Phytanic acid concentrations and diastereomer ratios in milk fat during changes in the cow’s feed from concentrate to hay and back

The phytanic acid content in milk correlates with the amount of green items in the cows’ feed. For this reason, the four-fold methyl-branched fatty acid has been suggested as a potential marker for the authentication of organic milk. In this study, we attempted to provide further support for this idea by studying the progression of the phytanic acid level and diastereomer ratio in milk fat by transition of the diets from high proportions of concentrate (typical “conventional” feed) to hay (typical “organic” feed in winter) and back to “conventional” feed. Milk samples taken from three cows were analyzed on both the phytanic acid concentration and diastereomer distribution. The cows were initially fed with “conventional” feed (ground feed with high portions (30–45?%) of concentrate), then the feed was changed within 1?week to 100?% hay (“organic” feed), and after ~6?weeks, the feed of two cows was changed back to the initial feed with concentrate (phase C_b). During the “conventional” feeding at the beginning of the experiment, the phytanic acid concentration was low (100–130?mg/100?g milk fat). When the feed was changed to hay (“organic” feed), the phytanic acid concentration immediately increased to a stable level of about 160?mg/100?g lipids. Changing back the feed to “conventional” feed, the phytanic acid concentration dropped immediately back below the value measured in the initial phase. Likewise, the SRR/RRR-diastereomer distribution of phytanic acid in the milk was an excellent indicator for the changes in the cows’ feed. While the SRR/RRR-diastereomer ratio was >1.5 during “conventional” feeding, it immediately decreased to equal amounts of both diastereomers when hay was supplied as feed. Abandonment of concentrate in conventional feeding increased the phytanic acid content but the SRR-diastereomer was still dominant and thus the SRR/RRR-diastereomer ratio was different to organic milk. Our results indicate that both parameters, i.e., the phytanic acid content and SRR/RRR-diastereomer ratio need to be measured for authentication of organic milk.     

Phenotypic and genotypic characterisation of Escherichia coli strains serogrouped as enteropathogenic E. coli (EPEC) isolated from pasteurised milk

Fifty-six Escherichia coli strains, serogrouped as EPEC, isolated from three different brands of pasteurised milk commercialised in Rio de Janeiro, Brazil, were tested for enteropathogenicity markers. Most of the strains (71.4%) were adherent to HEp-2 cells. The adherent strains were distributed among 7 EPEC serogroups (O26, O55, O111, O114, O125, O127, O128, O158). Although almost half of these strains (33.9%) presented unrecognisable adherence phenotypes, classical adherence patterns (localised-like, aggregative and diffuse adherence) described for E. coli and epidemiologically associated with diarrheagenic strains were observed. None of the strains showed typical localised adherence, usually associated with EPEC strains, but 4 of them displayed a localised-like adherence (LAL) phenotype, characterised by fewer and less compact microcolonies but that is still associated with diarrheagenic strains as well as strains of non-human origin. Indeed, 3 of these 4 strains were able to elicit the attaching-effacing lesion (FAS-positive), the central feature of EPEC pathogenesis, and hybridised with bfpA and eae DNA probes. The other LAL-positive strain hybridised with the bfpA probe but gave negative results for the eae probe and FAS assays. Interestingly, all LAL-positive strains produced amplicons of 200 bp in the PCR for bfpA, instead of the expected 326 bp fragment. PCR reactions for stx1 and stx2, two shiga-toxin-encoding genes, gave negative results. Typing of LEE-associated genes by PCR showed the profile eae (beta), tir (beta), espA (alpha) and espB (alpha) for one of the LAL-positive strain. The most prevalent adherence phenotype was the aggregative pattern which is observed in strains epidemiologically associated with persistent diarrhea. Additionally, one strain promoted complete detachment of the Hep-2 cell monolayer after 3 h of infection which might be related to the production of citotoxins, a feature that has been increasingly observed in clinical strains. The possession of EPEC-related O and H antigens is no longer deemed an essential characteristic of true pathogenic EPEC strains, emphasising the importance of routinely screen for virulence markers in E. coli strains isolated from foods. Our results are in accordance with data from the literature that demonstrate that environmental strains display atypical features but yet are capable of eliciting the classical A/E lesion and thus must be considered as potentially pathogenic. Further, our results demonstrate the potential of pasteurised milk as a vehicle for transmission of diarrheagenic E. coli in Brazil.     

Prevalence of non-aureus staphylococci species causing intramammary infections in Canadian dairy herds

Non-aureus staphylococci (NAS), the microorganisms most frequently isolated from bovine milk worldwide, are a heterogeneous group of numerous species. To establish their importance as a group, the distribution of individual species needs to be determined. In the present study, NAS intramammary infection (IMI) was defined as a milk sample containing ≥1,000 cfu/mL in pure or mixed culture that was obtained from a cohort of cows assembled by the Canadian Bovine Mastitis Research Network. Overall, 6,213 (6.3%) of 98,233 quarter-milk samples from 5,149 cows and 20,305 udder quarters were associated with an NAS IMI. Of the 6,213 phenotypically identified NAS isolates, 5,509 (89%) were stored by the Canadian Bovine Mastitis Research Network Mastitis Pathogen Collection and characterized using partial sequencing of the rpoB housekeeping gene, confirming 5,434 isolates as NAS. Prevalence of each NAS species IMI was estimated using Bayesian models, with presence of a specific NAS species as the outcome. Overall quarter-level NAS IMI prevalence was 26%. The most prevalent species causing IMI were Staphylococcus chromogenes (13%), Staphylococcus simulans (4%), Staphylococcus haemolyticus (3%), Staphylococcus xylosus (2%), and Staphylococcus epidermidis (1%). The prevalence of NAS IMI as a group was highest in first-parity heifers and was evenly distributed throughout cows in parities ≥2. The IMI prevalence of some species such as S. chromogenes, S. simulans, and S. epidermidis differed among parities. Overall prevalence of NAS IMI was 35% at calving, decreased over the next 10 d, and then gradually increased until the end of lactation. The prevalence of S. chromogenes, Staphylococcus gallinarum, Staphylococcus cohnii, and Staphylococcus capitis was highest at calving, whereas the prevalence of S. chromogenes, S. haemolyticus, S. xylosus, and S. cohnii increased during lactation. Although the overall prevalence of NAS IMI was similar across barn types, the prevalence of S. simulans, S. xylosus, S. cohnii, Staphylococcus saprophyticus, S. capitis, and Staphylococcus arlettae IMI was higher in tiestall barns; the prevalence of S. epidermidis IMI was lowest; and the prevalence of S. chromogenes and Staphylococcus sciuri IMI was highest in bedded-pack barns. Staphylococcus simulans, S. epidermidis, S. xylosus, and S. cohnii IMI were more prevalent in herds with intermediate to high bulk milk somatic cell count (BMSCC) and S. haemolyticus IMI was more prevalent in herds with high BMSCC, whereas other common NAS species IMI were equally prevalent in all 3 BMSCC categories. Distribution of NAS species IMI differed among the 4 regions of Canada. In conclusion, distribution differed considerably among NAS species IMI; therefore, accurate identification (species level) is essential for studying NAS epidemiology.     

Inulin and probiotic concentration effects on fatty and linoleic conjugated acids in cream cheeses

The aim of this study was to evaluate the changes in the lipid profile, especially in conjugated linoleic acid (CLA), in 12 cream cheese batches with different prebiotic (inulin) and probiotic (Bifidobacterium animalis and Lactobacillus acidophilus) concentrations stored for 45 days at 8 ± 0.5 °C and analyzed every 15 days. A Central Rotational Composite Design was used to establish the prebiotic and probiotic concentrations. The effect of the pre- and probiotic concentrations on CLA contents (mg g⁻¹ of fat) was verified by plotting the response surface. The linear, quadratic, and interaction coefficients of the multiple linear regression for both prebiotic and probiotics were not significant (p > 0.05). No significant changes were observed neither in the lipid profile nor in the CLA content among the different batches, in which fatty acid composition and CLA concentrations were similar to the milk used in the preparation of the cream cheese and also to the control batch.     

Effects of organic acids in total mixed ration and feeding frequency on productive performance of dairy cows

This study aimed to evaluate the effects of organic acid (OA; Mold-Zap, Alltech, Nicholasville, KY) inclusion in the total mixed ration (TMR) and feeding frequency of TMR for lactating dairy cows on intake, total-tract apparent digestibility, sorting index, feeding behavior, ruminal fermentation, milk yield and composition, nitrogen balance, and serum metabolites. Twenty-four lactating Holstein cows, 4 with rumen cannulas, with (mean ± standard error) 247 ± 22.2 d in milk, 672 ± 14.6 kg of body weight, and 31.1 ± 1.09 kg of milk yield at the beginning of the experiment were used. The cows were distributed in a balanced and contemporary 4 × 4 Latin square experimental design and randomly assigned in a 2 × 2 factorial arrangement to evaluate OA [0 (OA?) or 0.5 (OA+) L of Mold-Zap/tonne of TMR on a natural matter basis] and feeding frequency of TMR offered once a day (1×) or twice a day (2×). Each experimental period lasted 21 d, with 14 d for acclimation and 7 d for data collection. The treatments were tested for TMR, in which its temperature was recorded every 2 h through a 24-h period in each experimental period. Organic acid-treated TMR showed a lower temperature during the 24-h period compared with nontreated TMR. The OA and feeding frequency had no effect on intake and total-tract apparent digestibility of dry matter and nutrients, aside from a tendency to increase neutral detergent fiber digestibility in cows fed 2×. Also, cows fed 1× tended to select more particles between 19 and 8 mm and refused particles smaller than 4 mm, whereas cows fed OA tended to select more particles smaller than 4 mm. Cows fed OA had greater milk yield and milk protein and lactose yields, but tended to have higher 3.5% fat-corrected milk yield. Neither treatment influenced ruminal and serum variables nor milk fat yield and milk production efficiency. Cows fed OA spent less time idling and tended to have lower rumination time, and tended to have higher time spent drinking water and eating, whereas animals fed 1× spent more time drinking water. Under the conditions of this experiment, we conclude that it was possible to reduce the feeding frequency of TMR, without negative effects on dairy cow performance. However, the use of OA resulted in higher milk yield and mitigated TMR temperature rise regardless of feeding frequency. The effect of external factors such as collective stimulation of intake and stage of lactation on feeding frequency effect must be surveyed in further research.     

Chemo-Enzymatic Fluorescence Labeling Of Genomic DNA For Simultaneous Detection Of Global 5-Methylcytosine And 5-Hydroxymethylcytosine**

5-Methylcytosine and 5-hydroxymethylcytosine are epigenetic modifications involved in gene regulation and cancer. We present a new, simple, and high-throughput platform for multi-color epigenetic analysis. The novelty of our approach is the ability to multiplex methylation and de-methylation signals in the same assay. We utilize an engineered methyltransferase enzyme that recognizes and labels all unmodified CpG sites with a fluorescent cofactor. In combination with the already established labeling of the de-methylation mark 5-hydroxymethylcytosine via enzymatic glycosylation, we obtained a robust platform for simultaneous epigenetic analysis of these marks. We assessed the global epigenetic levels in multiple samples of colorectal cancer and observed a 3.5-fold reduction in 5hmC levels but no change in DNA methylation levels between sick and healthy individuals. We also measured epigenetic modifications in chronic lymphocytic leukemia and observed a decrease in both modification levels (5-hydroxymethylcytosine: whole blood 30 %; peripheral blood mononuclear cells (PBMCs) 40 %. 5-methylcytosine: whole blood 53 %; PBMCs 48 %). Our findings propose using a simple blood test as a viable method for analysis, simplifying sample handling in diagnostics. Importantly, our results highlight the assay‘s potential for epigenetic evaluation of clinical samples, benefiting research and patient management.     

Quantitative risk assessment for type A staphylococcal enterotoxin poisoning due to consumption of Minas Frescal cheese in Brazil

This study aimed to estimate the risk of staphylococcal toxin type A (SEA) poisoning from consuming Minas Frescal cheese (MFC) in Brazil. A Quantitative Microbiological Risk Assessment model was developed, focussing on the production of SEA while still in the raw material. The baseline scenario yielded a simulated mean concentration of SEA in the MFC portion of 16.20 ng. The concentration of Staphylococcus aureus in raw milk is proved to be the most influential parameter for the risk, followed by the serving size and the prevalence of toxin genes.     

Investigation of the Photocatalytic Hydrogen Production of Semiconductor Nanocrystal-Based Hydrogels

Destabilization of a ligand-stabilized semiconductor nanocrystal solution with an oxidizing agent can lead to a macroscopic highly porous self-supporting nanocrystal network entitled hydrogel, with good accessibility to the surface. The previously reported charge carrier delocalization beyond a single nanocrystal building block in such gels can extend the charge carrier mobility and make a photocatalytic reaction more probable. The synthesis of ligand-stabilized nanocrystals with specific physicochemical properties is possible, thanks to the advances in colloid chemistry made in the last decades. Combining the properties of these nanocrystals with the advantages of nanocrystal-based hydrogels will lead to novel materials with optimized photocatalytic properties. This work demonstrates that CdSe quantum dots, CdS nanorods, and CdSe/CdS dot-in-rod-shaped nanorods as nanocrystal-based hydrogels can exhibit a much higher hydrogen production rate compared to their ligand-stabilized nanocrystal solutions. The gel synthesis through controlled destabilization by ligand oxidation preserves the high surface-to-volume ratio, ensures the accessible surface area even in hole-trapping solutions and facilitates photocatalytic hydrogen production without a co-catalyst. Especially with such self-supporting networks of nanocrystals, the problem of colloidal (in)stability in photocatalysis is circumvented. X-ray photoelectron spectroscopy and photoelectrochemical measurements reveal the advantageous properties of the 3D networks for application in photocatalytic hydrogen production.