Guy Miranda

Milking of cows in late pregnancy: milk production during this period and during the succeeding lactation.

Fifteen lactating cows were milked throughout pregnancy, and the effects on milk performance were studied during this period and during the succeeding lactation, relative to 11 conventionally managed cows (2 months dry before calving) as controls. During the last 2 months of pregnancy, only nine cows did not dry off spontaneously. Protein and fat concentrations in milk increased rapidly, but the concentration of lactose, corrected for milk yield, did not change. The ratios of individual caseins to total protein decreased with the quantity of milk produced, but only for yields below approximately 6 kg/d. The relative proportion of kappa-casein tended to decrease in the last milkings. During the succeeding lactation (first 15 weeks after calving and first 6 weeks of grazing) continuously milked cows yielded 4 kg milk/d less than the cows of the other group. The protein content of their milk was higher (2-3 g/kg depending on the period) than that of the control group, and the lactose content tended (P less than 0.10) to be lower. Changes in the relative proportions of nitrogenous fractions with time were not different in the two groups. Differences between the two groups in the concentration of protein in milk, and in the concentration of glucose and non-esterified fatty acids in the plasma, suggest a better energy balance for the continuously milked cows during the succeeding lactation.

Kinetic studies of in vivo digestion of bovine unheated skim-milk proteins in the rat stomach

The in vivo action of gastric proteinases on bovine milk proteins was studied in rats fed with skim-milk, by analysing gastric contents after 30, 60 and 240 min of digestion. Gastric proteolysis was already marked after 30 min of digestion and liberated a large number of peptides with different molecular weights. alpha S1-Casein and beta-casein were still the main components of the stomach contents together with alpha-lactalbumin and beta-lactoglobulin, which were little degraded, even after 240 min of digestion. A statistical analysis (multivariate method), made on several parameters (such as pH, N, and NPN) showed changes in the stomach contents during the digestion. The amino acid composition of the protein fraction was close to that of the diet, whilst that of the non-protein fraction was very different, being between the amino acid composition of the endogenous proteins and that of the diet.

Analysis of the peptidoglycan hydrolase complement of Lactococcus lactis: identification of a third N-acetylglucosaminidase, AcmC.

ABSTRACT The peptidoglycan hydrolase (PGH) complement of Lactococcus lactis was identified by amino acid sequence similarity searching of the L. lactis IL-1403 complete genome sequence. Five PGHs that are not encoded by prophages were detected, including the previously characterized AcmA and AcmB proteins. Four of these PGHs, AcmA to AcmD, contain a catalytic domain homologous to that of enterococcal muramidase, but they have different domain structures. The fifth one (YjgB) has sequence similarity with the active-site domain of peptidoglycan-specific endopeptidases. The three new PGH-encoding genes identified in this study are all actively transcribed in L. lactis subsp. cremoris MG1363. The relative abundance of their transcripts varied during growth and was maximal during the early exponential growth phase. The three encoded proteins have peptidoglycan-hydrolyzing activities which are detected only at acidic pHs by zymography. Like AcmA and AcmB, AcmC has N-acetylglucosaminidase activity rather than the N-acetylmuramidase activity predicted by sequence similarity.

In vivo studies on the digestion of bovine caseins in the rat stomach

The action of gastric proteinases on bovine caseins was studied in vivo on rats fed skim-milk, or whole casein in water or whole casein in mineral solution, by analysing the gastric content after 30 min digestion. Clotting of the diet in the stomach greatly reduced the rate of gastric emptying. The proteolysis of caseins observed by gel electrophoresis appeared to follow a different pathway for the 3 different diets. The amino acid compositions of the trichloracetic acid sediments of the stomach contents did not differ between the 3 diets. The presence of free amino acids in the stomachs at significant levels (0·8–5 % of the total amino acid content) was observed.

Combining proteomic tools to characterize the protein fraction of llama (Lama glama) milk

Llamas belong to the Camelidae family along with camels. While dromedary camel milk has been broadly characterized, data on llama milk proteins are scarce. The objective of this study was thus to investigate the protein composition of llama milk. Skimmed llama milk proteins were first characterized by a 2D separation technique coupling RP‐HPLC in the first dimension with SDS‐PAGE in the second dimension (RP‐HPLC/SDS‐PAGE). Llama milk proteins, namely caseins (αs1‐, αs2‐, β‐, and κ‐caseins), α‑lactalbumin, lactoferrin, and serum albumin, were identified using PMF. Llama milk proteins were also characterized by online LC‐ESI‐MS analysis. This approach allowed attributing precise molecular masses for most of the previously MS‐identified llama milk proteins. Interestingly, α‐lactalbumin exhibits distinct chromatographic behaviors between llama and dromedary camel milk. De novo sequencing of the llama α‐lactalbumin protein by LC coupled with MS/MS (LC‐MS/MS) showed the occurrence of two amino acid substitutions (R62L/I and K89L/I) that partly explained the higher hydrophobicity of llama α‐lactalbumin compared with its dromedary counterpart. Taken together, these results provide for the first time a thorough description of the protein fraction of Lama glama milk.

Milk Proteins | Inter-Species Comparison of Milk Proteins: Quantitative Variability and Molecular Diversity

In the last few years, developments in molecular biology, genomics, and proteomics have allowed remarkable progress in the understanding of milk protein structure and function, highlighting the extreme complexity and large variability (qualitative and quantitative) of the milk protein fraction, across, but also within, species. Meaningful examples taken in different species, including non-eutherian mammals, are discussed in this article to show how genomes and genetic polymorphisms may modulate the milk protein fraction by affecting different cellular processes, mainly at the posttranscriptional level (splicing of primary transcripts, posttranslational modifications), making milk a more complex biological fluid than previously assumed. However, the repertoire of minor milk proteins remains to be characterized in most species. Numerous substantiated or potential bioactive protein components have been found and many others still remain to be identified either as intact protein or as derived peptides, encrypted in the sequence of milk proteins. This is probably one of the greatest challenges facing milk science in the next years to provide the food industry and consumers with the basis for health-promoting properties of these proteins and peptides.

Modelization of Gastric Digestion of Milk Proteins

In vivo digestion of proteins is initiated in the stomach by pepsins, and in some young animals by chymosin. It has been shown that milk coagulation in the stomach contributes to slowing down the degradation and it retains some peptides; casein degradation into small peptides takes place afterwards. β-lactoglobulin appears to be resistant to gastric proteolysis, but α-lactalbumin is hydrolyzed when stomach pH is about 3.5.

Combining different proteomic approaches to resolve complexity of the milk protein fraction of dromedary, Bactrian camels and hybrids, from different regions of Kazakhstan

Nutritional suitability of milk is not only related to gross composition, but is also strongly affected by the microheterogeniety of the protein fraction. Hence, to go further into the evaluation of the potential suitability of non-bovine milks in human/infant nutrition it is necessary to have a detailed characterization of their protein components. Combining proven proteomic approaches (SDS-PAGE, LC-MS/MS and LC-ESI-MS) and cDNA sequencing, we provide here in depth characterization of the milk protein fraction of dromedary and Bactrian camels, and their hybrids, from different regions of Kazakhstan. A total 391 functional groups of proteins were identified from 8 camel milk samples. A detailed characterization of 50 protein molecules, relating to genetic variants and isoforms arising from post-translational modifications and alternative splicing events, belonging to nine protein families (κ-, αs1-, αs2-, β-; and γ-CN, WAP, α-LAC, PGRP, CSA/LPO) was achieved by LC-ESI-MS. The presence of two unknown proteins UP1 (22,939 Da) and UP2 (23,046 Da) was also reported as well as the existence of a β-CN short isoform (946 Da lighter than the full-length β-CN), arising very likely in both genetic variants (A and B) from proteolysis by plasmin. In addition, we report, for the first time to our knowledge, the occurrence of a αs2-CN phosphorylation isoform with 12P groups within two recognition motifs, suggesting thereby the existence of two kinase systems involved in the phosphorylation of caseins in the mammary gland. Finally, we demonstrate that genetic variants, which hitherto seemed to be species- specific (e.g. β-CN A for Bactrian and β-CN B for dromedary), are in fact present both in Camel dromedarius and C. bactrianus.

Effect of Technological Treatments of Milk on Gastric Digestion

Technological treatments of milk modify its digestibility. Coagulation plays an important role during the first part of digestion which takes place in the stomach. To compare the effects of technological treatments of milk on the gastric emptying of proteins and peptides, three diets were studied: crude skim milk, pasteurized skim milk (95 °C, 45 s), and skim yoghurt. In the preruminant calf (monogastric) all effluents leaving the stomach during 12 h were collected and analyzed for amino acid composition, N emptying, NPN level, and identification of proteins and peptides (electrophoresis, HPLC).