The production of intrinsically labeled milk protein provides a functional tool for human nutrition research.

Oral or intravenous administration of labeled, free amino acids does not allow the direct assessment of protein digestion and absorption kinetics following dietary protein intake. Consequently, dietary protein sources with labeled amino acids incorporated within the protein are required. The aim of this study was to produce milk proteins intrinsically labeled with l-[1-(13)C]phenylalanine that would allow the assessment of protein digestion and absorption kinetics and the subsequent muscle protein synthetic response to dietary protein intake in vivo in humans. Two Holstein cows (body weight of 726 +/- 38 kg) were continuously infused with l-[1-(13)C]phenylalanine at 402 micromol/min for 44 to 48 h, during and after which plasma samples and milk were collected. After milk protein separation, casein was used in a subsequent human proof-of-principle experiment. Two healthy males (aged 61 +/- 1 yr; body mass index of 22.4 +/- 0.1 kg/m(2)) ingested 35 g of casein highly enriched with [1-(13)C] phenylalanine. Plasma samples were collected at regular intervals, and skeletal muscle biopsies were collected before and 6 h after casein ingestion. In the initial experiment, a total of 5.83 kg of l-[1-(13)C]phenylalanine-enriched milk protein (casein enrichment was 29.4 molar percent excess) was collected during stable isotope infusion in the cows. In the proof-of-principle study, ingestion of 35 g of intrinsically labeled casein resulted in peak plasma l-[1-(13)C]phenylalanine enrichments within 90 min after protein ingestion (9.75 +/- 1.47 molar percent excess). Skeletal muscle protein synthesis rates calculated over the entire 6-h period averaged 0.058 +/- 0.012%/h. The production of intrinsically labeled milk protein is feasible and provides dietary protein that can be used to investigate protein digestion and absorption and the subsequent muscle protein synthetic response in vivo in humans.

RNA extraction from cheese for analysis of in situ gene expression of Lactococcus lactis.

AIMS: The isolation of high-quality RNA from cheese is a prerequisite for analysis of in situ gene expression of dairy micro-organisms. METHODS AND RESULTS: A method for rapid isolation of bacterial cells from cheese using cold citrate buffer followed by mechanical cell disruption was developed. RNA was extracted from experimental ultrafiltration (UF) cheeses (at 2, 8, 24 h, 7 and 14 days) and from Cheddar cheese (from 1 day to 1 year). The quantity and quality of the extracted RNA was assessed. The transcript abundance of seven genes (tuf, gapB, purM, cysK, ldh, cit and gyrA) was estimated by reverse transcription real-time PCR. In UF cheeses, the quantity of RNA extracted increased from 0.2 to 24 microg g(-1), with an RNA Integrity Number (RIN) above 9. In the experimental Cheddar cheeses, the RNA extraction yield decreased from 67.7 microg g(-1) after 1 day to 23.7 microg g(-1) after 6 months, with RIN value above 9 during the first month. The transcript abundance of the seven genes demonstrated metabolic activity of lactococci after several weeks of ripening in both cheeses. SIGNIFICANCE AND IMPACT OF THE STUDY: The method described produced large quantities of high-quality RNA for future whole genome expression studies in cheese.

Microfiltration of raw whole milk to select fractions with different fat globule size distributions: process optimization and analysis.

We present an extensive description and analysis of a microfiltration process patented in our laboratory to separate different fractions of the initial milk fat globule population according to the size of the native milk fat globules (MFG). We used nominal membrane pore sizes of 2 to 12 microm and a specially designed pilot rig. Using this process with whole milk [whose MFG have a volume mean diameter (d43) = 4.2 +/- 0.2 microm] and appropriate membrane pore size and hydrodynamic conditions, we collected 2 extremes of the initial milk fat globule distribution consisting of 1) a retentate containing large MFG of d43 = 5 to 7.5 microm (with up to 250 g/kg of fat, up to 35% of initial milk fat, and up to 10% of initial milk volume), and 2) a permeate containing small MFG of d43 = 0.9 to 3.3 microm (with up to 16 g/kg of fat, up to 30% of initial milk fat, and up to 83% of initial milk volume and devoid of somatic cells). We checked that the process did not mechanically damage the MFG by measuring their zeta-potential. This new microfiltration process, avoiding milk aging, appears to be more efficient than gravity separation in selecting native MFG of different sizes. As we summarize from previous and new results showing that the physico-chemical and technological properties of native milk fat globules vary according to their size, the use of different fat globule fractions appears to be advantageous regarding the quality of cheeses and can lead to new dairy products with adapted properties (sensory, functional, and perhaps nutritional).

Advances in cooled semen technology.

In the horse industry, milk or milk-based extenders are used routinely for dilution and storage of semen cooled to 4-8 degrees C. Although artificial insemination (AI) with chilled and transported semen has been in use for several years, pregnancy rates are still low and variable related to variable semen quality of stallions. Over the years, a variety of extenders have been proposed for cooling, storage and transport of stallion semen. Fractionation of milk by microfiltration, ultrafiltration, diafiltration and freeze-drying techniques has allowed preparation of purified milk fractions in order to test them on stallion sperm survival. Finally, a high protective fraction, native phosphocaseinate (NPPC), was identified. A new extender, INRA96, based on modified Hanks' salts, supplemented with NPPC was then developed for use with cooled/stored semen. Four experiments were conducted to compare INRA96 and milk-based extenders under various conditions of storage. The diluted semen was maintained under aerobic conditions when stored at 15 degrees C, and anaerobic conditions when stored at 4 degrees C. In experiment 1, split ejaculates from 13 stallions were diluted either in INRA96 extender then stored at 15 degrees C or diluted in Kenney or INRA82 extenders and then stored at 4 degrees C for 24h, until insemination. In experiment 2, semen from two stallions was extended in INRA96 then inseminated immediately or stored at 15 degrees C for 3 days until insemination. In experiment 3, semen from three stallions was diluted in INRA96 then stored at 15 or 4 degrees C for 24h until insemination, finally, in experiment 4, split ejaculates from four stallions were diluted in INRA96 or E-Z Mixin extenders then stored at 4 degrees C for 24h until insemination. Experiment 1 demonstrated that at 15 degrees C, INRA96 extender significantly improved pregnancy rate per cycle compared to Kenney or INRA82 extenders at 4 degrees C after 24h of storage (57%, n=178 versus 40%, n=171, respectively; P<0.01). Experiment 2 showed that semen stored at 15 degrees C for 3 days can achieve pregnancy at a fertility rate per cycle of 48% (n=52) compared to 68% (n=50, immediate insemination, P=0.06). Experiment 3 demonstrated that INRA96 extender can be as efficient at 15 degrees C (54%, n=37) as at 4 degrees C (54%, n=35) after 24h of storage. Finally, experiment 4 showed that INRA96 extender used at 4 degrees C (59%, n=39) seems to improve fertility per cycle compared to E-Z Mixin at 4 degrees C (49%, n=39, P=0.25), but this result has to be confirmed. These results demonstrate that semen diluted in INRA96 extender and stored at 15 degrees C can be an alternative to semen diluted in milk-based extenders and stored at 4 degrees C for "poor cooler" stallions. Furthermore, INRA96 extender can be as efficient at 15 degrees C as at 4 degrees C, for preserving sperm motility and fertility.

Goats' milk of defective alpha(s1)-casein genotype decreases intestinal and systemic sensitization to beta-lactoglobulin in guinea pigs.

Contradictory results have been reported on the use of goats' milk in cows' milk allergy. In this study the hypothesis was tested, using a guinea pig model of cows' milk allergy, that these discrepancies could be due to the high genetic polymorphism of goats' milk proteins. Forty guinea pigs were fed over a 20 d period with pelleted diets containing one of the following: soyabean proteins (group S), cows' milk proteins (group CM), goats' milk proteins with high (group GM1) or low (group GM2) alpha(s1)-casein content. Parenteral sensitization to GM1 and GM2 proteins as also assessed. The sensitization was measured (1) by systemic IgG1 antibodies directed against bovine or caprine beta-lactoglobulin (beta-lg), alpha-lactalbumin (alpha-la) and whole caseins, and (2) by intestinal anaphylaxis measured in vitro in Ussing chambers, by the rise in short-circuit current (delta Isc) in response to milk proteins. Guinea pigs fed on CM and GM1 developed high titres (> 1500) of anti-beta-lg IgG1, with an important cross reactivity between goat and cow beta-lg. However, in guinea pigs fed on GM2, anti-goat beta-lg IgG1 antibodies were significantly decreased compared with GM1 guinea pigs (mean IgG1 titres were 546 and 2046 respectively), and the intestinal anaphylaxis was significantly decreased (3.5+/-4.5 microA/cm2) compared with that observed in GM1 guinea pigs (8.3+/-7.6 microA/cm2). Animals receiving GM1 or GM2 proteins via the parenteral route developed a marked sensitization. These results suggest that the discrepancies observed in the use of goats milk in cows' milk allergy could be due, at least in part, to the high genetic polymorphism of goats' milk proteins.

The digestion rate of protein is an independent regulating factor of postprandial protein retention.

To evaluate the importance of protein digestion rate on protein deposition, we characterized leucine kinetics after ingestion of "protein" meals of identical amino acid composition and nitrogen contents but of different digestion rates. Four groups of five or six young men received an L-[1-13C]leucine infusion and one of the following 30-g protein meals: a single meal of slowly digested casein (CAS), a single meal of free amino acid mimicking casein composition (AA), a single meal of rapidly digested whey proteins (WP), or repeated meals of whey proteins (RPT-WP) mimicking slow digestion rate. Comparisons were made between "fast" (AA, WP) and "slow" (CAS, RPT-WP) meals of identical amino acid composition (AA vs. CAS, and WP vs. RPT-WP). The fast meals induced a strong, rapid, and transient increase of aminoacidemia, leucine flux, and oxidation. After slow meals, these parameters increased moderately but durably. Postprandial leucine balance over 7 h was higher after the slow than after the fast meals (CAS: 38 +/- 13 vs. AA: -12 +/- 11, P < 0.01; RPT-WP: 87 +/- 25 vs. WP: 6 +/- 19 micromol/kg, P < 0.05). Protein digestion rate is an independent factor modulating postprandial protein deposition.

New genetic variants identified in donkey's milk whey proteins.

Novel genetic variants for donkey milk lysozyme and beta-lactoglobulins I and II have been identified by the combined use of peptide mass mapping and sequencing by tandem mass spectrometry in association with database searching. The novel donkey lysozyme variant designated as lysozyme B (Mr 14,631 Da) differed in three amino acid exchanges, N49 --> D, Y52 --> S, and S61 --> N, from the previously published sequence. Three novel genetic variants for donkey beta-lactoglobulins were identified. One of them is a type beta-lactoglobulin I with three amino acid exchanges at E36 --> S, S97 --> T, and V150 --> I (beta-lactoglobulin I B, Mr 18,510 Da). The two others are type beta-lactoglobulins II with two amino acid exchanges at C110 --> P and M118--> T (beta-lactoglobulin II B, Mr 18,227 Da) and with three amino acid exchanges at D96 --> E, C110 --> P, and M118 -->T (beta-lactoglobulin II C, Mr 18,241 Da). All these primary structures are closely related to those of homologous proteins in horse milk (percent identity >96%).

Characterization by ionization mass spectrometry of lactosyl beta-lactoglobulin conjugates formed during heat treatment of milk and whey and identification of one lactose-binding site.

The extent of the early stage of the Maillard-type reaction that impaired functional properties of whey proteins was evaluated by electrospray ionization mass spectrometry. Under conditions of mild heat treatment (63 degrees C for 20 s) applied to milk before whey separation at room temperature 23 degrees C), a modification of the relative molecular mass of beta-lactoglobulin (beta-LG) was observed that differed from that of the native form by 324. This specific modification of beta-LG occurred in acidified whey as well as in sweet whey and increased with the extent of the heat treatment. Incubation of purified beta-LG dissolved in milk ultrafiltration permeate or in lactose solution at 50 to 80 degrees C demonstrated the presence of a lactosyl residue that was covalently bound to beta-LG; beta-casein, used as a control, showed no mass modification. Studies of kinetics showed that a maximum of 35% of the beta-LG was lactosyl-beta-LG conjugate after heat treatment at 70 degrees C for 1 h. This study provides the first direct evidence of specific lactosylation of beta-LG during the initial stage of the Maillard reaction. One of the first lactose-binding sites was identified as a Lys47 by protease mapping and analysis by means of on-line liquid chromatography combined with mass spectrometry. In addition, collision-activated dissociation performed on the lactosylated peptide beta-LG (f 46-51) showed the rearrangement reactions occurring during the fragmentation process by electrospray. A mechanism is proposed.

Effect of milk fractions on survival of equine spermatozoa.

Milk-based semen diluents are known to be practical and effective in protecting equine spermatozoa during storage. Due to complex composition of milk, the components which are beneficial or harmful to spermatozoa are unknown. To address these unknowns the effect of various milk fractions on motility of stallion spermatozoa was evaluated. The fractions tested were native phosphocaseinate (NPPC), beta-casein, whey protein concentrate (WPC), alpha-lactalbumin, beta-lactoglobulin, microfiltrate, and ultrafiltrate. The standard reference diluents were INRA 82, commercial skim milk, and Hank's salts solution supplemented with Hepes, glucose, lactose (HGLL) supplemented with BSA. After 48 and 96 h storage at 4 or 15 degrees C some milk fractions (ultrafiltrate, microfiltrate, and alpha-lactalbumin fraction) decreased spermatozoal survival. Others (beta-lactoglobulin (BL) and native phosphocaseinate) were protective. Native phosphocaseinate (NPPC) at milk concentration afforted better protection than did the standard reference diluents. The optimal concentration of beta-lactoglobulin afforted significantly better protection than did BSA. The protection afforded by native phophocaseinate was not synergistic with beta-lactoglobulin. This implies a similar mechanism of protective action of these two components. Semen diluted in HGLL supplemented with NPPC (HGLL-NPPC) or in INRA 82 and stored 24 h at 15 degrees C or 4 degrees C, respectively, produced no difference of spermatozoal motility. However, fertility of semen stored in HGLL-NPPC (60%) was higher (p < 0.05) than that stored in INRA 82 (36%).

Acute postprandial changes in leucine metabolism as assessed with an intrinsically labeled milk protein.

Mechanisms of protein gain during protein feeding have been investigated using a combination of oral and intravenous labeled leucine in healthy young men. The oral labeled leucine was administered as a free oral tracer ([13C]- or [2H3]leucine) added to unlabeled whey protein or as whey protein intrinsically labeled with L-[1-13C]leucine. When the oral tracer was free leucine, it appeared in the plasma more rapidly than the unlabeled leucine derived from the whey protein, and this resulted in an artifactual 88% decrease of protein breakdown. When the oral tracer was protein bound, protein breakdown did not change significantly after the meal. In contrast, nonoxidative leucine disposal (i.e., protein synthesis) was stimulated by 63% by the meal. In conclusion, 1) an intrinsically labeled protein is more appropriate than an oral free tracer to study postprandial leucine kinetics under non-steady-state conditions and 2) protein gain after a single whey protein meal results solely from an increased protein synthesis with no modification of protein breakdown.

Production of large amounts of [13C]leucine-enriched milk proteins by lactating cows.

During protein metabolism kinetic studies, oral tracers are administered as labeled free amino acids and do not necessarily represent the metabolic fate of amino acids ingested as proteins. However, sufficient quantities of 13C-labeled proteins are not currently available. We here present a new methodology for producing large amounts of milk proteins intrinsically labeled with [13C]leucine. After surgical preparation, two lactating cows were infused with 80-90 g of L-[1-13C]leucine for 24-32 h, and milk was collected during and after the infusion. Casein and whey protein fractions were purified by membrane separation techniques. Arteriovenous balance across the udder indicated a very efficient extraction of leucine by the mammary gland. Five batches of pure casein and whey proteins, totaling 3854 g of protein of excellent bacteriological quality, were obtained. Two thirds of these proteins had [13C]leucine enrichments ranging from 10.5 to 19.4% ([13C]leucine atom percent excess). The overall tracer recoveries were 22 and 27% (cows 1 and 2, respectively). Thus, pure milk proteins were produced in large amounts with sufficient 13C enrichment to be used in human protein metabolism studies.