Structural Characterization and in Vitro Fermentation of Î²

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Structural Characterization and in vitro Fermentation of #-mannooligosaccharides Produced from Locust bean gum by GH-26 Endo-#-1,4-mannanase (ManB-1601) Praveen Kumar Srivastava, Deepesh Panwar, Harish Prashanth KV, and Mukesh Kapoor J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00123 • Publication Date (Web): 22 Feb 2017 Downloaded from http://pubs.acs.org on February 24, 2017

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Journal of Agricultural and Food Chemistry

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Structural Characterization and in vitro Fermentation of β-mannooligosaccharides

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Produced from Locust bean gum by GH-26 Endo-β-1,4-mannanase (ManB-1601)

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Praveen Kumar Srivastava†, Deepesh Panwar†,§, K. V. Harish Prashanth‡,§ and Mukesh

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Kapoor†,§,*

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†

Department of Protein Chemistry and Technology; ‡Department of Biochemistry; CSIR-Central

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Food Technological Research Institute, Mysuru-570 020, India; §Academy of Scientific and

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Innovative Research (AcSIR), CSIR-CFTRI Campus, Mysuru, India

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* Tel: +91-821-2515331, E-mail: [email protected]

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

1 ACS Paragon Plus Environment


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ABSTRACT

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Size exclusion chromatography of β-mannooligosaccharides (β-MOS) mixture, obtained from

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ManB-1601 hydrolysis of locust bean gum, resulted in separation of oligosaccharides with varied

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degree of polymerization (DP 2, 3 and 5). The oligosaccharides were structurally [ESI-MS,

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FTIR, XRD, TGA and NMR (1H and 13C)] and functionally (in vitro fermentation) characterized.

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DP2 oligosaccharide was composed of two species (A) mannopyranose β-1,4 mannopyranose

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and (B) α-1,6-galactosyl-mannopyranose, while DP3 oligosaccharide showed presence of only

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one species i.e. α-D-galactosyl-β-D-mannobiose. ManB-1601 was capable of cleaving near the

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branch points in the substrate resulting in oligosaccharides with galactose at the terminal position

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apart from attacking unsubstituted β-1,4 glycosidic linkages. DP2 and DP3 improved the growth

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of three out of seven species of Lactobacillus while, DP5 resulted in poor growth of all

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Lactobacillus spp. under in vitro conditions. DP2, DP3 and DP5 were found to inhibit the growth

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of E. coli, L. monocytogenes and S. typhi.

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Keywords: GH-26 endo-β-1,4-mannanase, Lactobacillus spp., β-mannooligosaccharides,

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Structural characterization, in vitro fermentation

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INTRODUCTION

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Currently, several researchers and leading food companies are developing functional food

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preparations

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xylooligosaccharides, pecticoligosaccharides, isomaltooligosaccharides, galactooligosaccharides,

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and fructooligosaccharides)/probiotics due the realization of the intricate relationship between

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food ingredients, beneficial gut microflora and health.1-8

containing

prebiotics

(arabinooligosaccharides, arabinoxylooligosaccharides,

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Mannooligosaccharides (MOS), including α-MOS and β-MOS, are a relatively new class

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of oligosaccharides, that have gained significant interest as a prebiotic.9,10 β-MOS are not acted

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upon by gastric or pancreatic enzymes and are used by a selected group of beneficial gut

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microflora for inducing several physiological changes.11 β-MOS can be produced by hydrolytic

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cleavage of mannans by endo-β-1,4-mannanase (EC 3.2.1.78) and show variability with respect

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to their degree of polymerization and presence/position of substituents.12-14 Recently, a number

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of reports have cited the prebiotic potential of crude β-MOS from guar gum, locust bean gum,

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konjac mannan and copra meal using endo-β-1,4-mannanase.10, 15-19 However, detailed studies

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encompassing purification, structural characterization and in vitro fermentation behaviour of β-

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MOS on probiotic populations need to be carried out in order to provide further information.

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ManB-1601 (UniProtKB-A0A0A1E3J1) obtained from Bacillus sp. CFR1601 is a GH26,

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thermostable, endo-β-1,4-mannanase having (β/α)8-barrel spatial arrangement.20-21 In the present

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study, attempts were made to understand the molecular properties [degree of polymerization,

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composition of monomer units, chemical structure and thermal properties] of β-MOS derived

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from locust bean gum after ManB-1601 hydrolysis; to analyze the cleavage pattern of ManB-

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1601 on locust bean gum and to evaluate the propensity of β-MOS towards fermentation by

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Lactobacillus spp. and food borne pathogens.



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MATERIALS AND METHODS

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Microbial culture and chemicals

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Locust bean gum, volatile free acid standard mix, fructo-oligosaccharides (FOS) [composed of

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glucose-(fructose)n with β−2→1 linkage between the fructose monomer units, fructose chain

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length: 2 to 60, average DP>10] and 3,5-dinitrosalicylic acid were purchased from Sigma-

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Aldrich (St. Louis, MO). Biogel-P2 was purchased from Bio-Rad Laboratories (Hercules, CA).

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Commercial mannobiose having degree of polymerization of two (CDP2) was purchased from

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Megazyme, Ireland. Microbial culture media and other ingredients were procured from Himedia

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(Bengaluru, India). Lactobacillus cultures were obtained from National Culture Collection of

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Industrial Microorganisms (NCIM) at CSIR-National Chemical Laboratory (NCL), Pune (India).

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Cultures of food borne pathogens (Escherichia coli ATCC 11775, Salmonella typhi ATCC

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25241 and Listeria monocytogenes ATCC 13932) were obtained from Microbiology and

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Fermentation Technology Department at CSIR-CFTRI, Mysuru. All other reagents used were of

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the highest purity available commercially.

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Production of β-MOS from locust bean gum using ManB-1601

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ManB-1601 was produced and purified as per the protocol described earlier.20,21 For the

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preparation of β-MOS, 1% (w/v) locust bean gum in 50 mM sodium phosphate buffer (pH 7, 80

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mL) was incubated with purified ManB-1601 (100 U/mL) under shaking conditions (200 rpm) at

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50 oC for 270 min.18 The hydrolysate obtained was kept in boiling water bath for 5 min to

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inactivate the enzyme. Enzyme resistant and insoluble fractions were removed by centrifugation

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at 3622 x g for 10 min at room temperature. Thereafter, three volumes of ethanol were added to

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the supernatant for precipitating high-molecular-mass fractions and centrifuged at 3622 x g for


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20 min. The supernatant containing β-MOS was flash evaporated and stored at -20 oC until

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further use.18

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Purification of β-MOS

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β-MOS mixture obtained after locust bean gum hydrolysis by ManB-1601 was fractionated on

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Biogel P-2 (bed volume, 100 mL) previously equilibrated with Milli-Q water at room

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temperature in accordance to our previous report.18 Elution was done using Milli-Q water at a

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flow rate of 10 mL/h.

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Characterization of purified β-MOS

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Electron spray ionization- mass spectrometry (ESI-MS)

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The molecular mass of purified β-MOS fractions was determined by ESI-MS under the

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following operational conditions: capillary voltage 3.5 kV, core voltage 100 V, source

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temperature 120 oC, desolvation temperature 300 oC, gas (nitrogen) 500 L/h and core gas (argon)

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50 L/h.

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Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD)

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IR spectra of β-MOS were determined using an IFS 25 FTIR spectrophotometer (Bruker,

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Ettlingen, Germany). β-MOS were ground and mixed with spectroscopic grade potassium

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bromide powder (KBr) and pressed into a pellet. The spectra of the samples were recorded in the

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range of 700-1500 cm-1. The XRD patterns of β-MOS were obtained using a SmartLab 3 kW X-

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ray diffractometer (Rigaku, Tokyo, Japan) with Cu Kα source at 2θ angle probed 5-75o at 0.02

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/min scanning speed.

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Thermogravimetric and differential thermogravimetric analysis

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Thermogravimetric (TGA) and differential thermal (DTA) analysis of β-MOS were carried out

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under continuous N2 flow on a STA 2500 Regulus differential scanning calorimeter (Netzsch



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Instruments, Burlington, MA). Samples were weighed (~2-5 mg) and heated from 30-700 oC at a

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heating rate of 10 oC/min in aluminium pans. The empty pan was used as reference.

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1

H and 13C NMR spectroscopy

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The NMR spectroscopic studies were carried out on an Avance AQS 500MHz

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spectrophotometer (Bruker, Reinstetten, Germany) NMR spectrometer. 5 mg of sample was

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dissolved in 600µl D2O and spectra were collected. 1H spectrum was collected at 500 MHz with

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spectral width kept at 10330 Hz. The signal for water occurs at 4.8 ppm frequency in 1H NMR

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spectra on the D2O solvent used. The water suppression pulse program zgpr was employed to

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collect the 1H spectrum with 16 scans. The

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10K scans.

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In vitro fermentation of β-MOS by lactobacilli and food borne pathogens

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Seed inoculum

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Seed inocula of Lactobacillus sp. [L. plantarum (NCIM 2372), L. fermentum (NCIM 2165), L.

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casei var. rhamnosus (NCIM 2125), L. brevis (NCIM 2090), L. acidophilus (NCIM 2285), L.

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casei (NCIM 2126) and L. helveticus (NCIM 2126)] and food borne pathogens [(E. coli (ATCC

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11775), S. typhi (ATCC 25241) and L. monocytogenes (ATCC 13932)] were prepared by

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cultivation in MRS (de Man, Rogosa and Sharpe) medium under static conditions and tryptic soy

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broth (TSB) under shaking (200 rpm) conditions, respectively for 12h at 37 oC.

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Batch fermentation

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Batch fermentation studies for Lactobacillus spp. and food borne pathogens were carried out in

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MRS and TSB media (devoid of glucose, 1 mL), respectively. The media were supplemented

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separately with either filter sterilized (0.22µ) β-MOS having degree of polymerization (DP2,

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DP3, DP5 and CDP2), FOS (positive control-1) or glucose (positive control-2) at a final

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C NMR spectra were collected at 125 MHz with


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concentration of 0.1% (w/v). Thereafter, MRS media was inoculated with 10 µl (A600nm~0.2) of

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inoculum obtained from respective Lactobacillus spp. and incubated under static conditions (37

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o

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respective food borne pathogens and incubated under shaking conditions (37 oC, 24h, 200 rpm).

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The fermented media was centrifuged (3500 rpm, 5 min, RT) and cells were resuspended in

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sterile phosphate buffer saline. Growth characteristics of the cultures were measured by

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determining the log colony forming units (CFU)/mL, absorbance (A600nm) and pH.

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SCFA analysis

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The cell free supernatants of Lactobacillus spp. were analysed for short chain fatty acid (SCFA)

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analysis. 100 mg of sodium chloride was added to each culture supernatant (450 µl) and the

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mixture was acidified by the addition of sulphuric acid (125 µl, 9 M). The acidified mixture was

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extracted by an equal volume of diethyl ether. 1 µl of sample (top layer) was subjected to gas

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chromatography on a Clarus 580 instrument (Perkin Elmer, Waltham, MA). The column used

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was a 30 m x 0.32 mm i.d., film thickness 0.25 µm, Elite-WAX cross bond- PEG (Perkin Elmer,

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Waltham, MA). Column conditions were 60 oC (2 min hold), 2 oC/min to 135 oC (0.5 min hold).

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Injector and detector were kept at 220 and 230 oC, respectively. Volatile free acid standard mix

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was used as the standard (Sigma-Aldrich, St. Louis, MO).

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Analytical procedures

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ManB-1601 activity was assayed by determining the release of reducing sugar equivalents from

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locust bean gum (0.5% w/v) in 50 mM sodium phosphate buffer (pH 7) at 55 oC using the

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dinitrosalicylic acid method.22 One unit of ManB-1601 activity was defined as the quantity of

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enzyme required for liberation of 1.0 µmol mannose/minute under the standard assay conditions.

C, 48h), while TSB media was inoculated with 10 µl (A600nm~0.2) of inoculum obtained from



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Total carbohydrate content of β-MOS was determined as glucose equivalents by the phenol-

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sulfuric acid method with slight modifications.23

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Statistical analysis

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Measurements pertaining to in vitro fermentation experiments (A600nm and pH) were carried out

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in duplicates and presented as arithmetic mean. Calculation for arithmetic mean was done using

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using Sigma plot 10.0. Analysis of variance (ANOVA) of data was performed with Origin Pro 7

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(Origin Lab Corporation, Northampton, MA) statistical software using Tuckey’s test at 0.01

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level.

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RESULTS AND DISCUSSION

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During the last two decades, non-digestible prebiotic oligosaccharides have garnered increased

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attention from both industry and academia due to their positive impact on the gut microbiome.4 It

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has been shown conclusively that DP and molecular structure of these prebiotic oligosaccharides

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play a vital role in their utilization by probiotic bacteria.24 Therefore, the overall aim of the

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present study was to establish the structure (glycosidic linkages, oligosaccharide composition

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and DP)-function (in vitro fermentability) relationship of β-MOS with probiotic Lactobacillus

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spp. and/or food borne pathogens.

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Production, purification and characterization of β-MOS

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Hydrolysis of locust bean gum by ManB-1601 for 270 min resulted in generation of β-MOS with

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an overall yield of 30% (w/v). Separation of crude β-MOS mixture by Biogel-P2

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chromatography resulted in five well separated peaks which were designated as P1, P2, P3, P4,

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and P5 (Figure 1A). Peak P1-P5 were identified as cationised molecules [M+Na]+ by ESI-mass

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spectroscopy (Figure 1B). The P1 and P2 peaks corresponded to a pentasaccharide (DP5) [P1-

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599, derived from cross cleavage of branched pentasaccharide; 25 P2- 851, [{(180x 5) - (4x18)} +


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23]. Peaks P3 (DP3), P4 (DP2) and P5 (monomer) corresponded to trisaccharide [527, {(180x 3)

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- (2x18)} + 23], disaccharide [365, {(180x 2) - (1x18)} + 23]; 707, two disaccharide residues

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with one sodium (342 + 342+23)] and mannose [203, (180 + 23)], respectively. The extent of

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hydrolysis of galactomannans by endo-β-1,4-mannanase derived either from legume seeds or

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microorganisms like fungi and bacteria and subsequent generation of oligosaccharides has been

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shown to be dependent noticeably on

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galactomannan and endo-β-1,4-mannanase family [as endo-β-1,4-mannanase from different

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families display different catalytic properties towards substituted mannans as seen in case of

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xylanases.26-29 Cerqueira et al.30 reported presence of oligosaccharides of DP ranging from 2 to 7

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after endo-β-1,4-mannanase hydrolysis of galactomannan obtained from Gleditsiatria canthos.

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Ghosh et al.10 showed production of mannose, mannobiose and mannotriose after endo-β-1,4-

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mannanase hydrolysis of copra meal. Similarly, endo-β-1,4-mannanase from Penicillium

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occitanis was able to produce mannose, mannobiose, mannotriose and mannotetrose from locust

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bean gum.31

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FTIR spectroscopy and XRD

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FTIR spectra of purified β-MOS were recorded in the carbohydrate region (700-1500 cm-1)

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(Figure 2). Peaks at 816, 811 and 817 cm-1 found in disaccharide (DP2), trisaccharide (DP3) and

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pentasaccharide (DP5) revealed the presence of anomeric configurations (α and β anomers). The

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glycosidic linkage of α-D-galactopyranose and β- D-mannopyranose units in DP2, DP3 and DP5

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β-MOS was observed at 875, 867 and 873 cm-1.32, 33 The peaks between 1037 to 1155 cm-1 in

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DP2, DP3 and DP5 β-MOS signified the stretching vibration of C-O and C-O-H bonds present in

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the glycosidic linkage of galactomannans.33, 34

frequency and pattern of galactose substitution in



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XRD studies were carried out only for DP2 and DP3 β-MOS, as the hygroscopic nature

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of DP5 β-MOS prevented the removal of bound water by lyophilisation and subsequent XRD

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studies. The broad characteristic peak of less intensity at 20o in DP2 (Figure 3A) and DP3

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(Figure 3B) β-MOS resulted due to more rotational freedom in the molecule, indicated their

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typical non-crystalline structure and amorphous nature. Similar results were found in case of

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chitosan oligosaccharides, konjac glucomannan-based films and guar gum.35-37

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Thermogravimetric (TGA) and differential thermal (DTA) analysis

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It is quintessential to understand the thermal behaviour of prebiotic molecules like β-

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mannooligosaccharides as many times they are added in food products which undergo thermal

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processing like roasting, frying and baking. In case, the prebiotic oligosaccharide is not able to

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withstand high temperatures encountered during thermal processing it is highly unlikely that it

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can deliver desirable attributes in the functional food. TGA and DTA is known to provide

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important information like determining the contents of oligosaccharides from the residual mass

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and also prediction for the ratio of bound water by applying mass loss calculation during first

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decomposition.38 The TGA (Figure 3C) and DTA (Figure 3D) graphs revealed three stages of

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mass loss for DP2 and DP3 β-MOS. TGA and DTA of DP5 were not carried out due to reasons

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stated above. The first mass loss (up to 12%) for DP2 and DP3 β-MOS occurred at 150 and 171

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o

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loss (up to 52%) occurred at the main chain fracture temperatures i.e. 314 and 318 oC for DP2

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and DP3 β-MOS, respectively.40 The final mass loss (up to 81%) of DP2 and DP3 β-MOS

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occurred at 510 and 516 oC, respectively which represented the decomposition temperatures and

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afterwards little residual mass of oligosaccharides was left.

C, respectively which might be due to loss of adsorbed or structural water.39 The second mass

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Nuclear Magnetic Resonance (NMR) analysis of DP2 and DP3

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P4 (DP2 oligosaccharide) (Figure 4A and B) and P3 (DP3 oligosaccharide) (Figure 5A and B)

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peaks were further analysed using NMR (500 MHz) for determination of their respective

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structures. P1 and P2 peaks were not structurally characterized due to their poor yield. Empirical

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rules for chemical shifts and comparative analysis of relative intensities of resonances were

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applied for making assignments and the results obtained were in agreement with earlier

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studies.12,13 1H and

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species of oligosaccharides. A) Mannopyranose β-1,4-mannopyranose (structure 4, figure 6):

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From 1H NMR spectra the disaccharide showed resonances at 5.2 ppm (MRα) and 4.7 ppm

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(MNRβ) which can be assigned to the anomeric protons of the reducing and non-reducing terminal

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mannose residues of mannopyranose β-1,4- mannopyranose, respectively.

13

C NMR spectra of DP2 oligosaccharides revealed the presence of two

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From 13C NMR, presence of glycosidic bond (β-1,4) was shown by the resonance of C4

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in reducing D-mannose at 81.1 ppm. However, in case of non-reducing D-mannose residue, C4

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resonance appeared at 76.3 thereby confirming the presence of mannopyranose β-1,4-

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mannopyranose. The spectrum of the disaccharide showed resonances at 91.9 ppm (MRα) and

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103.4 ppm (MNRβ) which can be assigned to the anomeric carbons of the reducing and non-

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reducing terminal mannose residues of mannopyranose β-1,4- mannopyranose, respectively. B)

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α-1,6-Galactosyl-mannopyranose (structure 3, figure 6): From 1H NMR spectra, the chemical

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shifts at 5.1 ppm and 4.6 ppm could be assigned to protons of galactose (H1) and mannose (H6)

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at the branch point of α-1,6-galactosyl-mannopyranose (Table 1). From

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showed two intense peaks around 93.6 and 99.9 ppm corresponding to C1 of D-mannose and D-

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galactose residues, respectively (Table 2). The resonances corresponding to ring carbons C2, C3,

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C4 and C5 are shown in Table 2. Interestingly, C6 δ value appeared around 66.4 ppm for D-

13

C NMR, this species



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mannose and 60.7 ppm (down field shift) for D-galactose, respectively. In the latter case, solitary

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C6 of D-galactose showed two resonances (α and β) (Table 2). Therefore, this disaccharide

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product of ManB-1601 depolymerized locust bean gum (structure 1, figure 6) could be

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concluded as α-1,6-galactosyl-mannopyranose. Considerable data was also obtained by spin-

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echo Fourier Transform (SEFT) analysis, 2D NMR, HSQC and HMBC with respect to DP2

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oligosaccharide so as to confirm the presence of two species of disaccharide (data not shown).

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The ratio of the integrals for the anomeric protons (1H NMR) assigned to the non-reducing

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residue to those assigned to the reducing residue was about ~1, as would be expected for a

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disaccharide. The generation of appreciable quantities of α-1,6-galactosyl-mannopyranose

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residues by ManB-1601, to the best of our knowledge, is the shortest substituted oligosaccharide

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reported to date produced from a bacterial endo-β-1,4-mannanase. 1

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H and 13C NMR spectrum of trisaccharide explicitly showed presence of α-D-galactosyl-

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β-D-mannobiose (structure 2, figure 6) devoid of any other trisaccharide (Figure 5). The

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assignments made for

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Table 2. A careful analysis of Biogel P2 profile (Peak 5) and disaccharide NMR data indicate

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that ManB-1601 might have the hydrolyzing capability of cleaving α-D-galactosyl-β-D-

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mannobiose to D-mannose (structure 5, figure 6) and α-1,6-galactosyl-mannopyranose.

13

C NMR spectrum of α-D-galactosyl-β-D-mannobiose are indicated in

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On the basis of the cleavage pattern of galactomannans or galactoglucomannan by endo-

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β-1,4-mannanase reported to date, we suggest the following three broad groups of endo-β-1,4-

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mannanase with respect to the type of oligosaccharides generated.

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First group: Endo-β-1,4-mannanase which give rise to substituted oligosaccharides with

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galactose at terminal position: can be formed with endo-β-1,4-mannanase from Aspergillus niger

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28

and Penicillium purpurogenum 29 wherein the shortest substituted oligosaccharide obtained in


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hydrolysis of galactomannan was a trisaccharide [α-D-galactosyl-β-D-mannobiose (GallMan2),

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endo-β-D-mannanase from Trichoderma reesei which produced GallMan2 (as the shortest

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substituted oligosaccharide i.e. trisaccharide) from galactoglucomannan of pine kraft pulp 41 and

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endo-β-1,4-mannanase from Bacillus subtilis, which had a more limited ability to hydrolyse

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galactomannans and the shortest substituted product was α-D-galactosyl-β-D-mannotetrose (Gal1-

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Man4) (mannopentose).27

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Second group: Endo-β-1,4-mannanase which give rise to substituted oligosaccharides with

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galactose at non-terminal position: can be formed of endo-β-1,4-mannanase from Aspergillus

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niger which hydrolysed carob galactomannan to a series of D-galactose-containing β-D-

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mannosaccharides with galactose substitution at non-terminal positions.28

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Third group: Endo-β-1,4-mannanase which give rise to unsubstituted oligosaccharides: can be

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formed of β-mannanase from Trichoderma reesei which showed release of un-substituted

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mannose, mannotriose and mannopentose from locust bean gum,12 and β-D-mannanases from

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lucerne seed which generated unsubstituted β-D-manno-biose, -triose, and -tetrose after the

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hydrolysis of carob galactomannan.28

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Most of the endo-β-1,4-mannanase reported to date might come under either of the above

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second or third group as steric hindrance by α-1,6 linked galactose in mannans do not allow

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endo-β-1,4-mannanase to cleave near branch points and consequently most of the generated

291

products are linear β-1,4 linked mannopyranose residues or β-1,4 linked mannopyranose residues

292

substituted with galactose at the non-terminal position(s).

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A schematic representation of the proposed depolymerization pattern of ManB-1601 on

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locust bean gum is shown in Figure 6 A and B on the basis of size-exclusion chromatography,

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ESI-MS, and NMR spectroscopy of locust bean gum derived β-MOS. Accordingly, ManB-1601



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might fall in the first group of classification mentioned above as it generates α-1,6-galactosyl-

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mannopyranose residues by cleaving near the branch points in galactomannans (cleavage site

298

shown by hollow arrow), apart from attacking internal β-1,4 glycosidic linkages (cleavage site

299

shown by solid arrow) (Figure 6A).

300

In vitro fermentation of β-MOS by lactobacilli and food borne pathogens

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The present study is the first report on prebiotic contributions of endo-β-1,4-mannanase derived β-

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MOS from locust bean gum with defined DP and chemical structure. Purified β-MOS (DP 2, 3 and

303

5) produced from the hydrolysis of locust bean gum by ManB-1601 were utilized for growth by

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various Lactobacillus spp. and inhibited the growth of various tested food borne pathogens.

305

In Lactobacillus spp., there were subtle differences in the extent of β-MOS fermentation.

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Utilization and growth by L. casei var. rhamnosus, L. fermentum and L. plantarum:

307

Feeding studies with DP2 and DP3: Supplementation of MRS media with DP2 and DP3 β-MOS,

308

showed higher growth when compared to respective controls (0.89 to 1.71 log CFU/mL and 1.71

309

to 3.4 fold A600nm when compared to FOS; 0.89 to 1.71 log CFU/mL and 0.03 to 1.35 fold A600nm

310

when compared to glucose and 1.43 to 2.75 log CFU/mL and 5.14 to 23 fold A600nm when

311

compared to media devoid of carbon source). A shift in media pH towards acidic side up to 1.1

312

units was observed in most cultures.

313

Feeding studies with DP5: Supplementation of MRS media with DP5 resulted in lowering of

314

growth parameters (up to 0.74 and up to 6 fold reduction in log CFU/mL and A600nm,

315

respectively) when compared to FOS or glucose. The growth obtained with L. fermentum and L.

316

plantarum was only slightly better (0.46 to 0.69 log CFU/mL and 3.2 fold A600nm) than negative

317

control. Similar to our results, Lactobacillus plantarum NRIC 1547, NRIC 1068 and L. sakei

318

NRIC 0126 showed better growth on oligosaccharides like lactosucrose, 1-kestose and FOS than


Page 15 of 36


319

glucose.42 A shift in media pH towards acidic side up to 0.8 units was observed in most strains

320

(Table 3).

321

Utilization and growth by L. acidophilus, L. casei, L. brevis and L. helveticus:

322

Feeding studies with DP2 and DP3: Supplementation of MRS media with DP2 and DP3, showed

323

no improvement in log CFU/mL and A600nm when compared to FOS except in L. casei where

324

marginal improvement (0.51 log CFU/mL and 1.1 fold A600nm) in growth parameters was found.

325

Surprisingly, in comparison to glucose, supplementation of MRS media with DP2 and DP3

326

resulted in lower (1.11 to 1.73 fold) A600nm and marginally higher (0.19 to 0.54) log CFU/mL. In

327

comparison to negative control, supplementation of MRS media with DP2 and DP3 resulted in

328

better growth (1.35 to 2.5 log CFU/mL and 3.9 to 8.28 fold A600nm). A shift in media pH towards

329

acidic side (up to 1.1 units) was observed in most cultures.

330

Feeding studies with DP5: Supplementation with DP5 resulted in lowering of growth parameters

331

(0.16 to 1.88 log CFU/mL and 2.6 to 7.2 fold A600nm, respectively) when compared to FOS or

332

glucose. The growth was higher (0.17 to 2.16 log CFU/mL and 1.42 to 2 fold A600nm) only when

333

compared to negative control. A shift in media pH towards acidic side (up to 0.8 units) was

334

observed in most cultures (Table 3).

335

The reasons for better growth response of various tested lactobacilli when fed with DP2

336

and DP3 β-MOS as compared with DP5 β-MOS could be the inherent difference in their

337

carbohydrate fermentation pathways and uptake mechanisms, composition of oligomeric units,

338

and chemical structure along with water solubility.43

339

oligosaccharides of shorter chain length have high water solubility and are quickly fermented,

340

while long chain NDOs are known to be steadily fermented.44 Moura et al.24 found that, L. brevis

341

preferred xylo-oligosaccharides with an average DP of 2 rather than DP 5-6. In other reports,

It has been shown earlier that



Page 16 of 36

342

mixed mannooligosaccharides from copra meal exhibited prebiotic effect by stimulating the

343

growth of Bifidobacterium infantis and Lactobacillus acidophilus.10

344

Utilization and growth by Lactobacillus spp. on commercial mannobiose (CDP2):

345

Presence of CDP2 (mannopyranose β-1,4 mannopyranose) in MRS media increased the log

346

CFU/mL values (0.02 to 0.36) of L. casei var. rhamnosus, L. fermentum, L. acidophilus and L.

347

brevis while the growth of L. casei, L. plantarum and L. helveticus was found to decrease (0.03

348

to 0.26 log CFU/mL) when compared with glucose. The CDP2 was more preferable (increase of

349

0.03 to 0.36 in log CFU/mL values) than FOS in case of L. casei var. rhamnosus, L. fermentum,

350

L. plantarum and L. helveticus. All the cultures exhibited higher growth (0.88 to 1.9 log

351

CFU/mL) on CDP2 than negative control. A shift in media pH (up to 0.6 units) towards acidic

352

side after fermentation was observed in most cultures.

353

The overall growth obtained on CDP2 was less in various Lactobacillus spp. than ManB-

354

1601 derived DP2 and DP3. The reason for this could be that disaccharides with linkages of 1-2,

355

1-4, and 1-6 are known to generate a high prebiotic index score while mannose containing

356

disaccharides give a low prebiotic index in lactobacilli45 and substituted galactose units in DP2

357

and DP3 could be amenable to α-galactosidases present in Lactobacillus spp.

358

for the substrate promiscuity between ManB-1601 derived DP2, and DP3 in terms of growth in

359

Lactobacillus spp. are difficult to comprehend, as the enzyme systems which are responsible for

360

cleavage of such β-MOS are not known.

361

Utilization and growth by E. coli, L. monocytogenes and S. typhi:

362

The growth of pathogens was lower (up to 0.70 log CFU/mL) in DP2, DP3, and DP5

363

supplemented TSB media when compared with glucose. However, the extent of pathogen

364

inhibition by β-MOS was different as it is known to vary with DP, molecular weight and type of

46, 47

The reasons


Page 17 of 36


365

functional group present in an oligosaccharide.48 CDP2 supplementation reduced the growth of

366

E. coli and S. typhi, (up to 0.14 log CFU/mL) but marginally promoted L. monocytogenes (0.06

367

log CFU/mL) when compared to glucose. All the food borne pathogens exhibited good

368

correlation between A600nm and log CFU/mL values.

369

Acetate was found as the major SCFA in all the tested probiotic strains and its presence

370

could be one of the reasons for the fall in pH of fermented broth. Our studies on in vitro

371

fermentation of guar gum degradation products by lactobacilli too showed acetate as the chief

372

SCFA component.18 Probiotic bacteria produce SCFAs as an end product of carbohydrate

373

fermentation mediated by glycolytic enzyme and they are known to display several physiological

374

benefits. Acetate in particular has been shown to be beneficial for human muscle, kidney, heart

375

and brain.49 Finally, the following conclusions can be made from in vitro fermentation studies:

376

1) DP3 oligosaccharide supported the growth of all the tested Lactobacillus spp.; 2) DP2

377

oligosaccharide supported the growth of all the tested Lactobacillus spp. except L. helveticus; 3)

378

CDP2 did not support the growth of L. casei, L. plantarum and L. helveticus; 4) DP5 did not

379

support the growth of L. plantarum and L. helveticus, L. casei var. rhamnosus, L. fermentum, L.

380

acidophilus and L. brevis; and, 5) DP2, DP3, and DP5 reduced the growth of E. coli, L.

381

monocytogenes and S. typhi but CDP2 reduced the growth of only E. coli and S. typhi.

382

In conclusion, our studies elucidated that ManB-1601 produced DP2, DP3 and DP5 β-

383

MOS from locust bean gum and belongs to a select group of endo-β-1,4-mannanase which can

384

produce substituted oligosaccharides with galactose at terminal position. In vitro fermentation

385

studies show that the DP2 and DP3 β-MOS were utilised efficiently by various lactobacilli for

386

growth and appear as potential prebiotic candidates when compared to the oligosaccharides (FOS

387

control) available on the market. At present, we are deciphering the hydrolytic action mechanism 17 ACS Paragon Plus Environment


Page 18 of 36

388

of ManB-1601 apart from key determinants responsible for the molecular cross-talk between β-

389

MOS and Lactobacillus spp. with an overall aim to develop potent functional foods based on β-

390

MOS.

391

ACKNOWLEDGEMENTS

392

We thank Prof. Ram Rajashekharan, Director, CSIR-CFTRI, for constant encouragement and

393

support. PKS and DP thank University Grants Commission (UGC), New Delhi, India, and CSIR,

394

New Delhi for providing Junior Research Fellowships. Authors acknowledge the support of

395

MLP 0116 project.

396

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397

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Page 25 of 36


FIGURE CAPTIONS Figure 1

(A) Biogel P-2 profile of β-MOS generated from locust bean gum by ManB-1601. P1, P2, P3, P4 and P5 represents the β-MOS eluted at fraction no. 31, 39, 49, 57 and 65, respectively. (B) ESI-MS of P1, P2, P3, P4 and P5 fractions obtained after Biogel P-2 chromatography of crude β-MOS derived from locust bean gum after ManB1601 hydrolysis.

Figure 2

FTIR spectra of β-MOS in the spectral region 700-1500 cm-1. (A) DP2, (B) DP3 and (C) DP5.

Figure 3

(A) and (B). XRD analysis of β-MOS DP2 and DP3, respectively. (C) and (D): TGA and DTA curves, respectively, of β-MOS DP2 and DP3,obtained by heating from room temperature at 10 °C/min under an N2 atmosphere.

Figure 4

NMR spectra of Biogel P-2 purified P4 fraction: A) 1H NMR B) 13C NMR

Figure 5

NMR spectra of Biogel P-2 purified P3 fraction: A) 1H NMR B) 13C NMR

Figure 6

Representation of the depolymerization pattern for locust bean gum by purified ManB-1601: Hollow arrows represent cleavage site near substituted region. Solid arrow represents cleavage site near unsubstituted region. Structure 1: Locust bean gum; structure 2: α-D-galactosyl-β-D-mannobiose; structure 3: α-1,6-galactosylmannopyranose; structure 4: mannopyranose β-1,4-mannopyranose; structure 5: Dmannose.



Page 26 of 36

Table 1 Chemical Shift Values Obtained from 1H NMR for Anomeric Protons for Mannobiose, α-1,6-Galactosyl-mannopyranose and α-D-Galactosyl-β-D-mannobiose

Type of unit

Chemical shift values (ppm) H1α H1β

H1

H1

H1

(non-

Gal

(man-

reducing)

branched)

Mannopyranose β-1,4 mannopyranose4

5.29 4.79

4.66

NA

NA

NA

NA

NA

5.06

4.62

NA

4.76

4.62

5.03

4.58

α-1,6-galactosyl-mannopyranose3 α-D-galactosyl-β-D-mannobiose2 NA: Not assigned; Superscripted numbers represent structure code as mentioned in figure 6


Page 27 of 36


Table 2 13C NMR Chemical Shift Values for Mannobiose, α-1,6-Galactosyl-Mannopyranose and αD-Galactosyl-β-D-Mannobiose

Chemical shift values (ppm)

Type of unit Mannopyranose β-1,4 mannopyranose4 β-D-mannopyranosyl non reducing end residue

C1

C2

C3

C4

C5

C6

103.39 71.42 72.29 76.28 73.73

59.86

91.89

70.78 72.19 81.08 74.51

62.07

β-D-mannopyranose residue, branched at O-6

93.55

70.25 72.54 76.59 76.13

66.43

α-D-galactopyranosyl residue

99.89

68.68 70.78 69.94

β-D-mannopyranosyl non reducing end residue

99.93

71.21 71.39 76.35 74.74

NA

β-D-mannopyranose residue, branched at O-6

93.55

70.21 72.50 76.52 76.14

66.41

α-D-galactopyranosyl residue

99.84

68.65 70.63 69.96

β-D-mannopyranose reducing end residue α-1,6-Galactosyl-mannopyranose3

NA

60.74/60.25

α-D-Galactosyl-β-D-mannobiose2

NA

60.72/60.24

NA: Not assigned; Superscripted numbers represent structure code as mentioned in figure 6



Page 28 of 36

Table 3 Growth Characteristics of Lactobacillus spp. and Food Borne Pathogens in Media Containing Purified β-Mannooligosaccharides. β-MOS (0.1% w/v)

Absorbance Log CFU/mL (A600nm) Lactobacillus casei var. rhamnosus DP2 0.73 ± 0.07a 8.0 DP3 0.72 ± 0.07a 7.73 6.3 DP5 0.12 ± 0.0b CDP2 0.61 ± 0.05a 7.2 c Positive control-1 0.42 ± 0.03 6.84 6.84 Positive control-2 0.69 ± 0.09a d Negative control 0.14 ± 0.0 6.3 Lactobacillus casei (NCIM 2126) DP2 0.58 ± 0.05a 7.15 a DP3 0.60 ± 0.06 7.5 DP5 0.10 ± 0.01b 7.16 CDP2 0.62 ± 0.06a 6.7 Positive control-1 0.54 ± 0.04a 6.99 Positive control-2 0.67 ± 0.08a 6.96 5 Negative control 0.07 ± 0.01c Lactobacillus fermentum (NCIM 2165) DP2 0.73 ± 0.06a 8.75 DP3 0.74 ± 0.07a 8.69 b DP5 0.13 ± 0.01 6.69 7.07 CDP2 0.66 ± 0.05a Positive control-1 0.40 ± 0.03c 7.04 a Positive control-2 0.79 ± 0.09 7.04 Negative control 0.04 ± 0.01d 6 Lactobacillus plantarum (NCIM 2372) DP2 0.80 ± 0.01a 7.95 DP3 0.92 ± 0.09a 8.37 DP5 0.13 ± 0.07b 6.3 a CDP2 0.68 ± 0.05 6.99

Final pH

6.0 ± 0.1 6.2 ± 0.1 6.5 ± 0.1 6.5 ± 0.2 6.3 ± 0.1 6.1 ± 0.2 6.5 ± 0.1 6.2 ± 0.2 6.2 ± 0.1 6.4 ± 0.1 6.7 ± 0.1 6.4 ± 0.1 5.9 ± 0.1 6.5 ± 0.1 6.0 ± 0.2 6.0 ± 0.1 6.5 ± 0.2 6.7±0.2 6.4 ± 0.1 5.8±0.2 6.8 ± 0.1 6.0 ± 0.2 6.0 ± 0.1 6.3 ± 0.1 6.7 ± 0.2

MOS (0.1% w/v)

Absorbance Log CFU/mL (A600nm) Lactobacillus acidophilus (NCIM 2285) DP2 0.43 ± 0.04a 7.24 DP3 0.50 ± 0.05a 7.43 b DP5 0.10 ± 0.01 5.47 CDP2 0.67 ± 0.04a 7.2 a Positive control-1 0.53 ± 0.05 7.35 Positive control-2 0.72 ± 0.05c 6.99 d Negative control 0.11 ± 0.01 5.3 Lactobacillus brevis (NCIM 2090) DP2 0.45 ± 0.04a 7.45 a DP3 0.48 ± 0.05 7.45 DP5 0.14 ± 0.01b 6.6 c CDP2 0.65 ± 0.06 6.98 Positive control-1 0.53 ± 0.05a 7.39 d Positive control-2 0.70 ± 0.03 6.96 Negative control 0.09 ± 0.01e 6.1 Lactobacillus helveticus (NCIM 2126) DP2 0.56 ± 0.06a 7.2 DP3 0.64 ± 0.06a 7.31 DP5 0.20 ± 0.02b 7.14 CDP2 0.85 ± 0.08c 7.47 Positive control-1 0.52 ± 0.05a 7.3 d Positive control-2 0.97 ± 0.07 7.5 Negative control 0.10 ± 0.01e 5.78 Listeria monocytogenes (ATCC 13932) DP2 0.7 ± 0.05a 7.73 DP3 0.5 ± 0.07b 7.97 DP5 0.3 ± 0.04c 7.39 CDP2 0.9 ± 0.06d 8.15

Final pH

6.0 ± 0.2 6.1 ± 0.1 6.3 ± 0.1 6.7 ± 0.2 6.3 ± 0.2 5.7 ± 0.2 6.5 ± 0.1 6.2 ± 0.2 6.2 ± 0.2 6.4 ± 0.1 6.6 ± 0.1 6.4 ± 0.1 5.6 ± 0.1 6.5 ± 0.1 6.2 ± 0.2 6.2 ± 0.2 6.3 ± 0.1 6.7±0.1 6.3 ± 0.2 5.7±0.1 6.5 ± 0.1 7.4 ± 0.2 7.6 ± 0.2 8.1 ± 0.1 6.5 ± 0.2 28

ACS Paragon Plus Environment

Page 29 of 36


Positive control-1 0.27 ± 0.01c 6.78 6.0 ± 0.2 Positive control-1 0.2 ± 0.03e 7.18 8.5 ± 0.2 a f Positive control-2 0.68 ± 0.08 7.04 6.2 ± 0.1 Positive control-2 0.9 ± 0.1 8.09 7.8 ± 0.2 Negative control 0.04 ± 0.01d 5.84 6.5 ± 0.1 Negative control 0.3 ± 0.04g 7.41 7.0 ± 0.1 Escherichia coli (ATCC 11775) Salmonella typhi (ATCC 25241) DP2 2.8 ± 0.05a 8.3 8.2 ± 0.2 DP2 2.0 ± 0.12a 7.72 8.3 ± 0.1 a a DP3 2.8 ± 0.1 8.6 8.5 ± 0.2 DP3 2.1 ± 0.08 7.64 8.3 ± 0.2 DP5 3.0 ± 0.1b 8.62 7.9 ± 0.2 DP5 2.1 ± 0.09a 7.59 8.7 ± 0.2 a a CDP2 2.8 ± 0.08 8.68 8.5 ± 0.1 CDP2 2.1 ± 0.13 7.68 8.3 ± 0.1 8.56 8.6 ± 0.2 Positive control-1 2.0 ± 0.1a 7.72 8.4 ± 0.1 Positive control-1 2.7 ± 0.02a Positive control-2 3.7 ± 0.1c 8.79 8.3 ± 0.2 Positive control-2 3.1 ± 0.13b 7.82 8.2 ± 0.1 8.49 8.5 ± 0.1 Negative control 2.0 ± 0.05a 7.58 8.5 ± 0.1 Negative control 2.7 ± 0.1a Positive control-1- MRS media containing fructo-oligosaccharides; Positive control-2: Glucose (0.1%w/v); Negative control- MRS media devoid of carbon source; Absorbance values of each lactobacilli sp. indicated with different superscripted letters (a, b, c, d, e, f, g) are statistically different (significant at 0.01 level), while values indicated with same superscripted letters are not statistically different (significant at 0.01 level); DP2: mannopyranose β-1,4 mannopyranose4 or α-1,6-galactosyl-mannopyranose3; DP3: α-Dgalactosyl-β-D-mannobiose2 (superscripted numbers represent structure code as mentioned in figure 6).



Page 30 of 36

Figure 1

A 2.5 P1

A480nm

2.0

P2

P3

P4 P5

1.5 1.0 0.5 0.0 0

20

40

TOF MS ES+ 243

49

22081402 19 (0.368) Cm (12:20) 850.6722 100

TOF MS ES+ 22081403 21 (0.407) Cm (20:23) 527.3005 867 100

P2

TOF MS ES+ 1.61e3

P3

%

P1

100

%

22081401 88 (1.681) Cm (84:91) 599.1530 100

80

Fraction no. B

39

31

60

% 599.7004

845.7578 851.7042

1173.9666

596.7271 680.0605

1030.3115

0

m/z 600

800

1000

1200

1400

1600

1800

200

400

600

800

1000

1200

1400

1600

1800

0

m/z 200

400

600

800

1000

1200

1400

1600

1800

65

57 22081404 41 (0.787) Cm (35:44) 365.5380 100

TOF MS ES+ 1.89e3

P4

22081405 46 (0.882) Cm (43:50) 100

%

400

m/z

203.6782

TOF MS ES+ 85

P5

%

200

0

366.5493

707.0107

381.5049

0

m/z 200

400

600

800

1000

1200

1400

1600

1800

223.7091 365.5768 605.4595

0


30

Page 31 of 36


Figure 2



Page 32 of 36

Figure 3

50

50

A

30

20

10

30

20

10

0

0

0

10

20

30

40

50

2θ

60

70

0

80

10

20

30

40

50

60

70

80

2θ

120

0

C Heat flow (mW/mg)

80

DP2

60

D

-5

100

Weight (%)

B

40

Intensity (coulumbs)

Intensity (coulumbs)

40

40

-10

171 318 516

-20

DP2

150 -25

314

DP3

20

DP3

-15

-30

510 0

-35 0

200

400

600

800

0

200

0

400

600

800

0

Temperature ( C)

Temperature ( C)


73

72

71

70

69 92

68

67 90

66 88

65 3.50

86

64 3.45

Mannobiose

84

63


2.52

4.2

3.40

82

4.1

3.35

80

62

61

4.0

3.30

78 76

60

59 73.738

3.55

4.3

74.517

4.4

76.590 76.283 76.134

4.5

81.085

3.60

4.68

2.05

1.22

0.54

4.6

59.860

94 3.65

3.41

3.70 2.59

2.08

3.75 4.7

4.622

4.663

4.791

5.060 5.057

5.292 5.284

3.951 3.945 3.926

4.101 4.083

Mannobiose

60.258

96 4.8

61.111 60.747

98 4.9

62.070

100 3.80

91.889

93.553 93.417

3.85 3.12

3.90 5.0

66.439

102 5.1

68.957 68.680

2.00

5.2

4.91

4.36

3.95

70.787 70.621 70.351 70.255 69.940

99.942 99.896

5.3

71.429

B 2.52

3.951 3.945 3.926 3.909 3.878 3.872 3.868 3.850 3.844 3.840 3.819 3.815 3.781 3.771 3.767 3.746 3.742 3.732 3.725 3.710 3.707 3.700 3.692 3.689 3.666 3.657 3.640 3.633 3.629 3.619 3.615 3.608 3.605 3.595 3.548 3.533 3.529 3.527 3.523 3.469 3.467 3.463 3.447 3.444 3.438 3.428 3.425 3.417 3.381 3.363 3.344 3.339 3.334 3.325 3.320 3.315 3.305 3.301

A

72.542 72.296 72.124

103.398

Page 33 of 36 Journal of Agricultural and Food Chemistry

Figure 4

ppm

ppm

ppm

ppm

33

75 98

70 96 94

65

3.40

92

3.35

90

60

3.30

88

3.25 3.20

86

ppm

84

55

ACS Paragon Plus Environment 2.70

82

50

2.65

80

2.60 2.55 ppm

0.39

1.68

3.8 1.12

3.61

3.9

2.568

4.0 3.82

4.1

2.615 2.600

4.2

1.03

4.620 4.616 4.589

4.766

5.033 5.031

3.987 3.984 3.923 3.917 3.898 3.853 3.846 3.828 3.818 3.814 3.789 3.762 3.747 3.742 3.732

4.068

Triose

1.77

1.66

4.3

0.33

3.45

4.4

0.30

4.5

1.85

3.50 0.77

4.6

0.98

3.67

3.55 0.51

4.7

Triose Mannotriose 76.520 76.359 76.205 76.141

3.60

2.00

4.8

43.068

93.558 93.413

0.15

1.00

4.9

60.727 60.243

100

3.65

4.54

3.70

1.35

5.00

1.12

5.0

66.414

B 99.934 99.841

3.747 3.742 3.732 3.727 3.721 3.717 3.707 3.684 3.676 3.670 3.653 3.648 3.641 3.633 3.622 3.618 3.609 3.602 3.597 3.588 3.577 3.563 3.523 3.517 3.504 3.498 3.435 3.415 3.405 3.396 3.378 3.368 3.361 3.356 3.343 3.313 3.309 3.299 3.294 3.290 3.280 3.276

A

72.501 71.396 71.215 70.639 70.384 70.213 69.968 69.688 69.157 68.655

74.747 74.516

Journal of Agricultural and Food Chemistry Page 34 of 36

Figure 5

ppm

78 ppm

45

ppm

34

Page 35 of 36


Figure 6



Page 36 of 36

Table of Contents Graphic


Structural Characterization and in Vitro Fermentation of Î²

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