Gram-scale chemical synthesis of galactosyllactoses and their impact on infant gut microbiota in vitro.

Galactooligosaccharides (GOS) are widely used as a supplement in infant nutrition to mimic the beneficial effects found in prebiotic human milk oligosaccharides (HMOs). However, the complexity of the GOS mixture makes it challenging to ascertain which of the GOS components contribute most to their health benefits. Galactosyllactoses (GLs) are lactose-based trisaccharides containing a ß-galactopyranosyl residue at the 3'-position (3'galactosyllactose, 3'-GL), 4'-position (4'-galactosyllactose, 4'-GL), or the 6'-position (6'-galactosyllactose, 6'-GL). These GLs are of particular interest as they are present in both GOS mixtures and human milk at early stages of lactation. However, research on the potential health benefits of these individual GLs has been limited. Gram quantities are needed to assess their health benefits but these GLs are not readily available at this scale. In this study, we report the gram-scale chemical synthesis of 3'-GL, 4'-GL, and 6'-GL. All three galactosyllactoses were obtained on a gram scale in good purity from cheap and commercially available lactose. Furthermore, in vitro incubation of GLs with infant faecal microbiota demonstrates that the GLs were able to increase the abundance of Bifidobacterium and stimulate short chain fatty acid production.

Infant Fecal Fermentations with Galacto-Oligosaccharides and 2'-Fucosyllactose Show Differential Bifidobacterium longum Stimulation at Subspecies Level.

The objective of the current study was to evaluate the potential of 2'-FL and GOS, individually and combined, in beneficially modulating the microbial composition of infant and toddler (12-18 months) feces using the micro-Matrix bioreactor. In addition, the impacts of GOS and 2'-FL, individually and combined, on the outgrowth of fecal bifidobacteria at (sub)species level was investigated using the baby M-SHIME® model. For young toddlers, significant increases in the genera Bifidobacterium, Veillonella, and Streptococcus, and decreases in Enterobacteriaceae, Clostridium XIVa, and Roseburia were observed in all supplemented fermentations. In addition, GOS, and combinations of GOS and 2'-FL, increased Collinsella and decreased Salmonella, whereas 2'-FL, and combined GOS and 2'-FL, decreased Dorea. Alpha diversity increased significantly in infants with GOS and/or 2'-FL, as well as the relative abundances of the genera Veillonella and Akkermansia with 2'-FL, and Lactobacillus with GOS. Combinations of GOS and 2'-FL significantly stimulated Veillonella, Lactobacillus, Bifidobacterium, and Streptococcus. In all supplemented fermentations, Proteobacteria decreased, with the most profound decreases accomplished by the combination of GOS and 2'-FL. When zooming in on the different (sub)species of Bifidobacterium, GOS and 2'-FL were shown to be complementary in stimulating breast-fed infant-associated subspecies of Bifidobacterium longum in a dose-dependent manner: GOS stimulated Bifidobacterium longum subsp. longum, whereas 2'-FL supported outgrowth of Bifidobacterium longum subsp. infantis.

2'-Fucosyllactose inhibits proliferation of Clostridioides difficile ATCC 43599 in the CDi-screen, an in vitro model simulating Clostridioides difficile infection.

Background: Clostridioides difficile is a Gram-positive anaerobic bacterium that can produce the toxins TcdA and/or TcdB and is considered an opportunistic pathogen. C. difficile is mainly transmitted as endospores, which germinate to produce the pathogenic vegetative cells under suitable conditions in the gut. To efficiently screen novel therapeutic- interventions against the proliferation of C. difficile within a complex microbial community, platforms are needed that facilitate parallel experimentation. In order to allow for screening of novel interventions a medium-to-high throughput in vitro system is desirable. To this end, we have developed the 96-well CDi-screen platform that employs an adapted simulated ileal effluent medium (CDi-SIEM) and allows for culturing of pathogenic C. difficile. Methods: C. difficile strain ATCC 43599 was inoculated in the form of vegetative cells and spores into the CDi-screen in the presence and absence of a cultured fecal microbiota and incubated for 48h. To demonstrate its utility, we investigated the effect of the human milk oligosaccharide 2'-Fucosyllactose (2'-FL) at 4 and 8 mg/mL on C. difficile outgrowth and toxin production in the CDi-screen. The test conditions were sampled after 24 and 48 hours. C. difficile -specific primers were used to monitor C. difficile growth via qPCR and barcoded 16S rRNA gene amplicon sequencing facilitated the in-depth analysis of gut microbial community dynamics. Results: C. difficile ATCC 43599 proliferated in CDi-SIEM, both when inoculated as spores and as vegetative cells. The strain reached cell numbers expressed as C. difficile genome equivalents of up to 10 8 cells per mL after 24h of incubation. 2'-FL significantly inhibited the outgrowth of the ATTC 43599 strain within a complex human gut microbial community in the CDi-screen. In addition, a dose-dependent modulation of the gut microbial community composition by 2'-FL supplementation was detected, with a significant increase in the relative abundance of the genus Blautia in the presence of 2'-FL. Conclusion: The CDi-screen is suitable for studying C. difficile proliferation in a complex gut ecosystem and for screening for anti-pathogenic interventions that target C. difficile directly and/or indirectly through interactions with the gut microbiota. Different doses of compounds such as in this study the dose of the human milk oligosaccharide 2'-FL can be screened for efficacy in the inhibition of C. difficile proliferation.

Heat sterilisation dramatically reduces filter efficiency of the majority of FFP2 and KN-95 respirators.

BACKGROUND: Because of the enormous demand for personal protective equipment and especially respiratory protective devices (respirators) during the initial phase of the corona pandemic shortages arose. Sterilisation of used respirators can reduce these shortages. In our study, respirator testing was carried out after only one sterilisation cycle. AIM: To determine if steam sterilisation and reuse could be safely applied for used respirators. METHODS: In a cabinet an aqueous solution of NaCl (0.02% w/v) was nebulized and passed through a sample of the material of a respirator. Passing particle concentrations were measured directly from the cabinet and via the filter material of the respirator for particles ≥ 0.3 µm, ≥ 0.5 µm and ≥ 1.0 µm. FINDINGS: only three out of ten steam sterilised respirators met the requirements of 94% filtration efficiency. CONCLUSION: The results prove that heat sterilisation cannot be generically applied for reuse of respirators safely.

Naturally Fermented Milk From Northern Senegal: Bacterial Community Composition and Probiotic Enrichment With Lactobacillus rhamnosus.

A variety of foods fermented with lactic acid bacteria serve as dietary staples in many African communities; yet, their bacterial profiles are poorly characterized. The integration of health-promoting probiotics into naturally fermented milk products could make a profound impact on human health. Here, we characterize the bacterial community composition of a naturally fermented milk product (lait caillé) from northern Senegal, prepared in wooden bowls (lahals) with a bacterial biofilm to steer the fermentation process. We incorporated a probiotic starter culture containing the most documented probiotic strain Lactobacillus rhamnosus GG (generic strain name yoba 2012) into the local fermentation process. Bar-coded 16S rRNA amplicon sequencing of lait caillé samples indicated that the bacterial community of lait caillé has high species richness with over 100 bacterial genera; however, few have high abundance. In contrast to the diverse bacterial compositions of other characterized naturally fermented milk products, the composition of lait caillé predominantly consists of the lactic acid bacteria Streptococcus and Lactobacillus, resembling the bacterial composition in regular yogurt. The bacterial community composition of lait caillé varies geographically based on the presence of some genera, including Lactoccoccus, Enterococcus, Bifidobacterium, and Bacillus, but this trend is not consistent within production communities. The diversity of bacterial communities is much higher in the lahal biofilm than in the naturally fermented milk products, which is in turn greater than in commercial yogurts. Addition of a starter culture with L. rhamnosus yoba 2012 to milk in lahals led to substantial growth of this probiotic bacterium during the fermentation process. Two independent quantitative PCR-analyses specific for L. rhamnosus yoba 2012 indicated a 20- to 60-fold increase in the total number of probiotic bacteria in the first batch after inoculation. A similar increase of the probiotic was observed in a variation of lait caillé prepared with carbohydrate-rich millet granules (thiakry) added prior to fermentation. This study shows the feasibility of integrating health-promoting probiotic strains into naturally fermented foods produced in regions with a high prevalence of malnutrition.

In Vitro Fermentation of Selected Prebiotics and Their Effects on the Composition and Activity of the Adult Gut Microbiota.

Recently, the concept of prebiotics has been revisited to expand beyond non-digestible oligosaccharides, and the requirements for selective stimulation were extended to include microbial groups other than, and additional to, bifidobacteria and lactobacilli. Here, the gut microbiota-modulating effects of well-known and novel prebiotics were studied. An in vitro fermentation screening platform (i-screen) was inoculated with adult fecal microbiota, exposed to different dietary fibers that had a range of concentrations (inulin, alpha-linked galacto-oligosaccharides (alpha-GOS), beta-linked GOS, xylo-oligosaccharides (XOS) from corn cobs and high-fiber sugar cane, and beta-glucan from oats), and compared to a positive fructo-oligosaccharide (FOS) control and a negative control (no fiber addition). All dietary fibers displayed prebiotic activity, with beta-glucan showing more distinct effects on the microbial composition and metabolism compared to the other fibers. Beta-glucan induced the growth of Prevotella and Roseburia with a concomitant increase in propionate production. Inulin and both forms of GOS and XOS had a strong bifidogenic effect on the microbial composition. A dose-response effect was observed for butyrate when exposed to beta-glucan and inulin. The findings of this study support the potential for alpha-GOS, XOS, and oat beta-glucan to serve as novel prebiotics, due to their association with the positive shifts in microbiome composition and short-chain fatty acid production that point to potential health benefits.

Aspergillus oryzae in solid-state and submerged fermentations. Progress report on a multi-disciplinary project.

We report the progress of a multi-disciplinary research project on solid-state fermentation (SSF) of the filamentous fungus Aspergillus oryzae. The molecular and physiological aspects of the fungus in submerged fermentation (SmF) and SSF are compared and we observe a number of differences correlated with the different growth conditions. First, the aerial hyphae which occur only in SSFs are mainly responsible for oxygen uptake. Second, SSF is characterised by gradients in temperature, water activity and nutrient concentration, and inside the hyphae different polyols are accumulating. Third, pelleted growth in SmF and mycelial growth in SSF show different gene expression and protein secretion patterns. With this approach we aim to expand our knowledge of mechanisms of fungal growth on solid substrates and to exploit the biotechnological applications.