Prebiotic Galactooligosaccharide Metabolism by Probiotic

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Prebiotic galactooligosaccharide metabolism by probiotic lactobacilli and bifidobacteria Taksawan Thongaram, Jennifer L Hoeflinger, Jo May Chow, and Michael Miller J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 03 May 2017 Downloaded from http://pubs.acs.org on May 4, 2017

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Journal of Agricultural and Food Chemistry

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Prebiotic Galactooligosaccharide Metabolism by Probiotic Lactobacilli and

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Bifidobacteria

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Taksawan Thongaram1†, Jennifer L. Hoeflinger1, JoMay Chow2, Michael J. Miller1*

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Department of Food Science and Human Nutrition and Department, 905 S Goodwin Ave Urbana, IL 61801; and 2Abbott Nutrition, 3300 Stelzer Rd Columbus, OH 43219

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*Correspondence: MJM; 905 S Goodwin Ave Urbana, IL 61801; telephone: (217) 244-1973;

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fax: (217) 265-0925; e-mail: [email protected]

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†Current Address: Department of Microbiology, Faculty of Science, Silpakorn University,

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Nakhonpathom, 73000, Thailand

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Running Title: GOS Utilization by Probiotic Bacterial Strains

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Author disclosures: TT, JLH, and MSK do not have any conflicts of interest. MJM has

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received grant funding from Abbott Nutrition. JMC is employed by Abbott Nutrition.

1 ACS Paragon Plus Environment


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ABSTRACT

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Galactooligosaccharide (GOS) are bifidogenic and lactogenic prebiotics; however,

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GOS utilization is strain-dependent. In this study, commercially-available bifidobacteria and

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lactobacilli probiotic strains were evaluated for growth in the presence of GOS. Several

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bifidobacteria and lactobacilli grew on GOS; however, the specific GOS oligomers utilized

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for growth differed. A subset of probiotic bifidobacteria and lactobacilli revealed three

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different GOS utilization profiles delineated by the degrees of polymerization (DP) of GOS:

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1) utilization of 2 DP GOS, 2) utilization of ≤ 3 DP GOS, and 3) utilization of all DP GOS.

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Specifically, Lactobacillus acidophilus NCFM (LA_NCFM) was found to efficiently

26

consume all GOS oligomers.

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supernatant of LA_NCFM correlated with accumulation of galactose. In a LacL-deficient

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LA_NCFM strain, GOS utilization was abolished. This is the first report of LacL’s role in

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GOS metabolism in LA_NCFM.

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delivering GOS with probiotic bifidobacteria and lactobacilli.

Extracellular β-galactosidase activity in the cell-free

In vitro GOS utilization should be considered when

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Keywords: galactooligosaccharide, probiotics,

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bifidobacteria

carbohydrate utilization, lactobacilli,


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INTRODUCTION

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Galactooligosaccharides (GOS) are galactose (gal) polymers containing either a gal or

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glucose (glu) monomer at the reducing end. Commercial GOS are produced by enzymatic

37

transgalactosylation of glu, gal, or lactose (lac) and have a degree of polymerization (DP) of

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two to eight monomers attached in either β-(1→2, 3, 4 or 6) linkages.1,2 GOS have GRAS

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(generally recognized as safe) status in the United States and are routinely added to food

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products as a functional food ingredient.2–4 Several studies report improvements in stool

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consistency5–8 and frequency8,9 with consumption of GOS. Increased interest in GOS is due

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to its ability to resist digestion in the proximal gastrointestinal tract (GIT).4 Greater than

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90% of GOS are available for bacterial fermentation in the distal GIT.4

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Prebiotics are nondigestible food ingredients that selectively stimulate the growth

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and/or activity of microbes in the colon and confer a beneficial effect on the host.10 Dietary

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supplementation of GOS is correlated with a dose-dependent increase in fecal

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bifidobacteria5,6,8,9,11–16 and lactobacilli5,9 in infants and adults. Some studies observed a

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corresponding decrease in fecal pH5,8,9 following GOS supplementation likely due to

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increased acetate and/or lactate production by the growth of resident bifidobacteria and

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lactobacilli. Several ex vivo fecal fermentation studies reported increased bifidobacteria17–23

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and lactobacilli18,20–23 counts in response to GOS inclusion.

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acetate17,19–23 and lactate17,23 within their closed fermentation systems was observed. Strain-

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dependent utilization of GOS by bifidobacteria24–28 and lactobacilli26,27 has been reported.

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Taken together, GOS are bifidogenic and lactogenic; however, in vivo and ex vivo studies do

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not exclude the possibility of cross-feeding within the fecal community. Previously, the in

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vivo lactobacilli community in neonatal piglets was GOS-responsive; however, in vitro

Similarly, an increase in



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fermentation experiments demonstrated this response was not due to the direct fermentation

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of GOS.29 It was hypothesized that the response in vivo was due to cross-feeding among

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GOS utilizers and lactobacilli.

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Commonly, GOS are delivered in combination with probiotic bifidobacteria and

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lactobacilli to promote the growth of these probiotics in the distal GIT, termed synbiotics.

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Probiotics are live microorganisms which when administered in adequate amounts confer

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beneficial effects on the host.30 Probiotic mechanisms of action have been extensively

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reviewed and include inhibition of pathogenic microorganisms31, promotion of intestinal

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barrier function32, and stimulation of the host’s immune response.33 The functional benefits

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of probiotics are strain-specific and should not be applied broadly. Likewise, the ability of

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probiotic bifidobacteria and lactobacilli to ferment GOS is strain-specific and dependent on

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the DP and glycosidic linkages of GOS.25,26,28,34 For example, the expression of several β-

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galactosidase genes in Bifidobacterium adolescentis YIT 401128 and Bifidobacterium longum

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subsp. infantis ATCC 1569734 were induced with GOS as the sole carbon source.

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Meanwhile, Viborg and colleagues35 reported substrate-dependencies of three β-

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galactosidases from B. longum subsp. infantis ATCC 15697. Specifically, β-galactosidase

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cleavage efficiency was dependent on the glycosyl residues present and glycosidic linkages

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in 2, 3, and 4 DP GOS oligomers.

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lactobacilli. The majority of research has focused on the probiotic, Lactobacillus acidophilus

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NCFM (LA_NCFM), which has been studied and utilized as a probiotic for several

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decades.36 LA_NCFM is capable of catabolizing a diverse number of oligosaccharides and

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prebiotic compounds, including GOS.37 Growth of LA_NCFM on GOS induced expression

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of the lac operon (LBA1460 - LBA1468) which includes two β-galactosidase genes (LacA

Far less is known regarding GOS utilization in


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and LacLM) and a lac permease (LacS).38 Further evaluation confirmed that a lacS-deficient

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LA_NCFM mutant was unable to grow on lactose and growth on GOS was lessened.38 This

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suggests that the LacS permease is not responsible for transport of all GOS oligomers.

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Watson and colleagues27 observed galactose accumulation in the growth media during

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growth of LA_NCFM on GOS. They speculated that galactose accumulation was due to a

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higher rate of galactose export overwhelming the rate of galactose uptake or intracellular

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metabolism. However, the function of an extracellular β-galactosidase could also lead to

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galactose accumulation in the growth media. Currently, it is unknown if LacA or LacLM

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participate in the cleavage of GOS in LA_NCFM and if either β-galactosidase functions

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extracellularly.

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Commercially-available probiotic bifidobacteria and lactobacilli were evaluated for

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their ability to utilize GOS as a sole carbon source. This study identified several probiotic

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candidates that could be combined with GOS in food products. Specifically, LA_NCFM was

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found to be a robust utilizer of GOS and this study confirmed that LacL is an extracellular β-

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galactosidase responsible for extracellular cleavage of GOS during utilization by LA_NCFM.

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MATERIALS AND METHODS

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Chemicals and Reagents Standard chemicals and reagents were purchased from Fisher Scientific (Hampton, NH) and were of analytical grade unless noted otherwise.

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Probiotic and human isolates of

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Bacterial Strains and Routine Growth Conditions.

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bifidobacteria (n = 12) and lactobacilli (n = 12) listed in Table 1 were cultured in deMan

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Rogosa Sharpe (MRS) broth (Difco, Detroit, MI) from frozen stocks (12.5% glycerol, v/v)

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and incubated at 37 °C for 24 h anaerobically (90% N2, 5% CO2 and 5% H2).

For

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bifidobacteria, all growth media was supplemented with 0.05% (w/v) L-cysteine.

All

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commercial strains were supplied by Abbott Nutrition. Additional strains were purchased

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from the American Type Culture Collection or isolated from a commercial probiotic product

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in order to provide taxonomic diversity.

110 111

Preparation of Purified Galactooligosaccharides. Commercially available Purimune™

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GOS (GOS-P, Ingredion, Westchester, IL) and Vivinal® GOS (GOS-V, FrieslandCampina

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Domo, Paramus, NJ) were processed to obtain ≥ 3 DP GOS molecules. Stock solutions were

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prepared by dissolving 1.5 g of GOS-P or GOS-V in 100 mL and applied to an XK 50/100

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column (GE Healthcare Life Sciences, Pittsburgh, PA) packed with Sephadex® G25 medium

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(Sigma-Aldrich, St. Louis, MO). The column was eluted with ultrapurified water at a rate of

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8 mL/min and 12 mL fractions were collected with a fraction collector, model #FC 203B

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(Gilson Inc., Middleton, WI). Stock solutions (1.5%) of glu, lac, or raffinose were used as

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mono-, di-, and trisaccharides standards.

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Carbohydrates were detected using the phenol–sulfuric acid assay.39

Briefly,

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fractions were diluted 1:25 (50 µL) and mixed with 150 µL of concentrated sulfuric acid in a

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96-well microtiter plate. Immediately, 30 µL of 5% (v/v) phenol in H2O was added and

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incubated in a static water bath at 80 °C for 30 min. After cooling to room temperature, the

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absorbance at 490 nm was measured. Based on carbohydrate analysis, fractions containing


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minimal mono- and disaccharides (fractions 30-55) were pooled and freeze-dried with a

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FreeZone 4.5 freeze-dryer (Labconco, Kansas, MO). Herein, pooled purified fractions of

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Purimune™ and Vivinal® GOS are referred to as GOS-PP and GOS-VP, respectively.

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Bifidobacteria and lactobacilli were

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Bacterial Growth on Galactooligosaccharides.

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evaluated for growth with GOS as a sole carbon source. Stationary phase bifidobacteria and

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lactobacilli were grown in MRS were subcultured twice in a semi-defined MRS (sMRS)

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broth40 containing 1% (w/v) glu. For bifidobacteria, sMRS was supplemented with 0.05%

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(w/v) L-cysteine. Twice subcultured bacterial cultures were centrifuged at 3220 x g for 10

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min, washed twice and resuspended in 10 mL of fresh carbohydrate-free sMRS. A 1% (v/v)

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bacterial inoculum was prepared by passage into a fresh aliquot of sMRS containing 1% (v/v)

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of a select carbohydrate. Carbohydrates tested were glu (positive control), lac, GOS-V,

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GOS-P, GOS-VP, GOS-PP, or carbohydrate-free sMRS (negative control). Honeycomb

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plates (Growth Curves USA, Piscataway, NJ) were prepared with 300 µL total volume; a 250

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µL aliquot of bacterial inoculum covered with 50 µl of mineral oil. Plates were incubated

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anaerobically at 37 °C for 48 h in a Bioscreen C machine (Growth Curves USA). Bacterial

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growth was monitored by measuring optical density at 600 nm (OD600) in 30 min intervals

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preceded by shaking at maximum speed for 30 s.

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replications were completed for each carbohydrate.

A minimum of three independent

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Carbohydrate utilization data were visualized by heatmap and dendrogram to show

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relatedness of bifidobacteria and lactobacilli strains by their pattern of growth. Heatmap and

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dendrograms were constructed in R (ver 3.3.1) using the gplots package ver 3.0.1.41

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β-galactosidase activity was determined using the

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β-galactosidase Activity Assay.

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chromogenic substrate, o-nitrophenyl-β-D-galactopyranoside (ONPG; Sigma-Aldrich) in the

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cell-free supernatant and permeabilized cells as previously described. Briefly, 1 mL of

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bifidobacteria or lactobacilli cells were harvested by centrifugation at 10,000 x g for 10 min,

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washed twice with 50 mM sodium phosphate buffer (pH 7), and resuspended in 1 mL of Z

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buffer (60 mM Na2HPO4.7H2O, 40 mM NaH2PO4.H2O, 10 mM KCl, 1 mM MgSO4.7H2O

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and 50 mM β-mercaptoethanol). The OD600 was measured, and cells were permeabilized by

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addition of 10 µL of 0.1% sodium dodecyl sulfate and 40 µL of chloroform and shaking for 5

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min. The β-galactosidase activity assay reaction mixture was a total of 1 mL; 100 µL of

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permeabilized cells, 200 µL of 10 mM ONPG, and 700 µL of 50 mM sodium phosphate

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buffer (pH 7). The reaction mixture was incubated at 37°C for 15 min and stopped by

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addition of 1 mL of 1 M Na2CO3. The OD420 and OD550 were measured, and the β-

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galactosidase activity is expressed in Miller Units as described by equation 1, where the

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OD600 is the initial optical density before permeabilization, OD550 is the optical density of

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cellular debris, OD420 measure the amount of o-nitrophenol released, T (min) is the reaction

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time, and V (mL) is the volume of permeabilized cells. Miller Units are defined as the

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increase in o-nitrophenol per bacterium per minute.42

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− = 1000 ∗

(.∗ ) !∗"∗ #

(1)

166 167

Matrix-assisted

Laser

Desorption/Ionization

Time-of-flight

Mass

Spectrometry

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(MALDI-TOF MS).

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supernatants were determined by MALDI-TOF MS.

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labeled with 2-aminobenzamide (2-AB) by the addition of 10 µL of 2-AB labeling reagent

The 24 h post-fermentation oligosaccharide profiles of cell-free Dried cell-free supernatants were


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(0.35 M 2-AB in glacial acetic acid and 1 M Na(CN)BH3 in dimethyl sulfoxide). The

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reaction mixture was incubated at 65 °C for 2.5 h. Following incubation, the 2-AB labeled

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samples were cooled to room temperature and cleaned with GlycoClean™ S-Cartridges

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(Prozyme, Hayward, CA) following the manufacturer’s instructions. The purified 2-AB

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labeled cell-free supernatants were vacuum dried.

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The 2-AB labeled cell-free supernatants were dissolved in 100 µL of MilliQ water,

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and 1 µL was mixed with 1 µL of 0.1% trifluoroacetic acid in water. This reaction mixture

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was combined with the matrix composed of 2 µL of 0.5 M 2,5-dihydroxybenzoic acid and

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0.1 M 5-methoxy salicylic acid in methanol and spotted on a MALDI plate. MALDI mass

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spectra were acquired using the 4800 Plus MALDI-TOF/TOF instrument (Applied

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Biosystems, Carlsbad, CA) equipped with 337 nm solid state laser and processed with the

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Data Explorer software (Applied Biosystems). The data are presented as a mass spectrum

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with peaks labeled according to their mass-to-charge (m/z) ratio: 485 (DP2), 647 (DP3), 809

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(DP4), 971 (DP5), and 1133 (DP6).

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Thin Layer Chromatography (TLC). The post-fermentation oligosaccharide profiles of

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three independent replicates of cell-free supernatants were determined by TLC.34 A 2 μL

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aliquot of 24 h cell-free supernatants were spotted onto silica gel TLC plates (Sigma-

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Aldrich). Glu, gal, and lac were included as detection standards. The TLC plates were

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developed with a 2:1:1 mixture of n-propanol, acetic acid, and distilled water. Plates were

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sprayed with 0.5% α-naphthol and 5% H2SO4 in ethanol and visualized by heating at 120 °C

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for 10 min.

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Bacterial genomic DNA of

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DNA Isolation, Manipulation, and Transformation.

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LA_NCFM was isolated using the Microbial DNA Isolation Kit (MO BIO, Carlsbad, CA)

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according to the manufacturer’s instructions. Plasmid DNA (pORI28) from E. coli was

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recovered with the QIAprep Spin Miniprep kit (QIAGEN, Valencia, CA). All DNA primers

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were purchased from Integrated DNA Technologies (Coralville, IA). PCR reactions were

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carried out using EconoTaq polymerase (Lucigen, Middleton, WI) and purified using the

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Qiagen PCR purification kit (QIAGEN, Valencia, CA) according to manufacturer’s

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instructions. Restriction enzymes (BamHI and XbaI) and T4 DNA ligase were purchased

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from New England Biolabs (Ipswich, MA).

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(Lactobacillus: 5 µg/mL) or erythromycin (E. coli and Lactobacillus: 150 µg/mL and 5

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µg/mL, respectively) were used for selection.

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prepared using 3.5x sucrose MgCl2 electroporation buffer as described elsewhere.43

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Electroporation was performed at 2.5 kV using the Electroporator 2510 (Eppendorf,

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Hauppauge, NY).

When necessary, chloramphenicol

Electrocompetent LA_NCFM cells were

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β-galactosidase (lacL) Gene Inactivation in Lactobacillus acidophilus NCFM. The β-

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galactosidase gene (lacL, ORF = LBA1467) of L. acidophilus NCFM (LA_NCFM) was

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inactivated by targeted homologous recombination using the erythromycin-resistant, non-

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replicative pORI28 vector.43,44 The primers lacLF_BamHI (5’-

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ATGCGGATCCTGCCGAACGAGCCATGTATG-3’) and lacLR_XbaI (5’-

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AATTTCTAGACCGGCATAAGATTCGTTTCC-3’) were used to amplify a 945 bp internal

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region of lacL from LA_NCFM. The lacL amplicon was cloned into pORI28 using the

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BamHI/XbaI sites (underlined in primer sequences). The resulting plasmid was transformed


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by electroporation into LA_NCFM containing pTRK669, a temperature-sensitive, repA+

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helper plasmid required for replication of pORI28. Inactivation of lacL in LA_NCFM was

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confirmed by junction fragment PCR. The 5’ junction fragment produced by amplification

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with the primers ori+ (5’-ATAATGAACTGTGCTGATTAC-3’) and lacL- (5’-

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GGATCGTAGACTAAGAGCGC-3’) had an expected size of 1385 bp. The 3’ junction

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fragment produced by amplification with the primers ori- (5’- TTCAATCGCCAACGAATC-

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3’) and lacL+ (5’-CACAATCCCAGTTCCTAGTG-3’) had an expected size 1315 bp. The

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resulting strain LA_NCFM ∆lacL was analyzed for its ability to utilize glu, gal, lac and GOS-

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P as sole carbon sources as described above. Utilization was compared to the parental strain,

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LA_NCFM.

227 228

RESULTS AND DISCUSSION

229 230

Purification of Commercially-available Galactooligosaccharides. GOS-P powder is 90-

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92% on a dry matter basis with 63-81% of ≥ 3 DP GOS. Similarly, GOS-V syrup is 74-76%

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on a dry matter basis with 59% of ≥ 2 DP GOS, excluding lac but including other 2 DP GOS

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molecules. Consistent with the manufacturer’s descriptions, GOS-P was largely composed

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of ≥ 3 DP GOS and eluted from the column between fractions 30 and 95 with its peak at

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fraction 59 (Figure 1A). Meanwhile, GOS-V was primarily composed of 2 DP GOS and

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eluted from the column between fractions 35 and 105 with its peak at fraction 70 (Figure

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1B). Based on the retention times of glu, lac, and raffinose, fractions 30 – 55 were collected

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to obtain high molecular weight (≥ 3 DP) GOS-P and GOS-V. The pooled fractions were

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labeled GOS-PP and GOS-VP and used to evaluate probiotic bifidobacteria and lactobacilli



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utilization of ≥ 3 DP GOS. Due to limitations of GOS-PP and GOS-VP recovered during

241

purification, GOS-PP and GOS-VP were only used for the fermentation experiments. All

242

other experiments were conducted with GOS-P.

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Overall growth was

244

Probiotic Fermentation of Prebiotic Galactooligosaccharides.

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reduced when GOS was added a sole carbon source rather than glu or lac (Figure 2). All

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strains, except L. jensenii ATCC 25258 (LJ_25258) and L. rhamnosus GG ATCC 53103

247

(LR_53103), were capable of growing in the presence of lac. Decreased growth was also

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observed when GOS-PP and GOS-VP were added rather than GOS-P and GOS-V,

249

respectively. On the contrary, a previous study demonstrated similar growth profiles of

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seven bifidobacteria regardless of GOS (55 and 85%) purity.24 Herein, GOS utilization was

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defined as growth to an OD600 > 0.25 when GOS-PP and GOS-VP were the sole carbon

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sources. Several bifidobacteria, including B. adolescentis ATCC 15703 (BA_15703), B.

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bifidum ATCC 11617 (BB_11617), B. longum subsp. infantis M63 (BI_M63), B. longum

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subsp. infantis S12 ATCC 15697 (BI_15697), B. breve M-16V (BB_M16V), B. breve ATCC

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15700 (BB_15700), and B. animalis subsp. lactis Bb-12 (BL_Bb12) were identified as GOS

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utilizers (Figure 2A). Specifically, BI_M63 and BI_15697 grew well in the presence of both

257

GOS-PP and GOS-VP and are consistently reported as strong GOS utilizers.24,27,28,34 B.

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animalis subsp. lactis ATCC 10140 (BL_10140), B. bifidum ATCC 29521 (BB_29521), B.

259

animalis subsp. lactis Bf-6 (BL_Bf6), and B. animalis subsp. animalis ATCC 25527

260

(BA_25527) were identified as non-GOS utilizers. Several lactobacilli, including L. reuteri

261

DSM 17938 (LR_17938), L. johnsonii ATCC 11506 (LJ_11506), L. plantarum LP-66

262

(LP_LP66), L. paracasei LCV-1 (LP_LCV1), L. johnsonii La-1 (LJ_La1), LA_NCFM, and


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L. acidophilus La-5 (LA_La5) were characterized as GOS utilizers (Figure 2B). The latter

264

three lactobacilli strains grew well in the presence of both GOS-PP and GOS-VP and

265

consistent with other work.27 L. rhamnosus HN001 DR20 (LR_DR20), L. gasseri ATCC

266

33323 (LG_33323), LJ_25258, and LR_53103 were non-GOS utilizers.

267

LR_53103 was reported as a GOS utilizer; however, the GOS preparation used was only

268

55% pure, and growth was likely due to residual gal.26 In this study, LR_53103 did not grow

269

on GOS-P, GOS-PP, and GOS-VP and the minimal growth on GOS-V (59% pure) was

270

presumably due residual glu or gal. The growth of probiotic bifidobacteria and lactobacilli in

271

the presence of GOS-PP and GOS-VP was highly strain-dependent and is consistent with

272

previous work.24–28 Also, intraspecies differential utilization of GOS-PP and GOS-VP were

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observed among B. bifidum and B. longum strains. Previously, variation in GOS utilization

274

has been reported amongst B. bifidum strains.24

275

bifidobacteria and 21 lactobacilli strains and reported widespread utilization of a purified

276

GOS-V preparation.27 The authors noted several intraspecies differences in purified GOS-V

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utilization, including B. breve, L. acidophilus, and L. fermentum; however, the two B.

278

bifidum and ten B. longum strains were utilizers. Herein, B. longum BB536 (BL_BB536) and

279

L. fermentum CECT 5716 (LF_5716) were capable of utilizing GOS-PP but not GOS-VP.

280

While GOS-P and GOS-V are both manufactured by transgalactosylation, the specific

281

conditions chosen by the manufacturers may result in complex β-linkage profiles.1 Perhaps

282

the β-linkages in the GOS-VP preparation are not cleaved by the BL_BB536 and LF_5716’s

283

β-galactosidases. Unfortunately, this study did not characterize GOS utilization by linkage

284

types, and further experimentation is needed to test this hypothesis.

Previously,

Watson and colleagues surveyed 12

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Post-fermentation Analysis of GOS-P by Select Probiotics. The oligosaccharide profile

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following growth on GOS-P was completed for a subset of probiotic bifidobacteria and

288

lactobacilli. The oligosaccharide profile of growth media (MRSC in Figure 3A and MRS in

289

Figure 3D) supplemented with 1% GOS-P analyzed by MALDI-TOF MS revealed distinct

290

signals with m/z values 485.08, 647.12, 809.14, 971.16, and 1133.22.

291

corresponded to GOS oligomers with a DP of 2 to 6, respectively. Consistent with the

292

purification experiment, GOS-P is primarily composed of 3 DP GOS which is displayed as

293

100% relative intensity in the growth media spectra (Figure 3A & D).

These peaks

294

Taken together with the growth data from above, the oligosaccharide profiles of two

295

bifidobacteria and three lactobacilli were analyzed by MALDI-TOF MS and TLC (Figures 3

296

& 4). Three distinct oligosaccharide profiles were observed; 1) bacteria which consumed

297

strictly ≤ 2 DP GOS, 2) bacteria which consumed ≤ 3 DP GOS, and 3) bacteria which

298

consumed all DP GOS. The oligosaccharide profile for BL_Bb12 and LR_DR20 showed a

299

preference for 2 DP GOS-P with no detectible consumption of ≥ 3 DP GOS-P (Figure 3B, E

300

& 4). Previously, BL_Bb12 was reported to consume ≥ 3 DP GOS as detected by high-

301

performance anion-exchange chromatography (HPAEC).27

302

have limitations in sensitivity. Small reductions in the major peak (set at 100%) or spot

303

intensity may go unnoticed, and each of these methods presents the isomeric diversity of

304

each DP of GOS as a single peak/spot. Previously, three bifidobacteria strains were observed

305

to differentially consume 3 DP GOS isomers and no strain consumed all fourteen isomers of

306

3 DP GOS.45 Herein, BI_M63 and LF_5716 preferentially consumed 2 & 3 DP GOS while ≥

307

4 DP GOS were not greatly utilized (Figure 3C, F & 4). The preference of BI_M63 for 2 &

308

3 DP GOS-P is consistent with other B. longum subsp. infantis strains.25,28,34 Contrary to

MALDI-TOF and TLC both


Page 15 of 31


309

Watson and colleagues, LF_5716 did not detectibly accumulate gal in the cell-free

310

supernatant in this study27. Lastly, LA_NCFM consumed all DP GOS-P oligomers and most

311

notably, 4 & 5 DP GOS-P (Figure 3G & 4). Since MALDI-TOF reports the relavtive

312

amounts of the various DPs and not absolute amounts, the LA_NCFM MALDI-TOF

313

spectrum understates the extent to which LA_NCFM consumes GOS-P.

314

LA_NCFM clearly consumes all GOS-P oligomers as these molecules are not detectable.

315

Additionally, LA_NCFM was observed to accumulate gal in the cell-free supernatant which

316

has been previously described (Figure 4).27 This finding would suggest that LA_NCFM’s

317

genome encodes an extracellular enzyme capable of liberating gal from high-DP GOS. It is

318

tempting to speculate that this excess gal could promote the growth of non-GOS utilizers in a

319

diverse bacterial community, such as the distal GIT.

By TLC,

320

Overall, the differences in GOS consumption reported here and elsewhere reaffirm

321

the conclusion that the ability for bifidobacteria and lactobacilli to consume various GOS

322

oligomers is strain-dependent. Furthermore, care should be taken when interpreting the GOS

323

literature as residual mono- and disaccharides remaining after transgalactosylation may mask

324

“true” GOS utilization. In this study, probiotic bifidobacteria and lactobacilli were identified

325

as GOS-utilizers by combining growth data on purified GOS-P and GOS-V preparations with

326

post-fermentation oligosaccharide profiles.

327

Gal-containing oligosaccharides are

328

β-galactosidase Activity of Select Probiotics.

329

catabolized by the glycosyl hydrolase, β-galactosidase (EC 3.2.1.23). Genes encoding β-

330

galactosidases are upregulated when bifidobacteria46,47 and lactobacilli37,38 are grown in the

331

presence of GOS. Herein, an increase in intracellular β-galactosidase activity corresponded



Page 16 of 31

332

with the exponential growth phase of select probiotic bifidobacteria and lactobacilli (Figure

333

5). The β-galactosidase activity was measured in permeabilized BL_Bb12, BI_M63, and

334

LF_5716 cells but not in the cell-free supernatant (Figure 5A, B & D). This suggests that

335

these strains are limited to ferment only the GOS oligomers that can be transported inside the

336

cell where it can be catabolized by intercellular β-galactosidases. Comparative genomics of

337

47 bifidobacteria strains revealed a large set of carbohydrate-active genes, including

338

catabolic enzymes and transporters.48 It is not surprising that no extracellular β-galactosidase

339

activity in BL_Bb12 and BI_M63 was detected as few encoded glycosyl hydrolases (~11%)

340

in bifidobacteria are predicted to be extracellular.48 LR_DR20 grew in the presence of GOS-

341

P without detectible β-galactosidase activity (Figure 5C).

342

consumption data (Figure 2B, 3E & 4) suggests that its β-galactosidase (LRH_RS01960) is

343

efficient at catabolism of lac and 2 DP GOS-P (contains 7-10% lac) but not higher DP GOS.

344

Therefore, the experimental conditions (6 h time points) in this study may have missed its

345

peak activity during growth on 2 DP GOS (Figure 5). Lastly, only LA_NCFM had β-

346

galactosidase activity in permeabilized cells and the cell-free supernatant (Figure 5E). The

347

extracellular β-galactosidase activity (575 Miller Units) in LA_NCFM occurred during

348

exponential growth and tapered off at the stationary growth phase. Previously, two β-

349

galactosidase genes, lacL and lacA, were upregulated during growth of LA_NCFM on

350

GOS.37,38 It was unknown whether lacL and/or lacA are able to catabolize GOS. In this

351

study, a lacL-deficient mutant was constructed in LA_NCFM.

352

abolished in the LA_NCFM_∆lacL strain as compared to the wild-type LA_NCFM (Figure

353

6A & B). Of note, the small initial increase in OD is likely due to utilization of 2 DP GOS

354

intracellularly rather than the activity of another extracellular β-galactosidase. While the

LR_DR20’s growth and

GOS-P utilization was


Page 17 of 31


355

LacS permease was found to transport lac and GOS,38 the growth of LA_NCFM_∆lacL on

356

lac was delayed (> 36 h) but not abolished (data not shown). Since LA_NCFM_ ∆lacL

357

retains a functional LacS permease and LacA β-galactosidase, the observed growth of

358

LA_NCFM_ ∆lacL on lac is likely due to intracellular cleavage of lac by LacA. Conversely,

359

it is unlikely that LacA contributes to ≥ 3 DP GOS-P utilization in LA_NCFM_ ∆lacL due to

360

this strain’s inability to transport large molecules of GOS-P intracellularly. This study did

361

not evaluate the role of LacA in the cleavage of intracellular GOS-P and further

362

experimentation is needed to determine if LacA is capable of cleaving GOS molecules.

363

Taken together, these results suggest that LacL is responsible for extracellular cleavage of

364

larger GOS oligomers allowing for intracellular transport of smaller GOS molecules and

365

LA_NCFM growth.

366 367

ACKNOWLEDGMENTS

368

We thank Todd R. Klaenhammer at North Carolina State University for providing the tools

369

to perform the lacL knockout in L. acidophilus NCFM and Mark S. Kuhlenschmidt for

370

technical assistance during the GOS purification.



371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416

Page 18 of 31

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(13) Davis, L. M. G.; Martínez, I.; Walter, J.; Hutkins, R. A dose dependent impact of prebiotic galactooligosaccharides on the intestinal microbiota of healthy adults. Int. J. Food Microbiol. 2010, 144 (2), 285–292. (14) Davis, L. M. G.; Martínez, I.; Walter, J.; Goin, C.; Hutkins, R. W. Barcoded pyrosequencing reveals that consumption of galactooligosaccharides results in a highly specific bifidogenic response in humans. PloS One 2011, 6 (9), e25200. (15) Walton, G. E.; van den Heuvel, E. G. H. M.; Kosters, M. H. W.; Rastall, R. A.; Tuohy, K. M.; Gibson, G. R. A randomised crossover study investigating the effects of galacto-oligosaccharides on the faecal microbiota in men and women over 50 years of age. Br. J. Nutr. 2012, 107 (10), 1466–1475. (16) Vulevic, J.; Juric, A.; Walton, G. E.; Claus, S. P.; Tzortzis, G.; Toward, R. E.; Gibson, G. R. Influence of galacto-oligosaccharide mixture (B-GOS) on gut microbiota, immune parameters and metabonomics in elderly persons. Br. J. Nutr. 2015, 114 (4), 586–595. (17) Rycroft, C. E.; Jones, M. R.; Gibson, G. R.; Rastall, R. A. A comparative in vitro evaluation of the fermentation properties of prebiotic oligosaccharides. J. Appl. Microbiol. 2001, 91 (5), 878–887. (18) Palframan, R. J.; Gibson, G. R.; Rastall, R. A. Effect of pH and dose on the growth of gut bacteria on prebiotic carbohydrates in vitro. Anaerobe 2002, 8 (5), 287–292. (19) Cardelle-Cobas, A.; Fernández, M.; Salazar, N.; Martínez-Villaluenga, C.; Villamiel, M.; Ruas-Madiedo, P.; de los Reyes-Gavilán, C. G. Bifidogenic effect and stimulation of short chain fatty acid production in human faecal slurry cultures by oligosaccharides derived from lactose and lactulose. J. Dairy Res. 2009, 76 (3), 317–325. (20) Hernot, D. C.; Boileau, T. W.; Bauer, L. L.; Middelbos, I. S.; Murphy, M. R.; Swanson, K. S.; Fahey, G. C. In vitro fermentation profiles, gas production rates, and microbiota modulation as affected by certain fructans, galactooligosaccharides, and polydextrose. J. Agric. Food Chem. 2009, 57 (4), 1354–1361. (21) Vester Boler, B. M.; Hernot, D. C.; Boileau, T. W.; Bauer, L. L.; Middelbos, I. S.; Murphy, M. R.; Swanson, K. S.; Fahey, G. C. Carbohydrates blended with polydextrose lower gas production and short-chain fatty acid production in an in vitro system. Nutr. Res. N. Y. N 2009, 29 (9), 631–639. (22) Beards, E.; Tuohy, K.; Gibson, G. Bacterial, SCFA and gas profiles of a range of food ingredients following in vitro fermentation by human colonic microbiota. Anaerobe 2010, 16 (4), 420–425. (23) Cardelle-Cobas, A.; Olano, A.; Corzo, N.; Villamiel, M.; Collins, M.; Kolida, S.; Rastall, R. A. In vitro fermentation of lactulose-derived oligosaccharides by mixed fecal microbiota. J. Agric. Food Chem. 2012, 60 (8), 2024–2032. (24) Hopkins, M.J.; Cummings, J.H.; Macfarlane, G.T. Inter-species differences in maximum specific growth rates and cell yields of bifidobacteria cultured on oligosaccharides and other simple carbohydrate sources. J. Appl. Microbiol. 1998, 85 (2), 381–386. (25) Barboza, M.; Sela, D. A.; Pirim, C.; Locascio, R. G.; Freeman, S. L.; German, J. B.; Mills, D. A.; Lebrilla, C. B. Glycoprofiling bifidobacterial consumption of galactooligosaccharides by mass spectrometry reveals strain-specific, preferential consumption of glycans. Appl. Environ. Microbiol. 2009, 75 (23), 7319–7325.



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(26) Mei, G.-Y.; Carey, C. M.; Tosh, S.; Kostrzynska, M. Utilization of different types of dietary fibres by potential probiotics. Can. J. Microbiol. 2011, 57 (10), 857–865. (27) Watson, D.; O’Connell Motherway, M.; Schoterman, M. H. C.; van Neerven, R. J. J.; Nauta, A.; van Sinderen, D. Selective carbohydrate utilization by lactobacilli and bifidobacteria. J. Appl. Microbiol. 2013, 114 (4), 1132–1146. (28) Akiyama, T.; Kimura, K.; Hatano, H. Diverse galactooligosaccharides consumption by bifidobacteria: implications of β-galactosidase--LacS operon. Biosci. Biotechnol. Biochem. 2015, 79 (4), 664–672. (29) Hoeflinger, J. L.; Kashtanov, D. O.; Cox, S. B.; Dowd, S. E.; Jouni, Z. E.; Donovan, S. M.; Miller, M. J. Characterization of the Intestinal Lactobacilli Community following Galactooligosaccharides and Polydextrose Supplementation in the Neonatal Piglet. PloS One 2015, 10 (8), e0135494. (30) FAO/WHO. Guidelines for the Evaluation of Probiotics in Food http://www.who.int/foodsafety/fs_management/en/probiotic_guidelines.pdf?ua=1 (accessed Oct 19, 2016). (31) Servin, A. L. Antagonistic activities of lactobacilli and bifidobacteria against microbial pathogens. FEMS Microbiol. Rev. 2004, 28 (4), 405–440. (32) O’Flaherty, S.; Klaenhammer, T. R. The role and potential of probiotic bacteria in the gut, and the communication between gut microflora and gut/host. Int. Dairy J. 2010, 20 (4), 262–268. (33) Gill, H.; Prasad, J. Probiotics, immunomodulation, and health benefits. Adv. Exp. Med. Biol. 2008, 606, 423–454. (34) Garrido, D.; Ruiz-Moyano, S.; Jimenez-Espinoza, R.; Eom, H.-J.; Block, D. E.; Mills, D. A. Utilization of galactooligosaccharides by Bifidobacterium longum subsp. infantis isolates. Food Microbiol. 2013, 33 (2), 262–270. (35) Viborg, A. H.; Katayama, T.; Abou Hachem, M.; Andersen, M. C. F.; Nishimoto, M.; Clausen, M. H.; Urashima, T.; Svensson, B.; Kitaoka, M. Distinct substrate specificities of three glycoside hydrolase family 42 β-galactosidases from Bifidobacterium longum subsp. infantis ATCC 15697. Glycobiology 2014, 24 (2), 208–216. (36) Sanders, M. E.; Klaenhammer, T. R. Invited review: the scientific basis of Lactobacillus acidophilus NCFM functionality as a probiotic. J. Dairy Sci. 2001, 84 (2), 319–331. (37) Andersen, J. M.; Barrangou, R.; Hachem, M. A.; Lahtinen, S. J.; Goh, Y.-J.; Svensson, B.; Klaenhammer, T. R. Transcriptional analysis of prebiotic uptake and catabolism by Lactobacillus acidophilus NCFM. PloS One 2012, 7 (9), e44409. (38) Andersen, J. M.; Barrangou, R.; Abou Hachem, M.; Lahtinen, S.; Goh, Y. J.; Svensson, B.; Klaenhammer, T. R. Transcriptional and functional analysis of galactooligosaccharide uptake by lacS in Lactobacillus acidophilus. Proc. Natl. Acad. Sci. U. S. A. 2011, 108 (43), 17785–17790. (39) Masuko, T.; Minami, A.; Iwasaki, N.; Majima, T.; Nishimura, S.-I.; Lee, Y. C. Carbohydrate analysis by a phenol-sulfuric acid method in microplate format. Anal. Biochem. 2005, 339 (1), 69–72. (40) Barrangou, R.; Altermann, E.; Hutkins, R.; Cano, R.; Klaenhammer, T. R. Functional and comparative genomic analyses of an operon involved in fructooligosaccharide


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utilization by Lactobacillus acidophilus. Proc. Natl. Acad. Sci. U. S. A. 2003, 100 (15), 8957–8962. Warnes, G. R.; Bolker, B.; Bonebakker, L.; Gentleman, R.; Huber, W.; Liaw, A.; Lumley, T.; Maechler, M.; Magnusson, A.; Moeller, S.; et al. gplots: Various R programming tools for plotting data. R Package Version 2009, 2 (4). Miller, J. H. Experiments in molecular genetics, 3rd ed.; Cold Spring Harbor Laboratory: New York City, NY, 1972. Luchansky, J. B.; Tennant, M. C.; Klaenhammer, T. R. Molecular cloning and deoxyribonucleic acid polymorphisms in Lactobacillus acidophilus and Lactobacillus gasseri. J. Dairy Sci. 1991, 74 (10), 3293–3302. Russell, W. M.; Klaenhammer, T. R. Efficient system for directed integration into the Lactobacillus acidophilus and Lactobacillus gasseri chromosomes via homologous recombination. Appl. Environ. Microbiol. 2001, 67 (9), 4361–4364. Peacock, K. S.; Ruhaak, L. R.; Tsui, M. K.; Mills, D. A.; Lebrilla, C. B. Isomerspecific consumption of galactooligosaccharides by bifidobacterial species. J. Agric. Food Chem. 2013, 61 (51), 12612–12619. O’Connell Motherway, M.; Kinsella, M.; Fitzgerald, G. F.; van Sinderen, D. Transcriptional and functional characterization of genetic elements involved in galacto-oligosaccharide utilization by Bifidobacterium breve UCC2003. Microb. Biotechnol. 2013, 6 (1), 67–79. Andersen, J. M.; Barrangou, R.; Abou Hachem, M.; Lahtinen, S. J.; Goh, Y. J.; Svensson, B.; Klaenhammer, T. R. Transcriptional analysis of oligosaccharide utilization by Bifidobacterium lactis Bl-04. BMC Genomics 2013, 14, 312. Milani, C.; Lugli, G. A.; Duranti, S.; Turroni, F.; Mancabelli, L.; Ferrario, C.; Mangifesta, M.; Hevia, A.; Viappiani, A.; Scholz, M.; et al. Bifidobacteria exhibit social behavior through carbohydrate resource sharing in the gut. Sci. Rep. 2015, 5, 15782.



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535

FIGURE LEGENDS

536

Figure 1. Chromatograms showing the separation of (A) Purimune™ GOS (GOS-P) and (B)

537

Vivinal® GOS (GOS-V) with a Sephadex®G-25 column at the flow rate 8 mL/min. Fractions

538

30 - 55 (outlined) were pooled and freeze-dried for bacterial fermentation experiments.

539

Glucose (glu), lactose (lac) and raffinose (raf) were run as mono-, di- and trisaccharide

540

standards, respectively.

541 542

Figure 2. Maximum bacterial growth (OD600) of probiotic (A) bifidobacteria and (B)

543

lactobacilli in the presence of 1% glucose (glu), lactose (lac), Purimune™ GOS (GOS-P),

544

purified Purimune™ GOS (GOS-PP), Vivinal® GOS (GOS-V), and purified Vivinal® GOS

545

(GOS-VP). Refer to Table 1for bacteria abbreviations. No growth was observed in the

546

absence of carbohydrates. Optical densities were corrected by subtracting blanks control

547

wells without added cells or carbohydrates. Dendrogram shows relatedness by fermentation

548

pattern. Average of three or more independent replicates.

549 550

Figure 3. Oligosaccharide profile of growth media containing 1% Purimune™ GOS (GOS-

551

P) and 24 h post-fermentation cell-free supernatants of probiotic bifidobacteria and

552

lactobacilli grown in the presence of GOS-P. (A) sMRSC, (B) B. animalis subsp. lactis

553

Bb12, (C) B. longum subsp. infantis M-63, (D) sMRS, (E) L. rhamnosus DR20, (F) L.

554

fermentum CECT5716, and (G) L. acidophilus NCFM. Mass spectrum with peaks labeled

555

according to their mass-to-charge (m/z) ratio and degrees of polymerization (DP) of GOS-P:

556

485 (DP2), 647 (DP3), 809 (DP4), 971 (DP5), and 1133 (DP6). The peak with the greatest

557

intensity is set to 100%.


Page 23 of 31


558 559

Figure 4. Thin layer chromatography (TLC) analysis of 24 h cell-free supernatants after

560

growth on 1% Purimune™ GOS (GOS-P). Lane 1: 1% glucose (glu); lane 2: 1% galactose

561

(gal); lane 3: 1%lactose (lac); lane 4: L. acidophilus NCFM (LA_NCFM); lane 5: L.

562

fermentum CECT5716 (LF_CECT5716); lane 6: L. rhamnosus DR20 (LR_DR20); lane 7: B.

563

longum subsp. infantis M-63 (BL_M63); lane 8: B. animalis subsp. lactis Bb-12 (BL_Bb12);

564

lane 9: sMRS + 1% GOS-P; lane 10: sMRSC + 1% GOS-P. TLC plate image was a

565

representative of three independent experiments.

566 567

Figure 5. β-galactosidase activity (Miller Units) in cell-free supernatant (red squares) and

568

permeabilized cells (green triangles) of (A) B. animalis subsp. lactis Bb12 (BL_Bb12), (B)

569

B. longum subsp. infantis M-63 (BI_M63), (C) L. rhamnosus DR20 (LR_DR20), (D) L.

570

fermentum CECT5716 (LF_CECT5716), and (E) L. acidophilus NCFM (LA_NCFM). β-

571

galactosidase activity was measured every 6 h during growth on Purimune™ GOS for 24 h.

572

Bacterial growth (OD600) is included for reference (blue diamonds). Data are reported as the

573

mean ± standard error of three independent replicates.

574 575

Figure 6. Growth curves of L. acidophilus NCFM (A) wild-type and (B) lacL gene

576

knockout strain grown in sMRS supplemented with 1% glucose (glu), galactose (gal), lactose

577

(lac) and Purimune™ GOS (GOS-P). Optical densities were corrected by subtracting blank

578

control wells without added bacteria or carbohydrates. Average of three independent

579

replicates.



Page 24 of 31

Table 1 Bacterial strains used in this study bacteria source/strain abbreviation origin bifidobacteria Bifidobacterium adolescentis ATCC 15703 BA_15703 adult intestine Bifidobacterium animalis subsp. animalis ATCC 25527 BA_25527 rat feces Bifidobacterium animalis subsp. lactis Bb-12 BL_Bb12 NA Bifidobacterium animalis subsp. lactis Bf-6 BL_Bf6 human feces Bifidobacterium animalis subsp. lactis DSM 10140 BL_10140 yogurt Bifidobacterium bifidum ATCC 11617 BB_11617 NA Bifidobacterium bifidum ATCC 29521 BB_29521 infant feces Bifidobacterium breve ATCC 15700 BB_15700 infant intestine Bifidobacterium breve M-16V BB_M16V infant feces Bifidobacterium longum BB536 BL_BB536 infant feces Bifidobacterium longum subsp. infantis M-63 BI_M63 human Bifidobacterium longum subsp. infantis S12 ATCC 15697 BI_15697 infant intestine lactobacilli Lactobacillus acidophilus La-5 LA_La5 NA Lactobacillus acidophilus NCFM LA_NCFM human Lactobacillus gasseri ATCC 33323 LG_33323 human Lactobacillus fermentum CECT 5716 LF_5716 human milk Lactobacillus jensenii ATCC 25258 LJ_25258 adult vagina Lactobacillus johnsonii ATCC 11506 LJ_11506 NA Lactobacillus johnsonii ACD-1/La-1 LJ_La1 NA Lactobacillus paracasei LCV-1 LP_LCV1 NA Lactobacillus plantarum LP-66 LP_LP66 NA Lactobacillus reuteri DSM 17938 LR_17938 probiotic1 Lactobacillus rhamnosus HN001 DR20 LR_DR20 cheddar cheese Lactobacillus rhamnosus GG ATCC 53103 LR_53103 human feces 1 Isolated from commercially available BioGaia ProTectis® drops (BioGaia, Stockholm, Sweden) NA = source of origin not publically available


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Figure 1.



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Figure 6.


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Prebiotic Galactooligosaccharide Metabolism by Probiotic

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