Production, Storage Stability, and Susceptibility Testing of Reuterin and Its Impact on the Murine Fecal Microbiome and Volatile Organic Compound Profile

Abstract

Background: Probiotics are generally considered as safe, but infections may rarely occur in vulnerable patients. Alternatives to live microorganisms to manage dysbiosis may be of interest in these patients. Reuterin is a complex component system exhibiting broad spectrum antimicrobial activity and a possible candidate substance in these cases.Methods: Reuterin supernatant was cultured from Lentilactobacillus diolivorans in a bioreactor in a two-step process. Storage stability at −20°C and effect of repeated freeze-thaw cycles were assessed by high performance liquid chromatography (HPLC). Antimicrobial activity was tested against Clostridium difficile, Listeria monocytogenes, Escherichia coli, Enterococcus faecium, Staphylococcus (S.) aureus, Staphylococcus epidermidis, Streptococcus (S.) agalactiae, Propionibacterium acnes, and Pseudomonas aeruginosae. Male BALBc mice were gavage fed with reuterin supernatant (n = 10) or culture medium (n = 10). Fecal volatile organic compounds (VOC) were assessed by gas chromatography mass spectroscopy; the microbiome was examined by 16S rRNA gene sequencing.Results: The supernatant contained 13.4 g/L reuterin (3-hydroxypropionaldehyde; 3-HPA). 3-HPA content remained stable at −20°C for 35 days followed by a slow decrease of its concentration. Repeated freezing/thawing caused a slow 3-HPA decrease. Antimicrobial activity was encountered against S. aureus, S. epidermidis, and S. agalactiae. Microbiome analysis showed no differences in alpha and beta diversity markers. Linear discriminant effect size (LEfSe) analysis identified Lachnospiraceae_bacterium_COE1 and Ruminoclostridium_5_uncultured_Clostridiales_ bacterium (in the reuterin medium group) and Desulfovibrio_uncultured_ bacterium, Candidatus Arthromitus, Ruminococcae_NK4A214_group, and Eubacterium_xylanophilum_group (in the reuterin group) as markers for group differentiation. VOC analysis showed a significant decrease of heptane and increase of 3-methylbutanal in the reuterin group.Conclusion: The supernatant produced in this study contained acceptable amounts of 3-HPA remaining stable for 35 days at −20°C and exhibiting an antimicrobial effect against S. aureus, S. agalactiae, and S. epidermidis. Under in vivo conditions, the reuterin supernatant caused alterations of the fecal microbiome. In the fecal, VOC analysis decreased heptane and increased 3-methylbutanal were encountered. These findings suggest the high potential of the reuterin system to influence the intestinal microbiome in health and disease, which needs to be examined in detail in future projects.

Plasmid-encoded toxin defence mediates mutualistic microbial interactions

“…It is important to consider that in natural environments, microbial growth is often asynchronous 33 and reuterin may already be present during microbial interactions. Therefore, we externally added reuterin to our experiments to create controlled conditions that closely resemble the rumen environment, where reuterin is prevalent during the growth of both bacterial species as seen in our measurements of the rumen environment and in other gut environments 34 – 37 . Our results demonstrate that in co-culture, the benefit on L. reuteri growth is observed only with E. faecalis strains containing 1,3-PD gene on plasmids and the benefit on E. faecalis growth is enhanced with the plasmid coding the 1,3-PD gene (Fig.…”

Application of the Reuterin System as Food Preservative or Health-Promoting Agent: A Critical Review

“…The 3-HPA content in the supernatant produced by the two-step process was stable at −20 °C for at least 35 days, and then it slowly decreased. Moreover, repeated freeze–thaw cycles (−20/4 °C) caused a slow decrease in 3-HPA with every cycle [ 23 ]. In addition, the degradation of 3-HPA to acrolein in aqueous solution was greater at high temperatures (37 °C and 40 °C), while there was very little degradation at 4 °C for 4 weeks [ 45 ].…”

“…Ingestion of reuterin may also exhibit other health promoting effects. Oral administration of the reuterin supernatant altered the fecal microbiome, decreased heptane and increased 3-methylbutanal in the feces of mice, suggesting the high potential of the reuterin system to relieve some diseases caused by the dysbiosis of intestinal flora [ 23 ]. Oral treatment of broilers with water supplemented with a combination of 5 mM 3-HPA and 0.08 mM microcin J25 promoted growth performance and improved health, presumably through alterations in the cecal microbiome and metabolome, indicating the potential of 3-HPA as a growth promoter instead of as an antibiotic in animals [ 95 ].…”

“…The reuterin system exhibits antimicrobial activities in different food matrices including dairy products and meat [ 19 , 20 ]. Moreover, the system also has some potential health-promoting effects, such as conjugating heterocyclic amines, regulating the gut microbiota and anti-infection [ 21 , 22 , 23 ]. However, many factors still limit the application of the reuterin system at industrial scales.…”

A mini-review: valuable allies for human health: probiotic strains of Limosilactobacillus reuteri

“…As a member of the gut microbiota, L. reuteri strains are involved in a complex interplay within the host and can exert different antimicrobial, immunostimulatory and anti-inflammatory activities (Abuqwider et al, 2022). In addition to lactic acid and different low-molecular mass compounds, L. reuteri produces a small aldehyde, known as reuterin, which is effective in inhibiting numerous bacterial pathogens (Castellani et al, 2021). At the same time, L. reuteri produces biofilm that provides a successful colonization of host-tissues, thus limiting the adhesion of pathogens (Grande et al, 2017).…”