Supplementation of sows with L-Arginine during gestating and lactation affects muscle traits of offspring related with postnatal growth and meat quality: From conception to consumption.

This study investigated the effect of dietary inclusion of 25 g/day of L-Arginine (n = 7) or iso­nitrogenous amounts of alanine (n = 6) from d 30 of gestation to d 28 of lactation of sows on performance, muscle traits and meat quality in offspring. From each litter, heaviest and smallest littermate of both sexes were reared from d 28 and slaughtered at d 140 in accordance with a 23factorial design. A response to L-Arginine were obtained on small females where L-Arginine increased birth weight, however this effect disappeared at weaning. L-Arginine increased daily gain by 7% and increased the cross-sectional area of the M. semitendinosus in small females by 14%, suggesting an increased lean ratio. Mechanistic studies showed firstly, that small female littermates had increased number of muscle fibres (myogenesis) after L-Arginine treatment (11%) and secondly increased total DNA (12%) as a consequence of satellite cell proliferation. Traits describing tenderness seem to be affected by L-Arginine but further studies are needed.

Short communication: Inhibition of angiotensin 1-converting enzyme by peptides derived from variants of bovine ß-casein upon apical exposure to a Caco-2 cell monolayer.

This study investigated the consequence of genetically contingent amino acid substitutions in bovine ß-casein (CN) genetic variants A1, A2, B, and I on the structure and bioactive potential of peptides following in vitro digestion. The ß-CN variants were digested in vitro using pepsin and pancreatin, and a peptide profile was obtained by liquid chromatography tandem mass spectrometry, revealing among others, the ß-casomorphin precursor peptides VYPFPGPIHN and VYPFPGPIPN, derived from variant A1/B and from A2/I, respectively. These 2 peptides were synthesized and assessed for angiotensin 1-converting enzyme (ACE) inhibitory capacity before and after incubation with a monolayer of Caco-2 intestinal cells. The VYPFPGPIHN was a stronger ACE inhibitor than VYPFPGPIPN, with the concentration needed to reach half-maximal inhibition (IC50) of 123 ± 14.2 µM versus 656 ± 7.6 µM. Exposure to a Caco-2 intestinal cell monolayer did not affect ACE inhibition by VYPFPGPIHN, but resulted in an almost 2-fold increase in inhibition by VYPFPGPIPN after incubation. Subsequent tandem mass spectrometric analysis identified the truncated peptide VYPFPGPIP, suggesting hydrolysis by a cell membrane associated peptidase. Thus, genetic variation in bovine ß-CN results in the generation of peptides that differ in bioactivity, and are differently affected by intestinal brush border peptidases.

Comparative proteomic analysis of casein and whey as prepared by chymosin-induced separation, isoelectric precipitation or ultracentrifugation.

Fractionation of bovine milk was performed using chymosin-induced separation, isoelectric precipitation or ultracentrifugation as separation techniques prior to gel-based proteomic analysis. This approach allowed for comparative display and identification of proteins partitioned into casein and whey, respectively. Initially, three different staining methods (silver staining, colloidal Coomassie Blue G-250 or fluorescent Flamingo Pink staining) for two-dimensional gel electrophoresis (2-DGE) analysis were compared for their suitability as staining agent, especially in relation to their suitability to reveal differences in the casein fractions. Fluorescent staining proved to be the most appropriate for this purpose, giving a high sensitivity, and using this staining method, characteristic 2-DGE fingerprints were obtained for each casein and whey fraction from each separation method. A number of protein spots in both casein and whey fractions varied with separation method and these spots were subsequently identified using tandem mass spectrometry (MS). In rennet casein, proteolytic fragmentation of caseins (α(s1)-, α(s2),-, ß- and κ-) was identified as a result of chymosin hydrolysis, whereas the 2-DGE profile of acid and ultracentrifuged casein was dominated by the presence of multiple isoforms of κ-caseins. Furthermore, casein remnants were identified in milk serum after ultracentrifugation. This study shows that gel-based proteomic analysis is suitable for characterisation of subtle variations in protein composition of milk fractions that occur as a consequence of different milk fractionation strategies.

Identification of peptides in milk as a result of proteolysis at different levels of somatic cell counts using LC MALDI MS/MS detection.

The somatic cell count (SCC) in milk is associated with increasing proteolytic degradation of caseins and it has been suggested that enzymes derived from somatic cells contribute to a lower yield and poorer quality of cheese. It is essential to increase the knowledge on naturally occurring milk proteinase activities to better understand how to improve the technological quality of milk. The aim of this work was to identify peptides actually present in milk as a result of proteolysis at different levels of SCC and to assign these peptides to potential responsible proteases where possible. Peptide fractions were prepared from acid whey by ultrafiltration at a molecular cut-off value of 10 000 Da. The peptides were separated using capillary reversed phase high performance liquid chromatography (RP-HPLC) and identified by matrix-assisted laser desorption/ionization-time of flight tandem mass spectrometry (MALDI-TOF MS/MS). Peptides identified ranged in mass from 1023 to 2000 Da, and originated from alphaS1-, alphaS2- or beta-casein. Possible responsible proteases that could be suggested when examining the peptide cleavage sites included plasmin, cathepsin B, D and leukocyte elastase. The results indicated that plasmin was primarily responsible for the observed proteolysis in milk at low cell count, whereas the cathepsins and elastase became implicated at elevated cell count. Specificity and activity of cathepsins and elastase has earlier mainly been studied in model systems, whereas less is known about their activities in milk itself. This is also the first indication of involvement of elastase in milk proteolysis through the unequivocal determination of cleavage sites.