Muramidases constitute a superfamily of enzymes that hydrolyse peptidoglycan (PGN) from bacterial cell walls. Recently, a fungal muramidase derived from Acremonium alcalophilum has been shown to increase broiler performance when added as a feed additive. However, the underlying mechanisms of action are not yet identified. Here, we investigated the hypothesis that this muramidase can cleave PGN to muramyl dipeptide (MDP), activating nucleotide-binding oligomerisation domain-containing protein 2 (NOD2) receptors in eukaryotic cells, potentially inducing anti-inflammatory host responses. Using Micrococcus luteus as a test bacterium, it was shown that muramidase from A. alcalophilum did not display antimicrobial activity, while it could cleave fluorescently labelled PGN. It was shown that the muramidase could degrade PGN down to its minimal bioactive structure MDP by using UPLC-MS/MS. Using HEK-Blue™-hNOD2 reporter cells, it was shown that the muramidase-treated PGN degradation mixture could activate NOD2. Muramidase supplementation to broiler feed increased the duodenal goblet cell and intraepithelial lymphocyte abundance while reducing duodenal wall CD3+ T lymphocyte levels. Muramidase supplementation to broiler feed only had moderate effects on the duodenal, ileal and caecal microbiome. It was shown that the newly discovered muramidase hydrolysed PGN, resulting in MDP that activates NOD2, potentially steering the host response for improved intestinal health.

Formaldehyde is a precursor of sugars, which are compounds essential in all forms of life and a necessary molecule for prebiotic processes. This work focuses on evaluating the stability of formaldehyde exposed to a high radiation field simulating prebiotic conditions on primitive Earth, such as the ocean or shallow waters. Formaldehyde may have been formed from reactions in the atmosphere and from rainout processes reached water bodies. In our experiments, we employed γ radiation and found that formaldehyde was labile towards radiation and decomposed even at low irradiation doses due to the fact that aldehyde/hydrate groups present in formaldehyde structure are very reactive under irradiation. However, after exposing this molecule to several doses of irradiation, we detected the formation of formic acid and glycolaldehyde – both of which are of prebiotic interest. We also observed formaldehyde regeneration by one of its radiolytic products: formic acid.

The percolation of water through waste landfill sites produces leachates with large amounts of pollutants. Clay barriers are often used to limit soil and underground water pollution. A better understanding of the interaction between ammonium smectite and carboxylic acids would contribute significantly to our understanding of such systems. The SWy-2 (Wyoming smectite) was exchanged with NH4+ and then batched with carboxylic acids (acetic, formic, chloroacetic and oxalic) in concentrations between 0.01 M and 1 M. The solid phases obtained were analysed chemically and characterized by infrared absorption spectroscopy (IR) and powder X-ray diffraction (XRD). Ionic chromatography was used for the quantitative measurement of ammonium ions in the solution after the interaction. For the four acids, the interaction was characterized by a cationic exchange of NH4+ to H3O+. A partial exchange to Al3+ due to a partial dissolution of the sample in strong acidic medium was observed with chloroacetic and oxalic acids for which adsorption of molecules on the clay sample occurs, mainly through H-bonding with the cation. Moreover, the intercalation of oxalic acid in the interlayer space was highlighted.

Management of intestinal microbiota of monogastric animals has increased in importance since the ban of growth promoting antibiotics in many countries. Organic acids have been used as alternatives to antibiotics by many feed manufacturers. Regardless of the wide usage, the effect, dose response and mode of action of acids on intestinal microbes is poorly understood. In this study, we investigated the effects of dietary supplementation of three commonly used products, namely formic acid (FA) (90%), dl-methionine (DLM) (99%) and liquid methionine hydroxy analogue-free acid (88%), on ileal microbiota of pigs. Laboratory simulation system, mimicking swine ileum, was used to study the products at various concentrations and combinations. Furthermore, selected combinations were tested in a piglet trial to confirm the findings made in in vitro studies. FA turned out to have a dual effect on ileal microbiota. At concentrations below 0.5%, it significantly stimulated bacteria, but at higher inclusion rates it was highly inhibitory. This finding, which was consistent in in vitro and in vivo studies, implies that reducing the dose of FA does not lead to a diluted inhibitory effect, but in fact, an opposite, stimulatory effect on intestinal microbiota. It is highly important that feed compounders acknowledge this finding. Unlike FA, the inhibitory effect of methionine hydroxy analogue on ileal bacteria was linearly dose dependent and significant at inclusion levels above 0.2%, in vitro. Partial replacement of methionine hydroxy analogue by FA, or FA by methionine hydroxy analogue, led to an unpredictable outcome due to the dual effects of FA; e.g., a minor inclusion of added FA changed the inhibitory effect of methionine hydroxy analogue into microbial stimulation by FA. Inhibition of ileal microbiota by methionine hydroxy analogue was detected only in in vitro studies, suggesting that intact methionine hydroxy analogue may not have reached the ileum, in live animals. Therefore, if the target is to ensure the inhibitory effect of FA, the FA level in feed should be kept above 0.6%, and not reduced, if methionine hydroxy analogue is used as a methionine source instead of DLM. DLM was totally inert with regard to bacterial growth and metabolism, both in vitro and in vivo. The results of these studies reveal the importance of knowing how each acid product works. Inconsistent results in animal trials may have been partly due to quadratic dose–response effects of products, and unpredictable product combination effects.

Four silages were prepared from perennial ryegrass: two unwilted, ensiled either without (UWN) or with formic acid additive (UWA) and two wilted for either 19 (WS) or 43 h (WL) prior to ensiling. The influence of conservation method on digestion in the rumen and the flow of organic mater (OM) and nitrogen (N) to the small intestine was studied using young steers fitted with rumen and duodenal T-piece cannulae. The silages were well preserved and of similar chemical composition despite a 1·5-fold increase in dry matter (DM) following wilting. The unwilted silages had a higher concentration of fermentation acids and a lower concentration of soluble N in the DM. Polyester bag studies showed that significantly more DM was potentially degradable but the degradation rate was slower in the rumen of steers fed UWA and WS silages (P < 0·05), however, rate of passage was not significantly different for the four treatments. Conservation method had little effect on post-feeding rumen ammonia concentration but feeding unwilted silages caused a greater depression in post-feeding rumen pH (P < 0·05). Wilting lowered N digestibility in the whole tract (P < 0·05), but had no effect on apparent digestion or degradability of N in the rumen as measured by the polyester bag method. Apparent OM digestibility in the whole tract was lower in steers given wilted rather than unwilted silages (P < 0·05), but disappearance of apparently digestible OM in the rumen was greatest for the 19 h wilted silage (WS, P < 0·05). It is concluded that, provided unwilted silage is well preserved, the use of formic acid has little effect on digestion of silage by steers and the effects of wilting are confined to a reduction in whole tract digestibility.