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Characterization of goat milk lactoferrin N-glycans and comparison with the N-glycomes of human and bovine milk
Abstract
Numerous milk components, such as lactoferrin, are recognized as health-promoting compounds. A growing body of evidence suggests that glycans could mediate lactoferrin's bioactivity. Goat milk lactoferrin is a candidate for infant formula supplementation because of its high homology with its human counterpart. The aim of this study was to characterize the glycosylation pattern of goat milk lactoferrin. After the protein was isolated from milk by affinity chromatography, N-glycans were enzymatically released and a complete characterization of glycan composition was carried out by advanced mass spectrometry. The glycosylation of goat milk lactoferrin was compared with that of human and bovine milk glycoproteins. Nano-LC-Chip-Q-TOF MS data identified 65 structures, including high mannose, hybrid and complex N-glycans. Among the N-glycan compositions, 37% were sialylated and 34% were fucosylated. The results demonstrated the existence of similar glycans in human and goat milk but also identified novel glycans in goat milk that were not present in human milk. These data suggest that goat milk could be a source of bioactive compounds, including lactoferrin that could be used as functional ingredients for food products beneficial to human nutrition. This article is protected by copyright. All rights reserved.
... In addition, the lactoferrin glycosylation and polypeptide chain modulate and influence its function [48]. Goat milk lactoferrin exhibits better coherence with the functional characteristics of human milk lactoferrin, making it a better choice for formula supplementation than the lactoferrin of cow milk [49]. Another class of whey protein is immunoglobulins, which are plasma cell-generated glycoproteins in mammalian milk that serve as antibodies in response to the immune system by binding to antigens, ultimately making them bioactive milk components. ...
... Lactoferrin from human milk and goat milk had two N-glycans not seen in cow milk. This research demonstrated substantial lactoferrin N-glycan homology between GM and HM, indicating that GM might be utilized as a source of functional elements, including lactoferrin, beneficial for supplementation of infant formula [49]. Human milk (1-2 mg/mL) contains roughly ten times more lactoferrin than cow or goat milk (0.02-0.2 mg/mL), where lactoferrin is considered to be the chief iron-binding protein present in HM [49]. ...
... This research demonstrated substantial lactoferrin N-glycan homology between GM and HM, indicating that GM might be utilized as a source of functional elements, including lactoferrin, beneficial for supplementation of infant formula [49]. Human milk (1-2 mg/mL) contains roughly ten times more lactoferrin than cow or goat milk (0.02-0.2 mg/mL), where lactoferrin is considered to be the chief iron-binding protein present in HM [49]. On the other hand, CM and GM have much higher transferrin levels than HM. ...
Goat milk contains an abundance of different macro and micro-nutrients. Compared with other milk, goat milk is a viable option due to its low allergy levels and is preferred for infants with cow milk allergies. A wide variety of goat milk-based products, including yoghurt, ice cream, fermented milk, and cheese, are available on the market. They are produced using effective processing technology and are known to exhibit numerous health benefits after consumption. However, goat milk consumption is limited in many nations (compared with cow, buffalo, camel, and sheep milk) due to a lack of awareness of its nutritional composition and the significance of its different byproducts. This review provides a detailed explanation of the various macronutrients that may be present, with special attention paid to each component, its purpose, and the health benefits it offers. It also compares goat milk with milk from other species in terms of its superiority and nutritional content, as well as the types, production methods, health advantages, and other beneficial properties of the various goat milk products that are currently available on the market.
... Therefore, a simple and rapid pretreatment method is required. Previous reports showed that peptide-N-glycosidase F (PNGase F) could release N-glycans from glycoprotein for quantitative analysis via LC-MS without derivatization [17,18]. For O-glycans, Kameyama et al. developed eliminative oximation that enables β-elimination of O-glycans from glycoproteins using hydroxylamine and 1, 8-diazabicyclo[5.4.0]undec-7-ene (DBU) to suppress O-glycan degradation [29]. ...
... element counts, C200H400N40O200; and adduct ions, [ 2+ . In-house mass lists were used based on previous reports of human milk glycans [15,[17][18][19][20][21][24][25][26][27]. The Fill Gaps node was used to obtain the LC-MS area of glycans in all samples. ...
Human milk is abundant in carbohydrates and includes human milk oligosaccharides (HMOs) and N/O-glycans conjugated to proteins. HMO compositions and concentrations vary in individuals according to the maternal secretor status based on the fucosyltransferase 2 genotype; however, the profile of N/O-glycans remains uninvestigated because of the analytical complexity. Herein, we applied a label-free chromatography–mass spectrometry (LC–MS) technique to elucidate the variation in the composition and concentration of N/O-glycans in human milk. We used label-free LC–MS to relatively quantify 16 N-glycans and 12 O-glycans in 200 samples of Japanese human milk (1–2 months postpartum) and applied high performance anion exchange chromatography with pulsed amperometric detection to absolutely quantify the concentrations of 11 representative HMOs. Cluster analysis of the quantitative data revealed that O-glycans and several HMOs were classified according to the presence or absence of fucose linked to galactose while N-glycans were classified into a different group from O-glycans and HMOs. O-glycans and HMOs with fucose linked to galactose were more abundant in human milk from secretor mothers than from nonsecretor mothers. Thus, secretor status influenced the composition and concentration of HMOs and O-glycans but not those of N-glycans in human milk.
... Konuspayeva et al. used gel permeation chromatography on Sephadex G-200 to purify lactoferrin from camel colostral whey and checked its purity by polyacrylamide gel electrophoresis (12.5%) [18]. 18 19 20 21 M (the human lactoferrin) Many researchers used SDS-PAGE to confirm the presence or for characterization of lactoferrin [19][20][21][22][23][24]. The purity of isolated lactoferrin was confirmed by SDS-PAGE. ...
... Younghoon et al. checked the purity of caprine lactoferrin using SDS-PAGE [20]. The purity of isolated lactoferrin from defatted bovine colostrums was also confirmed on the SDS-PAGE gel [ [24]. ...
The single chemical composition of mare milk, rich in whey proteins is similar to human milk. Lactoferrin is one of the important compounds contained in whey protein fraction, and has multiple biological functions such as antimicrobial and activation of human and animal immune system. Due to its strong antimicrobial activity, lactoferrin has potential pharmaceutical applications. The present work is mainly focused on the isolation and purification of the lactoferrin from mare’s milk. First, the lactoferrin from Kazakhstan mare milk has been purified by gel filtration Sephadex G-100 chromatography in two steps. The column of Sephadex G-100 was eluted with 0.01 M sodium phosphate buffer (pH 6.8). Lactoferrin enriched fractions were detected using UV absorbance at 280 nm and were identified in the first peak. Second, the purity of lactoferrin was checked by 12% SDS–PAGE and the molecular weight of lactoferrin (in the range of 80-82 kDa) was estimated using protein standard, which is recombinant human lactoferrin (expressed in rice, iron saturated, molecular weight 82.4 kDa).
... N-glycans are complex compounds due to their monosaccharide composition, peculiar branching, and linkage types (Stefansson & Novotny, 1994). Similar to free MOs, N-glycans derived from bovine milk are highly sialylated and slightly fucosylated, while human and goat milk N-glycans (GMNs) are highly fucosylated (Le Parc et al., 2014;Nwosu et al., 2012). Milk N-glycans have not been studied as extensively as MOs and were only recently described to be antipathogenic (Barboza et al., 2012;Rossi et al., 2002;Wang et al., 2017). ...
... The comparison of the carbohydrates of these milk types can provide information regarding the nutritional and functional properties of different kinds of milk as a reference for consumers. Up to now, structural comparison of free oligosaccharides and lactoferrin-derived N-glycans from these three types of milk, especially human and bovine milk, are substantial (Dong et al., 2016;Le Parc et al., 2014). However, the structural comparison of the N-glycome from whole skimmed milk and their functional comparison between free and N-linked MOs among human, bovine, and goat milk has not been reported yet. ...
N‐linked oligosaccharides (N‐glycans) derived from milk were recently found to be antipathogenic. This study compares the antimicrobial activity of N‐linked glycans and free oligosaccharides from human, bovine, and goat milk against Staphylococcus aureus. Milk N‐glycans showed a bactericidal/bacteriostatic effect on the pathogen when compared to free milk oligosaccharides, evidenced by the clear zone from the halo assay, with the order of human milk >goat milk >bovine milk. None of the free milk oligosaccharide samples were bactericidal/bacteriostatic, despite its positive results in growth curve and minimum inhibitory concentration (MIC) assays which are believed to be related to hyperosmosis. Both N‐glycans and free milk oligosaccharides can reduce the adhesion of Staphylococcus aureus to Caco‐2 cells, however, N‐glycans worked significantly more effective than free milk oligosaccharides. Structural analysis of all free oligosaccharide and N‐glycan samples showed the obvious interspecies differences, and the structure/function relationship of the respected N‐glycans is of interest for future study. The significant bactericidal/bacteriostatic activity possessed by human, bovine, and goat milk N‐linked glycans holds great potential as a novel substitute for antibiotics.
... Lf biological function is also influenced by glycosylation, the most common protein posttranslational modification affecting protein folding, immunogenicity, protein solubility, and resistance to proteolysis. Glycosylation is a species-specific and tissue-specific modification, e.g., hLf and recombinant human Lf (rhLf) contains two major glycosylation sites, Asn 138 and Asn 479 [37,38], but different N-glycan patterns [39]. Indeed, rLf expressed in cow has a lower content of sialic acid and fucose, showing high mannose-, hybrid-, and complex-type structures, while N-glycans from hLf are comprised entirely of highly branched, highly sialylated, and highly fucosylated complex-type structures [40]. ...
... Lf biological function is also influenced by glycosylation, the most common protein post-translational modification affecting protein folding, immunogenicity, protein solubility, and resistance to proteolysis. Glycosylation is a species-specific and tissue-specific modification, e.g., hLf and recombinant human Lf (rhLf) contains two major glycosylation sites, Asn 138 and Asn 479 [37,38], but different N-glycan patterns [39]. Indeed, rLf expressed in cow has a lower content of sialic acid and fucose, showing high mannose-, hybrid-, and complex-type structures, while N-glycans from hLf are comprised entirely of highly branched, highly sialylated, and highly fucosylated complex-type structures [40]. ...
Despite recent advances in cancer therapy, current treatments, including radiotherapy, chemotherapy, and immunotherapy, although beneficial, present attendant side effects and long-term sequelae, usually more or less affecting quality of life of the patients. Indeed, except for most of the immunotherapeutic agents, the complete lack of selectivity between normal and cancer cells for radio- and chemotherapy can make them potential antagonists of the host anti-cancer self-defense over time. Recently, the use of nutraceuticals as natural compounds corroborating anti-cancer standard therapy is emerging as a promising tool for their relative abundance, bioavailability, safety, low-cost effectiveness, and immuno-compatibility with the host. In this review, we outlined the anti-cancer properties of Lactoferrin (Lf), an iron-binding glycoprotein of the innate immune defense. Lf shows high bioavailability after oral administration, high selectivity toward cancer cells, and a wide range of molecular targets controlling tumor proliferation, survival, migration, invasion, and metastasization. Of note, Lf is able to promote or inhibit cell proliferation and migration depending on whether it acts upon normal or cancerous cells, respectively. Importantly, Lf administration is highly tolerated and does not present significant adverse effects. Moreover, Lf can prevent development or inhibit cancer growth by boosting adaptive immune response. Finally, Lf was recently found to be an ideal carrier for chemotherapeutics, even for the treatment of brain tumors due to its ability to cross the blood–brain barrier, thus globally appearing as a promising tool for cancer prevention and treatment, especially in combination therapies.
... From these studies, it had been demonstrated that humans' SNPs in the LF gene have been linked to cancer (Liu et al. 2002) and diarrhea (Mohamed et al. 2007). The impacts of LF gene SNPs on milk production traits and mastitis were studied in cattle (Li et al. 2004) and also in human beings (Le Parc et al. 2014). The four SNPs identified in this research, with low PIC values (0.25 < PIC < 0.50) indicating that they are highly polymorphic, deviated from the Hardy-Weinberg equilibrium (HWE) according to the analysis of allelic and genotypic frequencies (P < 0.05). ...
The sequence analysis of PCR product exhibited four novel SNPs in the promoter region of the LF gene at loci g.98T>C, g.143T>A, g.189AC>A, and g.346A>G. Each SNP yielded three genotypes; the genotypes TT (SNP1), AA (SNP3), and GG (SNP4) decreased SCC and increase milk quality traits such as density, protein, and milk yield (P < 0.01). The genotype CC (SNP2) and CA (SNP4) significantly (P < 0.01) decreased the milk quality parameters, while genotypes TC (SNP2) and GG (SNP4) showed significantly (P < 0.01) less SCC and increase lactose % in milk. Furthermore, screening of the LF promoter sequence explored the gain of four TF binding sites at locus g.98T˃C and three TF binding sites at g.346A˃G. However, the loss of four and two TF binding sites was seen at locus g.143T˃A and g.189C˃A, respectively. We can conclude from the present study that the GG, TT, and AA genotype might be utilized as genetic markers in marker-assisted selection for the breed improvement program of Beetal goats.
... 10,[170][171][172] HMOs, besides being a major carbon source for the infant gut microbiota, were shown as a nitrogen for those same bacteria. 173 The simplest HMOs are derivatized lactoses such as galactosyllactoses and fucosyl and sialyllactoses. The usual composition of detected HMOs follows the formula Lx/y-z (with L: lactose; x: Gal-GlcNac disaccharide units; y: fucoses and z: sialic acids). ...
Diet shapes our gut microbiome from the day we are born. The contribution of dietary non-protein nitrogen to normal and healthy nitrogen cycling in the infant gut is scarcely described. Herein, we review in vitro and in vivo findings that show the impact of Human Milk Nitrogen (HMN) on the gut microbiota that colonizes the gut in early human life. We describe that several non-protein nitrogen sources, that include creatine, creatinine, urea, polyamines and free amino acids, are key in establishing the bifidobacterium-dominated microbiome and thus are bifidogenic. Furthermore, several parts of HMN-related metabolism are associated with a healthy infant gut and commensal microbiota. We illustrate an overlap and great diversity in accessibility of HMN by large parts of the infant gut microbiota. This review nonetheless shows the importance of research on HMN and its effects on the activity and composition of the infant gut microbiota and its potential effect on early life infant health.
... It has been attributed with antimicrobial, immunomodulatory, antiparasitic, anti-cancer and wound healing activities. Goat milk oligosaccharides and glycosylation pattern of goat Lf have been found to be very much similar to that of humans (Le Parc et al., 2014). Due to its high digestibility, higher concentration of minerals like calcium and magnesium and immunological properties, goat milk is preferred as an alternative for infants and convalescents as a balanced nutrition. ...
Lactoferrin, a minor whey protein, possesses a multitude of biological functions including antimicrobial, immunomodulatory and antitumour activities. The present study focuses on the antitumour activity of lactoferrin isolated from Malabari goats. Lactoferrin was isolated from Malabari goat colostrum by Cation exchange chromatography. The purity of the isolated protein was confirmed by SDS-PAGE and its identity was ascertained by Western Blotting. Solid tumours were induced in hind limbs of Swiss Albino mice using Daltons lymphoma ascites cells. Animals which developed the tumours were divided into groups and administered different doses of Malabari goat lactoferrin (MgLf) intratumourally as a single dose. The tumour positive animals were sacrificed on day 10 post treatment to study the effects of MgLf on tumour growth kinetics by assessing the tumour weight: body weight ratio and tumour volume inhibition of the slaughtered animals. It was found that MgLf @150μg significantly lowered the tumour weight: body weight ratio and tumour volume of the treated animals.
... (2011) determined the molecular weight of bovine lactoferrin purified by gel filtration on Sephacryl S-300 and verified its purity by SDS PAGE [1]. Similarly, Le Parc et al. determined the molecular weight of goat milk lactoferrin to be 78 kDa by SDS-PAGE [22] . A study on bioactive proteins in camel milk has been conducted on many proteins including lactoferrin. ...
The present article exemplifies a novel method to isolate highly purified bioactive lactoferrin from camel milk. Cytotoxicity of lactoferrin against the Hela cells was used to evaluate its bioactivity. SDS-PAGE and LC-MS analysis was done for its identification and characterization. The purified camel milk lactoferrin was found to be 708 amino acids in length with a molecular weight of 77.3 kDa and a pI value of 8.24. This pH-dependent isolation procedure ensures the retention of bioactive lactoferrin from camel milk. The importance of the present work lies in its simplicity and scalability for manufacturing bioactive lactoferrin at an industrial level.
... The reason for this remains unclear as previous research did not show differences in the ability of bLF and pLF to degrade virulence factors and to decrease epithelial attachment of ETEC [26]. The recombinant production of pLF in CHO cells might introduce subtle changes to its structure, potentially caused by a different glycosylation pattern, as compared to native pLF, that might affect its in vivo behavior [45][46][47][48]. Further research will be needed to clarify this. ...
Enterotoxigenic Escherichia coli (ETEC) infections are one of the most prevalent causes of post-weaning diarrhea in piglets, resulting in morbidity, mortality and elevated use of antibiotics. The emergence and further spread of antimicrobial resistance together with the growing demand for high quality animal protein requires the identification of novel alternatives for antimicrobials. A promising alternative is lactoferrin, as we previously showed that it can both inhibit the growth and degrade bacterial virulence factors of porcine ETEC strains in vitro. Aiming to confirm these findings in vivo, we performed a small intestinal segment perfusion experiment in piglets. Here, we showed that lactoferrin could not only decrease ETEC-induced fluid secretion, but also their ability to colonize the small intestinal epithelium. Furthermore, while ETEC infection induced pro-inflammatory cytokine mRNA expression in this experiment, lactoferrin was not able to counteract these responses. In addition, a bacterial motility assay showed that lactoferrin can reduce the motility of ETEC. Our findings further support the use of lactoferrin as an alternative for antimicrobials and also show its potential for the prevention of ETEC infections in pigs.
... In addition to the presence of high-quality protein, goat milk comprises unsaturated fatty acids, vitamins, hormones, cytokines, enzymes, growth factors, and bioactive peptides, all of which assist to nourish and protect infants (95)(96)(97). Besides nutritional benefits of milk components, additional components such as antibodies, glycoproteins, and oligosaccharides can protect infants by preventing pathogen infections and supporting the growth of the intestinal epithelium (98). Various health benefits of goat milk are shown in Table 3. ...
Goat milk is considered to be a potential source of various macro- and micro-nutrients. It contains a good proportion of protein, fat, carbohydrates, and other nutritional components which help in promoting nutritional and desirable health benefits. Goat milk is considered to be superior in terms of numerous health benefits, and lower risk of allergy, when compared to the milk of other species. Several processing techniques such as pasteurization, ultrafiltration, microfiltration, and ultrasound have been employed to enhance the quality and shelf life of goat milk and its products. The diverse range of goat milk-based products such as yogurt, cheese, fermented milk, goat milk powder, and others are available in the market and are prepared by the intervention of advanced processing technologies. Goats raised in pasture-based feeding systems are shown to have a better milk nutritional composition than its counterpart. Goat milk contains potential bioactive components, which aids in the maintenance of the proper metabolism and functioning of the human body. This review gives insight into the key nutritional ingredients and bioactive constituents present in goat milk and their potential role in the development of various functional foods using different processing technologies. Goat milk could be considered as a significant option for milk consumption in infants, as compared to other milk available.
... The method of SDS-PAGE was reported to confirm the molecular weight and purity of caprine Lf (Younghoon et al., 2009;Le Parc et al., 2014;Abbas et al., 2015 andVijayan et al., 2017) and that of bovine Lf (Adam et al., 2008;Liang et al., 2011;Yafei et al., 2011;Moradian et al., 2014). Adam et al. (2008) Reports by Younghoon et al. (2009), Annabelle et al. (2014 and Abbas et al. (2015) confirmed molecular weight of caprine Lf to be 82kDa, 78kDa and 80kDa ...
Lactoferrin, well known as a minor whey protein, is an 80kDa iron-binding glycoprotein primarily present in milk. It exhibits an array of biological activities including antioxidant, antimicrobial, anticancer, metal binding and immunomodulatory properties. Many studies point out that whey proteins contain various bioactive peptides in their primary sequences whose immunoregulatory and therapeutic potentials have not been much explored. The present study focussed on the isolation and characterisation of lactoferrin (gLf) from the colostrum/milk of Malabari goats, followed by the preparation of its pepsin hydrolysate (gLPH) so as to assess their in vitro immunomodulatory potentials on peripheral blood mononuclear cells (PBMCs). Colostrum samples collected from Malabari goats maintained at University Goat and Sheep Farm, College of Veterinary and Animal Sciences, Mannuthy were processed and treated with ammonium sulphate to remove globulins from the samples. Fractions containing albumin and remaining proteins including lactoferrin after dialysis were loaded on to CM- Sephadex C-50 cation exchanger column and eluted with a step gradient of 0.4, 0.6 and 0.8M NaCl. The presence of gLf in the high OD280 value fractions eluted with 0.8M NaCl was confirmed by 12 per cent SDS-PAGE and Western blotting. The concentration of gLf as estimated by Lowry’s method was found to be 15.103 mg/L of colostrum. The gLf was hydrolysed by treatment with three per cent porcine pepsin under acidic conditions at 37 °C for four hours to form gLPH. The immunomodulatory potentials of gLf and gLPH were studied on bovine PBMCs which were cultured along with specific combinations of mitogen, phytohaemagglutinin (PHA)/gLf/gLPH at 37°C for 72h in five per cent CO2 humidified air. A wide range of concentrations of both gLf and gLPH were utilized to assess their cytoproliferative effect on PBMCs with or without mitogens. In the presence and absence of mitogen, higher concentrations of lactoferrin were found to significantly inhibit the proliferation of PBMCs whereas lower concentrations brought about significantly active proliferation of the cells. Maximum proliferation with or without mitogen was observed with 1.5 µg/mL of culture. The cell proliferation potentials of gLPH was higher than that of gLf. With gLPH, in the presence and absence of mitogen, maximum proliferation of PBMCs could be detected with the highest concentration of 50 µg/mL. The cells cultured with gLf and gLPH showing maximum cell proliferation were harvested and their RNA were isolated to study the expression of pro-inflammatory cytokines by real time PCR. It was demonstrated that gLf and gLPH showed potentiated anti-inflammatory activity by significantly inhibiting the expression of the pro-inflammatory cytokine genes IL-1β, IL-6 and TNF-α.
... In addition to Damascus goats or Shami which are considered dual-purpose breed, imported from Syria to Egypt by Ministry of Agriculture (Jnied et al., 2013). Goat milk is highly recommended for infants, elderly, and convalescents, because of its higher nutritional value, digestibility, immunological properties, and the hypo-allergenicity compared to others (Le Parc et al., 2014). Goat milk can be consumed as it is, but a large proportion is processed to cheese and other dairy products producing a large amount of whey (Hejtmánková et al., 2012;El-Tarabany et al., 2018). ...
... (2009) found that the molecular weight than reality (Lambert et al., 2005). When comparing the of caprine LF was 82 KDa, Annabelle et al. (2014) hololactoferrin. In breast milk the LF found is essentially determinate the molecular weight of goat milk LF by apolactoferrin. ...
Lactoferrin is an important protein in many biological applications as a potential cancer treatment agent. In this study, lactoferrin was purified from goat colostrum by ion exchange chromatography through CM-Sephadex C-50 column and gel filtration chromatography through Sephadex G-200 column. The purification fold and yield were (20.83 time and 62.50%) respectively. The purity of lactoferrin to homogeneity was examined by polyacrylamide gel electrophoresis under denaturing conditions (SDS-PAGE) with single bond. Some biochemical characteristics of the purified Lactoferrin were determined, The molecular weight of LF were 80 and 79.50kDa as determined by gel filtration and SDS-PAGE respectively. The percentage of carbohydrate content in goat Lactoferrin was 10.4%, mean while the Iron percentage was 123 ppm and as a saturated percent 7.8%.
... The amino acid sequence of human Lf was 70.8% and 69.7% homologous with those of bovine and caprine Lfs, respectively. Human Lf has 18 unique N-glycans that are not found in bovine milk, whereas caprine milk contains 24 unique N-glycans (Parc, Dallas, Duaut, Leonil, & Barile, 2014).These differences in the amino acid sequence and glycosylation level may also lead to the differences in Lf digestive patterns among species. ...
Immunoglobulin G (IgG) and lactoferrin (Lf) play an important role in the protection and maturation of the neonatal immune system; however, the survival of IgG and Lf during gastrointestinal digestion and their digestive differences among human, bovine, and caprine milk remain unclear. This study investigated the digestive behaviour of IgG and Lf by simulating infant digestion of the milk of three species. Both IgG and Lf in human milk showed higher resistance to digestion than those in the other two types of milk. In addition, caprine milk IgG survived for a longer period after gastrointestinal digestion than bovine milk IgG. These static in vitro digestion results showed that IgG and Lf from human and caprine milk are digested more slowly from those from bovine milk.
... Although bovine and caprine LTF have the same number of potential glycosylation sites (Baker and Baker, 2009), some differences do exist in LTF glycosylation pattern between these 2 species. For instance, caprine LTF contains fewer sialylated N-glycans (Le Parc et al., 2014). A previous study showed that glycans bound to LTF affect its digestibility (van Berkel et al., 1996). ...
Infant formula is used as a supplement for newborns. Although bovine milk-based infant formulas dominate the market, caprine milk-based infant formula has attracted increasing attention because of its lower allergenicity. This study compared the digestive peptidome of bovine and caprine milk serum proteins by using in vitro infant simulating conditions. The result showed that the degradation pattern of milk proteins was similar, whereas the digestive rates of milk proteins differed between bovine and caprine milks. Several proteins, such as α-lactalbumin (LALBA), β-lactoglobulin (LGB), serum amyloid A protein (SAA1), glycosylation-dependent cell adhesion molecule 1 (GLYCAM1), and lactotransferrin (LTF), released more peptides during digestion of caprine milk serum than during digestion of bovine milk serum; however, more peptides derived from αS1-casein (CSN1S1) were found in bovine digesta. In addition, antimicrobial-related peptides were mostly only found in caprine intestinal digesta. The results of this study may be useful in understanding the digestion characteristics of milk serum proteins and providing guidance on the improvement of infant formula.
... (2009) found that the molecular weight than reality (Lambert et al., 2005). When comparing the of caprine LF was 82 KDa, Annabelle et al. (2014) breast milk the LF found is essentially apolactoferrin. determinate the molecular weight of goat milk LF by Another study was done by Ella et al. (2009) about hLF SDS-PAGE as 78 KDa. ...
Lactoferrin is an important protein in many biological applications as a potential cancer treatment agent. In this study, lactoferrin was purified from goat colostrum by ion exchange chromatography through CM-Sephadex C-50 column and gel filtration chromatography through Sephadex G-200 column. The purification fold and yield were (20.83 time and 62.50%) respectively. The purity of lactoferrin to homogeneity was examined by polyacrylamide gel electrophoresis under denaturing conditions (SDS-PAGE) with single bond. Some biochemical characteristics of the purified Lactoferrin were determined, The molecular weight of LF were 80 and 79.50kDa as determined by gel filtration and SDS-PAGE respectively. The percentage of carbohydrate content in goat Lactoferrin was 10.4%, mean while the Iron percentage was 123 ppm and as a saturated percent 7.8%.
... Human milk is rich in LF (3-5 g/l in mature milk), which is 10-100 fold higher than that in cow and goat milk. 4 In addition, LF is the most prominent antimicrobial component in milk. 2 However, in this study, recombinant lactoferrin, bovine lactoferrin, and human lactoferrin showed limited inhibition of both SARS-CoV-2 and GX_P2V infection at the concentration of 1 mg/ml, suggesting that other components in breastmilk might play important roles in this inhibition (Fig. 1j). ...
Signal Transduction and Targeted Therapy (2020) 5:275 ; https://doi.org/10.1038/s41392-020-00408-z Dear Editor, Since severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA has been detected in human breastmilk, infants' safety with breastmilk feeding is of great concern for women with coronavirus disease 2019 (COVID-19). 1 It is known that milk has antiviral properties. 2 However, little is known about the antiviral property of human breastmilk to SARS-CoV-2 and its related pangolin coronavirus (GX_P2V). Here we present for the first time that whey protein from human breastmilk effectively inhibited both SARS-CoV-2 and GX_P2V by blocking viral attachment and viral replication at entry and even post entry. Moreover, human whey protein inhibited infectious virus production, as proved by the plaque assay. We found that whey protein from different species, such as cow and goat, also showed anti-coronavirus properties. Commercial bovine formula milk also showed similar anti-SARS-CoV-2 activity. Firstly, healthy human breastmilk samples collected in 2017 and stored properly at −80°C were tested for their potential effects on SARS-CoV-2 infection. Mothers provided informed consent. This study was approved by the ethics committees of the Medical Center and all samples were anonymized. The skimmed breastmilk was obtained after removal of the lipid fraction. Vero E6 cells were infected with a mixture of SARS-CoV-2 pseudovirus (650 TCID 50 /well) and human breastmilk (4 mg/ ml). Human breastmilk from eight donors showed a significant inhibition of more than 98% of the SARS-CoV-2 pseudovirus. As reported recently, a SARS-CoV-2-related pangolin coronavirus model (GX_P2V) 3 shares 92.2% amino acid identity in spike protein with SARS-CoV-2, which is a suitable model for SARS-CoV-2 infection research. We utilized GX_P2V (MOI: 0.01 in Vero E6 cells) as the model to study the effect of breastmilk on viral infection and also found similar results (Fig. 1a). The inhibition is concentration dependent with an EC 50 of 0.13 mg/ml of total protein (Fig. 1b and Supplementary Fig. S1) in the SARS-CoV-2 pseudovirus model. Consistent with the SARS-CoV-2 study, the GX_P2V model also showed inhibition with an EC 50 of about 0.5 mg/ml of total protein (Fig. 1c and Supplementary Fig. S2). Interestingly, human breastmilk did not show any cytotoxicity to Vero E6 cells (CC 50 > 3 mg/ml), and even promoted cell proliferation. These results indicated that human breastmilk showed high anti-SARS-CoV-2 and anti-GX_P2V property, but limited cytotoxicity to Vero E6 cells. We then assessed the impact of human breastmilk on infectious virus production in Vero E6. RT-qPCR analysis of the GX_P2V virus from supernatant showed that even 0.16 mg/ml of breastmilk significantly blocked viral production (Fig. 1d). Western blot of viral nucleoprotein also showed similar results (Fig. 1e). To investigate the infectious virus, we performed plaque assay. As shown in Fig. 1f, the plaque assays showed that live viruses were significantly lower in breastmilk treatment compared to the control group, which confirmed that breastmilk inhibit GX_P2V infection, replication, and production of infectious virions. Next, to rule out whether protein in breastmilk plays the important role of inhibiting viral infection, we inactivated the protein by applying a temperature of 100°C for 10 min and protease K digestion, respectively, and tested its role in viral inhibition. As shown in Fig. 1g, breastmilk with treatment by heat and protease K inhibited SARS-CoV-2 pseudovirus infection in both Vero E6 and A549 cells, indicating that the protein in breastmilk plays an important role through its antiviral properties. In addition, SARS-CoV-2 pseudovirus infection could be inhibited by commercial bovine formula milk (EC 50 = 1.67 mg/ml) in a dose-dependent manner (Fig. 1h). To investigate whether whey protein from other species could also inhibit SARS-CoV-2 and GX_P2V infection, we chose cow and goat whey protein to analyze their inhibition property. Luciferase assay and RT-qPCR revealed that both cow and goat whey protein inhibited the infectivity of SARS-CoV-2 pseudovirus and GX_P2V, although the inhibition efficiency was relatively lower compared to that of human breastmilk (Fig. 1i). These results indicated that human whey protein has a high concentration of antiviral factors than those from other species. It was reported that lactoferrin (LF) has broad antiviral effects. Human milk is rich in LF (3-5 g/l in mature milk), which is 10-100 fold higher than that in cow and goat milk. 4 In addition, LF is the most prominent antimicrobial component in milk. 2 However, in this study, recombinant lactoferrin, bovine lactoferrin, and human lactoferrin showed limited inhibition of both SARS-CoV-2 and GX_P2V infection at the concentration of 1 mg/ml, suggesting that other components in breastmilk might play important roles in this inhibition (Fig. 1j). It was also reported that IgA antibody from recovered COVID-19 patients inhibited SARS-CoV-2 infection in vitro. 5 As the human breastmilk samples were collected before the emergence of COVID-19, the breastmilk donors should have not been infected with SARS-CoV-2. To exclude the impact of IgA-related antibody on viral infection, we utilized the neutralized anti-IgA antibody (1 mg/ml) with different dilutions to mix with the breastmilk (1 mg/ml). As shown in Fig. 1k, the different dilutions of anti-IgA antibody did not influence the inhibitory activity of breastmilk to SARS-CoV-2 and GX_P2V infection, indicating that IgA antibody from breastmilk might not be the key factor inhibiting viral infection and other factors may be responsible for the anti-SARS-CoV-2 activity of whey protein, which merits further study. To study how human breastmilk inhibits viral infection, we did the viral attachment, entry, and post-entry experiments. As shown in Fig. 1l, human skimmed breastmilk could attach the cell surface to block viral binding and entry. Breastmilk also inhibited viral attachment (Fig. 1m and Supplementary Fig. S3a) and entry (Fig. 1n and Supplementary Fig. S3b) with more than 90% of inhibition rates during both SARS-CoV-2 pseudovirus and GX_P2V infection, suggesting that breastmilk blocks viruses from entry into the cytoplasm. Furthermore, we investigated the impact of human skimmed breastmilk on the affinity between SARS-CoV-2 S protein and ACE-2. As shown in Fig. S3c, the skimmed milk interferes with the affinity between these two proteins in a dose-dependent manner. However, when inactivated by 100°C for 10 min, the inhibitory activity of viral entry by inactivated breastmilk showed no significant difference compared to the breastmilk without heat treatment. In addition, breastmilk inhibited significantly GX_P2V replication in the present of breastmilk during viral post-entry Letter 2 Signal Transduction and Targeted Therapy (2020) 5:275 (Fig. 1o), suggesting that breastmilk inhibits not only viral entry but also viral replication. Furthermore, we also determined whether the possible mechanism of inhibiting SARS-CoV-2 replication is that breastmilk interferes with the RNA-dependent RNA polymerase (RdRp) activity of SARS-CoV-2. Surprisingly, breastmilk could significantly inhibit the RdRp activity of SARS-CoV-2 in a dose-dependent manner (Fig. 1p). These results suggest that breastmilk inhibits both SARS-CoV-2 and GX_P2V infection and replication and shed light on the protection of newborns by breastfeeding from mothers with or without SARS-CoV-2 infection. Collectively, we reported that human breastmilk inhibits SARS-CoV-2 virus infection in Vero E6 and A549 cell lines. These results suggest whey protein as a direct-acting inhibitor of SARS-CoV-2 and GX_P2V infection and replication by the mechanism of potentially impairing RdRp activity and slightly blocking the affinity of ACE-2 and SARS-CoV-2 S protein. Although our results are limited by the lack of a larger number of breastmilk samples and clinical study, we believe our findings give important clues for the safety of breastmilk feeding and further usage of breastmilk for antiviral drug development, which may be helpful for the development of recommendations on whether mothers with COVID-19 could breastfeed. ACKNOWLEDGEMENTS
... Altoghether, these results led to assume that the peaks with increased intensities refer to glycans from lactotransferrin (m/z 2966.5 and 2605.3) [36,41,42] and from serotransferrin (m/z 2792.4) [29,43], whereas peaks showing decreased intensity are associated to immunoglobulins. ...
... Since goat milk has 70% primary sequence homology with human lactoferrin while cow milk has 68% of the homology. Furthermore, latcoferrin has antioxidant, antibacterial, antiviral, and immunomodulation effect, so goat milk is more effective to help infant or baby's health by decreasing pathogen infection [18]. ...
Undernourished is one of the health problems faced by the world today, especially in Indonesia, with the percentage of undernourished children under 5 years old is 19.6% or 4,646,933 out of 23,708,844 children. For young children, breastmilk is the best food and complementary food after six months that is usually produced from cow milk is needed. Another problem comes, 2-7,5% children have the allergy of cow milk and choose soy milk as recommended by health workers, although it is more expensive (100,000,00 IDR or 6.9 USD/1000 grams) than cow milk (68,000,00 IDR or 4.74 USD/1000 grams). To solve this issue, goat milk is one of the good alternatives food for considering as a complementary food. Goat milk has beneficiary things such as treating malabsorption syndrome and undernourished children. Besides that, according to the Statistic Center Board/Badan Pusat Statistik (BPS), the number of the goat population in Indonesia is 18,879,596, more than the cow population which is 16,019,459. For further evaluating the advantage of goat milk, we conducted a review study. Methods: We searched all literature published in 2004-2014 on PubMed and Science Direct using the keywords "goat milk", "nutrition", and "content". Result: There were 311 journals that appeared in the selection, but we selected 10 journals which showed a clear comparison between goat and cow milk, especially in young children. After making the table with special consideration on population, place, age of the subject and nutrient content in both kinds of milk, we conclude all the studies. The studies showed that goat milk has the advantage not only for treating undernourished children but also many disorders and diseases such as malabsorption syndrome, malnutrition in infants and children, and cardiovascular (heart) disease, due to its small globule of fat which is a digestible and short and medium-chain fatty acid. Mineral content in goat milk such as Calcium, Phosphor, Magnesium, Natrium, Potassium, Chloride, Iron, and Zinc is higher than human's, that's all useful for preventing bone demineralization and iron deficiency anaemia which is still high in Indonesia. Conclusion: goat milk treated undernourished children and prevented several diseases and food disorders and it is suitable due to economic reasons compared to cow milk. Furthermore, we could state that cow milk seemingly could be a promising treatment for undernourished children in developing countries to reduce morbidity and mortality related to malnutrition-related diseases.
Keywords: Goat Milk, Complimentary Food, Undernourished
... (2009) found that the molecular weight than reality (Lambert et al., 2005). When comparing the of caprine LF was 82 KDa, Annabelle et al. (2014) breast milk the LF found is essentially apolactoferrin. determinate the molecular weight of goat milk LF by Another study was done by Ella et al. (2009) about hLF SDS-PAGE as 78 KDa. ...
Lactoferrin is an important protein in many biological applications as a potential cancer treatment agent. In this study, lactoferrin was purified from goat colostrum by ion exchange chromatography through CM-Sephadex C-50 column and gel filtration chromatography through Sephadex G-200 column. The purification fold and yield were (20.83 time and 62.50%) respectively. The purity of lactoferrin to homogeneity was examined by polyacrylamide gel electrophoresis under denaturing conditions (SDS-PAGE) with single bond. Some biochemical characteristics of the purified Lactoferrin were determined, The molecular weight of LF were 80 and 79.50kDa as determined by gel filtration and SDS-PAGE respectively. The percentage of carbohydrate content in goat Lactoferrin was 10.4%, mean while the Iron percentage was 123 ppm and as a saturated percent 7.8%.
... (2009) found that the molecular weight than reality (Lambert et al., 2005). When comparing the of caprine LF was 82 KDa, Annabelle et al. (2014) hololactoferrin. In breast milk the LF found is essentially determinate the molecular weight of goat milk LF by apolactoferrin. ...
Lactoferin was whey proteins it work as bioactive componed in this study we isolated it from whey if goat milk and added it to some cancer cell line .lactoferin inhibit the growth of four type of cancer cell line with power more than sodium methoxide which used as chemical therapy
... It was reported that human milk is rich in LF (3-5g/L in mature milk), which is 10-100 fold higher than that in cow and goat milk (20). In addition, LF is the most prominent antimicrobial component in milk (11). ...
Since the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in human breastmilk, little is known about the antiviral property of human breastmilk to SARS-CoV-2 and its related pangolin coronavirus (GX_P2V). Here we present for the first time that whey protein from human breastmilk effectively inhibited both SARS-CoV-2 and GX_P2V by blocking viral attachment, entry and even post-entry viral replication. Moreover, human whey protein inhibited infectious virus production proved by the plaque assay. We found that whey protein from different species such as cow and goat also showed anti-coronavirus properties. And commercial bovine milk also showed similar activity. Interestingly, the main antimicrobial components of breastmilk, such as Lactoferrin and IgA antibody, showed limited anti-coronavirus activity, indicating that other factors of breastmilk may play the important anti-coronavirus role. Taken together, we reported that whey protein inhibits SARS-CoV-2 and its related virus of GX_P2V. These results rule out whey protein as a direct-acting inhibitor of SARS-CoV-2 and GX_P2V infection and replication and further investigation of its molecular mechanism of action in the context of COVID-19.
... Goat colostrum lactoferrin contains 15 N-glycans, which are also present in human milk. So there is a significant degree of homology for lactoferrin N-glycans between human and goat colostrum [17]. ...
The review presents the composition of goat colostrum, antimicrobial, immunomodulatory and antioxidant properties of biologically active proteins of goat colostrum, as well as the prospects of their use in medicine. Due to the presence of a complex of biologically active proteins such as lactoferrin, lysozyme, lactoperoxidase, immunoglobulins, etc., goat colostrum increases the body's resistance to infectious diseases, strengthens the immune system, has antibacterial activity against a wide range of microorganisms, and has an antioxidant effect, thereby preventing or delaying chronic diseases associated with oxidative stress. In addition, goat colostrum bioactive proteins show antitumor activity, antiatherogenic properties, the ability to lower blood pressure and efficiency in the treatment of rheumatoid arthritis.
The development of infants needs an adequate amount of high-quality nutrition. Breastfeeding is the healthiest nutritional source for neonates throughout the initial months before weaning. Healthcare practitioners recommend that mothers utilize a breast milk substitute (BMS) if breast milk is unavailable. Infant formula is a breast-milk substitute that can meet all of the infant’s dietary requirements from birth to the introduction of supplemental feeding. These formulas are designed to provide adequate nutrition for infants. Still, some studies have shown the risks associated with infant formula consumption. The health issues include increased gastrointestinal diseases, respiratory infections, altered intellectual development, etc. Nutritionists and researchers are attempting to find ways to produce safe infant formula. Generally, different types of infant formulas, depending upon the added components and based upon cow milk, whey protein, elemental formula or amino-acid-based formulae, lactose-free formulae, etc., are available on the market. The current approaches and methods in developing infant formulae include supplementing the fat globular membrane, high-pressure pasteurization, and synthesis of human milk oligosaccharides in vitro, etc. This review paper will focus on various approaches to developing and formulating infant food. It further helps to disseminate current information to scientists and nutritionists about developing nutritious, risk-free infant food.
Bovine lactoferrin (bLF) is widely known as an iron-binding glycoprotein from the transferrin family. The bLF molecule exhibits a broad spectrum of biological activity, including iron delivery, antimicrobial, antiviral, immunomodulatory, antioxidant, antitumor, and prebiotic functions, thereby making it one of the most valuable representatives for biomedical applications. Remarkably, LF functionality might completely differ in dependence on the iron saturation state and glycosylation patterns. Recently, a violently growing demand for bLF production has been observed, mostly for infant formulas, dietary supplements, and functional food formulations. Unfortunately, one of the reasons that inhibit the development of the bLF market and widespread protein implementation is related to its negligible amount in both major sources—colostrum and mature milk. This study provides a comprehensive overview of the significance of bLF research by delineating the key structural characteristics of the protein and elucidating their impact on its physicochemical and biological properties. Progress in the development of optimal isolation techniques for bLF is critically assessed, alongside the challenges that arise during its production. Furthermore, this paper presents a curated list of the most relevant instrumental techniques for the characterization of bLF. Lastly, it discusses the prospective applications and future directions for bLF-based formulations, highlighting their potential in various fields.
Extensive research from large prospective cohort studies and meta-analytical investigations over recent decades have consistently indicated that dairy foods have protective effects, reducing the risk of colorectal cancer. Most of the literature has explored the potential role of milk minerals and vitamins in managing colorectal cancer. Yet, there is a paucity of a comprehensive summary of the anticancer attributes of milk protein components and their underlying mechanisms of action. Recent advancements have spotlighted the potential of whey proteins, including β-lactoglobulin, α-lactalbumin, serum albumin, and lactoferrin, as promising candidates for both the prevention and treatment of colorectal cancer. Notably, whey proteins have demonstrated a more pronounced capacity for suppressing carcinogen-induced tumors when compared to casein. Their strong binding affinity enables them to serve as effective carriers for small molecules or drugs targeting colon cancer therapy. Furthermore, numerous studies have underscored the anti-inflammatory and antioxidant prowess of whey proteins in cancer prevention. Additionally, whey proteins have been shown to trigger apoptosis, hinder tumor cell proliferation, and impede metastasis. This comprehensive review, therefore, not only substantiates the significance of incorporating whey protein components into a balanced daily diet but also underscores their potential in safeguarding against the onset and progression of colorectal cancer.
Goat milk and protein hydrolysate peptides have generated significant interest due to their diverse range of biological functions. To produce these peptides, casein (CP), and whey proteins (WP) were partially purified from goat milk and subsequently hydrolyzed with gastrointestinal endopeptidases such as pepsin, trypsin, and α‐chymotrypsin based on in silico selection to achieve gastrointestinal stable peptides. The hydrolysates were characterized using various techniques, including zeta sizer, polydispersity index (PDI), zeta potential, and reverse‐phase high‐performance liquid chromatography (RP‐HPLC). The resulting peptides were then purified using RP‐HPLC. To assess cytotoxicity, an MTT (3‐[4, 5‐dimethylthiazol‐2‐yl]‐2, 5‐diphenyl tetrazolium bromide) assay was performed with fluorescence microscopy. Finally, cytokine levels were determined using enzyme‐linked immunosorbent assay (ELISA). In comparison to WP and CP, the casein protein hydrolysates (CPH) and whey protein hydrolysates (WPH) peptides average particle size and PDI were decreased. The zeta potential of casein and whey peptides hydrolysates there were no differences. The RP‐HPLC analysis revealed the production of peptides through the hydrolysis of CP and WP. These hydrolysates peptides were found to possess diverse biological activities, as evidenced by their significant inhibition of α‐amylase, pancreatic lipase (PL), and angiotensin‐converting enzyme (ACE), as well as their antibacterial properties against selected pathogens. Further investigations were carried out to understand the mode of action of these peptides using fluorescence and scanning electron microscope (SEM) microscopy. This study provides the first evidence of gastrointestinal stable peptides derived from goat milk with inhibitory activity against α‐amylase, PL, and ACE, as well as antibacterial properties against specific pathogens. Additionally, the peptides demonstrated significant cytokine inhibition and low toxicity toward the HT‐29 cell line. This study strongly suggests that the biologically active peptides responsible for the health‐related bioactive properties described here, which are considered functional and nutraceutical ingredients, should be identified and validated in subsequent research, particularly the most effective hydrolysate peptides derived from goat milk protein. Goat milk is widely recognized as an excellent source of milk protein. In this study, bioactive peptides were produced using casein and whey proteins from goat milk, which exhibited inhibitory activity against angiotensin converting enzyme, α‐amylase, and pancreatic lipase, as well as antibacterial properties. Goat milk protein is a promising source for developing high‐quality protein products with excellent safety standards that have potential applications in the pharmaceutical and food industries.
Sephadex G-100 ب زمب زانمبن زنبكتةي ربعاتاربش صنلبعمىبمي تم زنمب زربتام ةيا كنبكت زي زممبش زربنع اةا زامربكترين ص زنبكت فاة ب ز ز بب ز زامبكتلوش ز يج كت بن زنة ز زاملبكيا ر كت 66.89% ب ن 55.86% )بن 46.13 ب ن 57.58 زنب ز زمبش ز ف زمبكت ز زاب بفش كت زن ز زىبكت ز )بعم امكمب ز س بر زار ر البكتفلي ي كءبكت ي انبر ةيانمبكترين زومب ب كي زار زلبكتشاس كش زنمبكتتن زمبرن اوشاا في SDS ب ز زهبهل ا زرب لش ينب ب رين ان ممبتمرين ك اةاربن ؛ ب ز ة ززبنل زهبرم ا ب زفا ثمبميسنبرتضبشنبص ب ب ز ار ل كت 79 ب زننب زنبمكت فام زنمب رام ز زامبكتلوش يج كت ب ن 79.5 ب زار ز ز ر زلبكتفلي ز ز ا ي زنمبكت ز ز زننبرام ز ز زنبمكت ز ز فام. ب زان ز ز ف ب انب زين ز ز بتمر ك زمي ز ز رنبا زنلبكتفاي ز ز كتش 11.08% ب زررب ز ز زنبةس ز ز زابرمن ز ز راةش مامب كت 169.3 ب جرلب لءبراتشماننبنر 12.09%. ب ا ا فمشانبشف ر ان اي ن :بيف ، ب رن بكت جي ، ب علل ، ب ةيار ، ب نصاف ، ب اصارن ب Abstract Lactoferrin isolated from whey cheese by using Ion exchange chromatography through CM-Sephadex C-50 before this stage the whey was skimmed by Cooling centrifugation and demineralized against distilled water using dialysis bag, after this stage gel filtration chromatography was used to purified lactoferrin in a high degree of purification. The purity of lactoferrin to homogeneity was examined by polyacrylamide gel electrophoresis under denaturing conditions (SDS-PAGE) with single band. The purification fold and yield were (55.86% time and 66.89%) and (57.58 and 46.13) respectively. Some biochemical characteristics of the purified Lactoferrin were determined. The molecular weight of LF were 79 and 79.5 KD as determined by gel filtration and SDS-PAGE respectively. The percentage of carbohydrate content in bovine Lactoferrin was 11.08% mean while the Iron percentage was 169.3 ppm and as a saturated percent 12.09%.
This study was conducted to genetically and environmentally characterize prolificacy (litter size and weight at birth; LSB and LWB and litter size and weight at weaning; LSW and LWW, respectively), milk yield at the 7th (MY7), 15th (MY15), 30th (MY30), 60th (MY60), 90th (MY90) day of lactation and monthly milk yield (MMY) and milk composition traits in Egyptian Zaraibi goats. A total of 443 and 421 records produced by 121 Zaraibi lactating goats were used to assess prolificacy and milk production traits, respectively. The milk composition traits were measured using 371 milk samples obtained at random from 53 goats. The fourth parity showed the highest values for LWB, LWW, and MMY (3.62, 18.15, and 28.99 kg, respectively). Milk composition traits revealed an inverse tendency, decreasing until the second month and then increasing until the seventh month. The heritability estimates ranged from 0.07 to 0.13, from 0.04 to 0.39, and from 0.07 to 0.33 for prolificacy, milk yield, and milk composition traits, respectively. Negatively high genetic correlations between MMY and all milk composition traits were found. MMY had the highest estimate of heritability (0.39 ± 0.07), this means that the genetic improvement of this trait could be achieved through direct selection.
Background
Nutrients in milk are abundant, and the rapid development of biomass spectrometry and bioinformatics algorithms has provided technical support to further elucidate nutrient content in milk such as N-glycoproteins, which are important post-translational modifications of proteins that have attracted significant research attention for their unique physicochemical and functional properties.
Scope and approach
We summarize the synthetic pathways of protein N-glycosylation, types of N-glycans, and primary distribution characteristics of N-glycoproteins in milk. Our review covers existing research of MS-based milk protein N-glycosylation in three dimensions: N-glycans, deglycosylated and intact N-glycopeptides obtained by enzymatic digestion of glycoproteins. A brief overview of protein N-glycosylation in dairy processing, absorption, and metabolism as well as biological functions is presented. The limitations and directions for future research on milk protein N-glycosylation are summarized.
Key findings and conclusions
Intact N-glycopeptides obtained by enzymatic digestion of glycoproteins in current studies of milk protein N-glycosylation are insufficiently characterized and the construction of linkages between N-glycans and glycosylation sites must be further enhanced as a prerequisite for exploring milk protein N-glycosylation. N-glycosylation in dairy processing stabilizes protein conformation. Evidence from in vivo and in vitro studies suggests that milk protein N-glycans can be metabolised by intestinal microorganisms, modulate gut microbiota, reduce pathogen adhesion, regulate immune function, alleviate necrotizing enterocolitis, and affect brain development, with different structures having specific biological functions. However, more research is needed to validate existing findings.
Glycans present in glycoproteins are structurally diverse and contribute to the carbohydrate pool of the milk. Goat milk is a leading non-bovine milk source, wherein glycan diversity of several glycoproteins remains unexplored. Herein, site-specific N-glycoprofiling of two major glycoproteins – immunoglobulin G (IgG) and lactoferrin (Lf) from goat milk was performed through RP-UHPLC Q-Tof MS/MS approach. IgG revealed diverse complex glycans that were predominantly biantennary type with differential core fucosylation, bisecting GlcNAc, and mono/di- sialylation (NeuAc/NeuGc). The N-glycan repertoire of Lf at four sites indicated the range of high mannose, complex and hybrid types with varying abundances. High mannose glycans were specifically observed at N252NT and N564DT sites. Majorly complex glycans with fully sialylated were found at N387VT site. While N495QT site revealed complex and hybrid types with differential core fucosylation and sialylation. The glycan features observed in these glycoproteins would pave way for effective utilization as bioactive ingredients.
Lactoferrin (Lf) from buffalo colostrum was probed for N-glycan diversity and abundances that were previously unknown. Out of four N-glycosylation sites present, two sites, N233NT and N545DT, exhibited predominantly high-mannose glycans. Site N281KS showed mainly sialylated hybrid types. A higher abundance of differentially sialylated hybrid glycans was present at N476QT. In addition, the genetic variant of Lf was identified. Overall, this study showed that high mannose and hybrid types are the major glycans in Lf from buffalo colostrum.
Lactoferrin isolated from whey cheese by using Ion exchange chromatography through CM-Sephadex C-50 before this stage the whey was skimmed by Cooling centrifugation and demineralized against distilled water using dialysis bag, after this stage gel filtration chromatography was used to purified lactoferrin in a high degree of purification. The purity of lactoferrin to homogeneity was examined by polyacrylamide gel electrophoresis under denaturing conditions (SDS-PAGE) with single band. The purification fold and yield were (55.86% time and 66.89%) and (57.58 and 46.13) respectively. Some biochemical characteristics of the purified Lactoferrin were determined. The molecular weight of LF were 79 and 79.5 KD as determined by gel filtration and SDS-PAGE respectively. The percentage of carbohydrate content in bovine Lactoferrin was 11.08% mean while the Iron percentage was 169.3 ppm and as a saturated percent 12.09%.
Bovine lactoferrin is a functional N-glycoprotein whose glycan moieties play an important role in its biological activities. Although bovine lactoferrin is rich in sialylated N-glycans, there are no reports on the differentiation between α2,3-linked and α2,6-linked sialylated N-glycan isomers. In this study, bovine lactoferrin was separated and purified by cation exchange chromatography, and > 95% pure protein was obtained. Following this, N-glycan derivatives of lactoferrin at different stages of lactation were qualitatively and quantitatively analyzed by MALDI-TOF-MS and HILIC-MS/MS. With prolonged lactation, the relative content of the sialylated and fucosylated bi-antenna complex N-glycan (Hex1Man3GlcNAc4GalNAc1Fuc1Neu5Ac1) changed the maximum, with 14.5%, 1.3%, and 0.9% contents in transitional milk, colostrum, and mature milk, respectively. Meanwhile, the contents of sialylated and fucosylated N-glycans in transitional milk were the highest. More importantly, the sialylated N-glycans-linked isomers could be distinguished by linkage-specific derivatization of sialic acid. There were 11 α2,3- and 13 α2,6-linked sialyation N-glycans isomers in the colostrum, which transformed to 4 and 3 N-glycan isomers, respectively, in the mature milk. These findings provide a foundation for an in-depth research on the structure–function relationship of bovine lactoferrin.
Before electrophoretic separation is performed, the samples must be dissolved in a lysis buffer (necessary to keep proteins dissolved and unbound during proteomic analyses during for a separation of proteins on polyacrylamide gels). The first step in preparing samples for proteomic analyses is their precipitation using e.g. acetone. The aim of precipitation is to obtain proteins from the sample and to remove the compounds interfering with 2-D electrophoresis. Due to difficulties in dissolving some colostrum and mare's milk samples in buffer lysis electrophoretic separation of this biological material was performed without acetone precipitation of proteins. To assess the effectiveness of the applied method, after two-dimensional separation of proteins (2-DE), the obtained gels were stained and archived. The preparation of mare's colostrum and milk samples for proteomic analyses, consisting of defatting, then precipitation of caseins and separation 2-DE, which was not preceded by precipitation of the samples with acetone, resulted in the loss of many protein spots which made it impossible to identify them later using the mass spectrometer.
Purpose
Tear fluid N‐Glycome from patients affected with vernal (VKC) and atopic keratoconjunctivitis (AKC) was investigated to identify specific changes in tears and to recognize possible glyco‐biomarkers.
Methods
The analysis of the N‐glycans was performed using matrix‐assisted laser desorption ionization mass spectrometry on single tear samples. Tears from control normal subjects (CTRL), VKC and AKC patients were processed and treated with peptide N‐glycosidase F (PNGase F) to deglycosylate N‐glycoproteins. Released N‐glycans were purified, permethylated, and analyzed by matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry and tandem mass spectrometry (MALDI‐TOF MS and MALDI‐TOF MS/MS).
Results
More than 150 complex N‐glycans, including highly fucosylated biantennary, triantennary, tetra‐antennary, and bisecting species, were observed in our spectra. Three distinct patterns for CTRL, VKC, and AKC patients were identified in terms of relative intensities for some N‐glycans structures. Major variations involved bisecting and hyperfucosylated glycoforms.
The most intense ions were associated with species at m/z 1907.0 (asialo, agalacto, bisected, biantennary structure—NGA2B) in CTRL MS profiles, at m/z 2605.3 and 2966.5 in VKC, and at m/z 2792.4 in AKC corresponding to a well‐known biantennary, disialylated N‐glycan. Several peaks were associated with structures bearing one or two Lewis X epitopes. Structures were confirmed by MS/MS analysis. Quantitative differences among the three groups were statistically significant.
Conclusions
Tear MS profiles are rich in specific glycoforms, particularly those with a high fucosylation degree, indicating both core and peripheral decoration. Tear N‐glycome analysis provided important information for a better comprehension of VKC and AKC alterations at the molecular level.
Bovine Lf (bLf) possesses five N-glycosylation sites having diverse glycans that alter over lactation and are extensively characterised in milk. However, detailed site-specific characterisation in colostrum is limited. In the present study, the glycan diversity in colostrum bLf from native and commercial preparations was determined by generating N-glycopeptides using trypsin and a combination of trypsin/chymotrypsin followed by RP-UHPLC-Q-TOF MS/MS. The glycan diversity at N300KS site is poorly understood and the analysis revealed the presence of mostly biantennary complex glycans with core fucosylation and differential sialylation. N252NS and N564DT sites were found to possess predominantly high mannose glycans. A range of complex, hybrid and high mannose glycans were determined at N387VT and N495QT sites. Glycan heterogeneity with relative abundance was determined on all glycosylation sites in bLf. In addition, the glycan diversity of lipocalin-2 (N85VT) that might have co-purified in the commercial colostrum bLf sample was also elucidated.
Studies on the possible alternative supplements to breastmilk are gaining research interests. Although milk from cow, goat, and mare is nutritious, its effects on the relationship between the immune system, metabolites, and gut microbiota remain unclear. This study aimed to comprehensively evaluate the effects of cow, goat, and mare milk on the immune system, metabolites, and gut microbiota of mice colonized by healthy infant feces using human milk as a standard. We examined the serum biochemistry parameters, immunity indicators, T cells, gut microbiota abundance, and metabolites. Results showed that the impact of human milk on alanine transaminase, glutamic oxaloacetic transaminase, total protein, globulin, and glucose values was different from the cow, goat, and mare milk types. The effects of mare milk on the percentage of CD4+ T, Th1, Th2, Th17, and Treg cells, and the levels of IL-2, IL-4, sIgA, and D-lactic acid in the serum of the human microbiota-associated mice were comparable to those of human milk. Also, bacterial 16S rRNA gene sequence analysis revealed that human milk enriched the relative abundance of Akkermansia and Bacteroides, cow milk increased the relative abundance of Lactobacillus, goat milk increased the relative abundance of Escherichia-Shigella, and mare milk improved the relative abundance of Klebsiella. Besides, mare milk was similar to human milk in the concentration of the metabolites we analyzed. Our findings suggest that mare milk can positively modulate the gut microbiota and immunity status of infants and thus could be a possible replacement for human milk.
Bovine milk-derived exosomes (BMDEs) have potential applications in the pharmaceutical industry as drug delivery carriers. A comprehensive analysis of protein glycosylation in exosomes is necessary to elucidate the process of targeted delivery. In this work, free oligosaccharides (FOSs), O-glycans, and N-glycans in BMDEs and whey were first analyzed through multiple derivation strategies. In summary, 13 FOSs, 44 O-glycans, and 94 N-glycans were identified in bovine milk. To analyze site-specific glycosylation of glycoproteins, a one-step method was used to enrich and characterize intact glycopeptides. A total of 1359 proteins including 114 glycoproteins were identified and most of these were located in the exosomes. Approximately 95 glycopeptides were initially discovered and 5 predicted glycosites were confirmed in BMDEs. Collectively, these findings revealed the characterization and distribution of glycans and glycoproteins in BMDEs, providing insight into the potential applications of BMDEs in drug delivery and food science.
Human milk contains a large variety of proteins, lipids, and microbes. A better understanding of the proteins, lipids, and microorganism species of human milk is the essential basis for formulation and improvement of infant formulas in the future. With the development of omics, great progress has been made in human milk composition. The identification and quantification of several human and mammalian milk protein systems and lipid profiles at the group and molecular species level have become a reality, providing useful information for the infant formula industry. In this chapter, the analytical methods and strategies and the bioinformatics of proteomics and lipidomics for human milk are discussed. The human milk microbiome, including the predominate microbes, plausible functions, and potential probiotics, are also discussed.
Sephadex G-100 ب زمب زانمبن زنبكتةي ربعاتاربش صنلبعمىبمي تم زنمب زربتام ةيا كنبكت زي زممبش زربنع اةا زامربكترين ص زنبكت فاة ب ز ز بب ز زامبكتلوش ز يج كت بن زنة ز زاملبكيا ر كت 66.89% ب ن 55.86% )بن 46.13 ب ن 57.58 زنب ز زمبش ز ف زمبكت ز زاب بفش كت زن ز زىبكت ز )بعم امكمب ز س بر زار ر البكتفلي ي كءبكت ي انبر ةيانمبكترين زومب ب كي زار زلبكتشاس كش زنمبكتتن زمبرن اوشاا في SDS ب ز زهبهل ا زرب لش ينب ب رين ان ممبتمرين ك اةاربن ؛ ب ز ة ززبنل زهبرم ا ب زفا ثمبميسنبرتضبشنبص ب ب ز ار ل كت 79 ب زننب زنبمكت فام زنمب رام ز زامبكتلوش يج كت ب ن 79.5 ب زار ز ز ر زلبكتفلي ز ز ا ي زنمبكت ز ز زننبرام ز ز زنبمكت ز ز فام. ب زان ز ز ف ب انب زين ز ز بتمر ك زمي ز ز رنبا زنلبكتفاي ز ز كتش 11.08% ب زررب ز ز زنبةس ز ز زابرمن ز ز راةش مامب كت 169.3 ب جرلب لءبراتشماننبنر 12.09%. ب ا ا فمشانبشف ر ان اي ن :بيف ، ب رن بكت جي ، ب علل ، ب ةيار ، ب نصاف ، ب اصارن ب Abstract Lactoferrin isolated from whey cheese by using Ion exchange chromatography through CM-Sephadex C-50 before this stage the whey was skimmed by Cooling centrifugation and demineralized against distilled water using dialysis bag, after this stage gel filtration chromatography was used to purified lactoferrin in a high degree of purification. The purity of lactoferrin to homogeneity was examined by polyacrylamide gel electrophoresis under denaturing conditions (SDS-PAGE) with single band. The purification fold and yield were (55.86% time and 66.89%) and (57.58 and 46.13) respectively. Some biochemical characteristics of the purified Lactoferrin were determined. The molecular weight of LF were 79 and 79.5 KD as determined by gel filtration and SDS-PAGE respectively. The percentage of carbohydrate content in bovine Lactoferrin was 11.08% mean while the Iron percentage was 169.3 ppm and as a saturated percent 12.09%.
Metabolomics deals with the identification and quantification of the small molecules involved in metabolic reactions. It has recently been used to understand the nutritional quality and authenticity of milk and milk products. Various analytical techniques such as nuclear magnetic resonance spectroscopy, liquid chromatography–mass spectrometry, gas chromatography–mass spectrometry and ultra‐performance liquid chromatography–quadrupole time‐of‐flight mass spectrometry have been successfully employed to identify and quantify the metabolites present in milk and milk products. This review focuses on the recent advances in metabolomics for quality and safety analyses of milk and milk products.
Lactoferrin, a minor whey protein present mainly in milk as well as in small quantities in most of the secretions of the body, has a wide range of biological activities to its credit including antimicrobial, antioxidant, anticancer and immunomodulatory properties. A low molecular weight peptideisolated from the pepsin hydrolysate of lactoferrin called aslactoferricin B, has been found to be more functionally active than its parent compound. The present work focussed on the isolation of lactoferrin from the colostrumof Malabari goats by cation exchange chromatography, followed by the assessment of its molecular weight by SDS-PAGE andits characterization by dot blot assay and western blotting.The concentration of lactoferrin as estimated by Lowry's method was found to be 15.103 mg/L of colostrum. Lactoferrin was hydrolysed by treatment with three per cent porcine pepsin under acidic conditions to form lactoferrin pepsin hydrolysate. The results of this study point to a single one step method to obtain pure lactoferrin from goats and further preparation of its pepsin hydrolysate
Nowadays, miniaturized liquid chromatography (LC) coupled to mass spectrometry is a leading analytical technique in several modern research areas. Its applications will keep in constant spreading, and new and miniaturized instrumental advances will continue to be necessary for the coming years. In this way, chip-based LC-MS technology is one of the most efficient strategies to i) address the extra column band broadening challenges of the capillary/nanoscale, and ii) provide portability, lower-cost and user-friendly character to the miniaturized LC-MS systems. Several commercial chip-based LC-MS are available, because of the notable developments in the manufacturing of the analytical chips throughout the last decades. Since the emerging of the first instruments based on MEMS LC-MS (microelectromechanical systems), diverse microfabrication technologies have been reported to engrave and model a wide gamma of materials and substrates. Within such a context, many strategies to on-chip fully-integration of the solvent delivering systems, injectors, chromatographic media, mass spectrometry interfaces, ionization sources, and mass analyzers have been explored and its feasibility demonstrated. Herein, we provide a comprehensive overview of the major chip technology developments, their state-of-art, and a consistent survey of the recent literature on it. Finally, a description about the most diverse applications of the fully integrated chip-based LC-MS platforms is summarized. In this case, showing remarkable examples using mono and bidirectional systems to the analysis of macro- and small molecules of biochemical, biological, pharmaceutical, clinical, forensic, environmental, foodstuff and quality control interest.
Very little is known about the effects of gestational diabetes mellitus (GDM) on lactation and milk components. Recent reports suggested that hyperglycemia during pregnancy was associated with altered breast milk immune factors. Human milk oligosaccharides (HMOs) and N-glycans of milk immune-modulatory proteins are implicated in modulation of infant immunity. The objective of the current study was to evaluate the effect of GDM on HMO and protein-conjugated glycan profiles in breast milk. Milk was collected at 2 wk postpartum from women diagnosed with (n = 8) or without (n = 16) GDM at wk 24-28 in pregnancy. Milk was analyzed for HMO abundances, protein concentrations, and N-glycan abundances of lactoferrin and secretory IgA (sIgA). HMOs and N-glycans were analyzed by mass spectrometry and milk lactoferrin and sIgA concentrations were analyzed by the Bradford assay. The data were analyzed using multivariate modeling confirmed with univariate statistics to determine differences between milk of women with compared with women without GDM. There were no differences in HMOs between milk from women with vs. without GDM. Milk from women with GDM compared with those without GDM was 63.6% lower in sIgA protein (P < 0.05), 45% higher in lactoferrin total N-glycans (P < 0.0001), 36-72% higher in lactoferrin fucose and sialic acid N-glycans (P < 0.01), and 32-43% lower in sIgA total, mannose, fucose, and sialic acid N-glycans (P < 0.05). GDM did not alter breast milk free oligosaccharide abundances but decreased total protein and glycosylation of sIgA and increased glycosylation of lactoferrin in transitional milk. The results suggest that maternal glucose dysregulation during pregnancy has lasting consequences that may influence the innate immune protective functions of breast milk.
Milk is an excellent source of well balanced nutrients and also exhibits a range of biological activities that influence digestion, metabolic responses to absorbed nutrients, growth and development of specific organs, and resistance to disease. Goat milk is as close to perfect food as possible in nature. Bioactive peptides have been defined as specific protein fragments that have a positive impact on body functions or conditions and may ultimately influence health. It can be generated during milk fermentation by the proteolytic activity of starter cultures. The beneficial health effects may be classified as antimicrobial, antioxidative, antithrombotic, antihypertensive and immunomodulatory. Sheep and goat milk proteins are also important sources of bioactive ACE inhibitory peptides and antihypertensive peptides. They can provide a non- immune disease defence and control of microbial infections. The activity of these biofunctional peptides is based on their inherent amino acid composition and sequence. The size of active sequences may vary from two to twenty amino acid residues. The total antibacterial effect in milk is greater than the sum of the individual contributions of immunoglobulin and nonimmunoglobulin defence proteins such as LF, LP, lysozyme, and other peptides. A variety of naturally formed bioactive peptides have been found in fermented dairy products, such as yoghurt, sour milk and cheese. BP have the potential to be used in the formulation of health-enhancing nutraceuticals, and as potent drugs with well defined pharmacological effects.
Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 strains are associated with haemor - raghic colitis and haemolytic uremic syndrome (HUS) in humans. Cattle are a reservoir of E. coli O157:H7. We studied the ability of bovine and human lactoferrin, two natural antimicrobial proteins present in milk, to inhibit E. coli O157:H7 growth and attachment to a human epithelial colorectal adenocarcinoma cell line (Caco-2). The direct antibacterial effect of bLF on E. coli O157:H7 was stronger than that of hLF. Nevertheless, both lactofer - rins had bacteriostatic effects even at high concentrations (10 mg/ml), suggesting blocking of LF activity by a yet undefined bacterial defence mechanism. Additionally, both lactoferrins significantly inhibited E. coli O157:H7 attachment to Caco-2 cells. However, hLF was more effective than bLF, probably due to more efficient binding of bLF to intelectin present on human enterocytes leading to uptake and thus removal of bLF from the extracellular environment. Inhibition of bacterial attachment to Caco-2 cells was at least partly due to the catalytic effect of lactoferrins on the type III secreted proteins EspA and EspB
Milk is traditionally considered an ideal source of the basic elemental nutrients required by infants. More detailed examination is revealing that milk represents a more functional ensemble of components with benefits to both infants and mothers. A comprehensive peptidomics method was developed and used to analyze human milk yielding an extensive array of protein products present in the fluid. Over 300 milk peptides were identified originating from major and many minor protein components of milk. As expected, the majority of peptides derived from β-casein, however no peptide fragments from the major milk proteins lactoferrin, α-lactalbumin and secretory immunoglobulin A were identified. Proteolysis in the mammary gland is selective-released peptides were drawn only from specific proteins and typically from only select parts of the parent sequence. A large number of the peptides showed significant sequence overlap with peptides with known antimicrobial or immunomodulatory functions. Antibacterial assays showed the milk peptide mixtures inhibited the growth of Escherichia coli and Staphylococcus aureus. The pre-digestion of milk proteins and the consequent release antibacterial peptides may provide a selective advantage through evolution by protecting both the mother's mammary gland and her nursing offspring from infection.
The goal of the current study was to identify proteins in goat milk before and at 18 h following intramammary challenge with lipopolysaccharide (LPS). Initial evaluation of protein profiles generated using 2-dimensional gel electrophoresis on skim milk samples from a group of 6 goats collected before challenge and at 18, 24, and 48 h after LPS challenge revealed little change in the abundance of casein proteins, and minimal changes in the presence or abundance of the plasma protein serum albumin, which is known to leak into milk during coliform mastitis in dairy cattle. Proteins in baseline milk samples and in milk from the same goats 18 h post-LPS challenge were excised from the gels, and peptides were sequenced using nano-flow liquid chromatography coupled with tandem mass spectrometry. Despite the overwhelming presence of casein proteins and β-lactoglobulin, the lower abundance proteins β-2-microglobulin, fatty acid-binding protein, serum albumin, and retinol-binding protein were detected in skim milk samples from healthy goats. Skim milk samples 18 h postchallenge were characterized by the sustained presence and abundance of the casein proteins, and by the presence of haptoglobin, serum amyloid A, lactoferrin, cathelicidin-1, and cathelicidin-3. No marked differences in the intensity of the spot corresponding to serum albumin were observed in gels of skim milk samples 18 h postchallenge, which could indicate that the breakdown of the blood-milk barrier during endotoxin mastitis may not be as profound in goats as has been observed in dairy cattle. Nonetheless, the occurrence of an inflammatory response was supported by elevated somatic cell counts in the goat milk following inoculation with endotoxin, as well as by the presence of both antimicrobial and acute phase proteins. The results provide information about the composition of proteins in goat milk as well as added knowledge of the host response during endotoxin mastitis in goats.
Human milk is a rich source of nutrients and energy, shaped by mammalian evolution to provide all the nutritive requirements of the newborn. In addition, several molecules in breast milk act as bioactive agents, playing an important role in infant protection and guiding a proper development. While major breast milk nutrients such as lactose, lipids and proteins are readily digested and consumed by the infant, other molecules, such as human milk oligosaccharides and glycosylated proteins and lipids, can escape intestinal digestion and transit through the gastrointestinal tract. In this environment, these molecules guide the composition of the developing infant intestinal microbiota by preventing the colonization of enteric pathogens and providing carbon and nitrogen sources for other colonic commensals. Only a few bacteria, in particular Bifidobacterium species, can gain access to the energetic content of milk as it is displayed in the colon, probably contributing to their predominance in the intestinal microbiota in the first year of life. Bifidobacteria deploy exquisite molecular mechanisms to utilize human milk oligosaccharides, and recent evidence indicates that their activities also target other human milk glycoconjugates. Here, we review advances in our understanding of how these microbes have been shaped by breast milk components and the strategies associated with their consumption of milk glycoconjugates.
Bovine milk oligosaccharides (BMOs) are recognized by the dairy and food industries, as well as by infant formula manufacturers, as novel, high potential bioactive food ingredients. Recent studies revealed that bovine milk contains complex oligosaccharides structurally related to those previously thought to be present in only human milk. These BMOs are microbiotic modulators involved in important biological activities, including preventing pathogen binding to the intestinal epithelium and serving as nutrients for a selected class of beneficial bacteria. Only a small number of BMO structures are fully elucidated. To better understand the potential of BMOs as a class of bio-therapeutics, their detailed structure analysis is needed. This study initiated the development of a structure library of BMOs and a comprehensive evaluation of structure-related specificity. The bovine milk glycome was profiled by high-performance mass spectrometry and advanced separation techniques to obtain a comprehensive catalog of BMOs, including several novel, lower abundant neutral and fucosylated oligosaccharides that are often overlooked during analysis. Structures were identified using isomer-specific tandem mass spectroscopy and targeted exoglycosidase digestions to produce a BMO library detailing retention time, accurate mass, and structure to allow their rapid identification in future studies.
The microbic colonization of human intestine begins at birth, when from a sterile state the newborn is exposed to an external environment rich in various bacterial species. The kind of delivery has an important influence on the composition of the intestinal flora in the first days of life. Thereafter, the microflora is mainly influenced by the kind of feeding: breast-fed infants show a predominance of bifidobacteria and lactobacilli, whereas bottle-fed infants develop a mixed flora with a lower number of bifidobacteria.The “bifidogenic effect” of human milk is not related to a single growth-promoting substance, but rather to a complex of interacting factors. In particular the prebiotic effect has been ascribed to the low concentration of proteins and phosphates, the presence of lactoferrin, lactose, nucleotides and oligosaccharides.The real prebiotic role of each of these substances is not yet clearly defined, with the exception of oligosaccharides which undoubtedly promote a bifidobacteria-dominant microflora.
Human milk lactoferrin (hmLF) is the most abundant glycoprotein present in human milk and displays a broad range of protective functions in the gut of newborn infants. hmLF is N-glycosylated, but little is known about the lactation stage-related development of the glycosylation phenotype. hmLF glycosylation from milk samples from five donors during the first 10 weeks of lactation was assessed and observed to be more diverse than previously reported. During this period dynamic changes in glycosylation were observed corresponding to a decrease in glycosylation in the second week followed by an increase in total glycosylation as well as higher order fucosylation thereafter. Gene expression analysis was performed in milk somatic cells from a sixth subject. It was found that fucosyltransferase expression increased during entire period, whereas expression of genes for the oligosaccharyl transferase complex decreased in the second week. The effect of hmLF glycosylation was examined for the protein's ability to affect bacterial binding to epithelial cells. hmLF significantly inhibited pathogen adhesion and purified hmLF glycans significantly reduced Salmonella invasion of colonic epithelial cells to levels associated with non-invasive deletion mutants. This study indicates that hmLF glycosylation is tightly regulated by gene expression and that glyco-variation is involved in modulating pathogen association.
N-Linked glycans of skim human milk proteins were determined for three mothers. N-Linked glycans are linked to immune defense, cell growth, and cell-cell adhesion, but their functions in human milk are undetermined. Protein-bound N-linked glycans were released with peptidyl N-glycosidase F (PNGase F), enriched by graphitized carbon chromatography, and analyzed with Chip-TOF MS. To be defined as N-glycans, compounds were required, in all three procedural replicates, to match, within 6 ppm, against a theoretical human N-glycan library and be at least 2-fold higher in abundance in PNGase F-treated than in control samples. Fifty-two N-linked glycan compositions were identified, and 24 were confirmed via tandem mass spectra analysis. Twenty-seven compositions have been found previously in human milk, and 25 are novel compositions. By abundance, 84% of N-glycans were fucosylated and 47% were sialylated. The majority (70%) of total N-glycan abundance was composed of N-glycans found in all three milk samples.
Human milk contains an unexpected abundance and diversity of complex oligosaccharides apparently indigestible by the developing infant and instead targeted to its cognate gastrointestinal microbiota. Recent advances in mass spectrometry-based tools have provided a view of the oligosaccharide structures produced in milk across stages of lactation and among human mothers. One postulated function for these oligosaccharides is to enrich a specific "healthy" microbiota containing bifidobacteria, a genus commonly observed in the feces of breast-fed infants. Isolated culture studies indeed show selective growth of infant-borne bifidobacteria on milk oligosaccharides or core components therein. Parallel glycoprofiling documented that numerous Bifidobacterium longum subsp. infantis strains preferentially consume small mass oligosaccharides that are abundant early in the lactation cycle. Genome sequencing of numerous B. longum subsp. infantis strains shows a bias toward genes required to use mammalian-derived carbohydrates by comparison with adult-borne bifidobacteria. This intriguing strategy of mammalian lactation to selectively nourish genetically compatible bacteria in infants with a complex array of free oligosaccharides serves as a model of how to influence the human supraorganismal system, which includes the gastrointestinal microbiota.
This review discusses the role of human milk glycans in protecting infants, but the conclusion that the human milk glycans constitute an innate immune system whereby the mother protects her offspring may have general applicability in all mammals, including species of commercial importance. Infants that are not breastfed have a greater incidence of severe diarrhea and respiratory diseases than those who are breastfed. In the past, this had been attributed primarily to human milk secretory antibodies. However, the oligosaccharides are major components of human milk, and milk is also rich in other glycans, including glycoproteins, mucins, glycosaminoglycans, and glycolipids. These milk glycans, especially the oligosaccharides, are composed of thousands of components. The milk factor that promotes gut colonization by Bifidobacterium bifidum was found to be a glycan, and such prebiotic characteristics may contribute to protection against infectious agents. However, the ability of human milk glycans to protect the neonate seems primarily to be due to their inhibition of pathogen binding to their host cell target ligands. Many such examples include specific fucosylated oligosaccharides and glycans that inhibit specific pathogens. Most human milk oligosaccharides are fucosylated, and their production depends on fucosyltransferase enzymes; mutations in these fucosyltransferase genes are common and underlie the various Lewis blood types in humans. Variable expression of specific fucosylated oligosaccharides in milk, also a function of these genes (and maternal Lewis blood type), is significantly associated with the risk of infectious disease in breastfed infants. Human milk also contains major quantities and large numbers of sialylated oligosaccharides, many of which are also present in bovine colostrum. These could similarly inhibit several common viral pathogens. Moreover, human milk oligosaccharides strongly attenuate inflammatory processes in the intestinal mucosa. These results support the hypothesis that oligosaccharides and other glycans are the major constituents of an innate immune system of human milk whereby the mother protects her infant from enteric and other pathogens through breastfeeding. These protective glycans may prove useful as a basis for the development of novel prophylactic and therapeutic agents that inhibit disease by mucosal pathogens in many species.
Acute gastrointestinal infections due to rotaviruses and other enteric pathogens are major causes of morbidity and mortality in infants and young children throughout the world. Breast-feeding can reduce the rate of serious gastroenteritis in infants; however, the degrees of protection offered against rotavirus infection vary in different populations. The mechanisms associated with milk-mediated protection against viral gastroenteritis have not been fully elucidated. We have isolated a macromolecular component of human milk that inhibits the replication of rotaviruses in tissue culture and prevents the development of gastroenteritis in an animal model system. Purification of the component indicates that the antiviral activity is associated with an acidic fraction (pI = 4.0-4.6), which is free of detectable immunoglobulins. Furthermore, high levels of antiviral activity are associated with an affinity-purified complex of human milk mucin. Deglycosylation of the mucin complex results in the loss of antiviral activity. Further purification indicated that rotavirus specifically binds to the milk mucin complex as well as to the 46-kD glycoprotein component of the complex. Binding to the 46-kD component was substantially reduced after chemical hydrolysis of sialic acid. We have documented that human milk mucin can bind to rotavirus and inhibit viral replication in vitro and in vivo. Variations in milk mucin glycoproteins may be associated with different levels of protection against infection with gastrointestinal pathogens.
Human milk contains a wide variety of proteins that contribute to its unique qualities. Many of these proteins are digested and provide a well-balanced source of amino acids to rapidly growing infants. Some proteins, such as bile salt–stimulated lipase, amylase, β-casein, lactoferrin, haptocorrin, and α1-antitrypsin, assist in the digestion and utilization of micronutrients and macronutrients from the milk. Several proteins with antimicrobial activity, such as immunoglobulins, κ-casein, lysozyme, lactoferrin, haptocorrin, α-lactalbumin, and lactoperoxidase, are relatively resistant against proteolysis in the gastrointestinal tract and may, in intact or partially digested form, contribute to the defense of breastfed infants against pathogenic bacteria and viruses. Prebiotic activity, such as the promotion of the growth of beneficial bacteria such as Lactobacilli and Bifidobacteria, may also be provided by human milk proteins. This type of activity can limit the growth of several pathogens by decreasing intestinal pH. Some proteins and peptides have immunomodulatory activities (eg, cytokines and lactoferrin), whereas others (eg, insulin-like growth factor, epidermal growth factor, and lactoferrin) are likely to be involved in the development of the intestinal mucosa and other organs of newborns. In combination, breast-milk proteins assist in providing adequate nutrition to breastfed infants while simultaneously aiding in the defense against infection and facilitating optimal development of important physiologic functions in newborns.
Caseins are phosphoproteins synthesised by mammary epithelial cells under multi-hormonal control as more or less large and stable particles, referred to as casein micelles and which appear like raspberries in electron micrographs. These spherical particles seem to be the result of aggregation of smaller discrete subunits or submicelles (Schmidt, 1982; Walstra, 1990), cemented by a calcium phosphate salt (colloidal calcium phosphate). Although this casein micelle model is widely accepted, it remains, in some circumstances, a topic of discussion and controversial debate. This issue will be dealt with in detail elsewhere in this book (Chapters 1 and 5). Casein micelles are present in the milk of all mammals and have a statistically broad distribution in size (Holt, 1985, 1992). In bovine milk, the most thoroughly studied milk to date, the casein micelle is considered to be comprised of submicelles, made of several casein molecules (15 to 20; Schmidt, 1982), arising from the expression of four single-copy autosomal genes which encode four distinct polypeptide chains (αs1-, β-, αs2- and κ-caseins).
The heterogeneity of caprine caseinmacropeptide (CMP) was determined by means of treatments with neuraminidase and acid phosphatase and analyses by anion exchange FPLC and reversed-phase (RP)-HPLC, with on-line and off-line electrospray ionization mass spectrometry. The main CMP components were two non-glycosylated and di-phosphorylated forms, as well as two other mono-phosphorylated species, each corresponding to a genetic variant of caprine 14% and 86–92%, respectively. Approximately 36% of caprine CMP was glycosylated. Based on the obtained molecular masses, the occurrence of tri-, di- and monosaccharide-containing di-phosphorylated CMP are reported, assuming that N-acetylgalactosamine, galactose, N-acetyl and N-glycolylneuraminic acids would constitute the main monosaccharides of caprine CMP. CMP microheterogeneity due to the genetic polymorphism was also observed in the glycosylated forms.
Lactoferrin is an iron-binding glycoprotein and is considered a major part of the non-specific disease resistance complex in the mammary gland. For cows, the influence of physiological factors on the lactoferrin concentration in milk has been reported. In addition, lactoferrin concentrations have been demonstrated to be proportional to somatic cell counts (SCC) in cows milk. In this study, we aimed to analyse the effects of lactational stage, lactation number and SCC in 19 goats throughout an entire lactational period. Lactoferrin concentrations in weekly composite milk samples were analysed with a competitive ELISA developed for caprine lactoferrin. Maximal lactoferrin concentrations were observed in the colostral samples (387±69μg/ml). In the following week, less than 20% of these concentrations were observed (62±25μg/ml) and thereafter until week 32 p.p., the weekly mean concentrations ranged between 10 and 28μg/ml. Toward the end of lactation, approximately during the 33rd week, the concentrations began to increase and were reaching about 3.2-fold higher values in week 44 (107±19μg/ml). SCC were only available in monthly intervals and could thus not be directly related to the weekly lactoferrin recordings. When classifying the individual goats according to the median of their SCC values obtained during midlactation, the goats with SCC medians >430,000 had higher lactoferrin milk concentrations during this time than the ones with SCC below this threshold (P
1.1. Action potentials and ionic membrane currents of the hypotrich ciliate Stylonychia mytilus were investigated in solutions of various Ba2+ and Ca2+ content, keeping the total concentrations of divalent cations constant.2.2. All ionic currents-early inward, delayed outward and the current upon membrane hyperpolarization-appeared to be affected by Ba2+.3.3. Both the inward and the outward steady-state rectification of the membrane were considerably reduced by Ba2+.4.4. Due to a reduced K+ outward current and possibly a decreased inward current inactivation an increasingly large net inward current became apparent in Ba2+ solutions.5.5. The maximum of the early inward current-voltage relationship was reduced and shifted along the voltage-axis in dependence of the Ba2+/Ca2+ concentration ratio.6.6. The changes in the membrane currents following gradual substitution of Ca2+ by Ba2+ appear to be responsible for the observed increase in both the amplitude and the duration of the action potentials.7.7. Our results suggest that the membrane currents are altered due to differences in the interaction of Ca2+ and Ba2+ with ionic channels.
Caprine milk contains a large amount of different sialylated and neutral lactose-derived oligosaccharides compared to cow or sheep milk. In addition, its oligosaccharide profile is very similar to human milk, which suggests it has similar physiological activity. Thus, the caprine milk oligosaccharide fraction is a very promising food ingredient for human nutrition applications, especially for the supplementation of infant formulas. In this research work, a two-step cross-flow filtration process was designed in order to recover the caprine milk oligosaccharides. Tubular ceramic membranes with molecular weight cut-offs of 50 and 1kDa, respectively, were employed in two separated, consecutive continuous diafiltration steps, in which the cumulated permeate from the first step was the initial feed in the second one. A final retentate containing more than 80% of the original oligosaccharides and less than 4% of the original lactose and protein was obtained.
Milk oligosaccharides (OS)—free complex carbohydrates—confer unique health benefits to the nursing neonate. Though human digestive enzymes cannot degrade these sugars, they provide nourishment to specific commensal microbes and act as decoys to prevent the adhesion of pathogenic micro-organisms to gastrointestinal cells. At present, the limited quantities of human milk oligosaccharides (HMO) impede research on these molecules and their potential applications in functional food formulations. Considerable progress has been made in the study of OS structures; however, the synthetic pathways leading to their synthesis in the mammary gland are poorly understood. Recent studies show that complex OS with fucose and N-acetyl neuraminic acid (key structural elements of HMO bioactivity) exist in goat milk. Polymorphisms in the CSN1S1 locus, which is responsible for synthesis of αs1-casein, affect lipid and casein micelle structure in goat milk. The present study sought to determine whether CSN1S1 polymorphisms also influence goat milk oligosaccharide (GMO) production and secretion. The GMO compositions of thirty-two goat milk samples, half of which were from genotype A/A (αs1-casein producers) and half from genotype O/O (αs1-casein non-producers), were determined with nanoflow liquid chromatography high-accuracy mass spectrometry. This study represents the most exhaustive characterization of GMO to date. A systematic and comprehensive GMO library was created, consolidating information available in the literature with the new findings. Nearly 30 GMO, 11 of which were novel, were confirmed via tandem mass spectrometric analyses. Six fucosylated OS were identified; 4 of these matched HMO compositions and three were identified for the first time in goat milk. Importantly, multivariate statistical analysis demonstrated that the OS profiles of the A/A and O/O genotype milks could be discriminated by the fucosylated OS. Quantitative analysis revealed that the goat milk samples contained 1.17 g/L of OS; however, their concentration in milks from A/A and O/O genotypes was not different. This study provides evidence of a genetic influence on specific OS biosynthesis but not total OS production. The presence of fucosylated GMO suggests that goat milk represents a potential source of bioactive milk OS suitable as a functional food ingredient.
Mice are the premier mammalian models for studies of human physiology and disease, bearing extensive biological similarity to humans with far fewer ethical, economic, or logistic complications. To facilitate glycomic studies based on the mouse model, we comprehensively profiled the mouse serum N-glycome using isomer-specific nano-LC/MS and -LC/MS/MS. N-glycans were identified by accurate mass MS and structurally elucidated by MS/MS. Porous graphitized carbon nano-LC was able to separate out nearly 300 N-linked glycan compounds (including isomers) from just over 100 distinct N-linked glycan compositions. Additional MS/MS structural analysis was performed on a number of novel N-glycans, revealing the structural characteristics of modifications such as dehydration, O-acetylation, and lactylation. Experimental findings were combined with known glycobiology to generate a theoretical library of all biologically-possible mouse serum N-glycan compositions. The library may be used for automated identification of complex mixtures of mouse N-glycans, with possible applications to a wide range of mouse-related research endeavors, including pharmaceutical drug development and biomarker discovery.
Determining protein-specific glycosylation in protein mixtures remains a difficult task. A common approach is to use gel electrophoresis to isolate the protein followed by glycan release from the identified band. However, gel bands are often composed of several proteins. Hence, release of glycans from specific bands often yields products not from a single protein but a composite. As an alternative, we present an approach whereby glycans are released with peptide tags allowing verification of glycans bound to specific proteins. We term the process in-gel nonspecific proteolysis for elucidating glycoproteins (INPEG). INPEG combines rapid gel separation of a protein mixture with in-gel nonspecific proteolysis of protein bands followed by tandem MS analysis of the resulting N- and O-glycopeptides. Here, in-gel digestion is shown for the first time with nonspecific and broad specific proteases such as pronase, proteinase K, pepsin, papain and subtilisin. Tandem MS analysis of the resulting glycopeptides separated on a porous graphitized carbon (PGC) chip was achieved via nanoflow liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (nano-LC/Q-TOF MS). In this study, rapid and automated glycopeptide assignment was achieved via an in-house software (Glycopeptide Finder) based on a combination of accurate mass measurement, tandem MS data and pre-determined protein I.D. (obtained via routine shotgun analysis).INPEG is here initially validated for O-glycosylation (kappa casein) and N-glycosylation (ribonuclease B). Applications of INPEG were further demonstrated for the rapid determination of detailed site-specific glycosylation of lactoferrin and transferrin following gel separation and INPEG analysis on crude bovine milk and human serum, respectively.
Whole goat κ-casein was prepared by chromatography of whole casein on hydroxyapatite. Chromatography of whole κ-casein on DEAE-cellulose separated 5 fractions. All of them were sensitive to chymosin. Their amino acid and carbohydrate composition, phosphate content and molecular weight were determined. Galactose, N-acetylgalactosamine, N-acetyl and N-glycolyl neuraminic acids were identified in whole κ-casein. It appears that goat κ-casein, like cow, buffalo and ewe κ-caseins, is composed of several fractions having identical peptide chains and differing in their carbohydrate contents. The main fraction, devoid of carbohydrate, was treated with chymosin. The para-κ-casein and caseinomacropeptide were isolated. Their amino acid composition and phosphate content were determined.
In the present study, lactoferrin was purified from caprine colostrum and its anticancer effects were investigated on various cancer cell lines, including lung, colon, cervix, stomach and breast cancer cells. The caprine lactoferrin (CLF) was purified by ultrafiltration and sequential chromatography using a CM-Sephadex C-50-120 ion-exchange column and affinity chromatography on Hi-TrapTM Heparin HP. The purified CLF exhibited antiproliferative effects on the five tested cancer cell lines in a dose-dependent manner, with 20–30% survival as compared to the control. Interestingly, the most intense inhibition of cell growth was observed on the ZR-75-1 breast cancer cells (IC50 = 27.5 µg/mL). Our findings suggest, for the first time, that purified CLF from caprine colostrum may constitute a novel anticancer agent for the food industry.
Physico-chemical characteristics of milk are related to its composition for a particular animal species. Sheep milk contains higher levels of total solids and major nutrient than goat and cow milk. Lipids in sheep and goat milk have higher physical characteristics than in cow milk, but physico-chemical indices (i.e., saponification, Reichert Meissl and Polenske values) vary between different reports. Micelle structures in goat and sheep milk differ in average diameter, hydration, and mineralization from those of cow milk. Caprine casein micelles contain more calcium and inorganic phosphorus, are less solvated, less heat stable, and lose -casein more readily than bovine casein micelles. Renneting parameters in cheese making of sheep milk are affected by physico-chemical properties, including pH, larger casein micelle, more calcium per casein weight, and other mineral contents in milk, which cause differences in coagulation time, coagulation rate, curd firmness, and amount of rennet needed. Renneting time for goat milk is shorter than for cow milk, and the weak consistency of the gel is beneficial for human digestion but decreases its cheese yield. Triacylglycerols (TAG) constitute the biggest part of milk lipids (nearly 98%), including a large number of esterified fatty acids. Sheep and goat milk also have simple lipids (diacylglycerols, monoacylglycerols, cholesterol esters), complex lipids (phospholipids), and liposoluble compounds (sterols, cholesterol esters, hydrocarbons). The average fat globule size is smallest (<3.5 m) in sheep milk followed by goat and cow milk. Five fatty acids (C10:0, C14:0, C16:0, C18:0, and C18:1) account for >75% of total fatty acids in goat and sheep milk. Levels of the metabolically valuable short and medium chain fatty acids, caproic (C6:0) (2.9%, 2.4%, 1.6%), caprylic (C8:0) (2.6%, 2.7%, 1.3%), capric (C10:0) (7.8%, 10.0%, 3.0%), and lauric (C12:0) (4.4%, 5.0%, 3.1%) are significantly higher in sheep and goat than in cow milk, respectively. Principal caseins (CN) in goat, sheep and cow milk are s1 -CN, s2 -CN, -CN and -CN. The main forms of caprine and ovine caseino-macropeptides (CMP), which are the soluble C-terminal derivatives from the action of chymosin on -casein during the milk clotting process of cheesemaking, have been identified and are a good source of antithrombotic peptides. Sheep and goat milk proteins are also important sources of bioactive angiotensin converting enzyme (ACE) inhibitory peptides and antihypertensive peptides. They can provide a non-immune disease defence and control of microbial infections. Important minor milk proteins include immunoglobulins, lactoferrin, transferrin, ferritin, proteose peptone, calmodulin (calcium binding protein), prolactin, and folate-binding protein. Non-protein nitrogen (NPN) contents of goat and human milks are higher than in cow milk. Taurine in goat and sheep milk derived from sulphur-containing amino acids has important metabolic functions as does carnitine, which is a valuable nutrient for the human neonate. Mineral and vitamin contents of goat and sheep milk are mostly higher than in cow milk.
Caprine milk is a nutritional and therapeutic food. The unique and beneficial characteristics of caprine milk that are superior to bovine milk include: better digestibility; greater buffering capacity; fat globules that are smaller in diameter and better distributed in the milk emulsion; higher content of short-chain fatty acids in the milk fat; higher content of zinc, iron and magnesium; stronger lactoperoxidase (antimicrobial) system as well as better immunological and antibacterial characteristics. The larger amounts of some minerals, such as calcium, zinc and magnesium, in caprine milk may influence the growth of lactic acid bacteria since they are a normal part of some enzymatic complexes involved in lactose fermentation. The higher whey protein content could also be significant because Lactobacillus acidophilus and bifidobacteria grow better in the presence of higher levels of some amino acids (valine, glycine, hystidine). The use of caprine and ovine milk in cheesemaking is well known, but the production of fermented caprine milk via probiotics has not yet been developed, although many studies have highlighted the requirements for production of that kind of healthy food. During fermentation caprine milk loses its characteristic ‘goaty’ taste, which is unacceptable to many consumers. Moreover, the nutritive value of caprine milk increases during fermentation. The rise in the number of goat farms in Croatia has created the need to find other products that can be produced using caprine milk. According to the present situation in Croatia, there is no real possibility of producing fermented caprine milk for the global market, but many studies of fermented caprine milk have been performed.
Free oligosaccharides are key components of human milk and play multiple roles in the health of the neonate, by stimulating growth of selected beneficial bacteria in the gut, participating in development of the brain, and exerting antipathogenic activity. However, the concentration of oligosaccharides is low in mature bovine milk, normally used for infant formula, compared with both human colostrum and mature human milk. Characterization of bovine milk oligosaccharides in different breeds is crucial for the identification of viable sources for oligosaccharide purification. An improved source of oligosaccharides can lead to infant formula with improved oligosaccharide functionality. In the present study we have analyzed milk oligosaccharides by high-performance liquid chromatography chip quadrupole time-of-flight mass spectrometry and performed a detailed data analysis using both univariate and multivariate methods. Both statistical tools revealed several differences in oligosaccharide profiles between milk samples from the two Danish breeds, Jersey and Holstein-Friesians. Jersey milk contained higher relative amounts of both sialylated and the more complex neutral fucosylated oligosaccharides, while the Holstein-Friesian milk had higher abundance of smaller and simpler neutral oligosaccharides. The statistical analyses revealed that Jersey milk contains levels of fucosylated oligosaccharides significantly higher than that of Holstein-Friesian milk. Jersey milk also possesses oligosaccharides with a higher degree of complexity and functional residues (fucose and sialic acid), suggesting it may therefore offer advantages in term of a wider array of bioactivities.
Human milk oligosaccharides (HMOs) are a family of structurally diverse unconjugated glycans that are highly abundant in and unique to human milk. Originally, HMOs were discovered as a prebiotic "bifidus factor" that serves as a metabolic substrate for desired bacteria and shapes an intestinal microbiota composition with health benefits for the breast-fed neonate. Today, HMOs are known to be more than just "food for bugs". An accumulating body of evidence suggests that HMOs are antiadhesive antimicrobials that serve as soluble decoy receptors, prevent pathogen attachment to infant mucosal surfaces and lower the risk for viral, bacterial and protozoan parasite infections. In addition, HMOs may modulate epithelial and immune cell responses, reduce excessive mucosal leukocyte infiltration and activation, lower the risk for necrotizing enterocolitis and provide the infant with sialic acid as a potentially essential nutrient for brain development and cognition. Most data, however, stem from in vitro, ex vivo or animal studies and occasionally from association studies in mother-infant cohorts. Powered, randomized and controlled intervention studies will be needed to confirm relevance for human neonates. The first part of this review introduces the pioneers in HMO research, outlines HMO structural diversity and describes what is known about HMO biosynthesis in the mother's mammary gland and their metabolism in the breast-fed infant. The second part highlights the postulated beneficial effects of HMO for the breast-fed neonate, compares HMOs with oligosaccharides in the milk of other mammals and in infant formula and summarizes the current roadblocks and future opportunities for HMO research.
Milk is an inherently expensive raw material for use as a food. To compete in the new millennium, dairy products will need to be based on special values that can only come from milk. These include traditional dairy products and health-giving products. Designer milks will be needed to give new, enhanced products and to improve the quality and value of traditional products. The use of milk for traditional products is likely to continue to be strong in western cultures. For these products, key issues are naturalness of supply, with “organic” milk being an important issue; and low-fat products, which may imply a need for lower fat milk. Health products are the most exciting new area for milk-based products. A number of components in milk are being recognised as conferring health benefits. These include minerals (calcium), peptides derived from milk proteins (ACE inhibitor peptide) and lipid components (conjugated linoleic acid). A number of harmful effects have been attributed to milk, often by groups with a vested interest, and often based on dubious data. We have investigated claims relating to diabetes, ischaemic heart disease and hypercholesterolaemia and been unable to substantiate any harmful effect. Designer milks that are improved raw materials can be approached through various combinations of genetics (including traditional genetics, marker-assisted selection and genetic modification of dairy cattle) and by farm and feed management. Examples are presented.
A cation-exchange chromatographic membrane was used for the rapid isolation of caprine and ovine lactoferrin (LF) from cheese whey. Caprine LF was isolated by one-step cation-exchange chromatography, but the isolation of ovine LF needed a further reversed-phase HPLC purification step. The antibacterial activity of these LFs, determined towards Micrococcus flavus and Escherichia coli, was compared with that of bovine LF. Of the apo-LFs the caprine protein had the highest activity. In all cases the holo-LFs showed low or negligible activity. The antibacterial properties of cationic peptides obtained by peptic hydrolysis of caprine LF were studied against E. coli and M. flavus, and the activity of these peptides was compared with that of bovine lactoferricin. In addition, the binding characteristics of bovine, ovine and caprine LF to bacterial cells of E. coli were investigated with an enzyme-linked binding assay using horseradish peroxidase-bovine LF as a conjugate. Ovine and bovine LF strongly inhibited the binding of the LF complex to the bacterial surface. The iron-free forms of these LFs showed a greater ability for binding to the bacterial cells than the iron-saturated forms. However, several cationic peptides and caprine LF did not inhibit the binding of the LF conjugate although they exhibited a marked antibacterial effect. The results of binding of the LF complex in the presence of LFs from different species and of membrane-active peptides are discussed in relation to the antibacterial activity of these proteins and peptides.
In this study, we measured the levels of 6′ sialyl lactose, 3′ sialyl lactose, 6′ sialyl lactosamine and disialyl lactose in the colostrum of individual Jersey and Friesian cows at first milking, individual Jersey and Friesian cows over the first five milkings, and also in colostrum pooled from Friesian or Jersey herds over the first 2 days post partum. Our results show significant differences between the breeds in the proportions of the sialyl oligosaccharides present, but not in the total concentration of the sialyl oligosaccharides. Friesian colostrum sialyl oligosaccharides contained a higher proportion of 6′ sialyl lactosamine while Jersey colostrum had higher proportions of 3′ sialyl lactose. The concentration of all measured sialyl oligosaccharides was highest in colostrum from the first milking post partum, dropping rapidly in successive milkings. We also measured the levels of selected individual oligosaccharides in milk (not separated by breed) over the entire milking season. The average concentrations over the season were 17 mg L−1 6′ sialyl lactose, 42 mg L−1 3′ sialyl lactose, 11 mg L−1 6′ sialyl lactosamine, 4 mg L−1 disialyl lactose, 11 mg L−1 free N-acetylneuraminic acid and 4 mg L−1N-acetylgalactosaminyl galactose.
The iron-binding protein characteristic of milk, here called lactoferrin, has been demonstrated to occur in saliva, nasal secretions, tears, bronchial mucus, hepatic bile, pancreatic juice, seminal fluid, female cervical mucus and urines.It is suggested that its iron-binding properties are of value in the defence of epithelial surfaces against infection.
Colostrum is the first natural food produced by female mammals during the first 24–36h directly after giving birth. Chemically, colostrum is a very complex fluid rich in nutrients, antibodies and growth factors. In cows the antibodies provide passive immunity to the new born calf, whereas the growth factors especially stimulate the growth of the gut. The other antimicrobial components of colostrum include lactoferrin, lysozyme and lactoperoxidase. Bovine colostrum has also been used as a raw material for immunonoglubulin-rich commercial products (immune milk preparations). These products can be given orally to patients who are suffering infections of the gastrointestial tract or in order to prevent these infections. Usually, however, the cows have to be hyperimmunized against microorganisms, if specific antibodies are required. Several animal studies have shown that the growth factors in bovine colostrum, especially insulin-like growth factors, stimulate cell growth in the gut. Bovine colostrum is also known to contain insulin, transforming growth factor β and related growth factors, but their function in colostrum is not fully understood. Small amounts of these growth factors can also be detected in normal milk. Growth factors as well as antimicrobial factors of colostrum may be used as potential components in clinical nutrition in the future.
The isolation of whey proteins from human and bovine milks followed by profiling of their entire N-glycan repertoire is described. Whey proteins resulting from centrifugation and ethanol precipitation of milk were treated with PNGase F to release protein-bound N-glycans. Once released, N-glycans were analyzed via nanoflow liquid chromatography coupled with quadrupole time-of-flight mass spectrometry following chromatographic separation on a porous graphitized carbon chip. In all, 38 N-glycan compositions were observed in the human milk sample while the bovine milk sample revealed 51 N-glycan compositions. These numbers translate to over a hundred compounds when isomers are considered and point to the complexity of the mixture. High mannose, neutral, and sialylated complex/hybrid glycans were observed in both milk sources. Although NeuAc sialylation was observed in both milk samples, the NeuGc residue was only observed in bovine milk and marks a major difference between human and bovine milks. To the best of our knowledge, this study is the first MS based confirmation of NeuGc in milk protein bound glycans as well as the first comprehensive N-glycan profile of bovine milk proteins. Tandem MS was necessary for resolving complications presented by the fact that (NeuGc:Fuc) corresponds to the exact mass of (NeuAc:Hex). Comparison of the relative distribution of the different glycan types in both milk sources was possible via their abundances. While the human milk analysis revealed a 6% high mannose, 57% sialylation, and 75% fucosylation distribution, a 10% high mannose, 68% sialylation, and 31% fucosylation distribution was observed in the bovine milk analysis. Comparison with the free milk oligosaccharides yielded low sialylation and high fucosylation in human, while high sialylation and low fucosylation are found in bovine. The results suggest that high fucosylation is a general trait in human, while high sialylation and low fucosylation are general features of glycosylation in bovine milk.
On addition of lactoferrin (LF) to skim milk, the turbidity decreases. The basic protein binds to the caseins in the casein micelles, which is then followed by a (partial) disintegration of the casein micelles. The amount of LF initially binding to casein micelles follows a Langmuir adsorption isotherm. The kinetics of the binding of LF could be described by first-order kinetics and similarly the disintegration kinetics. The disintegration was, however, about 10 times slower than the initial adsorption, which allowed investigating both phenomena. Kinetic data were also obtained from turbidity measurements, and all data could be described with one equation. The disintegration of the casein micelles was further characterized by an activation energy of 52 kJ/mol. The initial increase in hydrodynamic size of the casein micelles could be accounted for by assuming that it would go as the cube root of the mass using the adsorption and disintegration kinetics as determined from gel electrophoresis. The results show that LF binds to casein micelles and that subsequently the casein micelles partly disintegrate. All micelles behave in a similar manner as average particle size decreases. Lysozyme also bound to the casein micelles, and this binding followed a Langmuir adsorption isotherm. However, lysozyme did not cause the disintegration of the casein micelles.
Glycosylation is one of the most common yet diverse post-translational modifications. Information on glycan heterogeneity and glycosite occupancy is increasingly recognized as crucial to understanding glycoprotein structure and function. Yet, no approach currently exists with which to holistically consider both the proteomic and glycomic aspects of a system. Here, we developed a novel method of comprehensive glycosite profiling using nanoflow liquid chromatography/mass spectrometry (nano-LC/MS) that shows glycan isomer-specific differentiation on specific sites. Glycoproteins were digested by controlled non-specific proteolysis in order to produce informative glycopeptides. High-resolution, isomer-sensitive chromatographic separation of the glycopeptides was achieved using microfluidic chip-based capillaries packed with graphitized carbon. Integrated LC/MS/MS not only confirmed glycopeptide composition but also differentiated glycan and peptide isomers and yielded structural information on both the glycan and peptide moieties. Our analysis identified at least 13 distinct glycans (including isomers) corresponding to five compositions at the single N-glycosylation site on bovine ribonuclease B, 59 distinct glycans at five N-glycosylation sites on bovine lactoferrin, 13 distinct glycans at one N-glycosylation site on four subclasses of human immunoglobulin G, and 20 distinct glycans at five O-glycosylation sites on bovine κ-casein. Porous graphitized carbon provided effective separation of glycopeptide isomers. The integration of nano-LC with MS and MS/MS of non-specifically cleaved glycopeptides allows quantitative, isomer-sensitive, and site-specific glycoprotein analysis.
Figure
Overlaid chromatograms and associated structural assignments of glycopeptides from bovine κ-casein. Color denotes the site(s) of glycosylation from which the glycopeptide originated
A combined mass spectrometry (MS) and tandem mass spectrometry (MS/MS) approach implemented with matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI FTICR MS) in the negative ion mode is described for enhanced glycopeptide detection and MS/MS analysis. Positive ion mode MS analysis is widely used for glycopeptide characterization, but the analyses are hampered by potential charge-induced fragmentation of the glycopeptides and poor detection of the glycopeptides harboring sialic acids. Furthermore, tandem MS analysis (MS/MS) via collision-induced dissociation (CID) of glycopeptides in the positive ion mode predominantly yields glycan fragmentation with minimal information to verify the connecting peptide moiety. In this study, glycoproteins such as, bovine lactoferrin (b-LF) for N-glycosylation and kappa casein (k-CN) for O-glycosylation were analyzed in both the positive- and negative ion modes after digestion with bead-immobilized Pronase. For the b-LF analysis, 44 potential N-linked glycopeptides were detected in the positive ion mode while 61 potential N-linked glycopeptides were detected in the negative ion mode. By the same token, more O-linked glycopeptides mainly harboring sialic acids from k-CN were detected in the negative ion mode. The enhanced glycopeptide detection allowed improved site-specific analysis of protein glycosylation and superior to positive ion mode detection. Overall, the negative ion mode approach is aimed toward enhanced N- and O-linked glycopeptide detection and to serve as a complementary tool to positive ion mode MS/MS analysis.
While milk proteins have been studied for decades, strikingly little effort has been applied to determining how the post-translational modifications (PTMs) of these proteins may change during the course of lactation. PTMs, particularly glycosylation, can greatly influence protein structure, function, and stability and can particularly influence the gut where their degradation products are potentially bioactive. In this work, previously undiscovered temporal variations in both expression and glycosylation of the glycoproteome of human milk are observed. Lactoferrin, one of the most abundant glycoproteins in human milk, is shown to be dynamically glycosylated during the first 10 days of lactation. Variations in expression or glycosylation levels are also demonstrated for several other abundant whey proteins, including tenascin, bile salt-stimulated lipase, xanthine dehydrogenase, and mannose receptor.
Human milk contains large amounts of complex oligosaccharides that putatively modulate the intestinal microbiota of breast-fed infants by acting as decoy binding sites for pathogens and as prebiotics for enrichment of beneficial bacteria. Several bifidobacterial species have been shown to grow well on human milk oligosaccharides. However, few data exist on other bacterial species. This work examined 16 bacterial strains belonging to 10 different genera for growth on human milk oligosaccharides. For this propose, a chemically defined medium, ZMB1, was used, which allows vigorous growth of a number of gut-related microorganisms in a fashion similar to complex media. Interestingly, Bifidobacterium longum subsp. infantis, Bacteroides fragilis , and Bacteroides vulgatus strains were able to metabolize milk oligosaccharides with high efficiency, whereas Enterococcus , Streptococcus , Veillonella , Eubacterium , Clostridium , and Escherichia coli strains grew less well or not at all. Mass spectrometry-based glycoprofiling of the oligosaccharide consumption behavior revealed a specific preference for fucosylated oligosaccharides by Bi. longum subsp. infantis and Ba. vulgatus. This work expands the current knowledge of human milk oligosaccharide consumption by gut microbes, revealing bacteroides as avid consumers of this substrate. These results provide insight on how human milk oligosaccharides shape the infant intestinal microbiota.
Fat is present in milk as droplets of triglycerides surrounded by a complex membrane derived from the mammary epithelial cell called milk fat globule membrane (MFGM). Although numerous studies have been published on human or bovine MFGM proteins, to date few studies exist on MFGM proteins from goat milk. The objective of this study was thus to investigate the protein composition of the goat MFGM. Milk fat globule membrane proteins from goat milk were separated by 6% and 10% sodium dodecyl sulfate-PAGE and were Coomassie or periodic acid-Schiff stained. Most of MFGM proteins [mucin-1, fatty acid synthase, xanthine oxidase, butyrophilin, lactadherin (MFG EGF-8, MFG-E8), and adipophilin] already described in cow milk were identified in goat milk using peptide mass fingerprinting. In addition, lectin staining provided a preliminary characterization of carbohydrate structures occurring on MFGM proteins from goat milk depending on alpha(S1)-casein genotype and lactation stage. We provide here first evidence of the presence of O-glycans on fatty acid synthase and xanthine oxidase from goat milk. A prominent difference between the cow and the goat species was demonstrated for lactadherin. Indeed, whereas 2 polypeptide chains were easily identified by peptide mass fingerprinting matrix-assisted laser desorption/ionization-time of flight analysis within bovine MFGM proteins, lactadherin from goat milk consisted of a single polypeptide chain. Another striking observation was the presence of caseins associated with MFGM preparations from goat milk, whereas virtually no caseins were found in MFGM extracts from bovine milk. Taken together, these observations strongly support the existence of a singular secretion mode previously hypothesized in the goat.
Glycosylation is among the most common post-translational modifications that proteins undergo that may affect many of their activities. It may also modify the underlying energy landscape of glycoproteins in a way that their altered biophysical characteristics are linked to their bioactivity. Yet, the capability of glycosylation to modify thermodynamic and kinetic properties varies greatly between glycoproteins. Deciphering the 'glycosylation code' that dictates the interplay between the nature of the carbohydrates or the proteins and the biophysical properties of the glycosylated proteins is essential. In this article, we discuss how the size, number, and structure of glycans, as well as the attachment sites, may modulate the folding of glycoproteins. Understanding the cross-talks between the protein and the attached glycans at the molecular level may assist in tailoring the biophysical properties of proteins in general.
Annotation of the human serum N-linked glycome is a formidable challenge but is necessary for disease marker discovery. A new theoretical glycan library was constructed and proposed to provide all possible glycan compositions in serum. It was developed based on established glycobiology and retrosynthetic state-transition networks. We find that at least 331 compositions are possible in the serum N-linked glycome. By pairing the theoretical glycan mass library with a high mass accuracy and high-resolution MS, human serum glycans were effectively profiled. Correct isotopic envelope deconvolution to monoisotopic masses and the high mass accuracy instruments drastically reduced the amount of false composition assignments. The high throughput capacity enabled by this library permitted the rapid glycan profiling of large control populations. With the use of the library, a human serum glycan mass profile was developed from 46 healthy individuals. This paper presents a theoretical N-linked glycan mass library that was used for accurate high-throughput human serum glycan profiling. Rapid methods for evaluating a patient's glycome are instrumental for studying glycan-based markers.
Protein glycosylation involves the addition of monosaccharides in a stepwise process requiring no glycan template. Therefore, identifying the numerous glycoforms, including isomers, can help elucidate the biological function(s) of particular glycans. A method to assess the diversity of the N-linked oligosaccharides released from human serum without derivatization has been developed using on-line nanoLC and high resolution TOF MS. The N-linked oligosaccharides were analyzed with MALDI FT-ICR MS and microchip LC MS (HPLC-Chip/TOF MS). Two microfluidic chips were employed, the glycan chip (40 nL enrichment column, 43 x 0.075 mm(2) i.d. analytical column) and the high capacity chip (160 nL enrichment column, 140 x 0.075 mm(2) i.d. analytical column), both with graphitized carbon as the stationary phase. Both chips offered good sensitivity and reproducibility in separating a heterogeneous mixture of neutral and anionic oligosaccharides between injections. Increasing the length and volume of the enrichment and the analytical columns improved resolution of the peaks. Complex type N-linked oligosaccharides were the most abundant oligosaccharides in human serum accounting for approximately 96% of the total glycans identified, while hybrid and high mannose type oligosaccharides comprise the remaining approximately 4%.
Glycosylation produces a diverse and abundant repertoire of glycans, which are collectively known as the glycome. Glycans are one of the four fundamental macromolecular components of all cells, and are highly regulated in the immune system. Their diversity reflects their multiple biological functions that encompass ligands for proteinaceous receptors known as lectins. Since the discovery that selectins and their glycan ligands are important for the regulation of leukocyte trafficking, it has been shown that additional features of the vertebrate immune system are also controlled by endogenous cellular glycosylation. This Review focuses on the emerging immunological roles of the mammalian glycome.
Whole goat kappa-casein was prepared by chromatography of whole casein on hydroxyapatite. Chromatography of whole kappa-casein on DEAE-cellulose separated 5 fractions. All of them were sensitive to chymosin. Their amino acid and carbohydrate composition, phosphate content and molecular weight were determined. Galactose, N-acetylgalactosamine, N-acetyl and N-glycolyl neuraminic acids were identified in whole kappa-casein. It appears that goat kappa-casein, like cow, buffalo and ewe kappa-caseins, is composed of several fractions having identical peptide chains and differing in their carbohydrate contents. The main fraction, devoid of carbohydrate, was treated with chymosin. The para-kappa-casein and caseinomacropeptide were isolated. Their amino acid composition and phosphate content were determined.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
The mean lactoferrin (Lf) concentration determined by electroimmunodiffusion (EID) assay of whey preparations from 80 quarters of 20 normal lactating cows was 0.35 mg/ml. The mean alpha-lactalbumin (alpha-LAC) and bovine serum albumin (BSA) concentrations were 2.01 mg/ml and 0.29 mg/ml, respectively. The mean was significantly related to cell count (P smaller than 0.01), BSA (P smaller than 0.05), stage of lactation (P smaller than 0.05), and milk production (P smaller than 0.05). The Lf-milk production relationship was the only negative correlation. In 11 cows with mastitis, there was a significant (P smaller than 0.01) increase in mean Lf concentration in infected quarters from 0.55 mg/ml on day 1 of the infection to 1.89 mg/ml by day 3. By day 15 clinical signs had subsided and mean Lf concentrations had decreased to near day 1 values. On day 3 quarters infected with coliform bacteria (clinical mastitis generally more severe) had mean Lf values more than twofold greater than those quarters infected with species of Staphylococcus or Streptococcus (milder clinical signs). Noninfected (control) quarters of cows having coliform bacteria-infected quarters had slightly increased mean Lf concentrations, where Lf concentration in contral quarters of cows having quarters infected with gram-positive organisms remained unchanged.
Lactoferrin was measured immunologically in human colostrum and in milk from 2 to 197 days after parturition. Lactoferrin decreased during the first two weeks, paralleling a decrease in total and in whey protein nitrogen. Then the curve sloped more gradually. Colostrum, 2 to 5 days postpartum, 8 samples; transitional milk, 6 to 10 days, 10 samples; mature milk, 11 to 60 days, 43 samples; and mature milk, 61 to 197 days, 25 samples contained (mean ± standard deviation), .49 ±.06, .45±.08, .21±.05 and .16±.03 g of lactoferrin per 100 ml, respectively. From nitrogen distributions, acrylamide gel electrophoresis and immunological quantitation, human whey protein contained about 35.0% lactoferrin 46.4% α-lactalbumin, 10.6% serum albumin and 8.0% remaining proteins (globulins). Normal human milk, 11 to 60 days postpartum, contained about, 44 g casein, .21 g lactoferrin, .28 g α-lactalbumin, .06 g serum albumin and .05 g remaining proteins (globulins). The casein to whey protein ratio is about 1:1.4.