Influence of Lactobacillus plantarum WCFS1 on post-acidification, metabolite formation and survival of starter bacteria in set-yoghurt.

The objectives of this study were to evaluate the growth and survival of the model probiotic strain Lactobacillus plantarum WCFS1 in co-culture with traditional yoghurt starters and to investigate the impact of preculturing on their survival and metabolite formation in set-yoghurt. L. plantarum WCFS1 was precultured under sublethal stress conditions (combinations of elevated NaCl and low pH) in a batch fermentor before inoculation in milk. Adaptive responses of L. plantarum WCFS1 were evaluated by monitoring bacterial population dynamics, milk acidification and changes in volatile and non-volatile metabolite profiles of set-yoghurt. The results demonstrated that sublethal preculturing did not significantly affect survival of L. plantarum WCFS1. On the other hand, incorporation of sublethally precultured L. plantarum WCFS1 significantly impaired the survival of Lactobacillus delbrueckii subsp. bulgaricus which consequently reduced the post-acidification of yoghurt during refrigerated storage. A complementary metabolomics approach using headspace SPME-GC/MS and (1)H NMR combined with multivariate statistical analysis revealed substantial impact of sublethally precultured L. plantarum WCFS1 on the metabolite profiles of set-yoghurt. This study provides insight in the technological implications of non-dairy model probiotic strain L. plantarum WCFS1, such as its good stability in fermented milk and the inhibitory effect on post-acidification.

Minimal inhibitory concentrations of undissociated lactic, acetic, citric and propionic acid for Listeria monocytogenes under conditions relevant to cheese.

Minimal inhibitory concentrations (MICs) of undissociated lactic acid were determined for six different Listeria monocytogenes strains at 30 °C and in a pH range of 4.2-5.8. Small increments in pH and acid concentrations were used to accurately establish the growth/no growth limits of L. monocytogenes for these acids. The MICs of undissociated lactic acid in the pH range of 5.2-5.8 were generally higher than at pH 4.6 for the different L. monocytogenes strains. The average MIC of undissociated lactic acid was 5.0 (SD 1.5) mM in the pH range 5.2-5.6, which is relevant to Gouda cheese. Significant differences in MICs of undissociated lactic acid were found between strains of L. monocytogenes at a given pH, with a maximum observed level of 9.0 mM. Variations in MICs were mostly due to strain variation. In the pH range 5.2-5.6, the MICs of undissociated lactic acid were not significantly different at 12 °C and 30 °C. The average MICs of undissociated acetic acid, citric acid, and propionic acid were 19.0 (SD 6.5) mM, 3.8 (SD 0.9) mM, and 11.0 (SD 6.3) mM, respectively, for the six L. monocytogenes strains tested in the pH range 5.2-5.6. Variations in MICs of these organic acids for L. monocytogenes were also mostly due to strain variation. The generated data contribute to improved predictions of growth/no growth of L. monocytogenes in cheese and other foods containing these organic acids.

The protein and lipid composition of the membrane of milk fat globules depends on their size.

In bovine milk, fat globules (MFG) have a heterogeneous size distribution with diameters ranging from 0.1 to 15 µm. Although efforts have been made to explain differences in lipid composition, little is known about the protein composition of MFG membranes (MFGM) in different sizes of MFG. In this study, protein and lipid analyses were combined to study MFG formation and secretion. Two different sized MFG fractions (7.6±0.9 µm and 3.3±1.2 µm) were obtained by centrifugation. The protein composition of MFGM in the large and small MFG fractions was compared using mass-spectrometry-based proteomics techniques. The lipid composition and fatty acid composition of MFG was determined using HPLC-evaporative light-scattering detector and gas chromatography, respectively. Two frequently studied proteins in lipid droplet biogenesis, perilipin-2 and TIP47, were increased in the large and small MFG fractions, respectively. In the large MFG fraction, besides perilipin-2, cytoplasmic vesicle proteins (heat shock proteins, 14-3-3 proteins, and Rabs), microfilaments and intermediate filament-related proteins (actin and vimentin), host defense proteins (cathelicidins), and phosphatidylinositol were higher in concentration. On the other hand, cholesterol synthesis enzymes [lanosterol synthase and sterol-4-α-carboxylate 3-dehydrogenase (decarboxylating)], cholesterol, unsaturated fatty acids, and phosphatidylethanolamine were, besides TIP47, higher in concentration in the small MFG fraction. These results suggest that vesicle proteins, microfilaments and intermediate filaments, cholesterol, and specific phospholipids play an important role in lipid droplet growth, secretion, or both. The observations from this study clearly demonstrated the difference in protein and lipid composition between small and large MFG fractions. Studying the role of these components in more detail in future experiments may lead to a better understanding of fat globule formation and secretion.

Effect of sublethal preculturing on the survival of probiotics and metabolite formation in set-yoghurt.

The objective of this study was to investigate the effect of preculturing of Lactobacillus rhamnosus GG and Bifidobacterium animalis subsp. lactis BB12 under sublethal stress conditions on their survival and metabolite formation in set-yoghurt. Prior to co-cultivation with yoghurt starters in milk, the two probiotic strains were precultured under sublethal stress conditions (combinations of elevated NaCl and low pH) in a batch fermentor. The activity of sublethally precultured probiotics was evaluated during fermentation and refrigerated storage by monitoring bacterial population dynamics, milk acidification and changes in volatile and non-volatile metabolite profiles of set-yoghurt. The results demonstrated adaptive stress responses of the two probiotic strains resulting in their viability improvement without adverse influence on milk acidification. A complementary metabolomic approach using SPME-GC/MS and (1)H-NMR resulted in the identification of 35 volatiles and 43 non-volatile polar metabolites, respectively. Principal component analysis revealed substantial impact of the activity of sublethally precultured probiotics on metabolite formation demonstrated by distinctive volatile and non-volatile metabolite profiles of set-yoghurt. Changes in relative abundance of various aroma compounds suggest that incorporation of stress-adapted probiotics considerably influences the organoleptic quality of product. This study provides new information on the application of stress-adapted probiotics in an actual food-carrier environment.

Effect of the DGAT1 K232A genotype of dairy cows on the milk metabolome and proteome.

Diglyceride O-acyltransferase 1 (DGAT1) is the enzyme that catalyzes the synthesis of triglycerides from diglycerides and acyl-coenzyme A. The DGAT1 K232A polymorphism was previously shown to have a significant influence on bovine milk production characteristics (milk yield, protein content, fat content, and fatty acid composition). The mechanism of this influence has, however, not been elucidated. In this study, metabolomics ((1)H-nuclear magnetic resonance) and proteomics (laser chromatography-tandem mass spectrometry) were applied to determine the serum and lipid metabolite composition and milk fat globule membrane proteome of milk samples from cows with the DGAT1 KK and AA genotypes. The milk samples from cows with the DGAT1 KK genotype contained more stomatin, sphingomyelin, choline, and carnitine, and less citrate, creatine or phosphocreatine, glycerol-phosphocholine, mannose-like sugar, acetyl sugar phosphate, uridine diphosphate (UDP)-related sugar, and orotic acid compared with milk samples from cows with the DGAT1 AA genotype. Based on these results, we propose that the differences between the DGAT1 genotypes may be related to stomatin-sphingomyelin lipid rafts as well as structural (cell membrane) differences in epithelial cells of the mammary gland. In conclusion, our study shows that, in addition to previously described changes in triglyceride composition, cows differing in DGAT1 polymorphism differ in their milk proteome and metabolome, which may help in further understanding the effect of the DGAT1 K232A polymorphism on milk production characteristics.

Perspective on calf and mammary gland development through changes in the bovine milk proteome over a complete lactation.

Milk contains all the nutrients for the growth and development of the neonate. However, milk composition is not constant during lactation. To study the changes of the milk proteome over lactation, filter-aided sample preparation combined with dimethyl labeling followed by liquid chromatography tandem mass spectrometry was used to identify and quantify milk proteins from 4 cows. A total of 229 proteins were identified, of which 219 were quantified. An 80% overlap was found in identified and quantified proteins between the 4 individual cows during lactation. Over lactation, the number of quantified proteins changed slightly (less than 10%), whereas the concentration of proteins changed considerably. Transport proteins involved in lipid synthesis (fatty acid-binding protein, perilipin-2, butyrophilin) increased, whereas proteins related to cholesterol transport (apolipoprotein E) decreased. The changes of lipid synthesis proteins are in accordance with the increased milk fat yield over lactation, indicating the increase of de novo mammary fatty acid synthesis as lactation advances. The high abundance of immune-related proteins in early lactation indicates the important role of these proteins for immune system development of calves. The increase in immune-related proteins (immunoglobulins, osteopontin, lactoferrin) and the decrease of proteins related to milk component synthesis (α-lactalbumin, ß-lactoglobulin, fatty acid-binding protein, perilipin-2, butyrophilin) in late lactation can be associated with the protection of the mammary gland. In conclusion, the changes of proteins with different biological functions reflect not only the changing needs of calves but also the development and protection of the mammary gland over lactation.

Effect of shortening or omitting the dry period of Holstein-Friesian cows on casein composition of milk.

The aim of this study was to evaluate the effect of shortening or omitting the dry period of dairy cows on milk casein composition. For this study, we analyzed milk samples of 90 cows with a dry period of 0, 30, or 60d and either a glucogenic or a lipogenic ration in early lactation. Milk was sampled at 6 and 2wk prepartum and at 2, 6, and 12wk postpartum. Milk was analyzed for casein (CN) composition by capillary zone electrophoresis, and isoforms of κ-CN were measured by reversed phase-HPLC. Shortening the dry period from 60 to 30d reduced the αS1-CN fraction by 3.8% and increased the αS2-CN fraction by 5.5%. In milk from cows with a 0-d dry period, the glycosylated κ-CN fraction in late lactation increased from 8 to 12% between 6 and 2wk prepartum. After calving, the glycosylated κ-CN fraction in milk was higher for cows with a 0-d dry period (6.7%) compared with cows with a 60-d dry period (5.2%). The glycosylated κ-CN fraction at 2wk postpartum was negatively correlated with milk yield, suggesting that glycosylation was related to reduced productivity of mammary epithelial cells. In early lactation, the ß-CN fraction was reduced in milk of cows with a 0-d dry period. A lowered ß-CN fraction was associated with high somatic cell count and greater parity, indicating that it was the result of proteolytic activity. In conclusion, casein composition changes that result from shortening the dry period from 60 to 30d are not expected to affect processing characteristics of milk. Applying a 0-d dry period may affect processability of milk because of a higher glycosylated κ-CN fraction, and possibly because of higher proteolytic activity compared with a 60-d dry period.

Identification of lipid synthesis and secretion proteins in bovine milk.

Lactation physiology is a process that is only partly understood. Proteomics techniques have shown to be useful to help advance the knowledge on lactation physiology in human and rodent species but have not been used as major tools for dairy cows, except for mastitis. In this paper, advanced non-targeted proteomics techniques (Filter aided sample preparation and NanoLC-Orbitrap-MS/MS) were applied to study the milk fat globule membrane and milk serum fraction, resulting in the identification of 246 proteins. Of these, 23 transporters and enzymes were related to lipid synthesis and secretion in mammary gland and their functions are discussed in detail. The identification of these intracellular transporters and enzymes in milk provides a possibility of using milk itself to study lipid synthesis and secretion pathways. This full-scale scan of milk proteins by using non-targeted proteomic analysis helps to reveal the important proteins involved in lipid synthesis and secretion for further examination in targeted studies.

Changes in milk proteome and metabolome associated with dry period length, energy balance, and lactation stage in postparturient dairy cows.

The early lactation period of dairy cows, which produce high quantities of milk, is normally characterized by an insufficient energy intake to cover milk production and maintenance requirements. Mobilization of body reserves occurs to compensate this negative energy balance (NEB), and probably as a consequence there is a higher susceptibility to diseases and metabolic disorders. There are several diagnostic methods to detect NEB, usually involving ketosis related parameters. Due to the easy availability of milk this is a preferred matrix, but simple and robust predictors of NEB level are missing. To better understand the physiological mechanism of NEB, milk of cows subjected to different dry period lengths, in different energy balance status and lactation stage, were analyzed by untargeted metabolomics and proteomics techniques. Milk of cows in severe NEB showed higher concentrations of acute phase response proteins, unsaturated fatty acids, and galactose-1-phosphate. Improved energy balance (EB) resulted in higher concentration of cholesterol, cholesterol synthesis related proteins, and stomatin. The presence of stomatin and galactose-1-phosphate in milk was strongly dependent on the EB of the cows. These novel and interesting findings warrant more in-depth research to assess their applicability as robust indicators of NEB in milk and to clarify the role of stomatin and galactose-1-phophate in milk of dairy cows in NEB.

Characterization of milk fatty acids based on genetic and herd parameters.

The objective of this study was to characterize the fatty acids (FA) in milk based on genetic and herd parameters to investigate the origin of the different FA in milk. Milk samples of 1912 Dutch Holstein-Friesian cows were analysed for 39 different FA including odd and branched-chain fatty acids. The proportion of variation caused by genetic and herd effects was calculated. In addition, genetic and herd correlations among the fatty acids were estimated and a clustering technique was used to visualise these correlations. The results indicated that in Dutch milk C12:0 is not completely synthesised de novo but also partly blood derived. It was suggested that C20:0 in milk is formed from the action of elongase enzymes on C18:0 and that the odd-chain FA C5:0-C13:0 and a part of C15:0 and C17:0 are synthesised de novo while the other part of C15:0 and C17:0 is blood derived. Furthermore, this work gives an overview of the opportunities to change the concentration of individual FA both by breeding and feeding. It is clearly shown that the extent to which the individual FA can be changed varies greatly and is dependent on the origin of the different FA in milk.

Changes over lactation in breast milk serum proteins involved in the maturation of immune and digestive system of the infant.

Here we provide data from shot-gun proteomics, using filtered-aided sample preparation (FASP), dimethyl labeling and LC-MS/MS, to quantify the changes in the repertoire of human milk proteins over lactation. Milk serum proteins were analyzed at week 1, 2, 3 4, 8, 16, and 24 in milk from four individual mothers. A total of 247 proteins were identified, of which 200 proteins were quantified. The data supplied in this article supports the accompanying publication (Zhang et al., 2006) [1]. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (Vizcaíno et al., 2016) [2] via the PRIDE partner repository with the dataset identifier PXD003465.

Changes over lactation in breast milk serum proteins involved in the maturation of immune and digestive system of the infant.

UNLABELLED: To objective of this study was to better understand the biological functions of breast milk proteins in relation to the growth and development of infants over the first six months of life. Breast milk samples from four individual women collected at seven time points in the first six months after delivery were analyzed by filter aided sample preparation and dimethyl labeling combined with liquid chromatography tandem mass spectrometry. A total of 247 and 200 milk serum proteins were identified and quantified, respectively. The milk serum proteome showed a high similarity (80% overlap) on the qualitative level between women and over lactation. The quantitative changes in milk serum proteins were mainly caused by three groups of proteins, enzymes, and transport and immunity proteins. Of these 21 significantly changed proteins, 30% were transport proteins, such as serum albumin and fatty acid binding protein, which are both involved in transporting nutrients to the infant. The decrease of the enzyme bile salt-activated lipase as well as the immunity proteins immunoglobulins and lactoferrin coincide with the gradual maturation of the digestive and immune system of infants. The human milk serum proteome didn't differ qualitatively but it did quantitatively, both between mothers and as lactation advanced. The changes of the breast milk serum proteome over lactation corresponded with the development of the digestive and immune system of infants. BIOLOGICAL SIGNIFICANCE: Breast milk proteins provide nutrition, but also contribute to healthy development of infants. Despite the previously reported large number of identified breast milk proteins and their changes over lactation, less is known on the changes of these proteins in individual mothers. This study is the first to determine the qualitative and quantitative changes of milk proteome over lactation between individual mothers. The results indicate that the differences in the milk proteome between individual mothers are more related to the quantitative level than qualitative level. The correlation between the changes of milk proteins and the gradual maturation of the gastrointestinal tract and immune system in infants, contributes to a better understanding of the biological functions of human milk proteins for the growth and development of infants.

Dairy in a sustainable diet: a question of balance.

The demand for dairy products is growing rapidly, especially in emerging markets. Dairy products are nutrient rich and, therefore, an important food group for ensuring nutrient security in the future. In many countries, dairy contributes significantly to nutrient intake. Meta-analyses have shown that consumption of dairy may reduce the risk of chronic diseases and thereby lower healthcare costs. Milk production and processing contribute to greenhouse gas emissions, estimated at 2.7% (cradle-to-retail) of the world's total. Evaluating the position of dairy in the diet should take into account the impact of both nutritional and environmental factors. Local conditions are also important; in many parts of the world, the cow is an efficient converter of human-inedible resources into nutrient-dense food. Increased productivity of cows is a decisive factor in realizing sufficient milk production with optimal resource efficiency and minimal greenhouse gas emission. Models that optimize total diets, rather than individual food products, for their nutritional and environmental impact are the preferred approach for developing realistic alternative consumption strategies.

Bovine milk proteome in the first 9 days: protein interactions in maturation of the immune and digestive system of the newborn.

In order to better understand the milk proteome and its changes from colostrum to mature milk, samples taken at seven time points in the first 9 days from 4 individual cows were analyzed using proteomic techniques. Both the similarity in changes from day 0 to day 9 in the quantitative milk proteome, and the differences in specific protein abundance, were observed among four cows. One third of the quantified proteins showed a significant decrease in concentration over the first 9 days after calving, especially in the immune proteins (as much as 40 fold). Three relative high abundant enzymes (XDH, LPL, and RNASE1) and cell division and proliferation protein (CREG1) may be involved in the maturation of the gastro-intestinal tract. In addition, high correlations between proteins involved in complement and blood coagulation cascades illustrates the complex nature of biological interrelationships between milk proteins. The linear decrease of protease inhibitors and proteins involved in innate and adaptive immune system implies a protective role for protease inhibitor against degradation. In conclusion, the results found in this study not only improve our understanding of the role of colostrum in both host defense and development of the newborn calf but also provides guidance for the improvement of infant formula through better understanding of the complex interactions between milk proteins.

Influence of different proteolytic strains of Streptococcus thermophilus in co-culture with Lactobacillus delbrueckii subsp. bulgaricus on the metabolite profile of set-yoghurt.

Proto-cooperation between Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus is one of the key factors that determine the fermentation process and final quality of yoghurt. In this study, the interaction between different proteolytic strains of S. thermophilus and L. delbrueckii subsp. bulgaricus was investigated in terms of microbial growth, acidification and changes in the biochemical composition of milk during set-yoghurt fermentation. A complementary metabolomics approach was applied for global characterization of volatile and non-volatile polar metabolite profiles of yoghurt associated with proteolytic activity of the individual strains in the starter cultures. The results demonstrated that only non-proteolytic S. thermophilus (Prt-) strain performed proto-cooperation with L. delbrueckii subsp. bulgaricus. The proto-cooperation resulted in significant higher populations of the two species, faster milk acidification, significant abundance of aroma volatiles and non-volatile metabolites desirable for a good organoleptic quality of yoghurt. Headspace SPME-GC/MS and (1)H NMR resulted in the identification of 35 volatiles and 43 non-volatile polar metabolites, respectively. Furthermore, multivariate statistical analysis allows discriminating set-yoghurts fermented by different types of starter cultures according to their metabolite profiles. Our finding underlines that selection of suitable strain combinations in yoghurt starters is important for achieving the best technological performance regarding the quality of product.

The host defense proteome of human and bovine milk.

Milk is the single source of nutrients for the newborn mammal. The composition of milk of different mammals has been adapted during evolution of the species to fulfill the needs of the offspring. Milk not only provides nutrients, but it also serves as a medium for transfer of host defense components to the offspring. The host defense proteins in the milk of different mammalian species are expected to reveal signatures of evolution. The aim of this study is therefore to study the difference in the host defense proteome of human and bovine milk. We analyzed human and bovine milk using a shot-gun proteomics approach focusing on host defense-related proteins. In total, 268 proteins in human milk and 269 proteins in bovine milk were identified. Of these, 44 from human milk and 51 from bovine milk are related to the host defense system. Of these proteins, 33 were found in both species but with significantly different quantities. High concentrations of proteins involved in the mucosal immune system, immunoglobulin A, CD14, lactoferrin, and lysozyme, were present in human milk. The human newborn is known to be deficient for at least two of these proteins (immunoglobulin A and CD14). On the other hand, antimicrobial proteins (5 cathelicidins and lactoperoxidase) were abundant in bovine milk. The high concentration of lactoperoxidase is probably linked to the high amount of thiocyanate in the plant-based diet of cows. This first detailed analysis of host defense proteins in human and bovine milk is an important step in understanding the function of milk in the development of the immune system of these two mammals.