Myofibrillar protein oxidation affects filament charges, aggregation and water-holding

Hypochlorous acid (HClO) is a strong oxidant that is able to mediate protein oxidation. In order to study the effect of oxidation on charges, aggregation and water-holding of myofibrillar proteins, extracted myofibrils were oxidized by incubation with different concentrations of HClO (0, 1, 5, and 10 mM). Loss of free thiols, loss of histidine and formation of carbonyls were greater with increasing oxidation level and the particle size increased. Water-holding in the 5 and 10 mM HClO groups were greater than in the non-oxidized control. Isoelectric focusing (IEF) showed that the isoelectric point (pI) of oxidized proteins were lower compared to non-oxidized ones. The lower pI values of oxidized proteins suggests that oxidation increased the overall net negative charge of myofibrillar proteins solubilized for IEF. Here we propose a hypothesis that oxidation-induced increase in net negative charges is the driving force for improved water-holding in myofibrils, whereas protein cross-linking and aggregation have an opposing effect by decreasing the water-holding.Peer reviewe

Dataset on proteomic changes of whey protein after different heat treatment

Hereby we provide data from a shot-gun proteomics experiment, using filtered-aided sample preparation (FASP), and liquid chromatography with tandem mass spectrometry (LC-MS/MS), to relatively quantify the changes in the protein profile of whey proteins after heating milk at either 65 °C, 70 °C, 75 °C, 80 °C, or 85 °C for 30 min. The data supplied in this article supports the accompanying publication [1]. The raw mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier “PXD016436”.</p

Unravelling lactate-acetate and sugar conversion into butyrate by intestinal Anaerobutyricum and Anaerostipes species by comparative proteogenomics

Thed- andl-forms of lactate are important fermentation metabolites produced by intestinal bacteria but are found to negatively affect mucosal barrier function and human health. Both enantiomers of lactate can be converted with acetate into the presumed beneficial butyrate by a phylogenetically related group of anaerobes, includingAnaerobutyricumandAnaerostipesspp. This is a low energy yielding process with a partially unknown pathway inAnaerobutyricumandAnaerostipesspp. and hence, we sought to address this via a comparative genomics, proteomics and physiology approach. We compared growth ofAnaerobutyricum soehngeniion lactate with that on sucrose and sorbitol. Comparative proteomics revealed complete pathway of butyrate formation from sucrose, sorbitol and lactate. Notably, a gene cluster,lctABCDEFwas abundantly expressed when grown on lactate. This gene cluster encodes a lactate dehydrogenase (lctD), electron transport proteins A and B (lctCB), nickel-dependent racemase (lctE), lactate permease (lctF) and short-chain acyl-CoA dehydrogenase (lctG). Investigation of available genomes of intestinal bacteria revealed this new gene cluster to be highly conserved in onlyAnaerobutyricumandAnaerostipesspp. Present study demonstrates thatA. soehngeniiand several relatedAnaerobutyricumandAnaerostipesspp. are highly adapted for a lifestyle involving lactate plus acetate utilization in the human intestinal tract.Peer reviewe

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

Comparative genomics and proteomics of Eubacterium maltosivorans : functional identification of trimethylamine methyltransferases and bacterial microcompartments in a human intestinal bacterium with a versatile lifestyle

Eubacterium maltosivorans YIT is a human intestinal isolate capable of acetogenic, propionogenic and butyrogenic growth. Its 4.3-Mb genome sequence contains coding sequences for 4227 proteins, including 41 different methyltransferases. Comparative proteomics of strain YIT showed the Wood-Ljungdahl pathway proteins to be actively produced during homoacetogenic growth on H-2 and CO2 while butyrogenic growth on a mixture of lactate and acetate significantly upregulated the production of proteins encoded by the recently identified lctABCDEF cluster and accessory proteins. Growth on H-2 and CO2 unexpectedly induced the production of two related trimethylamine methyltransferases. Moreover, a set of 16 different trimethylamine methyltransferases together with proteins for bacterial microcompartments were produced during growth and deamination of the quaternary amines, betaine, carnitine and choline. Growth of strain YIT on 1,2-propanediol generated propionate with propanol and induced the formation of bacterial microcompartments that were also prominently visible in betaine-grown cells. The present study demonstrates that E. maltosivorans is highly versatile in converting low-energy fermentation end-products in the human gut into butyrate and propionate whilst being capable of preventing the formation of the undesired trimethylamine by converting betaine and other quaternary amines in bacterial microcompartments into acetate and butyrate.Peer reviewe

Development of omics-based protocols for the microbiological characterization of multi-strain formulations marketed as probiotics : the case of VSL#3

The growing commercial interest in multi-strain formulations marketed as probiotics has not been accompanied by an equal increase in the evaluation of quality levels of these biotechnological products. The multi-strain product VSL#3 was used as a model to setup a microbiological characterization that could be extended to other formulations with high complexity. Shotgun metagenomics by deep Illumina sequencing was applied to DNA isolated from the commercial VSL#3 product to confirm strains identity safety and composition. Single-cell analysis was used to evaluate the cell viability, and beta-galactosidase and urease activity have been used as marker to monitor the reproducibility of the production process. Similarly, these lots were characterized in detail by a metaproteomics approach for which a robust protein extraction protocol was combined with advanced mass spectrometry. The results identified over 1600 protein groups belonging to all strains present in the VSL#3 formulation. Of interest, only 3.2 % proteins showed significant differences mainly related to small variations in strain abundance. The protocols developed in this study addressed several quality criteria that are relevant for marketed multi-strain products and these represent the first efforts to define the quality of complex probiotic formulations such as VSL#3.Peer reviewe

Microbial propionate production from carbon monoxide a novel bioprocess

Introduction: The fermentation of CO-rich gases by carboxidotrophic microbes is a promising way to produce valuable organic compounds. Propionate is a value-added compound with numerous industrial applications, e.g. as an antifungal agent in food and feed, and as a building block to produce plastics and herbicides. Propionate is currently produced by petrochemical processes, but it can be produced from ethanol and acetate by some propionogenic bacteria. Ethanol and acetate are usually formed by acetogenic bacteria from CO-rich gases. Accordingly, propionate can be indirectly produced from CO-rich gases, representing a new approach on the realm of microbial CO conversion. Methodology: Four distinct synthetic co-cultures were constructed, consisting of: Acetobacterium wieringae (DSM 1911T) and Pelobacter propionicus (DSM 2379T); A. wieringae (DSM 1911T) and Anaerotignum neopropionicum (DSM 3847T); A. wieringae strain JM and P. propionicus (DSM 2379T); A. wieringae strain JM and A. neopropionicum (DSM 3847T). The physiology of CO conversion to propionate was accessed and a proteogenomic analysis was performed in the best performing co-culture to get insight into the involved biochemical pathways and microbial interactions within the synthetic consortium. Results: Propionate was produced by all the co-cultures, with the highest titer (~24 mM) measured in the co-culture composed of A. wieringae strain JM + A. neopropionicum, which also produced isovalerate (~4 mM), butyrate (~1 mM), and isobutyrate (~0.3 mM). In this synthetic consortium, A. wieringae strain JM converts CO to a acetate and ethanol via the Wood-Ljungdahl pathway; acetate can also be converted to ethanol through the action of aldehyde oxidoreductase (AOR); A. neopropionicum converts ethanol to propionate via the acrylate pathway. In addition, proteins related to amino acid metabolism and stress response were highly abundant during co-cultivation, which raises the hypothesis that amino acids are exchanged by the two microorganisms, and this results in isovalerate and isobutyrate production. Conclusions: This synthetic co-culture represents a new bioprocess for the microbial production of propionate from carbon monoxide, that couples the Wood-Ljungdahl and acrylate pathways. Furthermore, this symbiosis engages an interesting perspective on how C1-fixing and C3-producing microorganisms can be used to expand the product scope of gas fermentation.Portuguese Foundation for Science and Technology (FCT): POCI-01-0145-FEDER-031377; strategic funding of UIDB/04469/2020 unit; BioTecNorte operation (NORTE-01-0145-FEDER-000004); FCT doctoral grants PD/BD/128030/2016 and PD/BD/150583/2020. Netherlands Science Foundation (NWO): Project NWO-GK-07; Perspectief Programma P16-10; Gravitation Grant, Project 024.002.002.info:eu-repo/semantics/publishedVersio

Effect of Processing Intensity on Immunologically Active Bovine Milk Serum Proteins

Consumption of raw cow's milk instead of industrially processed milk has been reported to protect children from developing asthma, allergies, and respiratory infections. Several heat-sensitive milk serum proteins have been implied in this effect though unbiased assessment of milk proteins in general is missing. The aim of this study was to compare the native milk serum proteome between raw cow's milk and various industrially applied processing methods, i.e., homogenization, fat separation, pasteurization, ultra-heat treatment (UHT), treatment for extended shelf-life (ESL), and conventional boiling. Each processing method was applied to the same three pools of raw milk. Levels of detectable proteins were quantified by liquid chromatography/tandem mass spectrometry following filter aided sample preparation. In total, 364 milk serum proteins were identified. The 140 proteins detectable in 66% of all samples were entered in a hierarchical cluster analysis. The resulting proteomics pattern separated mainly as high (boiling, UHT, ESL) versus no/low heat treatment (raw, skimmed, pasteurized). Comparing these two groups revealed 23 individual proteins significantly reduced by heating, e.g., lactoferrin (log2-fold change = -0.37, p = 0.004), lactoperoxidase (log2-fold change = -0.33, p = 0.001), and lactadherin (log2-fold change = -0.22, p = 0.020). The abundance of these heat sensitive proteins found in higher quantity in native cow's milk compared to heat treated milk, renders them potential candidates for protection from asthma, allergies, and respiratory infections

Akkermansia muciniphila uses human milk oligosaccharides to thrive in the early life conditions in vitro

Akkermansia muciniphila is a well-studied anaerobic bacterium specialized in mucus degradation and associated with human health. Because of the structural resemblance of mucus glycans and free human milk oligosaccharides (HMOs), we studied the ability of A. muciniphila to utilize human milk oligosaccharides. We found that A. muciniphila was able to grow on human milk and degrade HMOs. Analyses of the proteome of A. muciniphila indicated that key-glycan degrading enzymes were expressed when the bacterium was grown on human milk. Our results display the functionality of the key-glycan degrading enzymes (alpha -l-fucosidases, beta -galactosidases, exo-alpha -sialidases and beta -acetylhexosaminidases) to degrade the HMO-structures 2 ' -FL, LNT, lactose, and LNT2. The hydrolysation of the host-derived glycan structures allows A. muciniphila to promote syntrophy with other beneficial bacteria, contributing in that way to a microbial ecological network in the gut. Thus, the capacity of A. muciniphila to utilize human milk will enable its survival in the early life intestine and colonization of the mucosal layer in early life, warranting later life mucosal and metabolic health.Peer reviewe

Conversion of dietary inositol into propionate and acetate by commensal Anaerostipes associates with host health

Here, the authors report an anaerobic metabolic pathway from the dominant gut butyrogen Anaerostipes, showing several strains of this genus to be capable of producing propionate from dietary myo-inositol that associates with reduced fasting-glucose levels in mice. We describe the anaerobic conversion of inositol stereoisomers to propionate and acetate by the abundant intestinal genus Anaerostipes. A inositol pathway was elucidated by nuclear magnetic resonance using [C-13]-inositols, mass spectrometry and proteogenomic analyses in A. rhamnosivorans, identifying 3-oxoacid CoA transferase as a key enzyme involved in both 3-oxopropionyl-CoA and propionate formation. This pathway also allowed conversion of phytate-derived inositol into propionate as shown with [C-13]-phytate in fecal samples amended with A. rhamnosivorans. Metabolic and (meta)genomic analyses explained the adaptation of Anaerostipes spp. to inositol-containing substrates and identified a propionate-production gene cluster to be inversely associated with metabolic biomarkers in (pre)diabetes cohorts. Co-administration of myo-inositol with live A. rhamnosivorans in western-diet fed mice reduced fasting-glucose levels comparing to heat-killed A. rhamnosivorans after 6-weeks treatment. Altogether, these data suggest a potential beneficial role for intestinal Anaerostipes spp. in promoting host health.Peer reviewe