Utilization of Ningxiang pig milk oligosaccharides by Akkermansia muciniphila in vitro fermentation: enhancing neonatal piglet survival

Abstract

Akkermansia muciniphila (A. muciniphila), an intestinal symbiont residing in the mucosal layer, shows promise as a probiotic. Our previous study found that the abundance of A. muciniphila was significantly higher in Ningxiang suckling piglets compared to other breeds, suggesting that early breast milk may play a crucial role. This study examines A. muciniphila's ability to utilize Ningxiang pig milk oligosaccharides. We discovered that A. muciniphila can thrive on both Ningxiang pig colostrum and purified pig milk oligosaccharides. Genetic analysis has shown that A. muciniphila harbors essential glycan-degrading enzymes, enabling it to effectively break down a broad spectrum of oligosaccharides. Our findings demonstrate that A. muciniphila can degrade pig milk oligosaccharides structures such as 3'-FL, 3'-SL, LNT, and LNnT, producing short-chain fatty acids in the process. The hydrolysis of these host-derived glycan structures enhances A. muciniphila's symbiotic interactions with other beneficial gut bacteria, contributing to a dynamic microbial ecological network. The capability of A. muciniphila to utilize pig milk oligosaccharides allows it to establish itself in the intestines of newborn piglets, effectively colonizing the mucosal layer early in life. This early colonization is key in supporting both mucosal and metabolic health, which is critical for enhancing piglet survival during lactation.

Keywords: Akkermansia muciniphila; Ningxiang pig; milk oligosaccharides; piglet survival; short-chain fatty acids.

Figure 1

Akkermansia muciniphila DSM 22959 growth in Ningxiang-pig colostrum. (A) A. muciniphila DSM 22959 growth was assessed over a 48 h period by measuring optical density at a wavelength of 600 nm (OD600). Bacteria were grown in brain heart infusion broth medium (BHI), or BHI supplemented with Ningxiang-pig colostrum (10% v/v). (B) A. muciniphila DSM 22959 was grown in vitro in the absence or presence of 10% v/v Ningxiang-pig colostrum until 48 h of growth; A. muciniphila was quantified by qPCR. (C–E) Changes in SCFA levels in fermentation solutions. Data are expressed as the mean ± standard deviation (n = 3). ^*p < 0.05, and ^***p < 0.001, Colostrum group compared with the CON group. ^#p < 0.05, and ^###p < 0.001 compared with the same treatment at different time.

Figure 2

Akkermansia muciniphila DSM 22959 growth in Ningxiang-pig purified PMO. (A) Schematic illustration of Ningxiang-pig purified PMO preparation. (B) A. muciniphila DSM 22959 growth was assessed over a 48 h period by measuring optical density at a wavelength of 600 nm (OD600). Bacteria were grown in brain heart infusion broth medium (BHI), or BHI supplemented with Ningxiang-pig purified PMO (1 mg/mL). (C) A. muciniphila DSM 22959 was grown in vitro in the absence or presence of 1 mg/mL Ningxiang-pig purified PMO until 48 h of growth; A. muciniphila was quantified by qPCR. (D–F) Changes in SCFA levels in fermentation solutions. Data are expressed as the mean ± standard deviation (n = 3). ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001, Colostrum group compared with the CON group. ^##p < 0.01, and ^###p < 0.001 compared with the same treatment at different time.

Figure 3

Identification of carbohydrate active enzymes (CAZymes) in A. muciniphila DSM 22959 genome. (A) Proportional Venn diagram showing the number of identified CAZyme encoding genes using the Diamond, HMMER and dbCAN_sub tools that are part of the dbCAN2 metaserver analysis. (B) The table shows the CAZymes and the presence of signal peptides, separated by CAZyme class, that were identified by at least two out of three dbCAN2 tools.

Figure 4

Domain architecture of putative MO targeting CAZymes in genome of A. muciniphila DSM 22959. (A) Simplified schematics of pathways involved in main HMO degradation (acidic trioses, neutral trioses and tetraoses). (B) CAZymes are divided in groups based upon their predicted enzyme activity: from top to bottom, α-sialidases, α-fucosidases, β-galactosidases and α/β-hexosaminidases. The displayed domains are those identified by HMMER, in number of amino acids, indicated at the right side of each protein. (C) The number of putative MO targeting CAZymes of A. muciniphila DSM 22959.

Figure 5

A. muciniphila DSM 22959 growth in acidic trioses, neutral trioses and tetraoses. (A) A. muciniphila DSM 22959 growth was assessed over a 48 h period by measuring optical density at a wavelength of 600 nm (OD600). Bacteria were grown in brain heart infusion broth medium (BHI), or BHI supplemented with neutral trioses (2-fucosyllactose [2-FL] and 3-fucosyllactose [3-FL]), tetraoses (lacto-N-tetraose [LNT], lacto-N-neotetraose [LNnT]), and acidic trioses (3-siallylactose [3-SL], 6-sialyllactose [6-SL]) (10 mM respectively). (B–D) Changes in SCFA levels in fermentation solutions. Data are expressed as the mean ± standard deviation (n = 3). ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001, Colostrum group compared with the CON group. ^##p < 0.01, and ^###p < 0.001 compared with the same treatment at different time.