Total Milk Protein Research Articles

Ameliorative Effect of Infused Watercress on Rat Galactopoiesis following Maternal Separation

Galactopoiesis is the maintenance of milk production. The process that regulated primarily by prolactin hormone. The research was conducted to evaluate galactopoiesis activity by measuring prolactin level and milk production continuity of lactating rats after oral administration of infused watercress. Twenty lactating rats were randomly divided into five groups (two control groups and three variations of infused watercress dose groups). Serum prolactin was measured using enzyme-linked immunosorbent assay. Milk yield was measured using indirect milk measurement method by Sampson and Jansen. Milk protein level was measured using Kjedahl method. The treatment of infused watercress (10 g/kg body weight), started from postnatal day 3 until 12, indicated ameliorative effect on rat galactopoiesis by increasing milk yield and prolactin levels, but not milk total protein.

DGAT1 polymorphism in Riverine buffalo, Swamp buffalo and crossbred buffalo.

This Research Communication describes the polymorphisms in the coding region of DGAT1 gene in Riverine buffalo, Swamp buffalo and crossbred buffalo, and associations between polymorphisms and milk production performance in Riverine buffalo. Two polymorphisms of DGAT1were identified, located in exon 13 and exon 17, respectively. The distribution of the genotypes of the two SNP loci in different buffalo population varied, especially the polymorphism located in exon 13 which was not found in the Swamp buffalo. Moreover, SNP located in exon 17 was a nonsynonymous switch resulting in the animo acid sequence changed from an arginine (Arg) to a histidine (His) at position 484. Both SNPs were in Hardy-Weinberg equilibrium, and the polymorphism of g.8330T>C in the exon 13 was significantly associated with peak milk yield, total milk yield and protein percentage. The C variant was associated with an increase in milk yield and peak yield but less in protein percentage compared with the T variant. The polymorphisms of g.9046T>C in exon 17 were significantly associated with fat percentage, in that the buffaloes with TT genotype had a significantly higher fat percentage than those with CC genotype. These findings reveal the difference in the genetic evolution of the DGAT1 between Riverine buffalo and Swamp buffalo, and provide evidence that the DGAT1 gene has potential effects for Riverine buffalo milk production traits, which can be used as a candidate gene for marker-assisted selection in buffalo breeding.

Milk raw materials from retrovirus infected cows: questions of safety and quality of produced products

The questions of quality control and safety of milk raw materials has always been relevant for the dairy industry. One of the most common cattle infection is bovine leukemia (causative agent - BLV), which often occurs in conjunction with a closely related infection -bovine immunodeficiency (causative agent - BIV). These retroviruses are considered to be species-specific, so the key issue for the dairy industry is the nutritional and raw value of milk of infected cows (without clinical signs). Milk of infected with leukemia cows has changed physical and chemical properties and microbiological parameters, but it is allowed to be processed after a single pasteurization. Thus, there is a possibility of getting of the infected with retroviruses cow's milk into the bulk milk, which can affect the quality of the processed products. Characteristics of BlV-positive cow's milk have not yet been studied. The aim of the research was the study of influence of BLV- and BLV- BIV-infected cows' milk impurities (10, 30 и 50%) on organoleptic, physicochemical and microbiological parameters of produced kefir drink. It is revealed that with increasing the mass fraction of infected with retroviruses cows' milk, acidity of composite samples increased of 1-3 °T and total viable bacteria's count rose by 1-3 orders of magnitude, while the total protein in milk decreased by 0.05-0.95% contrasted with an increasing of milk fat content by 0.3-0.9%. However, these characteristics were within normal limits. In samples containing 50% of milk infected with retroviruses cows, coliforms were detected in 0.1 ml, and the rate of total viable bacteria's count exceeded the permissible limits. Therefore, microbiological characteristics are determination markers of dairy raw materials with the high content of milk of the infected with retroviruses cows. The kefir drink was produced from pasteurized samples. It was found that the acidity of the product was lower by 1-10 °T, while the processing time increased by 4-50%, correlating with the mass fraction of infected with retroviruses cows' milk, compared to control. When the mass fraction of infected with retroviruses cows' milk was 30 and 50%, the processed product had a heterogeneous consistency with flake clots. At the same time, the lactic-acid-producing bacterium count in kefir drink decreased by 2-3 orders, and mold and coliforms were found in it. Thus, the presence more than 10% of milk from infected with retroviruses cows in raw materials leads to a decrease in its technological properties, and dose 30 and 50% leads to non-compliance of the processed product with hygienic standards.

ELECTROPHORETIC SYSTEMS FOR PREPARATIVE FRACTIONATION OF PROTEIN PRECURSORS OF BIOACTIVE PEPTIDES FROM COW’S MILK

The article considers the possibility of obtaining purified fractions-precursors of bioactive peptides from milk proteins by the method of preparative electrophoresis. To choose an electrophoretic system, a comparative study has been carried out of four methods of electrophoresis in polyacrylamide gel that are used to analyse milk proteins (disc-electrophoresis without disaggregating agents, and disc-electrophoresis in the presence of sodium dodecylsulfate in homogeneous and gradient gel, and electrophoresis in homogeneous gel with urea). Electrophoresis of the total milk protein has shown that none of these systems allows separating effectively all protein precursors of bioactive peptides. The next stage was obtaining two main groups of milk proteins – caseins and serum proteins for electrophoretic fractionation. With the help of analytical electrophoresis, it has been established that each of the obtained groups had a typical proteins composition. Then, the proteins groups obtained were fractionated by preparative electrophoresis using the four electrophoretic systems listed above. In this case, the casein proteins that differ in the primary structure (αS1-, αS2-, β-, and ϰ-caseins) can be effectively separated by preparative electrophoresis on the basis of a homogeneous gel system in the presence of urea. The composition of this electrophoretic system was simplified. Unlike the analytical variant of a homogeneous polyacrylamide gel system, the toxic 2-mercaptoethanol was excluded, and the urea concentration was reduced. For the fractionation of serum proteins, a disc-electrophoresis without disaggregating agents can be used as a basis. It allows obtaining the main precursors of bioactive peptides from milk serum proteins: β-lactoglobulin, α-lactalbumin, serum albumin, and immunoglobulins. The protein precursors obtained by preparative electrophoresis were used to develop the biotechnology of obtaining bioactive phosphopeptides and inhibitors of the angiotensin-converting enzyme.

Normal Human Lactation: closing the gap.

With the exception of infant growth, there are no well-defined parameters describing normal human lactation. This represents a major gap in the continuum of care that does not exist for other major organs. Biological normality occurs naturally and is characterized by well-integrated function. We have proposed a definition that highlights four key elements that describe parameters for biological normality: comfort, milk supply, infant health, and maternal health. Notwithstanding the current limitations, published data have been collated to provide preliminary markers for the initiation of lactation and to describe objective tests once lactation is established. Reference limits have been calculated for maternal markers of secretory activation, including progesterone in maternal blood and total protein, lactose, sodium, and citrate in maternal milk. Objective measurements for established lactation, including 3-hourly pumping and 24-hour milk production, together with pre-feed to post-feed milk fat changes (a useful indicator of the available milk removed by the infant) have been outlined. Considered together with the parameters describing normal function, this information provides a preliminary objective framework for the assessment of human lactation.

The Effects of Protein Type and Added Leucine on Myofibrillar Protein Synthesis Following Concurrent Exercise

Protein ingestion increases skeletal muscle protein synthesis (MPS) rates. The effect of protein on MPS rates is enhanced by prior exercise. However, the effect of different types of protein on MPS is unclear and has not been evaluated following concurrent exercise. PURPOSE: The objective of this study was to determine the capacity of total milk protein (MILK), whey (WHEY), micellar casein (CASEIN), and soy protein, without (SOY) and with additional leucine (SOY+LEU) to support postprandial myofibrillar protein synthesis (MyoPS) following concurrent exercise. METHODS: 72 young recreationally active males participated in this parallel group, double-blind, randomized controlled trial consisting of two arms. In arm #1, 48 subjects (23±0.3 y) ingested 45 g of carbohydrate with either 0 g protein (CHO), or 20 g MILK, WHEY, or CASEIN protein. In arm #2, 36 subjects (23±0.5 y) ingested 45 g of carbohydrate with 20 g WHEY, SOY, or SOY+LEU. A primed continuous infusion of L-[ring-13C ]-phenylalanine with blood and muscle biopsies was applied to evaluate postprandial MyoPS over 360 minutes after exercise. RESULTS: In arm #1, MyoPS did not differ between treatments (P=0.12) during the postprandial period after exercise (FSR: CHO=0.051±0.003; MILK=0.061±0.003; WHEY=0.056±0.002; CASEIN=0.062±0.005 %/h). When MILK, WHEY, and CASEIN were collapsed into a single group (PROTEIN), protein-carbohydrate co-ingestion resulted in greater MyoPS rates than carbohydrate alone (FSR: PROTEIN=0.060±0.002; CHO=0.051±0.003 %/h; P=0.042). In arm #2 MyoPS did not differ between treatments (P=0.85) after exercise (FSR: WHEY=0.056±0.002; SOY=0.056±0.004; SOY+LEU=0.058±0.004 %/h). CONCLUSION: Total milk protein, whey, and micellar casein (arm #1) do not differ in their capacity to support postprandial MyoPS rates when co-ingested with carbohydrate following concurrent exercise in young males. Similarly, whey and soy protein, irrespective of leucine enrichment of soy (arm #2), do not differ in their capacity to support postprandial MyoPS after concurrent exercise. Co-ingestion of protein with carbohydrate results in greater postprandial MyoPS rates than carbohydrate alone, and may therefore represent a nutritional strategy to support skeletal muscle remodeling following concurrent exercise.

Milk Composition for Admixed Dairy Cattle in Tanzania.

It is well established that milk composition is affected by the breed and genotype of a cow. The present study investigated the relationship between the proportion of exotic genes and milk composition in Tanzanian crossbred dairy cows. Milk samples were collected from 209 animals kept under smallholder production systems in Rungwe and Lushoto districts of Tanzania. The milk samples were analyzed for the content of components including fat, protein, casein, lactose, solids-not-fat (SNF), and the total solids (TS) through infrared spectroscopy using Milko-Scan FT1 analyzer (Foss Electric, Denmark). Hair samples for DNA analysis were collected from individual cows and breed composition determined using 150,000 single nucleotide polymorphism (SNP) markers. Cows were grouped into four genetic classes based on the proportion of exotic genes present: 25–49, 50–74, 75–84, and >84%, to mimic a backcross to indigenous zebu breed, F1, F2, and F3 crosses, respectively. The breed types were defined based on international commercial dairy breeds as follows: RG (Norwegian Red X Friesian, Norwegian Red X Guernsey, and Norwegian Red X Jersey crosses); RH (Norwegian Red X Holstein crosses); RZ (Norwegian Red X Zebu and Norwegian Red X N’Dama crosses); and ZR (Zebu X GIR, Zebu X Norwegian Red, and Zebu X Holstein crosses). Results obtained indicate low variation in milk composition traits between genetic groups and breed types. For all the milk traits except milk total protein and casein content, no significant differences (p < 0.05) were observed among genetic groups. Protein content was significantly (p < 0.05) higher for genetic group 75–84% at 3.4 ± 0.08% compared to 3.18 ± 0.07% for genetic group >84%. Casein content was significantly lower for genetic group >84% (2.98 ± 0.05%) compared to 3.18 ± 0.09 and 3.16 ± 0.06% for genetic group 25–49 and 75–84%, respectively (p < 0.05). There was no significant difference (p < 0.05) between breed types with respect to milk composition traits. These results suggest that selection of breed types to be used in smallholder systems need not pay much emphasis on milk quality differences as most admixed animals would have similar milk composition profiles. However, a larger sample size would be required to quantify any meaningful differences between groups.

Türkiye’de Üretilen Organik ve Konvansiyonel Sütlerin Bazı Fizikokimyasal Özellikleri ile Yağ Asitleri Kompozisyonu ve Antioksidan Kapasitesinin Belirlenmesi

In this study, determination of effect of production systems (conventional and organic) and time of milk collection on some physicochemical properties, fatty acids and antioxidant capacity of conventional and organic raw milk in produced Turkey were aimed. In this research, the milk samples was collected from nine conventional farms and nine organic farms at bimonthly years for one year. Fatty acid composition, antioxidant capacity, total phenolic matter, dry matter, fat, protein, ash, titratable acidity (lactic acid %) and pH values of organic and conventional milk were investigated. According to results of this research, the mean values of conventional and organic milk samples respectively for dry matter, fat, protein, ash, specific gravity, acidity and pH was determined as 12.06-11.97%, 3.67-3.50%, 3.33-3.34%, 0.67-0.66%, 1.0381-1.0381 g mL-1, 0.18-0.16% and 6.67-6.73, respectively. Conjugated linoliec acid proportions changed between 1.39% and 2.87% in organic milk, between 1.67% and 2.96% in conventional milk. Consequently, the farm production type did not have effects on the milk compassion (dry matter, fat, protein and ash), fatty acid composition, EC50 and total phenolic compounds. On the other hand, the significant variations in the fat, protein, fatty acid proportions, EC50, inhibition and total phenolic compound values were determined as regarding time of milk collection.

THE PREDICTED GENETIC PROGRESS IN DAIRY CATTLE POPULATIONS USING A VARIETY OF METHODS FOR EVALUATION AND SELECTION OF ANIMALS

THE PREDICTED GENETIC PROGRESS IN DAIRY CATTLE POPULATIONS USING A VARIETY OF METHODS FOR EVALUATION AND SELECTION OF ANIMALS

Inferring genetic parameters on latent variables underlying milk yield and quality, protein composition, curd firmness and cheese-making traits in dairy cattle

We studied the genetics of cheese-related latent variables (factors; Fs) for application in dairy cattle breeding. In total, 26 traits, recorded in 1264 Brown Swiss cows, were analyzed through multivariate factor analysis (MFA). Traits analyzed were descriptors of milk quality and yield (including protein fractions) and measures of coagulation, curd firmness (CF), cheese yields (%CY) and nutrient recoveries in the curd (REC). A total of 10 Fs (mutual orthogonal with a varimax rotation) were obtained. To assess the practical use of the Fs into breeding, we inferred their genetic parameters using single and bivariate animal models under a Bayesian framework. Heritability estimates (intra-herd) varied between 0.11 and 0.72 (F3: Yield and F7: κ-β-CN, respectively). The Fs underlined basic characteristics of the cheese-making process, milk components and udder health, while retaining 74% of the original variability. The first two Fs were indicators of the CY percentage (F1: %CY) and the CF process (F2: CFt), and presented similar heritability estimates: 0.268 and 0.295, respectively. The third factor was associated with the yield of milk and solids (F3: Yield) characterized by a low heritability (0.108) and the fourth with the cheese nitrogen (N) (F4: Cheese N) that conversely appeared to be characterized by a high heritability (0.618). Three Fs were associated with the proportion of the basic milk caseins on total milk protein (F5: as1-β-CN, F7: κ-β-CN, F8: as2-CN), also highly heritable (0.565, 0.723 and 0.397, respectively) and 1 factor with the phosphorylated form of the as1-CN (F9: as1-CN-Ph; 0.318). Moreover, 1 factor was linked to the whey protein α-LA (F10: α-LA; 0.147). An indicator factor of a cow’s udder health (F6: Udder health) was also obtained and showed a moderate heritability (0.204). Although the Fs were phenotypically uncorrelated, considerable additive genetic correlations existed among them, with highest values observed between F10: α-LA and F6: Udder health (−0.67) as well as between F9: as1-CN-Ph and F3: Yield (−0.60). Our results show the usefulness of MFA in dairy cattle breeding. The ability to replace a large number of variables with a few latent indicators of the same biological meaning marks MFA as a valuable tool for developing breeding strategies to improve cow’s cheese-related traits.

Effects of Oketani Breast Massage on Breast Discomfort, Breast Milk Amount, and Breast Milk Total Protein

Effects of Oketani Breast Massage on Breast Discomfort, Breast Milk Amount, and Breast Milk Total Protein

Genome-wide association studies to identify quantitative trait loci affecting milk production traits in water buffalo

Water buffalo is the second largest resource of milk supply around the world, and it is well known for its distinctive milk quality in terms of fat, protein, lactose, vitamin, and mineral contents. Understanding the genetic architecture of milk production traits is important for future improvement by the buffalo breeding industry. The advance of genome-wide association studies (GWAS) provides an opportunity to identify potential genetic variants affecting important economical traits. In the present study, GWAS was performed for 489 buffaloes with 1,424 lactation records using the 90K Affymetrix Buffalo SNP Array (Affymetrix/Thermo Fisher Scientific, Santa Clara, CA). Collectively, 4 candidate single nucleotide polymorphisms (SNP) in 2 genomic regions were found to associate with buffalo milk production traits. One region affecting milk fat and protein percentage was located on the equivalent of Bos taurus autosome (BTA)3, spanning 43.3 to 43.8 Mb, which harbored the most likely candidate genes MFSD14A, SLC35A3, and PALMD. The other region on the equivalent of BTA14 at 66.5 to 67.0 Mb contained candidate genes RGS22 and VPS13B and influenced buffalo total milk yield, fat yield, and protein yield. Interestingly, both of the regions were reported to have quantitative trait loci affecting milk performance in dairy cattle. Furthermore, we suggest that buffaloes with the C allele at AX-85148558 and AX-85073877 loci and the G allele at AX-85106096 locus can be selected to improve milk fat yield in this buffalo-breeding program. Meanwhile, the G allele at AX-85063131 locus can be used as the favorable allele for improving milk protein percentage. Genomic prediction showed that the reliability of genomic estimated breeding values (GEBV) of 6 milk production traits ranged from 0.06 to 0.22, and the correlation between estimated breeding values and GEBV ranged from 0.23 to 0.35. These findings provide useful information to understand the genetic basis of buffalo milk properties and may play a role in accelerating buffalo breeding programs using genomic approaches.

Variants in the 3' untranslated region of the ovine acetyl-coenzyme A acyltransferase 2 gene are associated with dairy traits and exhibit differential allelic expression.

The acetyl-CoA acyltransferase 2 (ACAA2) gene encodes an enzyme of the thiolase family that is involved in mitochondrial fatty acid elongation and degradation by catalyzing the last step of the respective β-oxidation pathway. The increased energy needs for gluconeogenesis and triglyceride synthesis during lactation are met primarily by increased fatty acid oxidation. Therefore, the ACAA2 enzyme plays an important role in the supply of energy and carbon substrates for lactation and may thus affect milk production traits. This study investigated the association of the ACAA2 gene with important sheep traits and the putative functional involvement of this gene in dairy traits. A single nucleotide substitution, a T to C transition located in the 3' untranslated region of the ACAA2 gene, was used in mixed model association analysis with milk yield, milk protein yield and percentage, milk fat yield and percentage, and litter size at birth. The single nucleotide polymorphism was significantly associated with total lactation production and milk protein percentage, with respective additive effects of 6.81 ± 2.95 kg and -0.05 ± 0.02%. Additionally, a significant dominance effect of 0.46 ± 0.21 kg was detected for milk fat yield. Homozygous TT and heterozygous CT animals exhibited higher milk yield compared with homozygous CC animals, whereas the latter exhibited increased milk protein percentage. Expression analysis from age-, lactation-, and parity-matched female sheep showed that mRNA expression of the ACAA2 gene from TT animals was 2.8- and 11.8-fold higher in liver and mammary gland, respectively. In addition, by developing an allelic expression imbalance assay, it was estimated that the T allele was expressed at an average of 18% more compared with the C allele in the udder of randomly selected ewes. We demonstrated for the first time that the variants in the 3' untranslated region of the ovine ACAA2 gene are differentially expressed in homozygous ewes of each allele and exhibit allelic expression imbalance within heterozygotes in a tissue-specific manner, supporting the existence of cis-regulatory DNA variation in the ovine ACAA2 gene. This is the first study reporting differential allelic imbalance expression of a candidate gene associated with milk production traits in dairy sheep.

Chemical Composition, Sensory Evaluation and Starter Activity in Cow, Soy, Peanut and Rice Milk

The aim of this work was to study the effect of mixing various levels of soy, peanut or rice milk with cow milk on the chemical composition, sensory attributes, starter activity and culture bacteria counts during fermentation. The results showed that no clear differences in titratable acidity, pH and redox potential (E h ) values were noticed between cow or soy milk while acidity and E h levels of peanut and rice milk were lower than those of cow milk. Total solids, fat, total protein and ash concentrations of cow milk were slightly higher than those of soy and rice milk. Peanut milk was richer in fat but poorer in ash than cow milk. Color, appearance, smell, taste, mouth feel, texture and body scores of cow milk were higher than those of soy, peanut or rice milk but rice milk gained the highest scores of color and appearance. Incorporation of cow milk with soy or peanut milk improved its sensory evaluation scores. Both acidity ratios and the development of acidity rates within fermentation were higher in cow milk than that of soy, peanut or rice milk. Culture bacteria not only were able to grow in soy, peanut or rice milk but also their numbers and viability were higher in them as compared with cow milk. Furthermore, incorporation of soy, peanut and rice milk with cow milk increased this influence.

Chemical Composition, Sensory Evaluation and Starter Activity in Cow, Soy, Peanut and Rice Milk

The aim of this work was to study the effect of mixing various levels of soy, peanut or rice milk with cow milk on the chemical composition, sensory attributes, starter activity and culture bacteria counts during fermentation. The results showed that no clear differences in titratable acidity, pH and redox potential (Eh) values were noticed between cow or soy milk while acidity and Eh levels of peanut and rice milk were lower than those of cow milk. Total solids, fat, total protein and ash concentrations of cow milk were slightly higher than those of soy and rice milk. Peanut milk was richer in fat but poorer in ash than cow milk. Color, appearance, smell, taste, mouth feel, texture and body scores of cow milk were higher than those of soy, peanut or rice milk but rice milk gained the highest scores of color and appearance. Incorporation of cow milk with soy or peanut milk improved its sensory evaluation scores. Both acidity ratios and the development of acidity rates within fermentation were higher in cow milk than that of soy, peanut or rice milk. Culture bacteria not only were able to grow in soy, peanut or rice milk but also their numbers and viability were higher in them as compared with cow milk. Furthermore, incorporation of soy, peanut and rice milk with cow milk increased this influence.

Effects of a diet containing dried grape pomace on blood metabolites and milk composition of dairy cows.

The effect of a diet containing 15% grape pomace (GP) on the general health status and milk quality of dairy cows was assessed by plasma biochemistry and total polyphenol (TP) content, milk polyphenols, milk composition and milk protein fractions. Among the polyphenols measured by liquid chromatography-mass spectroscopy in GP, in feed containing GP (GP+) or not containing GP (GP-), gallic acid and epicatechin were present in the highest concentrations (67.58 and 19.23 µg mL-1 , respectively). Higher amounts of TP were also detected in the blood plasma of GP+ cows (114.06 and 83.93 mg GAE L-1 , respectively) but not in their milk (233.17 and 245.75 mg GAE L-1 , respectively). Also a significant increase was found for lactose and β-lactoglobulin, although there was no effect on α-lactalbumin, albumin, secretory components and caseins. Inclusion of 15% GP in the diets of dairy cows is beneficial for overall normal blood constituent metabolism and helps to maintain cow health. The milk of cows fed with a GP diet preserves the normal levels of fat, protein and caseins, and has increased levels of components that make this milk a versatile ingredient material for the food industry (e.g. model whey powders, stability of lactose-rich powders). © 2016 Society of Chemical Industry.

Egg and Milk Proteins as Hidden Allergens in Food: 5-Year (2010 to 2014) Results of Food Allergen Monitoring in Piedmont, Italy

Cow's milk and egg allergies are two of the most common food allergies. Manufacturers of food products containing milk or eggs or their derivatives as an ingredient are required by European Union regulations to list their presence on the ingredient label. Under European Union legislation, member states are mandated to carry out food safety monitoring programs to verify compliance with food labeling requirements. Through the Regional Integrated Plan for Food Safety, the Piedmont (Italy) regional authority carries out an annual program to determine the presence of undeclared allergens in foods. In the 5-year period from 2010 to 2014, a total of 1,566 food samples were analyzed for the presence of hidden egg and milk proteins. The average positive percentage was 2.8% (3.6% egg and 2% milk proteins). Comparison between the allergen concentration and the published eliciting dose (ED) for egg proteins (0.03 mg) and for total milk proteins (0.1 mg) indicated a high risk of allergen exposure for sensitized consumers. The calculated exposure was up to 135× (for milk) the ED01 reported in the literature. Food manufacturers will need to improve their allergen control programs to reduce allergen exposure and risk.

Advances in ultra-high performance liquid chromatography coupled to tandem mass spectrometry for sensitive detection of several food allergens in complex and processed foodstuffs

Sensitive detection of food allergens is affected by food processing and foodstuff complexity. It is therefore a challenge to detect cross-contamination in food production that could endanger an allergic customer's life. Here we used ultra-high performance liquid chromatography coupled to tandem mass spectrometry for simultaneous detection of traces of milk (casein, whey protein), egg (yolk, white), soybean, and peanut allergens in different complex and/or heat-processed foodstuffs. The method is based on a single protocol (extraction, trypsin digestion, and purification) applicable to the different tested foodstuffs: chocolate, ice cream, tomato sauce, and processed cookies. The determined limits of quantitation, expressed in total milk, egg, peanut, or soy proteins (and not soluble proteins) per kilogram of food, are: 0.5mg/kg for milk (detection of caseins), 5mg/kg for milk (detection of whey), 2.5mg/kg for peanut, 5mg/kg for soy, 3.4mg/kg for egg (detection of egg white), and 30.8mg/kg for egg (detection of egg yolk). The main advantage is the ability of the method to detect four major food allergens simultaneously in processed and complex matrices with very high sensitivity and specificity.

Compared with Raw Bovine Meat, Boiling but Not Grilling, Barbecuing, or Roasting Decreases Protein Digestibility without Any Major Consequences for Intestinal Mucosa in Rats, although the Daily Ingestion of Bovine Meat Induces Histologic Modifications in the Colon

Background: Cooking may impair meat protein digestibility. When undigested proteins are fermented by the colon microbiota, they can generate compounds that potentially are harmful to the mucosa.Objectives: This study addressed the effects of typical cooking processes and the amount of bovine meat intake on the quantity of undigested proteins entering the colon, as well as their effects on the intestinal mucosa.Methods: Male Wistar rats (n = 88) aged 8 wk were fed 11 different diets containing protein as 20% of energy. In 10 diets, bovine meat proteins represented 5% [low-meat diet (LMD)] or 15% [high-meat diet (HMD)] of energy, with the rest as total milk proteins. Meat was raw or cooked according to 4 processes (boiled, barbecued, grilled, or roasted). A meat-free diet contained only milk proteins. After 3 wk, rats ingested a 15N-labeled meat meal and were killed 6 h later after receiving a 13C-valine injection. Meat protein digestibility was determined from 15N enrichments in intestinal contents. Cecal short- and branched-chain fatty acids and hydrogen sulfide were measured. Intestinal tissues were used for the assessment of protein synthesis rates, inflammation, and histopathology.Results: Meat protein digestibility was lower in rats fed boiled meat (94.5% ± 0.281%) than in the other 4 groups (97.5% ± 0.0581%, P < 0.001). Cecal and colonic bacterial metabolites, inflammation indicators, and protein synthesis rates were not affected by cooking processes. The meat protein amount had a significant effect on cecal protein synthesis rates (LMD > HMD) and on myeloperoxidase activity in the proximal colon (HMD > LMD), but not on other outcomes. The ingestion of bovine meat, whatever the cooking process and the intake amount, resulted in discrete histologic modifications of the colon (epithelium abrasion, excessive mucus secretion, and inflammation).Conclusions: Boiling bovine meat at a high temperature (100°C) for a long time (3 h) moderately lowered protein digestibility compared with raw meat and other cooking processes, but did not affect cecal bacterial metabolites related to protein fermentation. The daily ingestion of raw or cooked bovine meat had no marked effect on intestinal tissues, despite some slight histologic modifications on distal colon.

Simultaneous quantification of α-lactalbumin and β-casein in human milk using ultra-performance liquid chromatography with tandem mass spectrometry based on their signature peptides and winged isotope internal standards

In recent years, there is an increasing need to measure the concentration of individual proteins in human milk, instead of total human milk proteins. Due to lack of human milk protein standards, there are only few quantification methods established. The objective of the present work was to develop a simple and rapid quantification method for simultaneous determination of α-lactalbumin and β-casein in human milk using signature peptides according to a modified quantitative proteomics strategy. The internal standards containing the signature peptide sequences were synthesized with isotope-labeled amino acids. The purity of synthesized peptides as standards was determined by amino acid analysis method and area normalization method. The contents of α-lactalbumin and β-casein in human milk were measured according to the equimolar relationship between the two proteins and their corresponding signature peptides. The method validation results showed a satisfied linearity (R2>0.99) and recoveries (97.2–102.5% for α-lactalbumin and 99.5–100.3% for β-casein). The limit of quantification for α-lactalbumin and β-casein was 8.0mg/100g and 1.2mg/100g, respectively. CVs for α-lactalbumin and β-casein in human milk were 5.2% and 3.0%. The contents of α-lactalbumin and β-casein in 147 human milk samples were successfully determined by the established method and their contents were 205.5–578.2mg/100g and 116.4–467.4mg/100g at different lactation stages. The developed method allows simultaneously determination of α-lactalbumin and β-casein in human milk. The quantitative strategy based on signature peptide should be applicable to other endogenous proteins in breast milk and other body fluids.