An in-depth molecular characterization of the main milk proteins, caseins (CNs) and whey proteins, from Amiata donkey combining top-down proteomic analysis (LC-MS) and cDNA sequencing revealed multiple proteoforms arising from complex splicing patterns, including cryptic splice site usage and exon skipping events. Post-translational modifications, in particular phosphorylation, increased the variety and complexity of proteoforms. αs2-CN perfectly exemplifies such a complexity. With 2 functional genes, CSN1S2 I and CSN1S2 II, made of 20 and 16 exons respectively, nearly 30 different molecules of this CN were detected in the milk of one Amiata donkey. A cryptic splice site usage, leading to a singular shift of the open reading frame and generating two αs2-CN I isoforms with different C-terminal sequences, was brought to light. Twenty different αs1-CN molecules with different phosphorylation levels ranging between 4 and 9P were identified in a single milk sample, most of them resulting from exon skipping events and cryptic splice site usage. Novel genetic polymorphisms were detected for CNs (β- and αs-CN) as well as for whey proteins (lysozyme C and β-LG I). The probable new β-LG I variant, with a significantly higher mass than known variants, appears to display an N-terminal extension possibly related to the signal peptide sequence. This represents the most comprehensive report to date detailing the complexity of donkey milk protein micro-heterogeneity, a prerequisite for discovering new elements to objectify the original properties of donkey’s milk.

Malgré un net regain d’intérêt, les références techniques sur les systèmes valorisant les races locales bretonnes sont quasi inexistantes. Les éleveurs comme les restaurateurs s’intéressent à la spécificité des produits issus de ces races locales. Si leur qualité est empiriquement reconnue, elle n’a jamais été précisément décrite. Les éleveurs s’interrogent sur les qualités intrinsèques des produits laitiers (fromageabilité, crémeux du lait…), sur leurs qualités organoleptiques, et de façon plus générale, sur la notion de qualité globale des produits, à laquelle sont associées bien évidemment les pratiques de production. L’objectif de ce projet de recherche participative était de mieux connaître les qualités du lait des races locales de vaches laitières dans leurs diverses composantes : qualités intrinsèques mais aussi nutritionnelles et technologiques. Des prélèvements de laits de tank associés à des enquêtes ont été réalisés dans les exploitations participant au projet sur différentes races de vaches (6 en Bretonne Pie Noir et 4 en Froment du Léon). Nous avons effectué les prélèvements pendant trois périodes : avril, juillet et novembre 2017. Nous avons caractérisé les laits au niveau biochimique et technologique. Le lait de Bretonne Pie Noir et de Froment du Léon est plus riche en matières grasses et en protéines que le lait des principales races de vaches françaises. Les variants des protéines sont aussi assez caractéristiques de ces races. Ceci leur confère des propriétés technologiques particulières, comme une meilleure aptitude fromagère mais également une moins bonne aptitude à supporter les traitements thermiques. Ces laits sont également plus jaunes ce qui résulte de concentrations plus élevées en β-carotène et riboflavine, sans que cela soit totalement systématique. Les caractéristiques particulières de ces laits de vaches de race locales méritent d’être mieux valorisées et mises en avant dans des circuits courts de commercialisation.

Despite a clear resurgence of interest, technical references on systems promoting local Breton breeds are almost non-existent. Both breeders and restaurant owners are interested in the specificity of products from local breeds. While their quality is empirically recognized, it has never been precisely described. Farmers are wondering about the intrinsic qualities of dairy products (cheese capacity, creaminess of milk.), their organoleptic qualities, and more generally, the notion of overall product quality, to which are obviously associated production practices. The objective of this participatory research project was to improve our understanding of milk qualities of local breeds of dairy cows in their various components: intrinsic qualities but also nutritional and technological qualities. Samples of tank milk associated with surveys were carried out on farms participating in the project on different breeds of cows (6 in Bretonne Pie Noir and 4 in Froment du Leon). We took samples over 3 periods: April, July and November 2017. We characterized the milks at the biochemical and technological levels. The milk of Bretonne Pie Noir and Froment du Leon is richer in protein and fat than the milk of the main French cows. Protein variants are also quite characteristic of these breeds. This gives them particular technological properties, such as better cheese-making capacity, but also a poorer ability to resist to heat treatments. These milks are also yellower which results from higher concentrations of beta-carotene and riboflavin, without this being completely systematic. The particular characteristics of milk from local breed cows deserves to be better valued and put forward in short marketing circuits.

Here we describe a method based on Liquid Chromatography coupled with Mass Spectrometry (LC-MS) that provides an accurate determination of the six main bovine milk proteins, including allelic and splicing variants, as well as isoforms resulting from post-translational modifications, with an unprecedented level of resolution. Proteins are identified from observed molecular masses in comparison with theoretical masses of intact proteins indexed in an "in-house" database that includes nearly 3000 entries. Quantification was performed either from UV (214 nm) or mass signals. Thus, up to one hundred molecules, derived from the six major milk proteins, can be identified and quantified from an individual milk sample. This powerful and reliable method, initially developed as an anchoring method to estimate the composition of the six main bovine milk proteins from MIR spectra, is transferable to several mammalian species, including small ruminants, camels, equines, rabbits, etc., for which specific mass databases are available.

Exposure to fine-particulate air pollution is a major global health concern because it is associated with reduced birth weight and an increased risk of cardiovascular disease. Here we have investigated the potential for exposure to diesel exhaust during pregnancy to influence mammary gland development and milk composition. Female rabbits were therefore exposed by nose-only inhalation to either diluted diesel exhaust fumes (1 mg/m3) or clean air for 2h/day, 5 days/week, from the 3rd to the 27th days of pregnancy. On Day 28 of pregnancy, mammary glands were collected from twelve females (six controls and six diesel-exposed) and assessed for morphological and functional alterations. Milk samples were collected from eighteen dams (nine controls and nine diesel-exposed) during early (days 2 to 4) and established (days 13 to 16) lactation to verify the composition of fatty acids and major proteins and leptin levels. The mammary alveolar lumina contained numerous fat globules, and stearoyl CoA reductase expression was higher in mammary epithelia from diesel exhaust-exposed rabbits, which together suggested increased mammary lipid biosynthesis. Gas chromatography analysis of the composition of milk fatty acids revealed a sharp rise in the total fatty acid content, mainly due to monounsaturated fatty acids. Liquid chromatography-mass spectrometry analysis of milk samples enabled identification and quantification of the main rabbit milk proteins and their main phosphorylated isoforms, and revealed important changes to individual casein and whey protein contents and to their most phosphorylated isoforms during early lactation. Taken together, these findings suggest that repeated daily exposure to diesel exhaust fumes during pregnancy at urban pollution levels can influence lipid metabolism in the mammary gland and the lipid and protein composition of milk. As milk may contribute to metabolic programming, such alterations affecting milk composition should be taken into account from a public health perspective.

In a previous study on camel milk from Kazakhstan, we reported the occurrence of two unknown proteins (UP1 and UP2) with different levels of phosphorylation. Here we show that UP1 and UP2 are isoforms of camel αs2-CN (αs2-CNsv1 and αs2-CNsv2, respectively) arising from alternative splicing events. First described as a 178 amino-acids long protein carrying eight phosphate groups, the major camel αs2-CN isoform (called here αs2-CN) has a molecular mass of 21,906 Da. αs2-CNsv1, a rather frequent (35%) isoform displaying a higher molecular mass (+1,033 Da), is present at four phosphorylation levels (8P to 11P). Using cDNA-sequencing, αs2-CNsv1 was shown to be a variant arising from the splicing-in of an in-frame 27-nucleotide sequence encoding the nonapeptide ENSKKTVDM, for which the presence at the genome level was confirmed. αs2-CNsv2, which appeared to be present at 8P to 12P, was shown to include an additional decapeptide (VKAYQIIPNL) revealed by LC-MS/MS, encoded by a 3′-extension of exon 16. Since milk proteins represent a reservoir of biologically active peptides, the molecular diversity generated by differential splicing might increase its content. To evaluate this possibility, we searched for bioactive peptides encrypted in the different camel αs2-CN isoforms, using an in silico approach. Several peptides, putatively released from the C-terminal part of camel αs2-CN isoforms after in silico digestion by proteases from the digestive tract, were predicted to display anti-bacterial and antihypertensive activities.

BackgroundWhey acidic protein (WAP) is a major protein identified in the milk of several mammalian species with cysteine-rich domains known as four-disulfide cores (4-DSC). The organization of the eutherian WAP genes is highly conserved through evolution. It has been proposed that WAP could play an important role in regulating the proliferation of mammary epithelial cells. A bacteriostatic activity was also reported. Conversely to the other mammalian species expressing WAP in their milk, camel WAP contains 4 additional amino acid residues at the beginning of the second 4-DSC domain, introducing a phosphorylation site. The aim of this study was to elucidate the origin of this specificity, which possibly impacts its physiological functions.ResultsUsing LC-ESI-MS, we identified in Camelus bactrianus from Kazakhstan a phosphorylated whey protein, exhibiting a molecular mass (12,596Da), 32Da higher than the original WAP (12,564Da) and co-eluting with WAP. cDNA sequencing revealed a transition G/A, which modifies an amino acid residue of the mature protein (V12M), accounting for the mass difference observed between WAP genetic variants. We also report the existence of two splicing variants of camel WAP precursors to mRNA, arising from an alternative usage of the canonical splice site recognized as such in the other mammalian species. However, the major camel WAP isoform results from the usage of an unlikely intron cryptic splice site, extending camel exon 3 upstream by 12-nucleotides encoding the 4 additional amino acid residues (VSSP) in which a potentially phosphorylable Serine residue occurs. Combining protein and cDNA sequences with genome data available (NCBI database), we report another feature of the camel WAP gene which displays a very rare GC-AG type intron. This result was confirmed by sequencing a genomic DNA fragment encompassing exon 3 to exon 4, suggesting for the GC donor site a compensatory effect in terms of consensus at the acceptor exon position.ConclusionsCombining proteomic and molecular biology approaches we report: the characterization of a new genetic variant of camel WAP, the usage of an unlikely intron cryptic splice site, and the occurrence of an extremely rare GC-AG type of intron.

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.

Spontaneous lipolysis (SL) impairs the technological and sensory properties of milk and dairy products. Husbandry factors, and especially nutritional factors, are known to modulate SL levels, but the biochemical mechanisms involved remain unclear. We investigated effects of feeding levels and forage types on SL mechanisms. Thirty-two cows were divided into 4 groups according to their diets (corn silage or conserved grass). Two feeding levels were applied: "non-restricted", where cows were fed ad libitum, and "restricted", where cows were fed at 75% of their ad libitum dry matter intake. The trial was conducted in a split-plot design where "forage type" was the main factor and "feeding level" was the sub-factor. Cows were followed for 12.5 weeks: 4 pre-experimental weeks, then a 14-day transition period (to change the forage, and decrease feed intake), a first 3-week experiment period, then a 4-day transition period (to reverse the energy treatment) followed by a second 3-week experiment period. Milk samples were collected from morning and evening milkings at the end of each period to determine milk traits, fat globule size, protein and fatty acid profiles, lipoprotein lipase (LPL) activity, and SL. Data analysis using the SAS-software MIXED procedure found no statistically significant feeding-level x forage-type interactions on SL. Compared to non-restricted cows, feed-restricted cows tended to have higher SL levels in morning milks (0.60 vs 0.52 mEq/100 g fat) and even higher SL levels in evening milks (0.89 vs 0.58 mEq/100 g fat). Cows fed corn silage tended to have higher SL in morning milks (0.72 vs 0.40 mEq/100 g fat) but evening milks showed no forage-type effect. The low feeding level had no effect on milk fat content, fat globule size, LPL activity or protein profile. In both non-feed-restricted and feed-restricted cows, corn silage was associated with lower milk fat content (3.53 vs 3.91%), smaller globule size (d(4,3) : 3.40 vs 3.71 mu m), lower percent proteose peptone 5 and 8 s, but had no effect on LPL activity. Data analysis using the SAS-software REG procedure found that proteose peptone 5 (logarithm-converted) was strongly negatively correlated with SL, suggesting a potential inhibitor effect of proteose peptone 5. Certain milk components are therefore modulators of SL. In morning and evening milks of SL-sensitive cows (defined as SL level up to 0.60 mEq/100 g fat), SL was negatively correlated with C18:0 and fat content and positively correlated with cis9 C18:1/C18:0, suggesting a link between tissue mobilization, mammary metabolism and SL. To sum up, feeding restriction is associated with higher SL, probably due to higher tissue lipid mobilization. Further investigation is needed to determine the impact of corn silage on SL. Variations in proteose peptone 5 levels according to forage type could explain SL levels. These findings could serve for advisory practice and prevention education to farmers.. At a biochemical level, the findings could be used to create tools and tests to detect and monitor lipolysis cow-by-cow.

Genome-wide association studies (GWAS) were performed at the sequence level to identify candidate mutations that affect the expression of six major milk proteins in Montbéliarde (MON), Normande (NOR), and Holstein (HOL) dairy cattle. Whey protein (α-lactalbumin and β-lactoglobulin) and casein (αs1, αs2, β, and κ) contents were estimated by mid-infrared (MIR) spectrometry, with medium to high accuracy (0.59 ≤ R2 ≤ 0.92), for 848,068 test-day milk samples from 156,660 cows in the first three lactations. Milk composition was evaluated as average test-day measurements adjusted for environmental effects. Next, we genotyped a subset of 8080 cows (2967 MON, 2737 NOR, and 2306 HOL) with the BovineSNP50 Beadchip. For each breed, genotypes were first imputed to high-density (HD) using HD single nucleotide polymorphisms (SNPs) genotypes of 522 MON, 546 NOR, and 776 HOL bulls. The resulting HD SNP genotypes were subsequently imputed to the sequence level using 27 million high-quality sequence variants selected from Run4 of the 1000 Bull Genomes consortium (1147 bulls). Within-breed, multi-breed, and conditional GWAS were performed.

The objective of the study was to analyze milk protein composition (major proteins) of two genetic types of rabbits bred in Tizi-Ouzou (Algeria): the white population (PB) and the "synthetic" line (SS). Differences in size and litter weight at weaning were observed amongst the two types, which might be due to qualitative and/or quantitative variations in milk composition. Milk samples (n=7) from dams of the two genetic types were collected (10th of lactation) and analyzed by means of liquid chromatography associated to a mass spectrometer (LC-MS). We were thus able to identify and characterize the major milk proteins in both types of rabbits and determine their post-translational modification isoforms. Alpha-s1 and β represented 50% of the total milk proteins; αs2-like-, αs2and κcaseins represented respectively 13.5, 4 and 2.7%. Whey Acidic Protein was the main whey protein (14.5%) while lactoferrin represented 10% of the total milk proteins. Chromatographic profiles of milk proteins were similar between the two genetic types of rabbits. In the "synthetic" strain, a strong dispersal around the mean of the relative proportions of αs2 casein and the mix α-Lac + SA was observed (CVαs2 = 0.24 in PB vs. 0.46 in SS and CVα-Lac + SA = 0.19 vs. 0.274 in PB and in SS respectively).

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.

Les acteurs des filières laitières bovine, caprine et ovine françaises se sont regroupés dans le programme PhénoFinlait autour d’un but commun : caractériser la composition du lait en Acides Gras (AG) et protéines afin de la maîtriser. La quantification des AG et des protéines devait être possible à grande échelle et à moindre coût avant d’identifier des leviers permettant d’adapter cette composition à la demande. PhénoFinlait s’est organisé autour de trois objectifs : i) caractériser précisément la composition du lait, ii) phénotyper et génotyper une large population de femelles sur l’ensemble du territoire français et iii)identifier les leviers génétiques et alimentairespermettant de maîtriser cette composition. La spectrométrie dans le Moyen InfraRouge (MIR) a été choisie comme méthode de quantification à haut débit des composants du lait. Elle permet la quantification précise en routine de 15 à 27 AG, des quatre caséines et des deux protéines majeures du lactosérum. Une collecte de données de grande ampleur a été mise en œuvre dans plus de 1 500 élevages bovins, caprins et ovins. Les données de production laitière, les spectres MIR du lait, les informations sur le stade physiologique des femelles et sur la composition de l’alimentation des troupeaux ont été recueillies. Plus de 12 000 vaches, chèvres et brebis ont été génotypées. Finalement, plus de 800 000 données représentatives des situations de l’élevage français ont été stockées dans une base de données destinée à l’étude du déterminisme génétique de la composition en AG et en protéines du lait, et des facteurs d’élevage l’influençant.