Linking variation in the casein fraction and salt composition to casein micelle size in milk of Dutch dairy goats.

The casein (CN) composition, salt composition and micelle size varies largely between milk samples of individual animals. In goats, the link between those casein characteristics are unknown and could provide useful insights into goat casein micelle structure. In this study, the casein- and salt composition of 42 individual Dutch goats from 17 farms was studied and linked to casein micelle size. Micelle size, proportions of individual caseins, and protein content were associated with each other. Milk with smaller casein micelles was higher in protein content, salt content, and proportion of αs1-CN, but lower in αs2-CN and ß-CN. The higher salt content in milk with small casein micelles was mainly attributed to a higher protein content, but changes in casein composition might additionally contribute to differences in mineralization. The non-sedimentable casein content in goat milk correlated with non-sedimentable fractions of ß-CN and κ-CN and was independent of micelle size. Between large and small casein micelles, goat casein micelles showed more differences in casein and salt composition than bovine micelles, indicating differences in internal structure. Nevertheless, the casein mineralization in goat milk was similar to casein mineralization in bovine milk, indicating that mineralization of casein micelles follows a general principle. These results can help to better understand how composition and micelle structure in goat milk are related to each other, which may be useful to improve processing and product properties of goat milk in the future.

sRAGE-binding and antimicrobial bioactivities of soy and pea protein after heating and in vitro infant digestion.

During infant formula production, proteins are always heated, potentially affecting their digestibility and the bioactivities of resulting peptides. Although plant proteins are a promising dairy alternative for infant formula, they remain understudied, necessitating further investigations. Therefore, this research aimed to fill this gap by assessing the impact of different heating modes on soy protein (SP) and pea protein (PP), focusing on glycation levels, peptide formation during in vitro infant digestion, and immune protection potential (sRAGE-binding and antimicrobial activities) of the resulting peptides. Consequently, dry heating led to increased glycation and glycated peptide production, particularly with higher glycation in PP than SP. Moreover, PP exhibited an overall stronger sRAGE-binding capacity than SP, regardless of heating and digestion conditions. Regarding antimicrobial activity, both SP and PP-derived peptides displayed reduced effectiveness against Enterobacter cloacae after dry heating. Additionally, Staphylococcus epidermidis was differently inhibited, where PP-derived peptides showed inherent inhibition. The primary determinant of sRAGE-binding and antimicrobial potential in digestion-derived peptides was the protein source. Subsequent bioinformatics analysis predicted 519 and 133 potential antimicrobial peptides in SP and PP, respectively. This study emphasises the importance of protein source for infant formula to ensure infant health.

Differential effects of heating modes on the immunogenic potential of soy-derived peptides released after in vitro infant digestion.

During production of soy-based infant formula, soy protein undergoes heating processes. This study investigated the differential impact of heating modes on the immunogenic potential of peptides in soy protein digests. Wet or dry heating was applied, followed by in vitro gastrointestinal infant digestion. The released peptides were analyzed by LC-MS/MS. Bioinformatics tools were utilized to predict and identify potential linear B-cell and T-cell epitopes, as well as to explore cross-reactivity with other legumes. Subsequently, the peptide intensities of the same potential epitope across different experimental conditions were compared. As a result, we confirmed the previously observed enhancing effect of wet heating on infant digestion and inhibitory effect of dry heating. A total of 8,546 peptides were detected in the digests, and 6,684 peptides were with a score over 80. Among them, 29 potential T-cell epitopes and 27 potential B-cell epitopes were predicted. Cross-reactivity between soy and other legumes, including peanut, pea, chickpea, lentil, kidney bean, and lupine, was also detected. Overall, heating and digestion time could modulate the potential to trigger peptide-induced immune responses.

Glycation of goat milk with different casein-to-whey protein ratios and its effects on simulated infant digestion.

This research compared the effects of dry heating on the digestion of goat milk proteins with different casein-to-whey ratios (40% casein, C40 and 80% casein, C80). The glycation markers of heated samples were determined by LC-MS. Heating at 60 °C for 8 h induced early glycation while heating at 60 °C for 72 h induced advanced glycation. Unheated C80 samples showed a higher digestibility than unheated C40 samples, which may be due to their higher protein solubility. After dry heating for 72 h, no significant difference in digestibility was observed between C80 and C40 samples. Heating for 72 h decreased the digestibility of C40 samples compared to unheated samples, probably due to glycation, while protein aggregation was the main reason for the reduced digestibility of heated C80 samples. Overall, this study showed that dry heating for 72 h induced a lower digestibility of C80 and C40 samples, although with different underlying mechanisms.

Network analysis of the proteome and peptidome sheds light on human milk as a biological system.

Proteins and peptides found in human milk have bioactive potential to benefit the newborn and support healthy development. Research has been carried out on the health benefits of proteins and peptides, but many questions still need to be answered about the nature of these components, how they are formed, and how they end up in the milk. This study explored and elucidated the complexity of the human milk proteome and peptidome. Proteins and peptides were analyzed with non-targeted nanoLC-Orbitrap-MS/MS in a selection of 297 milk samples from the CHILD Cohort Study. Protein and peptide abundances were determined, and a network was inferred using Gaussian graphical modeling (GGM), allowing an investigation of direct associations. This study showed that signatures of (1) specific mechanisms of transport of different groups of proteins, (2) proteolytic degradation by proteases and aminopeptidases, and (3) coagulation and complement activation are present in human milk. These results show the value of an integrated approach in evaluating large-scale omics data sets and provide valuable information for studies that aim to associate protein or peptide profiles from biofluids such as milk with specific physiological characteristics.

Effects of protein genetic variants on their phosphorylation levels, milk composition, milk proteome, and milk coagulation ability in Chinese Holstein bovine milk.

Milk samples were collected from 3625 Chinese Holstein cows to assess the effects of κ-casein (κ-CN) and ß-lactoglobulin (ß-LG) genetic variants on its milk coagulation properties. The results show that Chinese Holstein cows have a higher frequency of the κ-CN AA and AB variants, and ß-LG of the AB and AA variants. Of these, κ-CN B variants, the ß-LG AA and BB variants were more frequent in milk showing good coagulation. The effects of the genetic variants on milk composition, milk proteome, and protein phosphorylation sites were studied. The results showed that higher concentrations of protein and dry matter were found in κ-CN BE variant. Moreover, large variations in milk proteome among different κ-CN and ß-LG variants were observed. Highly phosphorylated for κ-CN, especially Ser97, was observed in cows with the κ-CN BE variant, but no effect of ß-LG variants on phosphorylation site was found. Of the various factors examined, variation of κ-CN phosphorylation sites Ser97 may be the most important in affecting casein structure and milk coagulation ability. Some milk protein contents were found to be negative factors for milk coagulation. In summary, this study showed that κ-CN genetic variants contained different milk compositions and phosphorylation site Ser97 influenced milk coagulation.

Milk coagulation and gastric emptying in women experiencing gastrointestinal symptoms after ingestion of cow's milk.

BACKGROUND: Gastrointestinal symptoms after drinking milk are often attributed to lactose intolerance or cow's milk allergy. However, some individuals without either condition still report gastrointestinal symptoms after drinking milk. This may be caused by gastric emptying (GE) rate or gastric protein coagulation. This study aimed to compare GE rate and protein coagulation after milk consumption between individuals reporting gastrointestinal symptoms and those without symptoms using a novel gastric MRI approach. METHODS: Thirty women were included in this case-control study, of whom 15 reported gastrointestinal symptoms after drinking milk and 15 were controls. Participants underwent gastric MRI before and up to 90 min after consumption of 250 mL cow's milk. Gastric content volume and image texture of the stomach contents were used to determine GE and changes in the degree of coagulation. KEY RESULTS: GE half-time did not differ between the groups (gastrointestinal symptom group 66 ± 18 min; control group 61 ± 14 min, p = 0.845). The gastrointestinal symptom group reported symptoms from 30 min onwards and rated pain highest at 90 min. The control group reported no symptoms. Image texture analyses showed a significantly higher percentage of coagulum and lower percentage of liquid in the group in the GI symptom group (MD 11%, 95% CI [3.9, 17], p = 0.003). In vitro data suggests that pH and proteolytic enzyme activity influence the coagulum structure. CONCLUSIONS AND INFERENCES: Gastric milk coagulation and emptied fraction of stomach content may differ between individuals experiencing symptoms after milk consumption, possibly due to differences in pH and proteolytic enzyme activity.

Effect of pH and protein composition on proteolysis of goat milk proteins by pepsin and pancreatin.

The roles of protein composition, pH and enzymes in goat milk protein hydrolysis is still unclear and the proteolysis of low abundant goat milk proteins has received limited attention. The aim of this study was to study the impact of protein composition and proteolytic conditions on goat milk protein hydrolysis in a simplified digestion model. Both whole milk and infant formula were hydrolyzed at pH 2 and 4, using pepsin as well as pepsin combined with pancreatin. Intact proteins were separated from digests using spin filters, followed by bottom-up proteomics of the separated proteins. Results show that under all conditions, caseins are hydrolyzed quickly. Goat casein hydrolysis in infant formula was slightly faster than in goat whole milk, possibly due to less casein coagulation during pepsin hydrolysis at both pH 2 and 4. Several low abundant immunoactive goat milk proteins, especially immunoglobulins, GLYCAM-1 and osteopontin, resisted proteolysis more than high abundant proteins, independent of the pH and enzyme used for hydrolysis. Fast hydrolysis of casein and slow hydrolysis of immunoactive proteins may indicate a good balance between protein utilization and protection of the infant by goat milk proteins.

Corrigendum: The human milk proteome and allergy of mother and child: exploring associations with protein abundances and protein network connectivity.

[This corrects the article DOI: 10.3389/fimmu.2022.977470.].

The Effect of Milking Frequency, Breed, and Stage of Lactation on the Milk Fat Globule Size and Fatty Acid Composition in Sheep's Milk.

This study examined the effects of milking frequency, breed, and stage of lactation on the milk fat globules (MFG) size and fatty acids (FA) composition of sheep milk. Milk from Karagouniko (n = 13) and Chios (n = 13) ewes was sampled postpartum on the 93rd, 101st, 108th, 121st, 156th, and 188th days of lactation. On the 108th day, the ewes were divided randomly into two milking groups: Once daily at 06:00 a.m. or twice daily at 06:00 a.m. and 16:00 p.m. Morphometric characteristics of MFG and FA composition were determined for each sample. Once versus twice daily milking had no effect on MFG dimensions, which tended to vary according to breed (smaller MFG were secreted from Chios with p = 0.065), while the stage of lactation had a significant effect (p < 0.001). FA composition differed significantly according to the stage of lactation and breed. The FA profile of the Karagouniko breed showed higher concentrations of short-chain FA. The milk samples from late lactation were characterized by higher concentrations of mono-unsaturated FA (MUFA) compared to early and mid-lactation. Moreover, correlations were found between the average diameter of MFG and FA concentrations, where the size of MFG was positively correlated with saturated FA (SFA) and negatively correlated with MUFA.

Comparison of Whey Proteome and Glycoproteome in Bovine Colostrum and Mature Milk.

Milk samples were collected from 10 cows, in the colostrum (3-4 days) and mature (90 days) lactation stage, to assess the differential expression of all whey proteins and N-glycoproteins. In total, 240 whey proteins and 315 N-glycosylation sites on 214 glycoproteins were quantified. GO annotations, KEGG pathway analysis, and protein classification were performed to understand the similarities and differences of the biological functions of whey proteins and N-glycoproteins among different lactation stages in bovine milk. Furthermore, differential expression of whey proteins and whey N-glycosylated proteins was found between different lactation stages. The related changes of biological functions in differentially expressed proteins were discussed. For example, the increased frequency of glycosylation on lactoferrin and folate receptor alpha occurring in bovine colostrum may provide protection and stimulate development of the newborn calf. Our study thereby improves understanding of variations of glycosylation sites on milk glycoproteins among lactation stages.

Tolerance Induction in Cow's Milk Allergic Children by Heated Cow's Milk Protein: The iAGE Follow-Up Study.

Accelerating the induction of tolerance to cow's milk (CM) reduces the burden of cow's milk allergy (CMA). In this randomised controlled intervention study, we aimed to investigate the tolerance induction of a novel heated cow milk protein, the iAGE product, in 18 children with CMA (diagnosed by a paedriatric allergist). Children who tolerated the iAGE product were included. The treatment group (TG: n = 11; mean age 12.8 months, SD = 4.7) consumed the iAGE product daily with their own diet, and the control group (CG: n = 7; mean age 17.6 months, SD = 3.2) used an eHF without any milk consumption. In each group, 2 children had multiple food allergies. The follow-up procedures consisted of a double-blind placebo-controlled food challenge (DBPCFC) with CM t = 0, t = 1 (8 months), t = 2 (16 months), and t = 3 (24 months). At t = 1, eight (73%) of 11 children in the TG had a negative DBPCFC, versus four out of seven (57%) in the CG (BayesFactor = 0.61). At t = 3, nine of the 11 (82%) children in the TG and five of seven (71%) in the CG were tolerant (BayesFactor = 0.51). SIgE for CM reduced from a mean of 3.41 kU/L (SD = 5.63) in the TG to 1.24 kU/L (SD = 2.08) at the end of intervention, respectively a mean of 2.58 (SD = 3.32) in the CG to 0.63 kU/L (SD = 1.06). Product-related AEs were not reported. CM was successfully introduced in all children with negative DBPCFC. We found a standardised, well-defined heated CM protein powder that is safe for daily OIT treatment in a selected group of children with CMA. However, the benefits of inducing tolerance were not observed.

Raw milk kefir: microbiota, bioactive peptides, and immune modulation.

This study aims to characterize the microbiota and peptidomic composition of raw milk kefir, and to address the potential anti-allergic effects of raw milk kefir using validated research models for food allergy. Raw milk kefir was produced after incubation with a defined freeze-dried starter culture. Kefir was sampled during fermentation at seven time intervals. For comparison, kefir was also prepared from heat-treated milk. Peptide compositions were determined for the raw and heated milk, and kefir end products made from these milks. In a murine food allergy model, the two kefir end products were investigated for their allergy modulating effects. In both kefirs, we identified amplicon sequence variants identical to those in the starter culture, matching the bacteria Lactococcus lactis, Streptococcus thermophilus, Leuconostoc and the yeast Debaryomyces. In raw milk kefir, additional sequence variants of Lactococcus lactis and the yeasts Pichia and Galactomyces could be identified, which were absent in heated milk kefir. Analysis of peptide compositions in both kefirs indicated that the number and intensity of peptides drastically increased after fermentation. Heating of the milk negatively affected the diversity of the peptide composition in kefir. Only raw milk kefir suppressed the acute allergic skin response to the food allergen ovalbumin in sensitised mice. These effects coincided with differences in the T-cell compartment, with lower percentages of activated Th1 cells and IFNg production after treatment with kefir made from heated milk. The results of this study indicate specific properties of raw milk kefir that may contribute to its additional health benefits.

Heating affects protein digestion of skimmed goat milk under simulated infant conditions.

The objective of this research was to analyse the effects of heating on digestion of skimmed goat milk proteins. Most previous goat milk digestion studies evaluated the digestion only based on the supernatant. In this study, digestion of skimmed goat milk was studied in both supernatant and gastric clot. The results indicated that, compared to mild temperature heated samples (≤75 °C), samples heated at ≥80 °C showed more extensive gastric clot formation with a higher protein digestion rate, but also resulted in a larger amount of undigested whey proteins due to its severe aggregation. For the peptidome, ß-casein was the major source of bioactive peptides. The samples heated at 65 °C showed higher bioactive peptide abundances, whereas at temperatures higher than 75 °C, it was reduced due to cleavage into smaller peptides. Overall, this study showed that different heating temperatures induced different whey protein denaturation degrees, which affected their digestion in skimmed goat milk..

Maternal diet and human milk composition: an updated systematic review.

Context: Exclusive breastfeeding for 6 months after birth provides infants with the best start for life. A review by Bravi et al. summarized the importance of maternal diet as a determinant of human milk composition based on data up to 2015, but evidence on nutrient intake level was limited. Objective: We updated the review by Bravi et al., critically assessed differences in study designs and sampling methods, and graphically visualized trends and associations. Data sources: PubMed was systematically searched for articles published between January 2015 and March 2021. Data extraction: Article screening, selection, and data extraction was done by two independent researchers, including a risk of bias assessment based on 11 criteria. Articles were eligible when including: quantitative information, commonly used effect estimates, healthy mother-infant dyads. Results: Twenty seven observational and five intervention studies were identified (n = 7,138) and combined with results of Bravi et al. Fatty acids were still the most studied human milk components in relation to maternal diet (n = 17 studies) with maternal fish intake being predominantly positively associated with milk ALA (r = 0.28-0.42), DHA (r = 0.24-0.46), and EPA (r = 0.25-0.28) content. PUFAs from diet were generally positively correlated with their concentrations in milk, while SFA intake was negatively associated with several fatty acids in milk. Studies on associations with maternal diet and milk carbohydrates, proteins, vitamins and minerals were limited in number and varied in methods and results. Conclusion: This updated review shows that evidence on the association between maternal diet and human milk fatty acids is rapidly increasing, but still diversified in methodology and results. Further studies, preferably intervention studies, assessing diet and milk carbohydrates, proteins, vitamins and minerals are needed to be able draw conclusions on the importance of maternal diet for human milk composition as a whole.

Dietary d-Lactate Intake Facilitates Inflammatory Resolution by Modulating M1 Macrophage Polarization.

SCOPE: Given the d-lactate dehydrogenase (D-LDH) deficiency, L- but not d-lactate is assumed to be the physiological isomer in mammals. Paradoxically, many fermented foods (e.g., yogurt, sauerkraut, cheeses) often contain substantial amounts of d-lactate. In the present study, dietary d-lactate may be a previously unrecognized nutrient aiding in inflammatory resolution is hypothesized. METHODS AND RESULTS: The anti-inflammatory properties of d-lactate are evaluated in experimental colitis and endotoxemia. Oral administration of d-lactate favorably affects acute inflammation in two different mouse models. Analysis of lactate-the lactate receptor (the hydroxycarboxylic acid receptor 1 HCA1, formerly GPR81) signal axis in inflammation is performed in primary peritoneal macrophages and wild-type (WT) or GPR81 knockout (KO) mice. GPR81 KO mice are susceptible to endotoxic shock than WT mice, while d-lactate exerts its anti-inflammatory activities in a GPR81-dependent manner. Mechanistically, the activation of lactate-GPR81 axis may suppress LPS-TLR4 signaling to modulate M1 macrophage polarization. Although D-LDH deficiency in mammals impairs d-lactate clearance, it might prolong its plasma terminal half-life, and thus provide a pharmacokinetic advantage of d-lactate over l-lactate. CONCLUSION: This study highlights housekeeping function of the lactate-GPR81 axis in inflammation control, and suggests that dietary intake of d-lactate may underlie Metchnikoff's probiotic yogurt theory of life prolongation.

The human milk proteome and allergy of mother and child: Exploring associations with protein abundances and protein network connectivity.

Background: The human milk proteome comprises a vast number of proteins with immunomodulatory functions, but it is not clear how this relates to allergy of the mother or allergy development in the breastfed infant. This study aimed to explore the relation between the human milk proteome and allergy of both mother and child. Methods: Proteins were analyzed in milk samples from a subset of 300 mother-child dyads from the Canadian CHILD Cohort Study, selected based on maternal and child allergy phenotypes. For this selection, the definition of "allergy" included food allergy, eczema, allergic rhinitis, and asthma. Proteins were analyzed with non-targeted shotgun proteomics using filter-aided sample preparation (FASP) and nanoLC-Orbitrap-MS/MS. Protein abundances, based on label-free quantification, were compared using multiple statistical approaches, including univariate, multivariate, and network analyses. Results: Using univariate analysis, we observed a trend that milk for infants who develop an allergy by 3 years of age contains higher abundances of immunoglobulin chains, irrespective of the allergy status of the mother. This observation suggests a difference in the milk's immunological potential, which might be related to the development of the infant's immune system. Furthermore, network analysis showed overall increased connectivity of proteins in the milk of allergic mothers and milk for infants who ultimately develop an allergy. This difference in connectivity was especially noted for proteins involved in the protein translation machinery and may be due to the physiological status of the mother, which is reflected in the interconnectedness of proteins in her milk. In addition, it was shown that network analysis complements the other methods for data analysis by revealing complex associations between the milk proteome and mother-child allergy status. Conclusion: Together, these findings give new insights into how the human milk proteome, through differences in the abundance of individual proteins and protein-protein associations, relates to the allergy status of mother and child. In addition, these results inspire new research directions into the complex interplay of the mother-milk-infant triad and allergy.

Current progress of emerging technologies in human and animals' milk processing: Retention of immune-active components and microbial safety.

Human milk and commercial dairy products play a vital role in humans, as they can provide almost all essential nutrients and immune-active components for the development of children. However, how to retain more native immune-active components of milk during processing remains a big question for the dairy industry. Nonthermal technologies for milk processing are gaining increasing interest in both academic and industrial fields, as it is known that thermal processing may negatively affect the quality of milk products. Thermosensitive components, such as lactoferrin, immunoglobulins (Igs), growth factors, and hormones, are highly important for the healthy development of newborns. In addition to product quality, thermal processing also causes environmental problems, such as high energy consumption and greenhouse gas (GHG) emissions. This review summarizes the recent advances of UV-C, ultrasonication (US), high-pressure processing (HPP), and other emerging technologies for milk processing from the perspective of immune-active components retention and microbial safety, focusing on human, bovine, goat, camel, sheep, and donkey milk. Also, the detailed application, including the instrumental design, technical parameters, and obtained results, are discussed. Finally, future prospects and current limitations of nonthermal techniques as applied in milk processing are discussed. This review thereby describes the current state-of-the-art in nonthermal milk processing techniques and will inspire the development of such techniques for in-practice applications in milk processing.

Phosphoproteomics reveals that camel and goat milk improve glucose homeostasis in HDF/STZ-induced diabetic rats through activation of hepatic AMPK and GSK3-GYS axis.

Diabetes is a serious public health problem with global implications. Among many diabetes management therapies, non-pharmacological therapies such as those that focus on diet and exercise are gradually becoming more acceptable to patients. Within dietary management options, dairy products such as camel and goat milk are valued for their specific health benefits. The aim of this study was therefore to investigate the effect of camel and goat milk consumption on glucose homeostasis in high-fat diet and streptozotocin (HFD/STZ) induced diabetic rats. HFD/STZ-induced diabetic rats were fed with different milk for 35 days. Parameters related to glucose homeostasis, as well as hepatic proteome and phosphoproteome were investigated. The results of which showed that camel and goat milk consumption improved fasting glucose levels, glucose tolerance, and indicators related to lipid metabolism, while bovine and sheep milk consumption did not work. In addition, the hepatic phosphoproteome suggests that the ameliorative effect of both camel and goat milk was associated with the activation of AMPK. However, camel milk consumption further elevated the phosphorylation level of hepatic ACC, while goat milk consumption activated GSK3-GYS axis-related proteins. The present study investigated the possible mechanisms by which camel and goat milk consumption improves glucose homeostasis in HFD/STZ-induced diabetic rats and revealed their differences in the mechanism of antidiabetic effect.