Streptococcus (Str.) agalactiae is a contagious mastitis bacterium, often associated with cases of subclinical mastitis. Different mastitis bacteria have been evaluated previously from a diagnostic point of view, but there is a lack of knowledge concerning their effect on milk composition. Protein composition is important in achieving optimal yield and texture when milk is processed to fermented products, such as cheese and yoghurt, and is thus of great economic value. The aim of this in vitro study was to evaluate protein degradation mainly caused by exogenous proteases originating from naturally occurring Str. agalactiae. The samples were incubated at 37°C to imitate degradation caused by the bacteria in the udder. Protein degradation caused by different strains of Str. agalactiae was also investigated. Protein degradation was observed to occur when Str. agalactiae was added to milk, but there were variations between strains of the bacteria. Caseins, the most economically important proteins in milk, were degraded up to 75% in milk inoculated with Str. agalactiae in relation to sterile ultra-high temperature (UHT) milk, used as control milk. The major whey proteins, α-lactalbumin and β-lactoglobulin, were degraded up to 21% in relation to the sterile control milk. These results suggest that different mastitis bacteria but also different strains of mastitis bacteria should be evaluated from a milk quality perspective to gain knowledge about their ability to degrade the economically important proteins in milk.

Bulk tank ewe's milks with low (<500,000 ml−1), medium (1,000,000–1,500,000 ml−1) and high (>2,500,000 ml−1) somatic cell counts (SCC) from three breeds were used to manufacture hard ewes’-milk cheese. Physico-chemical analysis and capillary electrophoresis of fresh cheeses and cheeses that had been ripened for 1, 2, 3 and 6 months were carried out. The results showed that high SCC levels in milk affected the moisture content of only freshly made cheeses and the pH, fat content and fat acidity of ripened cheeses. Regarding proteolysis, the levels of all β-CNs in freshly made cheeses were significantly lower as the SCC values increased and the Castellana breed was the most affected by SCC levels because a significant decrease in all α-CNs was also observed as SCC levels rose. Analysis of the casein profile by principal component analysis (PCA) revealed that there were no clear differences according the SCC up to the third month. However in the third and sixth months cheeses with low levels of SC were closely grouped and characterised by the highest levels of intact caseins. Regarding the effect of breed, the results point to a more intense proteolytic activity in the Assaf breed, whose more matured cheeses showed the highest content of casein proteolytic fragments.

Bulk tank ewe's milks from Assaf, Castellana and Churra breeds categorized within three different Somatic Cell Count (SCC) groups (LSCC: <500 000; MSCC: 1 000 000 to 1 500 000; and HSCC: 2 500 000 to 3 000 000 cells ml−1) were used to investigate changes in capillary electrophoresis protein profiles and cheese-making properties. The results do not reveal a significant effect of SCC on total casein contents, because the sum of β-caseins decreased as SCC increased; no statistically significant differences were observed for the sum of α-caseins, and the values of κ-casein were higher in the HSCC milk. However, the soluble proteins other than α-lactalbumin and β-lactoglobulin increased with SCC. Regarding the effect of breed, the Assaf breed had the lowest contents of κ-CN, αs1-I-CN, αs1-II-CN 1-CN, αs1-III-CN, β1-CN and β2-CN. The protein profile was significantly correlated with curd textural properties. αs1-I-CN was the most influential variant because it was positively correlated with a large number of textural parameters. Cheese yield was positively correlated with all casein variants except αs1-III-CN, showing that the milk from local breeds were more suitable for cheese-making due to their higher contents of all the casein variants. Regarding curd texture properties LSCC milk curds showed more cohesiveness, associated with its lower content of αs1-III-CN and Castellana milk curds showed the highest values for firmness owing to their higher content of αs1-I-CN.

Ewes' milk samples with low (<500000 ml−1), medium (1000000–1500000 ml−1) and high (>2500000 ml−1) somatic cell counts (SCC) were used to manufacture hard ewes' cheese using the Zamorano cheese manufacturing protocol. Cheeses that had been ripened for 1, 2 and 3 months were used to obtain isoelectric ovine casein that was analysed by capillary electrophoresis. The texture of the cheeses during ripening was determined instrumentally using the Warner-Bratzler maximum shear force and assessed for sensory qualities by consumers using hedonic tests. The study revealed that the pH value and the lactose content of the milk were affected by high SCC and that the coagulation properties were dependent on the somatic cell content. The protein and moisture contents of the cheeses were unaffected by SCC but a significant increase of pH with ripening time were observed in high-SCC cheeses. The results also pointed to a significant increase in proteolysis related to SCC levels, showing that intact casein, both αs1 and β-casein, decreased as the SCC of milk increased, and that the proteolytic fragments, mainly I-αs1, increased with SCC levels. Significant differences in texture were found among the samples, the cheeses made with high levels of SCC being significantly less compact at each ripening time. The differences in texture were detected by the consumers, who reported defects in cheeses made with high levels of SCC. Indeed, high SCC cheeses were significantly less well accepted.

A sensitive capillary electrophoresis mobility shift assay (CEMSA) to analyze RNA/peptide interactions has been developed. Capillary electrophoresis (CE) has been adapted for investigating the interaction between variously methylated 17-nt analogs of the yeast tRNAPhe anticodon stem and loop domain (ASLPhe) and 15-amino-acid peptides selected from a random phage display library (RPL). A peptide-concentration-dependent formation of RNA/peptide complex was clearly visible during CEMSA. In the presence of peptide, the UV-monitored CE peak for ASLPhe with three of the five naturally occurring modifications (2′-O-methylcytidine (Cm32), 2′-O-methylguanine (Gm34) and 5-methylcytidine (m5C40) shifted from 18.16 to 20.90 min. The mobility shift was observed only for methylated RNA. The negative effects of diffusion, electroosmotic flow and adhesion of molecules to the capillary internal wall were suppressed by using a buffer containing a sieving polymer and a polyacrylamide-coated capillary. Under these conditions, well-shaped peaks and resolution of RNA free and bound to peptide were achieved. Peptide tF2, the most populated ligand in the RPL, specifically bound triply methylated ASLPhe in a methylated nucleoside-dependent manner. CE was found to be an efficient and sensitive method for the qualitative analysis of RNA–peptide interaction and should be generally applicable to the study of RNA–peptide (protein) interactions.

The efficiency of reversed-phase HPLC, capillary electrophoresis (CE), PAGE and isoelectric focusing with immunoblotting in separating ovine caseins has been evaluated. The assessment was carried out by employing electrospray ionization–mass spectrometry (ESI–MS) and matrix-assisted laser desorption ionization–time of flight as reference tools for identifying protein components. Ovine casein was fractionated by HPLC into four major peaks. With ESI–MS, each peak contained components belonging to only one of the four casein families. On-line liquid chromatography–ESI–MS allowed us to determine each fraction's composition by detecting thirteen αs1-, eleven αs2-, seven β-, and three κ-casein (CN) components. The αs1-CN and αs2-CN consisted of eight and two protein chains respectively of lengths differing through the deletion of one or more peptide sequences; they were also discretely phosphorylated as κ-CN and β-CN. By CE at pH 2·5, each casein fraction was as heterogeneous as that resulting from ESI–MS for the single HPLC-derived fractions. The separation of αs1-CN and αs2-CN proved to be excellent, with the exception of a co-migration of κ0-CN with a minor αs1-CN component and of a glycosylated κ-CN form with low-phosphorylated αs1-CN and β-CN components. Dephosphorylation of whole casein was used to reduce the heterogeneity of the native fractions and by applying currently used analytical techniques it was possible to visualize the protein moiety difference along the CE profile. CE, HPLC, and immunoblotting were all equally capable of effecting an accurate separation of the four dephosphorylated casein families. The spectra obtained by ESI–MS directly on dephosphorylated whole ovine casein samples contained the signals of the four casein families and the relative αs1-CN variants, the non-allelic αs1-CN and αs2-CN forms, dimeric κ-CN and other newly formed peptides. We suggest using this procedure for rapid characterization of whole casein.

Marginal Zn deficiency is thought to be prevalent in both developed and developing countries. However, the extent of Zn deficiency is not known, due to the lack of a reliable diagnostic indicator. Blood plasma and erythrocyte concentrations of metallothionein (MT) reflect Zn status, but measurement of MT is dependent on the availability of sensitive immunoassays. Our aim was to show whether measurement of T lymphocyte MT-2A mRNA, using a competitive reverse transcriptase (RT)–polymerase chain reaction (PCR) assay, could indicate Zn status in human subjects in a residential Zn-depletion study. In the study, the Zn intake of seven volunteers was maintained at 13·7 mg/d for 5 weeks (baseline) followed by 4·6 mg/d for 10 weeks (marginal intake) and then 13·7 mg/d (repletion) for 5 weeks. The quantitative assay was developed using standard techniques and concentrations of MT-2A mRNA were normalized by reference to β-actin mRNA which was also measured by competitive RT–PCR assay. An alternative method of measuring the PCR product using capillary electrophoresis with laser-induced fluorescence detection was also evaluated. There was considerable inter-individual variation in MT-2A mRNA concentration and the mean level at the end of the baseline period was 10·3 (SE 3·7) fg MT-2A mRNA/pg β-actin mRNA, which then decreased by 64 % during the low Zn intake period. After repletion, MT-2A mRNA returned to baseline concentrations. In contrast, plasma Zn was unchanged by marginal Zn intake or repletion. The effect of low Zn in all individuals was consistent. We conclude that this assay is a sensitive method of evaluating marginal changes in dietary Zn intake.

A centrifugal method for extracting apoplastic sap from roots of lupin (Lupinus angustifolius) and pea (Pisum sativum) plants, and a method to analyse malic dehydrogenase in the sap using capillary electrophoresis, are described. Osmolality of apoplastic sap was relatively constant at relative centrifugal forces (RCFs) between 600 and 3000 g for lupin, and between 600 and 4000 g for pea. Electropherograms of a marker enzyme (malic dehydrogenase) and other components in apoplastic and symplastic saps revealed that contamination occurred at 7000 g. It is suggested that apoplastic sap expelled from plant roots at RCF between 600 and 3000 g is free from symplastic contamination, and is regarded as being of apoplastic origin. The proposed method was used to measure apoplastic pH changes in the plant roots in response to external pH, ammonium, nitrate and vanadate.