Exploration of Alginate oligosaccharide (AOS) promoting Lactobacillus reuteri Y15 growth and production of potential beneficial metabolites and their mechanisms

Pengfei  Zhang

doi:10.60867/00000053

The gut microbiota plays a pivotal role in host health, with lactic acid bacteria (LAB) such as Lactobacillus reuteri (L. reuteri) emerging as key probiotics due to their ability to enhance gut barrier function, modulate inflammation, improve glucose metabolism and regulate lipid metabolism. Alginate oligosaccharides (AOS) which derived from alginate, have gained attention as promising prebiotics, yet their interaction with L. reuteri and underlying mechanisms remain poorly understood. This study focused on the interaction between AOS and L. reuteri Y15 (LRY15). To investigate the strain - specific effects of AOS on LRY15, L. reuteri Y20 (LRY20) (a subtype of LRY15, serving as an intra - species control within L. reuteri), BC - 1 (a Gram - positive bacterium) and ECD8 (a Gram - negative bacterium) were selected as controls, establishing a comparative framework covering intra - species, Gram - positive, and Gram - negative dimensions. The research comprised five phases. Firstly, growth curve analysis showed that AOS (1, 5 and 10 mg/ml) significantly promoted the growth of LRY15 (20–30% increase in OD₆₀₀ at 24 h), with its OD₆₀₀ at 8 h being markedly higher than that of LRY20, BC - 1, and ECD8; LRY20, as a subtype of LRY15, showed a similar growth trend at 8 h but no significant differences at 24 h. So, LRY15 was selected as the subject for this experiment; AOS inhibited BC-1 growth, while ECD8 showed no obvious growth response in four AOS concentrations. Secondly, metabolomic data revealed that principal component analysis (PCA) plots clearly separated different strains at 8 h and 24 h, distinguished AOS treatments within the same strain and differentiated supernatant and bacterial cell metabolomes. KEGG pathway enrichment and metabolite analysis indicated that AOS enhanced the utilization of genetic material - related metabolites (e.g., adenine, thymine), amino acids (e.g., L - alanine), nitrogen - containing substances and increased glycolytic products (e.g., lactic acid) in LRY15, collectively supporting its growth. For BC-1 and ECD8, these metabolites were also supported their bacterial growth. Thirdly, proteomic analysis demonstrated that proteins corresponding to these metabolites were significantly up - regulated in LRY15 but down - regulated in BC - 1 and ECD8. Fourthly, regarding AOS transport and utilization, glycoside hydrolase was up - regulated in LRY15, down - regulated in BC - 1 and not find in ECD8; Mannose-6-phosphate isomerase showed no difference among the three strains. Given that LRY15 lacks mannose enzyme II of the specific PTS transport system and most ABC transporters are related to amino acids, it was speculated that AOS might be transported via the MFS family. The mechanism may be glycoside hydrolase in LRY15 hydrolyses AOS to produce mannose, which is then transported into the bacterium via the MFS family and converted into fructose - 6 - phosphate, entering glycolysis under the action of mannose-6-phosphate isomerase. In contrast, BC - 1 and ECD8 rely on the PTS transport system. Fifthly, AOS stimulated LRY15 to produce beneficial metabolites, including polyunsaturated fatty acids (alpha-linolenic acid, docosahexaenoic acid and eicosapentaenoic acid) and 3-hydroxypropanal. Also, the content of oxidized glutathione was much lower in LRY15 with AOS treatment. AOS enhanced LRY15’s antioxidant capacity, suggesting it promoted the production or utilization of antioxidant - related substances in LRY15. This study clarifies the mechanisms by which AOS acts as a potent prebiotic, facilitating LRY15 growth via MFS-mediated mannose utilization and driving the production of functional metabolites. It addresses the critical gap in understanding AOS-L. reuteri interactions, highlights strain-specific metabolic flexibility of LRY15 and provides a foundation for developing next-generation probiotic therapies and functional foods. The findings underscore the potential of AOS-LRY15 interactions in gut health and biopharmaceutical applications.

Exploration of Alginate oligosaccharide (AOS) promoting Lactobacillus reuteri Y15 growth and production of potential beneficial metabolites and their mechanisms

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