Human milk oligosaccharides (HMOs), as the third largest components in human milk, are thought to be important for the development of infants for various reasons. Previous in vitro studies support the idea that HMOs help to build up the human gut microbiota in infants. It has been shown that some of the intestinal microorganisms found in feces of infants grow on single HMOs as sole carbon substrate. However, so far, the growth effect of specific HMO fractions whose pattern is strongly influenced by the mothers Lewis blood group and secretor status on gut bacteria has not been investigated yet.In this study, HMO fractions were isolated from human milk samples with known Lewis blood group specificity (Lewis a, Lewis b and Lewis negative). Human milk was first delipidated and deproteinized followed by an exclusion of lactose by Sephadex G25 Chromatography. The separation of HMO fractions into neutral (n) and acidic (a) fractions was achieved by FPLC-anion exchange chromatography. Resulting fractions and, in addition, single HMO standards as well as glucose, galactose and lactose were used for growth study of microorganisms.The HMO composition of the samples and the Lewis specificity were analyzed by HPAEC-PAD in combination with MALDI-TOF-MS. Fourteen fractions with Lewis a-, Lewis b- or Lewis-negative specificity and five single HMOs (2 -FL, 3-FL, LNT, LNnT and 6 -SL) were tested for their properties to function as growth substrate. According to previous studies and the existence of intestinal microorganisms in infants gut, we selected seven kinds of intestinal microorganisms for the study: Lactococcus lactis subsp. lactis, Lactobacillus acidophilus, Bifidobacterium longum subsp. infantis, Bifidobacterium longum subsp. longum, Bacteroides vulgatus, Cronobacter sakazakii and Akkermansia muciniphila. The growth studies were performed under anoxic condition using specific medium with 1 mM carbon substrates instead of glucose. Growth curves were measured by OD500nm value using spectrophotometer. The carbon substrates in the medium were analyzed by RP-HPLC and the metabolic products (fatty acids) were analyzed by HPLC.From growth studies of L. lactis and L. acidophilus on glucose, galactose and lactose, we found that L. acidophilus grew on glucose, galactose and lactose; L. lactis grew on glucose, low concentration of galactose or lactose. From growth studies of seven microorganisms on 1 mM glucose, galactose, lactose, neutral or acidic HMO fractions and single HMO standards, we found that L. lactis grew well on lactose and on Lewis fractions LeA1n, LeA3n, LeA3a, LeB2n as well as on 3-FL, LNT and 6 -SL; L. acidophilus grew well on lactose, LeA1n, LeA2n, LeA3n, LeB1n, LeB2n, LeA1a, LeB2a, single 2 -FL, 3-FL and 6 -SL; B. infantis grew well on most of the carbon substrates, B. longum could only grow well on some of the substrates. B. vulgatus grew well on lactose, LeA1n, LeA2n, LeB1n, LeB2n, LeB2n, Le0n, Le0a, 2 -FL, LNT and LNnT. C. sakazakii could grow on glucose, lactose and 3-FL, but most carbon substrates seem to have no effect nor inhibitory effect on the growth. A. muciniphila grew well on glucose, lactose, LeA2n, LeA3n, LeB1n, LeB2n, Le0n, LNT and LNnT. Most neutral HMO fractions had better growth effects than acidic HMO fractions on bacterial growth.In order to analyze the carbon substrates before and after cultivation, 2-AA was used for labeling and RP-HPLC was used for quantification. From the analysis of substrates, we found that B. infantis could metabolize almost all the carbon substrates, including mono-, di-saccharides, HMO fractions, and single HMO standards. B. longum could only metabolize some kinds of the HMO fractions, or metabolized some components to some extent.From the analysis of metabolic products using HPLC (analysis for fatty acid detection), we found that both B. infantis and B. longum consumed the carbon substrates, including mono-, di-saccharides, HMO fractions and single HMOs; they mainly produced lactic acid, acetic acid and some formic acid. The amounts of fatty acids produced by B. infantis were mostly larger than those from B. long.In conclusion, HMOs can positively affect the growth of intestinal microorganisms. B. infantis and B. vulgatus grew well on almost all the HMO fractions. B. longum, L. lactis, L. acidophilus grew well on some of HMO fractions. C. sakazakii could grow on only a few carbon substrates whereas single HMOs and Lewis specific fractions seem to have no effect. A. muciniphila grew well on most of the neutral HMO fractions. The results indicated that most neutral HMO fractions had better growth effects than acidic HMO fractions on bacterial growth. In addition, there were no distinct differences between the effects of the various Lewis blood group specific neutral HMO fractions.
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