Total Milk Protein Content Research Articles

Development and single laboratory validation of a targeted liquid chromatography-triple quadrupole mass spectrometry-based method for the determination of insulin like growth factor-1 in different types of milk samples.

A simple and reliable targeted liquid chromatography-electrospray-tandem mass spectrometry (LC-MS/MS) method was developed and validated through the selection of two biomarker peptides for the identification and determination of bovine insulin like growth factor-1 (IGF-1) in milk samples. Two urea-based sample extraction procedures were tested. The validation results provided detection limits at the 1-5ng IGF-1/mL level as a function of the milk matrix, precision ranged from 3 to 8% and the method accuracy in the different milk matrices was assured. Finally, IGF-1 was measured in milk samples obtained by treatment with eleven different technological processes: IGF-1 concentrations were spread over a wide range from 11.2±0.3ng/mL to 346±8ng/mL with a median of 57.0±0.2ng/mL. The highest amount of IGF-1 was found in fresh whole milk samples and no significant correlation was found between the total milk protein content and the IGF-1 concentration level.

Determination of hidden milk allergens in meat-based foodstuffs by liquid chromatography coupled to electrospray ionization and high-resolution tandem mass spectrometry

The issue of deliberate addition of antigenic proteins to foodstuffs for ameliorating bulk properties or the unintentional cross-contamination poses potentially life-threatening health problems to susceptible subjects. Even the intake of food products declaring the absence of allergens on their labels could lead to severe risks for sensitive consumers due to the presence of the so-called “hidden allergens”. Thus, the quantification of low-abundant proteins as putative allergens has become mandatory. Herein, we present a sensitive and selective analytical method based on reversed-phase liquid chromatography coupled to electrospray ionization and hybrid orbitrap high-resolution mass spectrometry (RPLC-ESI-HRMS) and tandem MS, identifying, and quantifying allergenic milk proteins in complex meat-based foodstuffs from direct measurement of tryptic peptides. Two signature peptides of α-S1-casein and β-lactoglobulin, i.e., FFVAPFPEVFGK (m/z 692.8682+) and TPEVDDEALEK (m/z 623.2952+), respectively, were chosen to search for hidden allergens in meat-based samples such as cooked meat, sausages, and sterilised pâté. The marker peptides were identified and were exploited for method validation including recovery, matrix effect, precision, linearity, method variation, limit of detection, and limit of quantification. The undeclared occurrence of milk allergens as total milk protein content (TCMP) was verified in commercial meat products; beef and pork pâté were meat-based products which require a major alert because up to 22 μgTCMP/g of matrix i.e. more than 10 times the action level was determined.

Concentration of osteopontin in human milk and associated factors in Chinese populations from 2011 to 2013

To analyze content of human milk osteopontin(OPN) and to explore associated factors in Chinese populations. The samples and data were extracted from the database for human milk composition in China between 2011 and 2013. A sub-sample of 459 mothers was randomly selected after stratification according to lactation stage, and human milk OPN concentrations were determined by ultra performance liquid chromatography-tandem mass spectrometer(UPLC/MS). The average OPN concentration(M(P25, P75)) in breast milk was 44.0(30.1-72.0) mg/L within 0-330 days postpartum. OPN concentrations were independent of lactation stage, which were 45.6(31.8, 80.7) mg/L in colostrum, 41.3(29.2, 70.0) mg/L in transitional milk and 46.9(30.2, 71.9) mg/L in mature milk, corresponding to 0.40%、0.42% and 0.65% of the total milk protein content(OPN/protein%). The percentage of OPN to total protein in milk showed an increasing trend with lactation progression(r=0.21, P<0.001). Multivariate analysis showed that sleep quality of mothers within one week prior to milk collection was correlated with the breast milk OPN level(P=0.04). The OPN level in breast milk from mothers with good sleep quality was significantly higher than that from mothers with poor sleep quality(46.5 mg/L vs.34.7 mg/L). The median level of milk OPN concentration in mothers from Yunnan was higher than mothers from Beijing(50.5 mg/L vs.36.1 mg/L, P=0.03). Maternal age, mode of delivery, prepregnancy body mass index, weight gain during pregnancy, passive smoking and outdoor activities 24 hours prior to milk collection were not correlated with milk OPN concentration. OPN concentration in breast milk was not related to preterm birth. Also, milk OPN concentration did not correlate with diarrhoea, respiratory disease, or allergic disease in infants during two weeks before milk collection. The concentration of OPN in breast milk of Chinese woman may be similar among different lactation stages. Geographic region and sleep quality of mothers may be related to the milk OPN concentration.

A reference method for determining the total allergenic protein content in a processed food: the case of milk in cookies as proof of concept

The establishment of a reference method for the determination of the allergen protein content in a processed food material has been explored. An analytical approach was developed to enable the comparability of food allergen measurement results expressed in a decision-relevant manner. A proof of concept is here presented, resulting in quantity values for the common measurand, namely ‘mass of total allergen protein per mass of food’. The quantities are determined with SI traceability to enable the comparability of reported results. A method for the quantification of total milk protein content in an incurred baked food at a concentration level clinically relevant is presented. The strategy on how to obtain the final analytical result is outlined. Challenges associated with this method are discussed, in particular the optimal extraction of the marker proteins, the complete digestion and release of the peptides in an equimolar fashion, the use of conversion factors to translate the amount of measured proteins into total milk protein and the estimation of the uncertainty contributions as well as of the combined uncertainty of the final result. The implementation of such a reference method for the determination of the total allergen content in a processed food is an important step, which will provide comparable measurement data of relevance to risk assessors.Graphical abstract

Genes encoding equine β-lactoglobulin (LGB1 and LGB2): Polymorphism, expression, and impact on milk composition.

β-lactoglobulin is one of the most abundant milk whey proteins in many mammal species, including the domestic horse. The aim of this study was to screen for polymorphism in the equine LGB1 and LGB2 gene sequences (all exons, introns, and 5’-flanking region) and to assess potential relationship of particular genotypes with gene expression levels (measured in milk somatic cells) and milk composition traits (protein, fat, lactose, and total β-lactoglobulin content). Direct DNA sequencing analysis was performed for twelve horse breeds: Polish Primitive Horse (PPH), Polish Coldblood Horse (PCH), Polish Warmblood Horse (PWH), Silesian, Hucul, Fjording, Haflinger, Shetland Pony, Welsh Pony, Arabian, Thoroughbred, and Percheron—and revealed the presence of 83 polymorphic sites (47 and 36 for LGB1 and LGB2 genes, respectively), including eight that were previously unknown. Association analysis of the selected polymorphisms, gene expression, and milk composition traits (conducted for the PPH, PCH, and PWH breeds) showed several statistically significant relationships; for example, the two linked LGB1 SNPs (rs1143515669 and rs1144647991) were associated with total milk protein content (p < 0.01). Our study also confirmed that horse breed had significant impact on both gene transcript levels (p < 0.01) and on milk LGB content (p < 0.05), whereas an influence of lactation period was seen only for gene relative mRNA abundances (p < 0.01).

5\u2019-flanking variants of equine casein genes (CSN1S1, CSN1S2, CSN2, CSN3) and their relationship with gene expression and milk composition

Genes encoding casein proteins are important candidates for milk composition traits in mammals. In the case of the domestic horse, our knowledge of casein genes is limited mainly to coding sequence variants. This study involved screening for polymorphism in 5’-flanking regions of four genes encoding equine caseins (CSN1S1, CSN1S2, CSN2, and CSN3) and making a preliminary assessment of their effect on the gene expression (on the mRNA and protein levels) and milk composition traits in selected horse breeds. Altogether, 23 polymorphisms (21 described previously SNPs and two novel InDels) were found in the studied sequences, the majority of which are common in various horse breeds. Statistical analysis revealed that some are putatively associated with gene expression or milk composition — for example, the c.-2047_-2048insAT polymorphism (CSN1S1) turns out to be related to the total milk protein content in Polish Primitive Horse (p < 0.05), whereas c.-2105C>G SNP (CSN2) is related to beta-casein relative mRNA level and milk lactose concentration in the Polish Coldblood Horse breed (p < 0.05). We have also found significant effects of horse breed and lactation time-point on gene expression and mare’s milk composition. Our study indicates that the 5’-regulatory regions of genes encoding casein proteins are interesting targets for functional studies of their expression and the composition traits of mare’s milk.

Genetic polymorphism in β-lactoglobulin gene of some sheep breeds in the Kingdom of Saudi Arabia (KSA) and its influence on milk composition

β-Lactoglobulin (β-Lg) is one of the most important proteins in mammals’ milk. It plays a crucial role in milk quality. The polymorphism of β-Lg gene can be used as a marker system. To analyze the genotype distribution of β-Lg gene in some sheep breeds reared in Taif region of Saudi Arabia and its influence on milk composition, sixty (60)animals belonging to four sheep breeds named Noami, Sawakni, Harry and Nagdi, were utilized. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) test was performed and genetic polymorphism was detected by the digestion of a 452 bp PCR fragment of exon II of β-Lg gene with the endonuclease RsaI. The results revealed that Noami and Sawakni breeds belong toβ-Lg-A genotype while Harry and Nagdi belong to genotype β-Lg-B. Sequence analysis of a 340 bp fragment in the promoter region of β-Lg showed polymorphismamong the examined breeds. Analysis of milk composition in the different breeds indicated that the total protein content of milk was the highest in Noami breed followed by Sawakni, Nagdi and Harry. Concerning milk total fat and total solids contents, Harry breed was the highest, while no significant difference was evident among different breeds in lactose or non-fat solid contents. These results indicate the feasibility of PCR-RFLP test for differentiating sheep breeds and the existence of a significant relationship between β-Lg-A genotype and total milk protein content while no clear association between β-Lg genotypes and other milk content was proved. Key words: Sheep, β-lactoglobulin, genetic polymorphism, PCR-RFLP, milk composition.

포유모돈에 있어 복합효소제 함유 식물성 단백질 공급원이 생산성과 돈유성상에 미치는 영향

ABSTRACT A total of thirty sows (Landrace × Yorkshire) were used to determine the effects of plant protein source containing multienzyme on performance, nutrients digestibility and milk characteristics. A feeding trial was conducted for 21 days from parturition to weaning. Experimental diets were supplied for 1 week before the parturition day and throughout the experimental period. Dietary treatments included: 1) Control (CON; basal diet), 2) CGLT (included corn gluten) and 3) FSPM(included fermented soy protein containing multienzyme). Through the entire experimental period, backfat loss and return-to-estrus intervals were not affected by the treatments (P>0.05). Nitrogen digestibility was increased significantly (P<0.05) in FSPM treatment compared to CON treatment. Blood urea nitrogen (BUN) concentration was increased significantly (P<0.05) in FSPM treatment compared to CON treatment. At the initial period, total protein content of milk was higher significantly (P<0.05) in FSPM treatment compared to CGLT treatment and at the final period, total fat content of milk was higher significantly (P<0.05) in FM treatment compared to CON treatment. Rectal temperature showed similar tendency of change among treatments. The final piglet body weight, weight gain and ADG were higher significantly in FSPM treatment compared to CON treatment. On diarrhea rate in piglet, just one piglet occurred in CGLT treatement. In conclusion, 2.5% dietary plant protein source containing multienzyme suppelmentation improved N digestibility, BUN concentration, fat and protein contents in milk and weight gain in piglet.(

포유모돈 사료에 코코넛 분말지방 첨가가 모돈의 생산성 및 모유성상에 미치는 효과

본 시험은 복합효소제 함유 식물성단백질공급원이 포유모돈의 생산성, 영양소 소화율 및 돈유성상에 미치는 효과를 알아보고자 실시하였다. 모돈은 건강상태 및 산차를 고려하여 3처리, 처리당 10두씩 30두를 공시하였다. 사료는 1차로 분만 일주일 전부터 급여하고 2차로 분만 후부터 21일령 이유시까지 급여하였다. 시험설계는 1)CON(basal diet), 2)CGLT(included corn gluten) 및 3) FSPM(included fermented soy protein containing multienzyme)로 하였으며, 분만후 자돈은 모돈에 복당 10마리씩 균일하게 배치하였다. 포유모돈의 영양소 소화율에서는 질소 소화율에서 FSPM 처리구가 CON 처리구와 비교하여 유의적으로 높았다(P<0.05). 포유모돈의 혈중 요소태질소 함량에서는 종료시에 FSPM 처리구가 CGLT 처리구보다 유의적으로 높았으며(P<0.05) 변화량에 있어서는 FSPM 처리구가 CON 및 CGLT 처리구보다 유의적으로 높았다(P<0.05). 포유모돈의 모유내 지방 함량에서는 종료시에 FSPM 처리구가 CON 처리구보다 유의적으로 높았으며(P<0.05), 모유내 단백질 함량에서는 개시시에 FSPM 처리구가 CGLT 처리구와 비교하여 유의적으로 높았다(P<0.05). 포유모돈의 직장온도 변화에 있어서는 세 처리 모두 유사한 변화를 보였다. 포유자돈의 성장률에서는 종료시체중, 증체량 및 일당증체량에서 FSPM 처리구가 CON 처리구와 비교하여 유의적으로 높았으며(P<0.05) CGLT 처리구는 CON 처리구보다 높았으며 FSPM 처리구보다 낮았다.포유자돈에서의 설사발생에 있어서는 0~5일째에 CGLT 처리구에서 한 마리가 발생하였다. 본 시험결과 사료내 0.25% 복합효소제 함유 식물성단백질공급원의 급여는 포유모돈의 질소 소화율과 혈중 요소태질소 함량, 모유내 단백질과 지방 함량을 향상시켰으며 포유자돈의 증체량 또한 향상시켰다. A total of thirty sows(Landrace×Yorkshire) were used to determine the effects of plant protein source containing multienzyme on performance, nutrients digestibility and milk characteristics. A feeding trial was conducted for 21 days from parturition to weaning. Experimental diets were supplied for 1 week before the parturition day and throughout the experimental period. Dietary treatments included: 1) Control(CON; basal diet), 2) CGLT(included corn gluten) and 3) FSPM(included fermented soy protein containing multienzyme). Through the entire experimental period, backfat loss and return-to-estrus intervals were not affected by the treatments(P>0.05). Nitrogen digestibility was increased significantly(P<0.05) in FSPM treatment compared to CON treatment. Blood urea nitrogen(BUN) concentration was increased significantly (P<0.05) in FSPM treatment compared to CON treatment. At the initial period, total protein content of milk was higher significantly(P<0.05) in FSPM treatment compared to CGLT treatment and at the final period, total fat content of milk was higher significantly(P<0.05) in FM treatment compared to CON treatment. Rectal temperature showed similar tendency of change among treatments. The final piglet body weight, weight gain and ADG were higher significantly in FSPM treatment compared to CON treatment. On diarrhea rate in piglet, just one piglet occurred in CGLT treatement. In conclusion, 2.5% dietary plant protein source containing multienzyme suppelmentation improved N digestibility, BUN concentration, fat and protein contents in milk and weight gain in piglet.

The influence of texture improver type and its addition level on rheological properties of goat’s milk yogurt

Set yogurt from goat?s milk with addition of two texture improvers DSE 6693 and DSE 6694 from NZMP New Zealand, added in the amount of 1, 2and 3% and control yogurt without addition of texture improvers were produced. In yogurt determined sensory quality, pH, apparent viscosity and rheological properties. Flow curves obtained for produced yogurts were described by three rheological models: Ostwald de Waele, Herschel-Bulkley (H-B) and Casson. Addition of texture improver caused an increase in total solids and total protein content of milk for yogurt. Yogurt with texture improvers gave better sensory quality than control yogurt. The yogurt with 1% addition of DSE 6693 and with 2% addition of DSE 6694 had the best sensory quality. The increase in addition level of texture improvers caused an increase in apparent viscosity, consistency coefficient K, yield stress (except H-B model) and deviation from Newtonian flow (decrease of flow index n). Effect of type of improver was rather negligible, whereas its level addition considerably influenced the evaluated rheological parameters.

Kappa-casein gene study with molecular markers in female buffaloes (Bubalus bubalis)

Caseins comprise make up about 80% of the total protein content of milk and present polymorphism with changes in the amino acid sequence. Within this abundance of proteins, kappa-casein is noteworthy, since it has been associated with differences in milk yield, composition and processing. The objective of this study was to observe the existence of polymorphism in the kappa-casein gene in female buffaloes. For this purpose, blood samples from 115 female buffaloes, collected with vacutainer by needle punctionure of the jugular vein, were used. for genomic DNA extraction was done from blood samples. The PCR-RFLP and SSCP techniques demonstrated that the studied animals were monomorphic for the kappa-casein gene. Only allele B was observed in these animals, which was present in homozygosis. Therefore, it was not possible to quantify the gene action on milk yield and its constituents. The monomorphism observed in the population studied would allow the development of a method to identify mixtures of cow and buffalo milk in mozzarella cheese production, especially because, in cattle, the kappa-casein gene is polymorphic.

Direct biosensor immunoassays for the detection of nonmilk proteins in milk powder.

The low prices of some nonmilk proteins make them attractive as potential adulterants in dairy products. An optical biosensor (BIACORE 3000) was used to develop a direct and combined biosensor immunoassay (BIA) for the simultaneous detection of soy, pea, and soluble wheat proteins in milk powders. Affinity-purified polyclonal antibodies raised against the three protein sources were immobilized in different flow channels (Fcs) on the biosensor chip (CM5). Dissolved milk powders were injected (20 microL injections at 20 microL min(-1)) through the serially connected Fcs, and the antibody-bound plant proteins were detected directly. The total run time between samples, including a regeneration step with 5 microL of 10 mM HCl, was 5 min. The limits of detection in milk powder were below 0.1% of plant protein in the total milk protein content. The antibodies also recognized some proteins from other plant sources, which made this BIA even more suitable as a broad screening assay for nonmilk proteins.

Human proteome enhancement: high-recovery method and improved two-dimensional map of colostral fat globule membrane proteins.

The human milk fat globule membrane protein composition is still largely unknown, although it counts for 2-4% of the total milk protein content and contains several important biologically active components. The aim of this work was to create a two-dimensional electrophoresis (2-DE) map of the human milk fat globule membrane proteins, both integral and membrane-associated, and to identify and characterize as many protein components as possible. A new protocol for the solubilization and extraction of the human milk fat globule membrane proteins with a double extraction procedure is presented, and the results compared with the extraction methods reported in the literature. The proteins were separated, in the first dimension, by isoelectric focusing (IEF) in the pH range 3-10 on strips of 13 cm length and, in the second dimension, by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 11.5% T homogeneous gels. A reproducible 2-DE map of integral and membrane-associated proteins was obtained and the first 23 spots, representing the major components, were identified by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometric analysis and/or by amino acid sequencing.

An ultraviolet spectrophotometric method to determine milk protein content in alkaline medium

An ultraviolet (UV) spectrophotometric method to estimate the total milk protein content is proposed. The method is based on the capacity of strong alkaline solutions to change the spectrum of the amino acid tyrosine to higher wavelength values in the UV region. In the range between 248 and 256 nm the absorbance is a linear function of the wavelength and the slope coefficient is directly proportional to the protein concentration. Thus, the measurements of absorbance at two wavelengths were used to estimate the protein content. For samples where the ratio of lipid to protein was ≤0.05, the following general expression can be used: [Protein] (g litre −1) = (A 248−A 256) ( A ̊ 248− A ̊ 256) where A 248 and A 256 are the absorbance values of the sample at 248 and 256 nm and A ̊ 248 and A ̊ 256 are the values for the standard protein at the same wavelengths. Due to the interference of the fat globules, when the ratio lipid/protein is ≥0.05, it is necessary to introduce a correction coefficient, as follows: [Protein]={ (A 248−A 256) ( A ̊ 248− A ̊ 256 }·{ 1 [1+a·exp(b( A 256 A 248 ))] } where a = 1.76 × 10 5 and b = 12.29

Relation of Milk Fat-Depressing Rations and Subclinical Mastitis to Milk Proteins

Two continuous feeding trials involving 28 cows were used to study the influence of a pelleted, high-grain ration, of dietary supplementation with unsaturated oils (safflower and cod liver oil), and of subclinical mastitis under these conditions, on milk total proteins, casein, whey proteins, solids-notfat (SNF), and fat. Milk samples from 512 udder quarters were analyzed. Total milk protein and casein contents were elevated by each nutritional regime. Whey protein and SNF contents increased while feeding the high-grain ration, but both showed only minor changes during the oil supplementation. A net gain in milk protein synthesis of 15% was obtained during the high-grain regime, but protein yields during the oil regime were lower because of reduced milk production. Milk fat percentages and fat yields were depressed by each regime.Ratios of casein content to total protein content of milk were significantly related to Wisconsin Mastitis Test (WMT) scores (r = —0.45). Intraclass correlation coefficients within udder quarters for WMT scores on whey protein per cent, SNF per cent, casein yield, milk yield, total protein yield, fat per cent, casein per cent, whey protein yield, and total protein per cent were, in decreasing order, 0.45, —0.41, —0.34, —0.33, —0.29, —0.19, —0.18, —0.14, and 0.08, respectively.