Probiotic metabolism of human milk oligosaccharides (HMOs) and prebiotics

Thongaram, Taksawan

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Description

Title

Probiotic metabolism of human milk oligosaccharides (HMOs) and prebiotics

Author(s)

Thongaram, Taksawan

Issue Date

2015-01-21

Director of Research (if dissertation) or Advisor (if thesis)

Miller, Michael J.

Doctoral Committee Chair(s)

Donovan, Sharon M.

Committee Member(s)

• Miller, Michael J.
• Jin, Yong-Su

Department of Study

Food Science & Human Nutrition

Discipline

Food Science & Human Nutrition

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Probiotics
• Human Milk Oligosaccharides (HMOs)
• Prebiotics
• Lactic Acid Bacteria

Abstract

Human milk contains a high concentration of complex oligosaccharides (HMOs) that are believed to confer physiological benefits to infants such as immunomodulation and prevention of pathogen attachment. In addition, it has been postulated that HMOs serve as prebiotics by promoting the growth of bifidobacteria in the infant gastrointestinal tract (GIT). In this study, the first aim was to investigate the probiotic metabolism of HMOs and HMO precursors. Growth parameters were determined by inoculating glucose-grown cultures into basal deMan Rogosa Sharpe (MRS) (no added glucose) with 1% carbohydrate (+0.5 g/l L- cysteine for bifidobacteria) and measuring growth over 48 h. Cultures were grown in microtiter plates, which were incubated under 90% N2, 5% CO2 and 5% H2 at 37oC. Results indicated that: (1) N-acetyl-D-glucosamine (GlcNAc) was widely used by the lactobacilli, but B. breve ATCC15700 was the only bifidobacteria strain that could utilize this carbohydrate, (2) none of the bifidobacteria and very few lactobacilli could utilize either free L-fucose (L. rhamnosus GG and L. rhamnosus DR20) or sialic acid (L. plantarum LP‐66), (3) none of the lactobacilli could ferment the HMOs: 3’-Sialyllactose (3’-SL), 6’-Sialyllactose (6’-SL), 2’- Fucosyllactose (2’-FL) and 3’- Fucosyllactose (3’-FL), yet four lactobacilli demonstrated moderate growth with LNnT, (4) amongst the bifidobacteria strains, only B. infantis ATCC 15697 and B. infantis M-63 were able to ferment 3'-SL, 6’-SL, 2’-FL and 3’-FL, (5) when B. infantis M‐63 was grown with 3’-SL, no sialic acid accumulated in the growth media, but when it was grown with 2’-FL, the 44% of the L‐fucose liberated from 2’-FL remained in the media iii and (6) B. infantis, B. breve, L. acidophilus, L. plantarum and L. reuteri were able to ferment Lacto-N-neotetraose (LNnT), which was confirmed by High Performance Liquid Chromatography (HPLC) analysis. Thus, there are differences in utilization profiles of milk oligosaccharides among lactobacilli and bifidobacteria strains, information that may aid in the development of future synbiotic formulations. The second aim was to investigate the consumption of LNnT by selected lactobacilli and bifidobacteria. We found that LNnT was a growth factor for B. infantis, B. breve, L. acidophilus, L. plantarum and L. reuteri. In this study, HPLC and Thin Layer Chromatography (TLC) results confirmed that amongst the tested strains, L. acidophilus NCFM was found to be the most efficient lactobacillus strain to utilize LNnT. In addition, we characterized the consumption of LNnT in L. acidophilus NCFM and further investigated a -galactosidase gene involved in LNnT utilization by L. acidophilus NCFM. β-galactosidase lacL gene knockout in L. acidophilus NCFM and subsequent carbohydrate utilization analysis demonstrated that LNnT was unable to be utilized by the knockout strain, confirmimg that β-galactosidase lacL gene is required for LNnT utilization. Additionally, growth curves of the lacL knockout strain showed a reduced growth rate and a longer lag phase on lactose, suggesting that the β- galactosidase lacL gene plays a significant role in LNnT and lactose utilization in L. acidophilus NCFM. In the third aim, the consumption of galactooligosaccharides (GOS) by lactobacilli was investigated by comparing with selected bifidobacteria in order to determine the metabolism of GOS, utilization patterns and potential of probiotic and prebiotic combinations. Matrix- assisted laser desorption/ionization time-of-flight (MALDI-TOF) and TLC analysis of cell- iv free supernatants taken during growth of five probiotic bacteria suggested differences in the utilization of Purimune™ GOS (GOS-P). L. rhamnosus DR20 and B. lactis Bb-12 preferentially utilized disaccharides, while L. fermentum and B. infantis utilized mostly di- and trisaccharides over larger degree of polymerization (DP) GOS. Among the tested strains, only L. acidophilus NCFM showed extracellular and intracellular β-galactosidase activity. Interestingly, L. acidophilus NCFM showed a preference to consume GOS with DP 2-6 and released galactose very efficiently from GOS-P. L. acidophilus NCFM lacL gene knockout and subsequent carbohydrate utilization analysis demonstrated that GOS was not utilized by the knockout strain, confirmimg that β-galactosidase lacL gene is required for GOS utilization. Our results suggest that the -galactosidase lacL gene is involved in GOS consumption by L. acidophilus NCFM, revealing that the role of functional lacL -galactosidase is important for the metabolism of lactose and complex carbohydrates for the survival of intestinal lactobacilli in GI tract.

Graduation Semester

2014-12

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Copyright 2014 Taksawan Thongaram

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