al latte con una percentuale inferiore di κ-CN B (LOWB). Tuttavia, l'incremento di resa osservato per il formaggio Montasio è stato attribuito a un maggior contenuto di grasso del latte HIGHB in confronto con il latte LOWB. La probabilità del latte HIGHB di dare un formaggio con una resa del 5% superiore a quella del latte LOWB variava dal 51 al 67% per il Montasio, ma è stata inferiore al 21% per Asiago e Caciotta. Il rapporto tra le varianti A e B di κ-CN non ha quindi influito in modo rilevante sulla resa casearia industriale, quando la composizione del latte era bilanciata per la composizione caseinica, ed è possibile supporre pertanto che vi sia un effetto indiretto delle varianti di κ-CN sulla resa casearia, a causa del più elevato contenuto di κ-CN associato alla variante B. I valori di ereditabilità per αS1-CN%, κ-CN% e β-CN% erano simili e variabili da 0.61 al 0.70, mentre l’ereditabilità di αS2-CN%, γ-CN% e β-LG% erano 0.28, 0.29 e 0.33, rispettivamente. Quando l’effetto dell’aplotipo CSN2-CSN3 e del genotipo al locus BLG sono stati inclusi nel modello, le stime di ereditabilità di tutte le frazioni proteiche sono divenute simili suggerendo che la sintesi di proteine del latte sia sottoposta a un controllo genetico da parte di geni specifici che controllano il livello generale di proteina del latte. Le correlazioni genetiche tra il contenuto delle 5 frazioni caseiniche e tra le frazioni caseiniche e le frazioni sieriche erano generalmente basse. In generale, tutte le frazioni caseiniche erano anche moderatamente positivamente correlata con WH, suggerendo che vi sia una regolazione generale del livello di proteina del latte che coinvolge contemporaneamente TCN e WH. Quando l’effetto dell’aplotipo CSN2-CSN3 e del genotipo al locus BLG sono stati inclusi nel modello, le correlazioni genetiche tra i contenuti delle frazione proteiche sono aumentate significativamente, supportando l’ipotesi che tutte le frazioni siano oggetto di una regolazione generale. Il contenuto di κ-CN del latte non è risultato essere geneticamente correlato con RCT, αS1- and αS2-CN hanno mostrato una correlazione sfavorevole con RCT, mentre un aumento della β-CN nel latte sarebbe a favore di RCT più brevi. Coaguli più consistenti sono stati associati ad un maggior contenuto di κ-CN e β-CN e ad un minor contenuto di αS1-, αS2-, e γ-CN. I risultati ottenuti confermano la mancanza di un’associazione favorevole tra TCN e MCP, sottolineando l’esigenza di utilizzare altri caratteri, come il contenuto delle frazioni proteiche, per il miglioramento genetico delle proprietà casearie del latte.

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第14228号
農博第1737号
新制||農||964(附属図書館)
学位論文||H20||N4416(農学部図書室)
UT51-2008-Q697
京都大学大学院農学研究科農学専攻
(主査)教授 松村 康生, 教授 内海 成, 教授 北畠 直文
学位規則第4条第1項該当

A total of 853 milk samples with different phenotypes of $ kappa$-casein ($ kappa$-CN) and $ beta$-lactoglobulin ($ beta$-LG) and different preheating temperatures of 30, 70, 75 and 80$ sp circ$C were used for the making of individual laboratory scale Cheddar type cheese and for the determination of coagulating properties. Data obtained from milk input, cheese output and chemical analyses were used to calculate actual, 37% moisture adjusted and Van Slyke's theoretical yields and yield efficiency. Least squares analyses of data indicated that higher 37% moisture adjusted yields and yield efficiencies were obtained with milk types VII, VIII and IX, which have the B gene for $ kappa$-CN when compared to milk types I, II and III, which have the A gene for $ kappa$-CN irrespective of preheating temperatures. Moisture adjusted yield, 10.49%, was the highest when milk type VII containing $ kappa$-CN BB and $ beta$-LG AA phenotypes was preheated at 30$ sp circ$C, whereas milk type IX, which has phenotype BB for $ kappa$-CN and BB for $ beta$-LG, had the highest adjusted yields with values of 11.36, 11.91 and 11.99% when preheated at 70, 75 and 80$ sp circ$C, respectively. When cheese was made from milk preheated at 30$ sp circ$C, total solids (64.19%), fat (35.31%) and protein (25.82%) were highest in cheese obtained from milk types IX, VII and IX, respectively, all of which have the B gene for $ kappa$-CN. These three components (61.54%, 30.85% and 24.47%) were lowest in cheese made from milk types III, III and I, respectively, all of which have the A gene for $ kappa$-CN. Cheese with moisture content close to 39% were produced by milk types I and II preheated at 30$ sp circ$C. by milk types III, IV, V, VI, VII and VIII preheated at below 70$ sp circ$C and by milk type IX preheated at below 75$ sp circ$C. (Abstract shorted by UMI.)

O leite instável não ácido (LINA) caracteriza-se pela perda da estabilidade da caseína ao teste do etanol, sem apresentar acidez acima de 18 °D. Este tipo de leite não é transportado para a indústria devido à subjetividade e imprecisão do teste do etanol, o qual não diferencia os tipos de leite, sendo eles: leite normal, LINA e o leite ácido. Este trabalho teve como objetivo contribuir para a compreensão dos fenômenos físico-químicos envolvidos na estabilidade e instabilidade das micelas de caseína em LINA e leite estável bovino e, desenvolver testes alternativos para a identificação e diferenciação de leite estável, ácido e LINA. Para este estudo foram utilizadas 58 vacas da raça Jersey e 130 vacas da raça Holandês em lactação. Teste de mastite subclinica para os animais da raça Jersey revelou 67% de positividade. O principal agente microbiano isolado do leite foi o Staphylococcus coagulase positivo. Não foi evidenciada relação entre a mastite subclínica e o LINA. Os atributos químicos do leite apresentaram diferenças significativas entre as raças avaliadas, independente da estabilidade do leite. Animais com períodos de lactação mais longos apresentaram maior instabilidade da caseína independente da raça estudada. Quanto a acidez, pH, contagem de células somáticas e contagem de bactérias totais não houve diferenças significativas para a estabilidade e raças avaliadas. O LINA apresentou todos os parâmetros avaliados dentro dos padrões exigidos pela legislação brasileira e não apresentando variações significativas com o leite estável. Testes de estabilidade em distintas concentrações de etanol mostrou que 72% (v/v) de etanol diferenciam claramente leite estável e o LINA. Leite estável e LINA apresentaram estabilidade térmica, mostrando que os dois testes não estão correlacionados. O perfil eletroforético das proteínas de leite estável e LINA não apresentou diferenças evidentes, indicando que a instabilidade não está associada a modificações qualitativas ou quantitativas nas caseínas do leite. Os resultados da análise de cálcio e fósforo total, micelar e solúvel, indicaram que o teor de cálcio na fração solúvel é consideravelmente maior no LINA, evidenciando que o cálcio iônico pode ser um fator importante na estabilidade do leite. Além disso, a relação Ca/P foi de 1,1/1 e 1,6/1 em leite estável e LINA, respectivamente. O maior teor de cálcio em relação ao fósforo no LINA pode contribuir para a sua desestabilização da micela de caseína na presença de etanol. O presente estudo deu origem a uma patente de invenção e sistema logístico para coleta de leite, a fim de evitar desperdícios indevidos e interpretações equivocadas acerca do tipo de leite. Particularmente, os métodos patenteados permitem que sejam realizados testes em campo que identificam e diferenciam de forma rápida e eficiente as amostras de leite estável, LINA e leite ácido.
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Universidade de Caxias do Sul, UCS.
The unstable non-acid milk (UNAM) is characterized by the instability of caseins in the ethanol test and low acidity (≤ 18 °D). This kind of milk is not transported to the industry due to the subjectivity and low precision of the ethanol test, which is not able to differenciate normal (stable), UNAM, and acid milk. The objective of the present work was to contribute for the understanding of the physic-chemical fenomena involved in the stability and instability of the casein micela in UNAM and stable bovine milk, and to develop reliable tests for the identification and diferenciation of stable, unstable, and acid milks. This study included 58 Jersey and 130 Holland cows in lactation. The subclinical mastitis test of Jersey animals resulted in 67% positivity. The most prevalent microbial agent isolated from subclinical mastitis milks was coagulase-positive staphylococci. It was not evidence any relation between subclinical mastitis and UNAM. Significant differences were detected between Jersey and Holland milks, but these were not related with milk stability. Animals with longer lactation period showed highest frequency of UNAM, independent of cow´s race. No significant differences for pH, acidity, somatic cell count, and total bacterial count, were detected between races and stable milk and UNAM. The UNAM showed all the parameter within the patterns stablished by Brazilian legislation, with not significant differences from stable milks. Stability tests in different ethanol concentrations showed that 72% (v/v) ethanol clarely discriminated UNAM and stable milk. Both UNAM and stable milk exhibited the same behavior at high temperatures, indicating that ethanol and thermal stability are not correlated. No differences were detected between the protein electrophoretic profile of stable milk and UNAM, indicating that the instability is not associated with qualitative or quantitative modification of milk caseins. The analysis of total, micelial and soluble calcium and phosphorus showed that calcium concentration in the soluble fraction is higher in UNAM than in stable milk, indicating that ionic calcium can be an important factor in milk stability. Moreover, Ca/P relation was 1.1/1 and 1.6/1 in stable milk and UNAM, respectively. The highest Ca/P relation in UNAM can contribute for the destabilization of the casein micela in the presence of ethanol. The present study originates an invention patent for the evaluation of milk stability, and a logical system for milk transport, that can reduce losses by the correct determination of milk quality. The patented method allows the rapid and efficient identification and differenciation of stable, UNAM and acid mik in the rural property and industry.

The percent in-vitro digestibility of proteins in human milk was determined by three different methods and compared with the in-vitro digestibility of proteins in Enfamil, Similac and Isomil. In-vitro digestibility was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), pH drop and a simultaneous dialysis method. Significant differences (P<0.05) were observed in the in-vitro protein digestibility of human milk and the three infant formulas. Estimated percent in-vitro protein digestibility of each of the samples was also found to be significantly affected by the method of determination. The in-vitro protein digestibility of all four samples estimated by simultaneous dialysis was lower than results obtained by SDS-PAGE and pH drop methods. Except for Isomil, results obtained by the pH drop method were lower than those determined by SDS-PAGE. The in-vitro protein digestibility of Enfamil and Similac was found to be greater than that of human milk by all three methods. Except for results obtained by SDS-PAGE, the in-vitro digestibility of proteins in Isomil was found to be greater than for the proteins in human milk. These results indicate that the proteins in human milk are not as extensively hydrolyzed in-vitro as the proteins in powdered infant formulas.
Master of Science

Systemic tolerance to BLG was studied in a) male and female mice heterozygous for the BLG transgene derived from crossing homozygous BLG transgenic males with wild-type females, b) BLG-transgenic and non-transgenic offspring derived from mating male and female heterozygous for the BLG transgene with wild type partners and c) BLG-transgenic and non-transgenic mice derived from back crossing onto a CBA/Ca MHC background. Using either a BLG-specific ELISA (for antibody responses) or footpad thickening assay (for T cell responses) the immune response to the BLG antigen was assessed. Hypo-responsiveness to both ovine and bovine BLG was observed at the antibody level but not the T cell level for mice transgenic for BLG as compared to wild-type and non-transgenic littermates. Antibody tolerance could not be attributed to expression of the gene during pregnancy and lactation since all mice tested were virgin mice. These experiments also confirm that suckling on BLG-containing milk was not responsible for the antibody hypo-responsiveness seen in BLG-transgenic mice. Male and female mice heterozygous for the BLG transgene were mated to wild type partners such that the offspring fell into eight classes: male or female, suckled or non-suckled on 'transgenic' milk and heterozygous or wild-type for the transgene. Antibody and T cell data indicated that suckling 'transgenic' milk did not induce oral tolerance to ovine BLG in either transgenic or non-transgenic offspring. In contrast, voluntary ingestion of bovine BLG by wild-type mice for 24 hours or 21 days resulted in both antibody and T cell tolerance. Hypo-responsiveness could not be induced by transferring transgenic marrow into lethal irradiated normal recipients, but similarly irradiated transgenic recipients were still hypo-responsive after reconstitution with normal bone marrow.

In this research, beta-lactoglobulin was chemically modified by attaching different levels of stearic acid to the protein. The effect of this modification on hydrophobic!ty, emulsifying and foam properties, digestibility and allergenicity of the protein was investigated. It was found that the effect of fatty acid attachment or lipophilization depended on the amount of fatty acids attached to the protein. Incorporation of the hydrophobic ligands led to increased hydrophobic interactions, resulting in a decreasing solubility with extent of incorporation. Furthermore, the surface hydrophobicity measurements showed that the two fluorescence probes 8-anilinonaphthalene-l-sulfonate (ANS) and cis-parinaric acid (CPA) used for the surface hydrophobicity measurements were not equivalent This may support the. observation by earlier workers that ANS measures aromatic hydrophobicity and CPA aliphatic hydrophobicity. The studies on surface functional properties i.e. emulsifying and foaming properties, indicated that there was some improvement in these functional properties at low and medium levels of incorporation which decreased as the extent of fatty acid attachment further increased. The improvement, of these functional properties could be attributed to improved amphiphilicity of the proteins at these levels of incorporation. This research also showed that both high solubility and high ANS surface hydrophobicity is needed for the best emulsifying properties. In vitro digestibility studies showed a decrease in digestibility of the modified proteins with increased lipophilization. From the passive cutaneous anaphylaxis experiments, it was found that the level of fatty acid attachment to the protein had a significant effect on its ability to elicit IgE antibodies. Increased ability to elicit IgE antibodies was observed at a low level of fatty acid. When a medium level of fatty acid was attached the ability to elicit antibodies was reduced and almost completely destroyed when a higher level of fatty acid was incorporated. The above observations could be explained by the fact that the low level incorporation of fatty acid led to changes in the protein structure which exposed more allergenic sites. The almost complete destruction of the allergenicity could be attributed to denaturation of the protein which reduced or destroyed available allergenic sites. The antigenicity or binding of the modified proteins to the IgG antibodies raised against the native protein was studied by both direct and competitive enzyme linked immunosorbent assay. It was found that at low and medium levels of incorporation, the proteins demonstrated increased binding ability compared to the native protein. This was attributed to the increased exposure of antigenic sites on the protein with fatty acid incorporation. However, the protein with high level of incorporated fatty acid showed decreased binding ability.
Land and Food Systems, Faculty of
Graduate

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.

Orientador: Marcelo Cristianini
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: O tratamento de homogeneização a ultra alta pressão (HUAP) é uma tecnologia que vem sendo utilizada tanto para inibir o crescimento de microorganismos, como para alterar as propriedades físico-químicas e funcionais do leite e de sua fração proteica. O objetivo deste estudo foi avaliar algumas das principais alterações físico-químicas de leite desnatado submetido a diferentes níveis de HUAP. Além disso, avaliar solubilidade, propriedades de aeração e emulsificação de caseína e proteínas de soro isoladas de leite processado por HUAP. Leite cru desnatado foi submetido a 3 níveis de pressão de homogeneização (100 MPa, 200 MPa e 300 MPa) onde foram avaliadas as alterações físico-químicas do leite (pH, composição centesimal, estabilidade ao álcool, luminosidade, desnaturação de proteínas do soro, hidrofobicidade e viscosidade) e as propriedades funcionais da caseína e proteínas do soro (solubilidade, aeração e emulsificação). O processo apresentou boa repetibilidade e aumento de temperatura de no máximo 55°C. As medidas de pH e nitrogênio não proteico (NNP) foram as únicas variáveis que não apresentaram alteração estatisticamente significativa para nenhum dos níveis de pressão. Estabilidade a precipitação com álcool, luminosidade e hidrofobicidade apresentaram aumento a partir de 100 MPa. A desnaturação de proteínas do soro ocorreu somente a partir de 200 MPa, aumentando ainda mais com uso de 300 MPa. A única variável que apresentou alteração somente no nível de 300 MPa foi a viscosidade. A caseína e as proteínas do soro foram isoladas por acidificação a pH 4,6 e centrifugação, o sobrenadante e precipitado foram neutralizados e liofilizados. Foram avaliadas as seguintes propriedades: solubilidade, capacidade de formação de espuma, estabilidade de espuma, capacidade emulsificante e estabilidade de emulsão. A caseína não sofreu alteração de solubilidade, já as propriedades de aeração e emulsificação apresentaram melhora de performance, sendo estatisticamente significativas a partir de diferentes níveis de pressão de acordo com a propriedade avaliada. As proteínas do soro apresentaram diminuição da solubilidade nos níveis de 100 MPa e 200 MPa, as propriedades de aeração foram melhoradas em 300 MPa, e as propriedades de emulsificação não foram influenciadas. O tratamento de leite fluido por tecnologia de HUAP promove alterações significativas em propriedades funcionais de caseína e proteínas do soro. As alterações estão relacionadas à desestruturação das micelas de caseína, desnaturação de proteínas do soro, interação das micelas entre si e com as proteínas do soro, e com o aumento da hidrofobicidade e capacidade de hidratação das mesmas
Abstract: The ultra high-pressure homogenisation (UHPH) is a recent technology used to inhibit microorganisms¿ growth, as to modify the physical-chemical properties of milk and its protein fraction. The purpose of this study was to evaluate the main physical-chemical characteristics caused by different levels of high-pressure homogenisation in skim milk. Besides to evaluate solubility, foaming properties and emulsifying properties of casein and whey proteins isolated from milk processed by UHPH. Raw skimmed milk was submitted to 3 pressure levels (100 MPa, 200 MPa e 300 MPa), the processing parameters and the modifications on milk physical-chemical properties has been evaluated. The process presented good repeatability and the maximum temperature increase was 55°C. The measurements of pH and non-protein nitrogen (NPN) were the only variables that did not present any statistically significant change. Ethanol stability, lightness (L*) and hydrophobicity showed an improvement or increment from 100 MPa to 300 MPa. Whey protein denaturation occurred only from 200 MPa to 300 MPa. The only variable that presented changes only at 300 MPa was viscosity. Casein and whey proteins were isolated by acidification at pH 4.6 and centrifugation, further they were neutralized and freeze-dried. The following properties were evaluated: solubility, foaming capacity, foam stability, emulsifying capacity and emulsion stability. The freeze-dried casein did not show any modification on solubility, however all foaming and emulsifying characteristics presented improved performance, being statistically significant for different pressure levels according to each analysed property. UHPH decreased the solubility of the whey proteins obtained from milk treated at 100 MPa and 200 MPa, foaming properties increased for the freeze-dried protein obtained from milk treated at 300 MPa, and no influence were noted on the emulsifying properties. The treatment of fluid milk by UHPH was able to promote significant alterations on the functional properties of casein and whey proteins. The modifications were related to casein micelles disruption, whey proteins denaturation, interaction among the micelles and with denaturated whey proteins, and also with increase of molecular hydrophobicty and water retention
Mestrado
Mestre em Tecnologia de Alimentos

Thesis (Ph. D.)--Ohio State University, 2004.
Title from first page of PDF file. Document formatted into pages; contains xii, 113 p.; also includes graphics. Includes bibliographical references (p. 99-113).

Thesis (Ph.D.) -- University of Maryland, College Park, 2009.
Thesis research directed by: Dept. of Animal and Avian Sciences. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.

Cardiovascular diseases (CVD) remain one of the leading causes of death in the world. Despite the decreasing incidence of CVD in Western countries, due to the aging population, the prevalence is still increasing. High blood pressure (BP) is the most important modifiable risk factor for CVD, due to its high mortality rate. The impact of diet on CVD and its risk factors is well described. Epidemiological studies have demonstrated an inverse association between high milk consumption and lower BP. Milk is a complex, nutrient-dense food containing an array of essential nutrients. It is, however, of great scientific interest to determine which constituent is responsible for this beneficial effect. Milk proteins have been shown to exert angiotensin-converting enzyme inhibitory (ACEi) effect, which is a key enzyme in BP regulation. After an extensive literature search, a meta-analysis was performed to examine the impact of the casein-derived lactotripeptides on BP. This study demonstrated hypotensive effects in humans, specifically in Japanese individuals. A clear gap in the current scientific knowledge was well-designed randomised controlled trials to examine the effects of intact milk proteins on BP and other novel and classic risk markers for CVD. The findings from my randomised, controlled, double-blind, chronic intervention study showed that whey protein consumption (2 x 28 gld) for 8 weeks reduced 24-h ambulatory BP, peripheral and central BP assessed by applanation tonometry as well as improved endothelial function (flow-mediated dilation› FMD) compared to control (2 x 27 gld maltodextrin) in 38 hypertensive participants. Whey protein also reduced fasted total cholesterol (TC) and triacylglycerol (TAG) compared with control, while Ca-caseinate (2 x 28 glday) improved endothelial function (FMD) and lowered fasted TC compared with control. Both whey protein and Ca-caseinate decreased adhesion molecules compared to control (sICAM-l was decreased by whey protein and VCAM-l by Ca-caseinate). These changes were achieved without significant changes in body weight or habitual diet of the participants. The acute study also demonstrated beneficial effects of dairy proteins after two high-fat meal challenges in 27 mildly hypertensive adults: whey protein (28 glmeal) decreased BP between the two test meals compared with Ca-caseinate (28 glmeal) and control (27 glmeal), and also improved arterial stiffness (augmentation index) and maintained endothelial function (FMD) over the 8-h postprandial period. Furthermore, although whey protein increased insulin response to a similar magnitude to the carbohydrate-based control, lower postprandial glycaemia was maintained by whey protein. Furthermore, Ca-caseinate lowered postprandial TAG response compared with whey protein. The purported molecular mechanisms underlying the impacts of dairy proteins on the cardiovascular system still remain unclear. The in vitro ACEi experiment presented in this thesis confirmed the hypotensive effects of whey protein over Ca-caseinate and control, however serum samples failed to confirm this ACEi activity. In summary, milk proteins, particularly whey protein, had important fasted and postprandial benefits to the cardiovascular system compared with control, however concomitant consumption of dairy proteins may be of further benefit due to their different kinetics in the gastrointestinal system. Future research should address the impacts of milk proteins in type 2 diabetic patients with impaired glucose metabolism .

The main objective of this PhD thesis was to study the association between udder health [focusing on subclinical cases of bovine mastitis identified by somatic cell count (SCC) and bacteriological analyses] and several milk quality and technological traits related to the cheese-making process, and blood serum proteins, as possible immune response indicators. To achieve our goal, the work was splitted in 4 chapters. Two datasets were used: for the 1st chapter, milk samples from 1,271 Brown Swiss cows from 85 herds were used. In the subsequent 3 chapters, milk and blood samples were collected from 1,508 dairy specialized and dual-purpose cows of 6 different breeds (Holstein Friesian, Brown Swiss, Jersey, Simmental, Rendena and Grey Alpine) housed in 41 multi-breed herds. The aim of the 1st chapter was to determine the effects of very low to very high SCC on milk yield, composition, coagulation properties [including traditional milk coagulation properties (MCP) and new curd firming model parameters (CFt)], cheese yield (CY) and recovery of milk nutrients in the curd (REC) at the individual cow level. The objective of the 2nd chapter was to investigate the association between blood serum proteins [i.e., total protein, albumin, globulin and the ratio of albumin-to-globulin (A:G)] and milk SCC. Since several factors should be considered to appropriately interpret serum proteins concentration in blood, we explored the effect of herd productivity (defined according to the average net energy of milk yielded daily by the cows), breed, and individual cow factors (i.e., stage of lactation and parity) on blood traits. In chapters 3 and 4, pathogen-specific information was included in the analysis to gain a better understanding of the specific changes in the traits previously investigated. Subclinical cases of mastitis were confirmed by bacteriological analysis and multiplex-PCR assays. In particular, in the 3rd chapter we investigated the association between pathogen-specific cases of subclinical mastitis and several milk quality and technological traits (i.e., milk yield, composition, detailed protein profile, coagulation properties and cheese-related traits). Based on the results of the 2nd chapter, the 4th chapter studied the association between pathogen-specific cases of subclinical mastitis and blood serum proteins, that in chapter 2 showed a correlation with SCC in milk. Results of chapter 1 confirmed the negative effect of high SCC on milk yield, composition, MCP, CFt, CY and REC traits. As somatic cell score (SCS) increased, a linear loss of milk production and variations in milk composition (e.g., casein-to-protein ratio, lactose and pH) were observed. These changes decreased the quality and clotting ability of the processed milk, which showed a slower coagulation time and a weaker curd firmness. This, in turn, affected the cheese processing (as confirmed by reductions in the CY and the recovery of milk nutrients in the curd). Our findings showed nonlinear trends for some milk traits with respect to SCS, highlighting the negative effect of very low SCC on some milk technological traits. Our 2nd chapter showed that cows in high producing herds had greater serum albumin concentrations. Breed differences in serum protein profile could be associated with individual genetic variation and could also be explained by the different selective breeding programs to which breeds have been subjected. Changes in blood serum proteins were observed throughout the entire lactation and according to the parity order. Linear relationships between blood serum proteins and SCS confirmed the importance of SCC as an indicator of mammary gland inflammation. Moreover, our results highlighted the potential use of blood serum proteins as indicators of immune response of the mammary gland to infections and their analysis represents a possible initial screening test to identify animals which need further clinical investigations. Such non-genetic factors affecting variation in blood serum proteins should also be considered in future genetics/genomics investigations. Results of the 3rd chapter revealed that compared with normal milk, all culture-positive samples and culture-negative samples with medium to high SCC presented significant variations in the casein-to-protein ratio and lactose content. Given that no differences were observed comparing milk infected by contagious, environmental and opportunistic pathogens, our findings suggested an effect of inflammation rather than infection. The greatest impairment in milk yield and composition, clotting ability and cheese production was observed for milk samples with the highest SCC (i.e., culture-positive samples where contagious pathogens were recovered, and culture-negative samples with high SCC), revealing a discrepancy between inflammatory status and bacteriological results, and thus confirming the important role of SCC as udder health indicator. Culture-negative samples with high SCC were possibly undergoing a strong inflammatory response and pathogens could not be isolated because engulfed by macrophages. In the 4th chapter, culture-negative samples with high milk SCC, which we hypothesized to be infected by contagious bacteria engulfed by neutrophils, and milk samples infected by contagious and environmental bacteria were associated with greater globulin content (and lower A:G) in blood. In accordance with the results in chapter 3 for milk traits, variation in blood serum proteins seemed to be associated with inflammation rather than infection, as globulin significantly increased in the blood of cows whose milk samples had the highest SCC, independently from intramammary infection pathogens.
L’obiettivo principale di questa tesi di dottorato è stato quello di studiare l'associazione tra stato sanitario della mammella [con particolare riferimento a casi subclinici di mastite bovina identificati attraverso conta delle cellule somatiche (SCC) e analisi batteriologica] e una serie di caratteri qualitativi e tecnologici del latte legati al processo di caseificazione, e le proteine del siero, quali possibili indicatori di risposta immunitaria dell’animale. Per raggiungere tale obiettivo, il lavoro è stato suddiviso in 4 capitoli. Due diversi datasets sono stati utilizzati: nel 1° capitolo sono stati utilizzati campioni di latte raccolti da 1,271 bovine di razza Bruna provenienti da 85 allevamenti. Nei successivi 3 capitoli, i campioni di latte e di sangue sono stati raccolti da 1,508 bovine da latte e a doppia attitudine di 6 diverse razze (Frisona, Bruna, Jersey, Pezzata Rossa, Rendena e Grigio Alpina) provenienti da 41 allevamenti multi-razza. Nel 1° capitolo di questa tesi sono stati analizzati, a livello individuale, gli effetti di un contenuto variabile di SCC nel latte (da molto basso a molto alto) sulla produzione di latte, la composizione chimica, le proprietà di coagulazione [includendo le proprietà di coagulazione tradizionali (MCP) e nuovi parametri modellizzati di consistenza della cagliata (CFt)], la resa casearia (CY) e il recupero di nutrienti nella cagliata (REC). Lo scopo del 2° capitolo è stato quello di studiare l'associazione tra proteine del siero [proteine totali, albumina, globulina e il rapporto tra albumina e globulina (A:G)] e SCC nel latte. Tuttavia, per interpretare in modo appropriato la concentrazione delle proteine nel sangue, devono essere presi in considerazione diversi fattori. Pertanto, è stato valutato l'effetto del livello produttivo dell’allevamento (definito sulla base dell'energia netta del latte prodotta in media giornalmente dalle bovine), della razza, dello stadio di lattazione e dell’ordine di parto sulle proteine ematiche. Nei capitoli 3 e 4, sono state incluse nelle analisi informazioni a livello patogeno-specifico allo scopo di acquisire una migliore comprensione dei cambiamenti precedentemente osservati nei caratteri tecnologici e di qualità del latte e nei parametri ematici esaminati. I casi subclinici di mastite sono stati confermati attraverso analisi batteriologica e saggi PCR in multiplex. In particolare, l'obiettivo del 3° capitolo è stato quello di studiare l'associazione tra i casi di mastite subclinica a livello patogeno-specifico e i diversi caratteri qualitativi e tecnologici del latte (produzione, composizione chimica, profilo proteico dettagliato, proprietà di coagulazione e caratteri legati al processo di caseificazione). Sulla base dei risultati ottenuti nel capitolo 2, nel 4° capitolo è stata valutata l'associazione tra i casi di mastite subclinica a livello patogeno-specifico e le proteine del siero, che nel capitolo 2 erano risultate correlate alle SCC nel latte. I risultati ottenuti nel capitolo 1 hanno confermato l'effetto negativo di un alto contenuto di SCC sulla produzione di latte, la composizione e i caratteri MCP, CFt, CY e REC. All’aumentare del punteggio di cellule somatiche (SCS), sono state osservate una diminuzione lineare della quantità di latte prodotto e alcune variazioni nella composizione (in particolare nel rapporto tra caseina e proteina, nel contenuto di lattosio e nel pH). Questi cambiamenti hanno causato una riduzione della qualità e dell’attitudine casearia del latte trasformato, caratterizzato da una coagulazione più lenta e una ridotta consistenza del coagulo. Di conseguenza, tali variazioni hanno avuto ripercussioni negative sul processo di caseificazione, ovvero ridotta resa in formaggio e minor recupero di nutrienti nella cagliata. I risultati ottenuti hanno inoltre evidenziato andamenti non lineari per alcuni caratteri del latte rispetto ad SCS, mettendo in evidenza l'effetto negativo di un contenuto molto basso di SCC su alcuni caratteri tecnologici del latte. Nel 2° capitolo è stato dimostrato che le bovine allevate in allevamenti ad alta produttività presentavano una maggiore concentrazione di albumina sierica. Le differenze nel profilo proteico osservate tra le diverse razze potrebbero essere associate alla variazione genetica individuale e ai diversi programmi di selezione a cui tali razze sono state sottoposte. Variazioni del contenuto di proteine ematiche sono state riportate all’avanzare della lattazione e a seconda dell’ordine di parto. Le relazioni lineari tra proteine del siero e SCS hanno confermato l'importanza delle SCC come indicatore di infiammazione della mammella. I risultati ottenuti hanno evidenziato inoltre il potenziale uso delle proteine del siero come indicatori di risposta immunitaria della ghiandola mammaria alle infezioni e la loro analisi rappresenta un possibile test iniziale di screening per identificare animali che hanno bisogno di ulteriori indagini cliniche. Tali fonti di variazione non-genetiche delle proteine del siero dovrebbero essere prese in considerazione anche in future analisi genetiche e genomiche. I risultati del 3° capitolo hanno mostrato che, rispetto al latte di bovine sane, tutti i campioni di latte risultati positivi all’esame batteriologico e i campioni che non hanno evidenziato crescita batterica ma con un contenuto medio-alto di SCC presentavano significative variazioni nel rapporto tra caseina e proteina, nonchè nel contenuto di lattosio. Poiché non sono state osservate differenze significative confrontando latte infetto da patogeni contagiosi, ambientali e opportunisti, i risultati ottenuti hanno evidenziato un deterioramento del latte dovuto alla risposta infiammatoria dell’animale piuttosto che all'infezione stessa. Un peggioramento più pronunciato per quanto riguarda produzione e composizione chimica del latte, attitudine alla coagulazione e resa in formaggio è stato osservato per i campioni di latte con il più alto contenuto di SCC (ovvero i campioni infettati da patogeni contagiosi e i campioni risultati negativi all’esame batteriologico ma con un alto contenuto di SCC). Questo ha rivelato una discrepanza tra stato infiammatorio e risultati batteriologici, confermando così il ruolo importante delle SCC quale indicatore dello stato di salute della mammella. É possibile che, nei campioni risultati negativi all’esame batteriologico ma con un alto contenuto di SCC, una risposta infiammatoria intensa abbia impedito l’isolamento degli agenti patogeni in quanto internalizzati dai macrofagi. Nel capitolo 4, i campioni che non hanno evidenziato crescita batterica ma con un alto contenuto di SCC, che abbiamo ipotizzato essere infettati da batteri contagiosi internalizzati dai neutrofili, e i campioni di latte infettati da batteri contagiosi e ambientali sono risultati associati a un maggior contenuto di globulina (e a un valore inferiore del rapporto A:G) nel sangue. In accordo con i risultati relativi ai caratteri del latte ottenuti nel capitolo 3, la variazione del profilo proteico del sangue sembra essere associata al processo infiammatorio piuttosto che all'infezione. Infatti è stata osservata una concentrazione elevata di globulina nel sangue di bovine il cui latte presentava un contenuto elevato di SCC, indipendentemente dal tipo di patogeno causa di infezione.