Major Milk Proteins Research Articles

A hypomorphic mutation in the mouse Csn1s1 gene generated by CRISPR/Cas9 pronuclear microinjection.

Caseins are major milk proteins that have an evolutionarily conserved role in nutrition. Sequence variations in the casein genes affect milk composition in livestock species. Regulatory elements of the casein genes could be used to direct the expression of desired transgenes into the milk of transgenic animals. Dozens of casein alleles have been identified for goats, cows, sheep, camels and horses, and these sequence variants are associated with altered gene expression and milk protein content. Most of the known mutations affecting casein genes’ expression are located in the promoter and 3’-untranslated regions. We performed pronuclear microinjections with Cas9 mRNA and sgRNA against the first coding exon of the mouse Csn1s1 gene to introduce random mutations in the α-casein (Csn1s1) signal peptide sequence at the beginning of the mouse gene. Sanger sequencing of the founder mice identified 40 mutations. As expected, mutations clustered around the sgRNA cut site (3 bp from PAM). Most of the mutations represented small deletions (1–10 bp), but we detected several larger deletions as well (100–300 bp). Functionally most mutations led to gene knockout due to a frameshift or a start codon loss. Some of the mutations represented in-frame indels in the first coding exon. Of these, we describe a novel hypomorphic Csn1s1 (Csn1s1c.4-5insTCC) allele. We measured Csn1s1 protein levels and confirmed that the mutation has a negative effect on milk composition, which shows a 50 % reduction in gene expression and a 40–80 % decrease in Csn1s1 protein amount, compared to the wild-type allele. We assumed that mutation affected transcript stability or splicing by an unknown mechanism. This mutation can potentially serve as a genetic marker for low Csn1s1 expression.

Longitudinal Changes in the Concentration of Major Human Milk Proteins in the First Six Months of Lactation and Their Effects on Infant Growth.

Our knowledge related to human milk proteins is still limited. The present study determined the changes in multiple human milk proteins during the first six months of lactation, investigated the influencing factors of milk proteins, and explored the impact of milk proteins on infant growth. A total of 105 lactating women and their full-term infants from China were prospectively surveyed in this research. Milk samples were collected at 1–5 days, 8–14 days, 1 month, and 6 months postpartum. Concentrations of total protein and α-lactalbumin were measured in all milk samples, and concentrations of lactoferrin, osteopontin, total casein, β-casein, αs−1 casein, and κ-casein were measured in milk from 51 individuals using ultra performance liquid chromatography coupled with mass spectrometry. The concentration of measured proteins in the milk decreased during the first six months of postpartum (p-trend < 0.001). Maternal age, mode of delivery, maternal education, and income impacted the longitudinal changes in milk proteins (p-interaction < 0.05). Concentrations of αs−1 casein in milk were inversely associated with the weight-for-age Z-scores of the infants (1 m: r −0.29, p 0.038; 6 m: r −0.33, p 0.020). In conclusion, the concentration of proteins in milk decreased over the first six months postpartum, potentially influenced by maternal demographic and delivery factors. Milk protein composition may influence infant weights.

Gastric Digestion of Milk Proteins in Adult and Elderly: Effect of High-Pressure Processing.

Reduced physiological capability of the human gastrointestinal tract with increasing age has recently attracted considerable attention to the potential of novel technologies to modify food digestion. Thus, the aim of this study was to investigate gastric digestion of milk proteins after application of high-pressure processing (HPP) at 400 MPa 15 min, 600 MPa 5 min and 600 MPa 15 min using two static in vitro models of adults (INFOGEST) and the elderly in comparison to a fresh untreated raw milk. Peptides distribution classified based on the number of amino acids (AA) (<10, 11–15, 16–20, 21–30, >30 AA) were investigated after 0, 5, 10 and 30 min of digestion using LC–MS and multivariate data analysis. Our results show significantly less efficient protein digestion of all investigated milks in the elderly model indicated by higher percentages of longer peptides during digestion, except for the HPP milk 400 MPa 15 min, which indicated an improved and comparable digestion in the elderly as in the adult model. Furthermore, increasing the pressurization time at 600 MPa did not have a significant effect on the peptides profile during the digestion. More efficient digestion of whey proteins in HPP milks, with the majority of peptides in the 16–20 AA range, compared to fresh milk was also noticed. According to the findings of this study, HPP at 400 MPa 15 min showed the most efficient digestion of major milk proteins and thus may be considered a suitable process to improve bioaccessibility of milk proteins, especially in products intended for the elderly.

Influence of the kappa casein genotype on the technological properties of cow milk of Simmental and Alatau breeds.

The main objective of dairy farming is to obtain highly productive animals that produce milk with high protein content and have good technological properties. Currently, with the development of molecular genetics and molecular biology, it is possible to identify genes directly or indirectly associated with the milk production of animals. The identification of variants of such genes that are preferable from the standpoint of selection will allow, apart from the conventional selection of animals, to perform selection at the level of deoxyribonucleic acid technologies, i.e. according to genotype. The paper includes the study of milk productivity indicators and physicochemical quality indicators of milk from cows of Simmental and Alatau breeds by loci of the kappa casein gene, and also determines the cheese suitability and technological properties of milk of cows of Simmental and Alatau breeds with different genotypic affiliations for kappa casein for the production of semi-hard cheeses. The determination of polymorphism by the kappa casein gene was carried out with the use of the polymerase chain reaction method for genotyping of the major milk proteins of kappa casein gene. In a comparative aspect, the interrelation of genotypes with milk productivity was studied, the frequencies of allelic polymorphism of cows of Simmental and Alatau breeds in the Republic of Kazakhstan were identified, the nature of milk productivity was determined for each genotype. Scientifically based data on the assessment of biological value, technological properties, cheese suitability of milk of cows of Simmental and Alatau breeds are obtained.

The ultrafiltration molecular weight cut-off has a limited effect on the concentration and protein profile during preparation of human milk protein concentrates

Optimizing protein intake for very low birth weight (<1,500 g) infants is fundamental to prevent faltering postnatal growth with the potential association of impaired neurodevelopment. The protein content of human milk is not sufficient to support the growth of very low birth weight infants. To meet their elevated protein requirements, human milk is currently fortified using typically bovine milk-based protein isolates (>85% on a dry basis). However, these products have several limitations for use in this vulnerable population. To overcome the shortcomings of bovine milk-based protein supplement, a human milk protein concentrate (HMPC) was developed. In preliminary attempts using 10 kDa ultrafiltration (UF) membranes, it was not possible to reach the protein content of commercial protein isolates, presumably due to the retention of human milk oligosaccharides (HMO). Consequently, it was hypothesized that the use of a UF membrane with a higher molecular weight cut-off (50 kDa rather than 10 kDa) could improve the transmission of carbohydrates, including HMO, in the permeate, thus increasing the protein purity of the subsequent HMPC. The results showed that permeate fluxes during the concentration step were similar to either UF molecular weight cut-off, but the 50-kDa membrane had a higher permeate flux during the diafiltration sequence. However, it was not sufficient to increase the protein purity of the human milk retentate, as both membranes generated HMPC with similar protein contents of 48.8% (10 kDa) and 50% (50 kDa) on a dry basis. This result was related to the high retention of HMO, mainly during the concentration step, although the diafiltration step was efficient to decrease their content in the HMPC. As the major bioactive proteins (lactoferrin, lysozyme, bile salt stimulated lipase, and α1-antitrypsin) in human milk were detected in both HMPC, the 50-kDa membrane seems the most appropriate to the preparation of HMPC in terms of permeation flux values. However, improving the separation of HMO from proteins is essential to increase the protein purity of HMPC.

Generation of Lactose- and Protease-Positive Probiotic Lacticaseibacillus rhamnosus GG by Conjugation with Lactococcus lactis NCDO 712.

Lacticaseibacillus rhamnosus GG (LGG) is the most studied probiotic bacterium in the world. It is used as a probiotic supplement in many foods, including various dairy products. However, LGG grows poorly in milk, as it neither metabolizes the main milk carbohydrate lactose nor degrades the major milk protein casein effectively. In this study, we made L. rhamnosus GG lactose and protease positive by conjugation with the dairy Lactococcus lactis strain NCDO 712 carrying the lactose-protease plasmid pLP712. A lactose-hydrolyzing transconjugant colony was obtained on agar containing lactose as the sole source of carbohydrates. By microscopic analysis and PCR with LGG- and pLP712-specific primers, the transconjugant was confirmed to have originated from LGG and to carry the plasmid pLP712. The transconjugant was named L. rhamnosus LAB49. The isolation of plasmids revealed that not only pLP712 but also other plasmids had been transferred from L. lactis into LGG during conjugation. With plasmid-specific PCR primers, four additional lactococcal plasmids were detected in LAB49. Proteolytic activity assay and SDS-PAGE analysis verified that L. rhamnosus LAB49 effectively degraded β-casein. In contrast to its parental strain, LGG, the ability of LAB49 to metabolize lactose and degrade casein enabled strong and fast growth in milk. As strains with new properties made by conjugation are not regarded as genetically modified organisms (GMOs), L. rhamnosus LAB49 could be beneficial in dairy fermentations as a probiotic starter culture.IMPORTANCE Probiotic strain Lacticaseibacillus rhamnosus GG (LGG) is widely sold on the market as a probiotic or added as a supplement in dairy foods because of its benefits in human health. However, due to the deficiency of lactose and casein utilization, LGG does not grow well in milk. On the other hand, lactose intolerance and cow's milk protein allergy are the two major problems related to milk consumption. One option to help with these two conditions is the use of probiotic or lactose- and casein-hydrolyzing bacteria in dairy products. The purpose of this study was to equip LGG with lactose/casein-hydrolyzing ability by bacterial conjugation. As a result, we generated a non-GMO LGG derivative with improved properties and better growth in milk.

Covalent and non-covalent interactions of cyanidin-3-O-glucoside with milk proteins revealed modifications in protein conformational structures, digestibility, and allergenic characteristics.

Currently, there is a need to explore the effects of different types of protein-anthocyanin complexations, as well as the possible changes in the nutrition and allergenicity of the formed complexes. Here, we systematically investigated the covalent and non-covalent interactions between cyanidin-3-O-glucoside (C3G) and two major milk proteins, α-casein (α-CN) and β-lactoglobulin (β-LG). Fluorescence quenching data showed that, under non-covalent conditions, C3G quenched the fluorescence of the two proteins via a static process, with the interaction forces being revealed; for covalent products, decreased fluorescence intensities were observed with red shifts in the λmax. Multiple spectroscopic analyses implied that C3G-addition induced protein structural unfolding through transitions between the random coil and ordered secondary components. With a two-stage simulated gastrointestinal (GI) digestion model, it was seen that covalent complexes, not their non-covalent counterparts, showed reduced protein digestibility, ascribed to structural changes resulting in the unavailability of enzyme cleaving sites. The GI digests displayed prominent 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation-scavenging abilities (3.8-11.1 mM Trolox equivalents per mL digest), in contrast to the markedly reduced 1,1-diphenyl-2-picrylhydrazyl radical-scavenging capacities. Additionally, covalent protein-C3G complexes, but not their non-covalent counterparts, showed lower IgE-binding levels in comparison to the native control. This study provides new understanding for the development of anthocyanin-milk protein systems as functional ingredients with health-beneficial properties.

Capillary electrophoresis with dual C4D/UV detection for simultaneously determining major metal cations and whey proteins in milk.

A reliable and simple CE method with dual C4D and UV detection modes for simultaneous determination of major metal cations and whey proteins in milk samples was developed. Sample pretreatment comprised dilution, acidification to pH 4.55 with 10 mM AcOH and centrifugation. The complete separation of metal cations K+, Ca2+, Na+, and Mg2+ and whey proteins α-Lac, and β-Lg could be achieved respectively within 10 min and 20 min in a simple BGE composed of 1.0 M AcOH, 12 mM l-His and 2 mM 18-crown-6 with pH 2.74 at a voltage/current of +15 kV/12.5 μA. The samples were injected hydrodynamically by a pressure of 50 mbar for 5 s, the excitation voltage and excitation frequency of the C4D detector were 80 V and 1000 kHz, respectively and the detection wavelength of UV detection was set at 200 nm. In cation analysis, the range of the detection limit was 0.05-0.10 mg L-1 for C4D detection and 0.10-0.50 mg L-1 for UV detection, respectively, and the relative standard deviations (RSD%, n = 5) of intraday and interday analysis were 0.37-0.55% and 0.46-0.79% for the relative migration time, and 2.51-4.12% and 3.65-4.91% for the peak area, respectively. In whey protein analysis, the detection limits of β-Lg and α-Lac analysis were 5 mg L-1 and 3 mg L-1, respectively and the relative standard deviations (RSD%, n = 5) of intraday and interday analysis were 0.29-0.31% and 0.43-0.48% for the migration time and 2.89-3.25% and 3.29-4.18% for the peak area, respectively. The content of four major metal cations and two whey proteins in various types of milk samples was obtained. The results indicated that the content of metal cations varied little in milk samples of different brands and prices, while the content of whey proteins, as thermosensitive active proteins, varied greatly among different heat-treated milk samples.

Determination of A2 β-casein and total β-casein in cow milk and milk powder by capillary zone electrophoresis

β-casein (β-CN) is one of the major casein proteins in cow milk. There are 13 different variants documented for β-CN in cow milk, among which A1 and A2 are the major variants. The separation and quantitation of A2 β-CN are imperative for A2 dairy products. A new capillary zone electrophoresis (CZE) method with UV detection at 214 nm was established for the separation and quantification of the A2 variant and total β-CN content in cow milk and milk powders. The separation of β-CN variants was achieved on bare fused silica capillaries (50 μm×30/40 cm (effective/total length)). The separation buffer was a mixture of 4 mol/L urea, 0.2% (mass fraction) hydroxypropyl methylcellulose, 140 mmol/L citric acid, and 50 mmol/L disodium hydrogen phosphate buffer (pH 2.7). The corrected peak areas and the concentrations of total β-CN and the A2 variant showed good linearity, with correlation coefficients (r2) ranging from 0.9968 to 0.9997. The intra-day precisions for A2 β-CN and total β-CN determination in four samples (two pasteurized milk samples and two milk powder samples) were in the ranges of 2.4%-4.7% and 2.6%-4.8%, respectively. The inter-day precisions for A2 β-CN and total β-CN determination in four samples were in the ranges of 4.0%-6.3% and 3.9%-6.7%, respectively. The recoveries of A2 and total β-CN ranged from 85.5% to 106.4%. With the established CZE method, the A2 β-CN variant and total β-CN protein in liquid and powder bovine milk products could be separated and accurately quantified. By calculating the A2 β-CN content in the total β-CN, the quality of A2 dairy products can be evaluated, and this in turn would aid in the protection of consumer rights.

A molecular docking and molecular dynamics simulation study on the interaction between cyanidin-3-O-glucoside and major proteins in cow's milk.

The objective of this study was to investigate the molecular interaction and complex stability of four major cow's milk (CM) proteins (α-LA, β-LG, αs1 -CA, and β-CA) with cyanidin-3-O-glucoside (C3G) using computational methods. The results of molecular docking analysis revealed that hydrogen bond and hydrophobic interaction were the main binding forces to maintain the stability of the C3G-CM protein complexes. Molecular dynamics simulation results showed that all complexes except for C3G-αs1 -CA were found to reach equilibrium within 50ns of simulation. αs1 -CA and β-CA switched to a more compact conformation after binding with C3G. Additionally, the radius of gyration, number of hydrogen bond, radial distribution function, and interaction energy showed that β-CA is the best C3G carrier protein among the four CM proteins. This study can provide valuable information for CM proteins to serve as C3G delivery carriers. PRACTICAL APPLICATIONS: Anthocyanins (ACNs) are flavonoid-based pigments that play an important functional role in regulating human's health. Cow's milk (CM) proteins are the most representative protein-based carriers that can improve the short-term bioavailability and stability of ACNs. Thus, it is important to study the interactions between ACNs and CM proteins at the molecular level for the development of effective ACNs delivery carriers. Our study showed that caseins (αs1 -CA and β-CA) had more hydrophobic and hydrogen-bonding sites with cyanidin-3-O-glucoside (C3G) than whey proteins using computational methods. Among the four CM proteins, β-CA was the best C3G carrier protein showing the best interaction stability with C3G. Thus, it is helpful for us to screen effective ACNs carriers from multiple protein sources by computational methods.

Effectiveness of antibody specific for heat-processed milk proteins and incurred calibrants for ELISA-based quantification of milk in dark chocolate matrices

Although undeclared milk is prevalent in chocolate products, processing-induced alterations of milk proteins and matrix composition prevent reliable quantification of this allergen by current enzyme-linked immunosorbent assays (ELISA). High affinity antibodies (Abs) are required to detect milk protein epitopes that may be modified by manufacturing procedures. We therefore developed an Ab specific for heat-processed milk proteins (HPMP), which might allow enhanced milk quantification in processed complex matrices, including chocolate. When characterizing HPMP-Ab by immunoblot analysis, greater signal intensities were observed with heat modified proteins. Using a sandwich (s)-ELISA, HPMP-Ab reacted with high affinity to purified major milk proteins, proteins extracted from milk-derived ingredients and reference materials subjected to several thermal processing procedures. Milk-incurred cookies (0–17.7 ppm) and tempered dark chocolate samples (62% cacao: 0–134 ppm; 100% cacao: 0–75 ppm) were assessed using HPMP-Ab in a revised s-ELISA. To optimize protein extraction, samples were defatted with hexane; cookies were then extracted using a high salt buffer (HSB)/1% Tween (T)-20 while chocolate samples were extracted using HSB/1%T-20/5% polyvinylpyrrolidone (PVP)/1% fish gelatin. Despite reliable detection of milk proteins using incurred cookies and tempered 62% dark chocolate samples, as shown by acceptable dynamic ranges of the HPMP-Ab binding curves, high baseline absorbance/background persisted using 100% dark chocolate samples. The HPMP-Ab ELISA was highly specific; no cross reactivity was observed when testing a broad range of food commodities using incurred cookies as calibrants. The HPMP-Ab demonstrated the capacity to detect declared milk proteins in products containing a variety of milk-derived ingredients and undeclared milk proteins in products with precautionary labeling when used to analyze chocolate market samples (up to 85% cacao). Milk proteins were also detected in cross-contact samples produced on equipment used to manufacture milk chocolate, utilizing tempered 62% dark chocolate samples as calibrants. Our results establish the benefits of using HPMP-Ab and matrix-specific incurred calibrants for milk quantification in chocolate matrices by ELISA.

The effect of atmospheric cold plasma treatment on the antigenic properties of bovine milk casein and whey proteins

Casein, β-lactoglobulin and α-lactalbumin are major milk protein allergens. In the present study, the structural modifications and antigenic response of these bovine milk allergens as induced by non-thermal treatment by atmospheric cold plasma were investigated. Spark discharge (SD) and glow discharge (GD), as previously characterized cold plasma systems, were used for protein treatments. Casein, β-lactoglobulin and α-lactalbumin were analyzed before and after plasma treatment using SDS-PAGE, FTIR, UPLC-MS/MS and ELISA. SDS-PAGE results revealed a reduction in the casein and α-lactalbumin intensity bands after SD or GD treatments; however, the β-lactoglobulin intensity band remained unchanged. FTIR studies revealed alterations in protein secondary structure induced by plasma, particularly contents of β-sheet and β-turn. The UPLC-MS/MS results showed that the amino acid compositions decreased after plasma treatments. ELISA of casein and α-lactalbumin showed a decrease in antigenicity post plasma treatment, whereas ELISA of β-lactoglobulin showed an increase in antigenicity. The study indicates that atmospheric cold plasma can be tailored to mitigate the risk of bovine milk allergens in the dairy processing and ingredients sectors.

Quantitative LC-MS/MS analysis of high-value milk proteins in Danish Holstein cows

High-value milk proteins, which can be obtained by optimized fractionation procedures, are ideal ingredients in many food applications. Thus, a simple and robust analytical method is required for the identification and quantification of these individual milk proteins. Here, we present a liquid chromatography–tandem mass spectrometry (LC-MS/MS) method using multiple reaction monitoring (MRM) to simultaneously detect and measure target peptides of two major milk proteins, α-lactalbumin (α-LA) and β-casein (β-CN), in raw milk samples from 662 Danish Holstein cows. The MRM quantification of α-LA and β-CN was achieved with limit of detection (LOD) of 0.14 and 0.16 g/L, respectively and reproducibility of the assay <15%. By this newly established MRM-based method, the concentration of α-LA and β-CN in an individual cow's milk ranged from 0.5 to 1.9 (average 1.1) g/L, and from 7.5 to 23.4 (average 15) g/L, respectively. There was no significant effect of parity, whereas significantly increasing concentrations of α-LA and β-CN were observed through lactation (P < 0.001). This shows a considerable biological variation of these two ingredient milk proteins, providing potential varying outputs of fractionation in the dairy streams.

Assessment of allergenicity of irradiated dairy products in a Balb/c mice model

The objective of the presentin vivostudy was to analyze the changes in the immune response of the sensitized Ваlb/c mice against milk allergens in lyophilized cow’s milk (LCM) and whey curd (LC) before and after gamma irradiation (10 kGy). The results showed lower levels of IgE in the group treated with irradiated LCM (ICM) compared to the group treated with non-irradiated LCM (NiCM). Hence, it could be suggested that gamma irradiation influenced the epitopes of the major milk proteins and was associated with lower allergenicity of the lyophilized irradiated milk. The gamma irradiation in the whey curd, however, did not significantly change the level of IgE antibodies in IC (treated with irradiated LC) compared to NiC (treated with non-irradiated LC) group.

Negative regulation of αS1-casein (CSN1S1) improves β-casein content and reduces allergy potential in goat milk

Compared with other milk proteins, αS1-casein (encoded by CSN1S1) is associated with higher rates of allergies in humans. Although some studies have revealed that CSN1S1 affects a variety of cellular physiological processes such as immune response and proliferation in various cells, whether CSN1S1 regulates other milk proteins in ruminants is not known. In this study, we observed a negative Pearson correlation between the contents of αS1-casein and β-casein in goat milk. Thus, we used isolated primary goat mammary epithelial cells along with adenoviral infection or small interference RNA to alter abundance of CSN1S1 and examine its role in milk protein synthesis regulation. Overexpressing CSN1S1 through adenoviral transfection decreased β-casein mRNA (CSN2) and protein abundance, whereas interference of CSN1S1 resulted in a significant increase in β-casein abundance. CSN1S1 reduced phosphorylation level of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 5a (STAT5a). The transcription factor STAT5a activates CSN2 transcription by binding with its promoter region and then promotes β-casein synthesis. Furthermore, CSN1S1 inhibited CSN2 promoter activity and β-casein synthesis by modulating the JAK2/STAT5a signaling pathway. No changes in abundance were detected for αS2-casein (CSN1S2), κ-casein (CSN3) and major whey proteins (LALBA, BLG). Overall, our results underscored a mechanism whereby CSN1S1 inhibits β-casein synthesis via inhibition of STAT5a. The data suggested that knocking down CSN1S1 not only reduced the content of αS1-casein, but also increased the abundance of major milk proteins including β-casein. Thus, the present study provides a theoretical basis for manipulating αS1-casein mRNA to improve quality of ruminant milk for human consumption.

Quantification of the kokumi peptide, γ-glutamyl-valyl-glycine, in cheese: Comparison between cheese made from cow and ewe milk

Recent studies have shown that several types of cheese contain kokumi γ-glutamyl dipeptides, and the kokumi tripeptide, γ-glutamyl-valyl-glycine (γ-Glu-Val-Gly), is a component of various fermented foods. The quantification of γ-Glu-Val-Gly in various types of cheese was herein conducted by HPLC-tandem mass spectrometry followed by derivatization with 6-aminoquinoyl-N-hydroxysuccinimidyl-carbamate. The γ-Glu-Val-Gly concentrations were between 0.35 and 0.59 μg/g in cheese made from ewe milk, but were not detected in cheese made from cow milk. The amino acid sequences of major milk proteins showed that the β-caseins of sheep had the Val-Gly sequence at the 9-10 position, whereas β-caseins of cows contained a Pro-Gly sequence at the same position. The Val-Gly sequence was absent in other caseins of sheep and cattle. These results suggest that the different γ-Glu-Val-Gly concentrations present in cheese made from cow and ewe milk are due to differences in the amino acid sequences of caseins.

β-Galactosidase gene codon optimization results in post-transcriptional enhancement of expression.

lacZ encodes for β-galactosidase within the galactose operon of bacterial cells. When used as a reporter gene, bacterial "β-galactosidase" expression is often insufficient for detection in mammalian cells. We intended to optimize the lacZ codon usage according to the most frequently used codons for the seven major proteins in cow's milk, in order to pave a way for the enhancement of transgenic genes expression in eukaryotes. We constructed modified lacZ (named olacZ) according to optional codons used for proteins expressed in cow's milk. The expression of lacZ and olacZ was then compared in HC11 (a murine mammary gland epithelial line), 293T, HeLa, Cos7, and NIH 3T3 cells. While there was no significant difference at the mRNA level between lacZ and olacZ (P>0.05). The quantification of β-galactosidase activity and in situ staining experiments showed a 1.2-fold to 3.3-fold expression improvement when comparing olacZ with lacZ. The levels of β-galactosidase expression at the protein levels from olacZ were approximately 9.2-fold and 2.4-fold respectively for Cos7 and HC11 cells. Furthermore, a 1.9-fold tendency of enhanced expression of olacZ in mammary gland during lactation was observed in transgenic-olacZ mice. This study demonstrates an alternative choice for improving lacZ reporter expression in eukaryotes, especially in the mammary gland of cattle or goats.

Novel Single Nucleotide Polymorphisms of Lysozyme (C-Type) Gene in Donkey (Equus Asinus) Populations in Marmara Province of Turkey

The major antimicrobial proteins in donkey milk are lysozyme, lactoferrin, lactoperoxidase and immunoglobulins. Lysozyme has an important role in the host defense by way it inhibits the pathogenic bacteria. The aim of this study is to investigate the Lysozyme (LYZ) gene polymorphism in 82 donkeys reared in Thrace region of Turkey. 716 bp long partial 5‘ UTR, exon 1, intron 1, exon 2 regions of LYZ gene were amplified and PCR products were analyzed via DNA sequencing. Three novel single nucleotide polymorphisms (SNPs) were identified as g.1782775A&gt;G, g.1782924A&gt;G and g.1782960T&gt;C in the first intron of LYZ gene. The partial DNA sequence of LYZ gene in donkeys was reported in the present study and sequences of LYZ were entered to NCBI Genbank database with the accession number: MK984689-MK984692. This SNP may have an effect on immune system and milk traits in donkeys and additional studies are needed to confirm this assumption for donkey breeding.

A single-step genomic evaluation for milk production in Egyptian buffalo

The objectives of this study were to estimate genetic parameters and identify candidate genes for milk production in Egyptian buffalo using single-step method. Daily milk yield of 174,619 records from 698 buffalo cows were used. Animals with high and low deviated performance were selected for genotyping with Axiom® Buffalo Genotyping array 90 K. Variance components were estimated using AIREMLF90 for single-step GBLUP. Accuracy of prediction was calculated using predicted ability approach. The percentage of genetic variance explained by a window segment of each 30 adjacent SNPs was calculated. The top 20 window segments for additive genetic variance were chosen for QTL and candidate genes retrieving. Heritability estimate for daily milk yield was moderate (0.20 ± 0.03). The accuracy of prediction (0.61) seems to be suitable under current challenges. The top ten window segments with largest effects are located on chromosomes 1, 2, 3, 4, 6, 9, 11, 12 and 16 and explained around 6% of additive genetic variance. These genomic regions are coincided with previously reported QTL in different cattle breeds and harbored many propitious candidate genes for milk production, for example: the Oct1 protein encoded by POU2F1 gene on chromosome 6 is known to have a role for the regulation of major milk protein genes expression and development of mammary gland. These results indicated that a more reliable estimation of genetic parameters from population with small size can be obtained with a single-step genomic evaluation. The identified genomic regions with highest proportion of additive genetic variance harbor promising candidate genes with biological roles associated with milk production. Our findings provided a solid basis for improving milk production in Egyptian buffalo with reasonable accuracy, since traditional methods is hindered by the lack of sufficient pedigree information. A further research including more animals, records, and genotypes is required to validate our findings.

Genomic Analysis of Milk Protein Fractions in Brown Swiss Cattle

Simple SummaryMilk protein fractions are hugely important in the dairy industry because of the key role they play in milk technological properties. The selection of cows for milk protein fractions may, therefore, improve both the nutritional and technological characteristics of milk, and, consequently, the processing efficiency and value of the dairy product. This study estimated the genetic parameters of the major milk protein fractions (four caseins, and two whey proteins) determined variously as: (i) milk content (g/100g milk), (ii) percentage of milk nitrogen (%N) and (iii) daily yield (g/d) in Brown Swiss dairy cattle. The results showed that the (co)variances and genetic parameter estimates differed according to how the proteins were measured. These results provide useful information for developing selection strategies in dairy cattle breeding programs aimed at improving both the nutritional and technological properties of milk.Depending on whether milk protein fractions are evaluated qualitatively or quantitatively, different genetic outcomes may emerge. In this study, we compared the genetic parameters for the major milk protein fractions—caseins (αS1-, αS2-, β-, and к-CN), and whey proteins (β-lactoglobulin, β-LG; α-lactalbumin, α-LA)—estimated using the multi-trait genomic best linear unbiased prediction method and expressed variously as milk content (g/100g milk), percentage of milk nitrogen (%N) and daily yield per cow (g/d). The results showed that the genetic parameter estimates varied according to how the milk protein fractions were expressed. Heritability estimates for the caseins and whey protein fractions expressed as daily yields were lower than when they were expressed as proportions and contents, revealing important differences in genetic outcomes. The proportion and the content of β-CN were negatively correlated with the proportions and contents of αS1-CN, αS2-CN, and к-CN, while the daily yield of β–CN was negatively correlated with the daily yields of αS1-CN and αS2-CN. The Spearman’s rank correlations and the coincidence rates between the various predicted genomic breeding values (GEBV) for the milk protein fractions expressed in different ways indicated that these differences had a significant effect on the ranking of the animals. The results suggest that the way milk protein fractions are expressed has implications for breeding programs aimed at improving milk nutritional and technological characteristics.