the differential proteome of the probiotic …...ncfm(ncfm)isalowgcratio(38.4%),gram-positive...

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The Differential Proteome of the Probiotic Lactobacillus acidophilus NCFM Grown onthe Potential Prebiotic Cellobiose Shows Upregulation of Two beta-GlycosideHydrolases

van Zanten Gabriella Christina Sparding Nadja Majumder Avishek Lahtinen Sampo J SvenssonBirte Jacobsen Susanne

Published inBioMed Research International

Link to article DOI1011552015347216

Publication date2015

Document VersionPublishers PDF also known as Version of record

Link back to DTU Orbit

Citation (APA)van Zanten G C Sparding N Majumder A Lahtinen S J Svensson B amp Jacobsen S (2015) TheDifferential Proteome of the Probiotic Lactobacillus acidophilus NCFM Grown on the Potential PrebioticCellobiose Shows Upregulation of Two beta-Glycoside Hydrolases BioMed Research International 2015[347216] httpsdoiorg1011552015347216

Research ArticleThe Differential Proteome of the ProbioticLactobacillus acidophilus NCFM Grown on thePotential Prebiotic Cellobiose Shows Upregulation ofTwo 120573-Glycoside Hydrolases

Gabriella C van Zanten12 Nadja Sparding2 Avishek Majumder2

Sampo J Lahtinen3 Birte Svensson2 and Susanne Jacobsen2

1Department of Food Science Faculty of Science University of Copenhagen Rolighedsvej 26 1958 Frederiksberg C Denmark2Enzyme and Protein Chemistry Department of Systems Biology Technical University of DenmarkSoslashltofts Plads Building 224 2800 Kongens Lyngb Denmark3DuPont Nutrition and Health Sokeritehtaantie 20 02460 Kantvik Finland

Correspondence should be addressed to Birte Svensson bisbiodtudk

Received 16 July 2014 Revised 22 September 2014 Accepted 23 September 2014

Academic Editor Borja Sanchez

Copyright copy 2015 Gabriella C van Zanten et alThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Probiotics prebiotics and combinations thereof that is synbiotics are known to exert beneficial health effects in humans howeverinteractions between pro- and prebiotics remain poorly understood at the molecular level The present study describes changes inabundance of different proteins of the probiotic bacterium Lactobacillus acidophilusNCFM (NCFM) when grown on the potentialprebiotic cellobiose as compared to glucose Cytosolic cell extract proteomes after harvest at late exponential phase of NCFMgrownon cellobiose or glucose were analyzed by two dimensional difference gel electrophoresis (2D-DIGE) in the acidic (pH 4ndash7) andthe alkaline (pH 6ndash11) regions showing a total of 136 spots to change in abundance Proteins were identified by MS or MSMSfrom 81 of these spots representing 49 unique proteins and either increasing 15ndash139-fold or decreasing 15ndash78-fold in relativeabundance Many of these proteins were associated with energy metabolism including the cellobiose related glycoside hydrolasesphospho-120573-glucosidase (LBA0881) and phospho-120573-galactosidase II (LBA0726) The data provide insight into the utilization of thecandidate prebiotic cellobiose by the probiotic bacterium NCFM Several of the upregulated or downregulated identified proteinsassociated with utilization of cellobiose indicate the presence of carbon catabolite repression and regulation of enzymes involvedin carbohydrate metabolism

Dedicated to Susanne Jacobsen who sadly has passed away

1 Background

Probiotics are defined as ldquolive microorganisms that whenadministered in adequate amounts confer a health benefit onthe hostrdquo [1] Reported beneficial effects include preventionand reduced severity of respiratory infections [2]modulationof immune responses [3] diminished symptoms of irritablebowel disease [4] and a remedy against antibiotic-associated

[4] and infectious diarrhea [5] Lactobacillus acidophilusNCFM (NCFM) is a low GC ratio (384) Gram-positivelactic acid bacterium with well-documented probiotic effects[6] NCFMhas a genome of 20Mbwith 1864 predicted openreading frames (ORFs) including different genes and geneloci involved in carbohydrate metabolism [7] Furthermorea large number of identified proteins are dedicated to energymetabolism [8] Noticeably cDNA microarray analyses of

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 347216 9 pageshttpdxdoiorg1011552015347216

2 BioMed Research International

different oligosaccharide transporters support that NCFM iscapable of utilizing a wide range of carbohydrates includingknown prebiotics such as fructooligosaccharides (FOSs)galactooligosaccharides (GOSs) and potential prebiotics forexample cellobiose [9 10]

Prebiotics are food ingredients not digested by the hostwhich selectively stimulate bacterial species of the gut micro-biota that confer health benefits which include stimulationof mineral absorption improvement of the intestinal barrierand modulation of immune functions [11] Since cellobiose(4-O-120573-d-glucopyranosyl-d-glucose) is not digested in thehuman upper gastrointestinal tract it is available for bac-terial fermentation in the colon [12] In in vitro experi-ments in the presence of human fecal bacteria cellobiosewas reported to increase production of short-chain fattyacids (SCFA) [13 14] which are important for mainte-nance of epithelial cell homeostasis [15] and possess anti-inflammatory [16] as well as anticarcinogenic effects [17]In mice cellobiose thus improved clinical and pathologicalmarkers of experimentally induced colitis [18] indicating thatcellobiose may exert prebiotic effects also in humans

A synergistic effect can be achieved when a probioticand a prebiotic are administered together as a synbiotic [19]Reported beneficial outcomes include decreased postopera-tive infection rates [20] and improvements of liver functionin patients with liver failure [21] The combination of NCFMand lactitol is thus found to improve mucosal integrityintestinal motility and gut microbiota composition of elderlysubjects [22 23] A simulated model of the human coloninoculated with fecal bacteria was used to demonstrate thesynbiotic potential of NCFM in combination with cellobiose[14] Recently this combination was demonstrated to increasebifidobacteria and lactobacilli both regarded as beneficial inhealthy humans [24]

Recently transcriptomic analysis of cellobiose meta-bolism in Clostridium acetobutylicum a low G+C Gram-positive bacteria found in a number of ecological nichessuch as soil and feces indicated the presence of multi-ple cellobiose-inducible operons [25] Differential transcrip-tomics and functional genomics have been used to studygenes of NCFM involved in uptake of cellobiose [26] how-ever molecular responses at the protein level were notexplored Effects of prebiotics on probiotic are not wellstudied however humanmilk oligosaccharides were recentlyshown to increase the adhesion of Bifidobacterium longumsubsp infantitis [27] Changes in adhesion were correlated tochanges at the transcriptional level for genes involved in hostinteraction including some encoding so-calledmoonlightingproteins that is proteins having multiple and apparentlyunrelated activities in different locations in the cell Thepresent study investigates changes in abundances of individ-ual NCFM proteins as elicited by growth on cellobiose byusing 2D-DIGE and mass spectrometry

2 Methods

21 Growth Conditions Freeze dried commercial Lactobacil-lus acidophilus NCFM (NCFM Danisco USA Inc MadisonUS-WI) was revived in semisynthetic medium for lactic

acid bacteria (LABSEM) [28] supplemented with 1 (wv)of either cellobiose or glucose (Sigma-Aldrich BroslashndbyDenmark) To avoid carry-over effects three cycles of subcul-tivationwere performed (40mL) Cells in the late exponentialphasewere harvested by centrifugation (3200 g 10min 4∘C)Growth experiments were done in quadruple Cells wereharvested at OD

60020 and 07 for glucose and cellobiose

respectably washed twice in 09 NaCl (pH 55 4∘C) andcentrifuged (10000 g 5min 4∘C)The pellets were dried (2 hSpeedVac Savant SC110Awith vacuum unit UVS400A GMIInc Ramsey US-MN) and stored at minus80∘C until use

22 Preparation and Fluorophore-Labelling of Cytosolic Pro-tein Samples Extraction of cytosolic proteins was done bymechanical grinding as described [29] Protein concentra-tions were determined using the 2D Quant Kit (GE LifeSciences) and samples were stored at minus80∘C

The CyDye DIGE Fluor minimal dyes (GE Life Sciences)Cy3 or Cy5 (200 pmol) were used to label aliquots offour biological replicates (50 120583g each) interchangeablyaccording to Ettan DIGE System manual (GE Life Sciences)using the dye swapping approach Internal standards wereprepared by mixing protein samples (25 120583g from each)and labeling with Cy2 (400 pmol) Samples for analysis atpH 4ndash7 were mixed with the internal sample and adjusted to450 120583Lwith rehydration buffer (7Murea 2M thiourea 3 3-((3-cholamidopropyl)-dimethylammonio)1-propansulfonate(CHAPS) 15 pharmalyte pH 4ndash7 1 bis(2-hydroxyethyl)disulfide (HED) 100mM DTT and 10mM Tris-HCl) forimmobilized pH gradient (IPG) strips (linear pH 4ndash7 24 cm)and incubated in the dark for 30min at room temperatureFor analysis at pH 6ndash11 samples were mixed as above and thevolume was adjusted to 120120583L with rehydration buffer (7Murea 2M thiourea 3 CHAPS 1 pharmalyte solution pH6ndash11 25 HED 10 isopropanol 5 glycerol and 10mMTris-HCl)

23 2D-DIGE For the acidic proteome isoelectric focusing(IEF) was done at a total of 75 kVh (6 h at 30V 6 h at 60V1 h at 500V 1 h at 1000V and 2 h gradient to 8000V andhold at 8000V until a minimum of 75 kVh was reached)using IPG strips (pH 4ndash7 24 cm GE Life Sciences) on EttanIPGphor (GE Life Sciences) For the alkaline region strips(pH 6ndash11 18 cm GE Life Sciences) were rehydrated in 7Murea 2M thiourea 3 CHAPS 1 pharmalyte solution pH6ndash11 25 HED 10 isopropanol 5 glycerol and 10mMTris-HCl overnight prior to loading of the sample using thecup-loading method [30 31] The IEF was done at a total of40 kVh (10min at 30V 5 h at 100V 5 h at 600V 3 h gradientto 4000V and 2 h gradient to 8000V and hold at 8000V untila minimum of 40 kVh was reached) IPG strips were subse-quently equilibrated in 5mL equilibration buffer (6M urea30 glycerol 50mM Tris-HCl pH 88 001 bromophenolblue and 2 SDS) with 1 DTT (15min) followed by 5mLequilibration buffer with 25 iodoacetamide (15min) Sep-aration in the second dimension was performed with 125SDS-PAGE gels on Ettan DALT twelve Electrophoresis Unit(GE Life Sciences) as previously described [8]

BioMed Research International 3

4 7

97

10

pH(k

Da)

(a)

6pH

11

(kD

a)

97

10

(b)

4 7pH

(kD

a)

97

10

(c)

6pH

11

(kD

a)

97

10

(d)

Figure 1 Representative 2D-DIGE fluorescent gel images of Lactobacillus acidophilus NCFM soluble cytosolic proteins for (a) the acidicregion pH 4ndash7 and (b) the alkaline region pH 6ndash11 2D-DIGE Coomassie Brilliant Blue gel images showing numbers indicating spots pickedfor identification by mass spectrometry are shown for (c) the acidic region pH 4ndash7 and (d) the alkaline region pH 6ndash11

24 Image Analysis Immediately after the second dimen-sion DIGE gels were imaged at excitationemission wave-lengths of Cy2 (488520 nm) Cy3 (532580 nm) and Cy5(633670 nm) at 100 120583m resolution (Typhoon 9410 VariableMode Imager GE Life Sciences Uppsala Sweden) Theobtained images were analyzed using Progenesis SameSpotsv 40 software (Nonlinear Dynamics Ltd) Alignment ofgel images was performed by automated calculation of 20manually assigned vectors Differentially expressed proteins(threshold of ge 15-fold difference in spot intensity) werechosen based on power calculation of 80 (minimum samplesize required to accept the outcome of a statistical test) andthe 119902-value was set at 005 (119902 le 005) giving a false discoveryrate of 5 (119875 le 005) Prior to spot identification gels werestained with colloidal Coomassie Brilliant Blue as describedpreviously [32]

25 In-Gel Digestion and Protein Identification Spots of vary-ing relative intensity were manually excised and subjected toin-gel tryptic digestion [8] MS and MSMS were performedas previously described [8] Spectra were searched againstthe NCBInr database for bacteria (20100323 10606545

sequences 3615943919 residues) usingMASCOT20 software(httpwwwmatrixsciencecom) integrated with BioToolsv31 (Bruker-Daltonics) The parameters for the search weremonoisotopic peptide mass accuracy of 80 ppm fragmentmass accuracy of 07Da missed cleavage of maximum onecarbamidomethylation of cysteine and partial oxidation ofmethionine Known keratin and autocatalytic trypsin peakswere removed and the signal to noise ratio was set at 1 6A Mascot score of ge 80 (119875 le 005) was used to confirmproteins identified by peptide mass fingerprint and shouldhave a minimum of six matched peptides For MSMS basedidentification of proteins a Mascot score of ge 40 (119875 le 005)was used for each peptide

3 Results

31 Acidic andAlkaline Differential Proteome of NCFMGrownon Cellobiose The present investigation of cytosolic pro-teomes of NCFM grown on cellobiose a potential synbioticcombination and glucose identified a total of 136 differen-tially abundant spots in 2D-DIGE (Figure 1) Of these 108(spots 1ndash108) were in the acidic region (pH 4ndash7 Figure 1(c))


AA 58BCPC 96

CE 38

CIM 38

CP 19DM 38

EM 346

FP 19HP 77

PS 38

PF 19

PPNN 96

RF 77

TR 19

UF 154

Figure 2 Functional classification of the differentially abundantproteins presented in Tables S1 and S2 shown as percentage of thetotal number of unique proteins Functional categories are AAamino acid metabolism BCPC biosynthesis of cofactors prostheticgroups and carriers CIM central intermediate metabolism CEcell envelope CP cellular processes DM DNA metabolism EMenergymetabolism FP fatty acid and phospholipidmetabolismHPhypothetical proteins PF protein fate PPNN purines pyrimidinesnucleosides and nucleotides PS protein synthesis RF regulatoryfunctions TR transport and UF unknown function

and 28 spots in the alkaline region (pH 6ndash11 Figure 1(d))respectively This difference is in accordance with the cytoso-lic proteome distribution of NCFM [8 29] Among the 136spots 81 showed higher (15ndash139-fold) and 55 lower (15ndash78-fold) relative abundance Proteins were identified withconfidence by MS andor MSMS in 81 of 136 picked spotsrepresenting 49 unique proteins The 81 spots correspond to64 and 17 identifications from the acidic and alkaline regionsrespectively (Tables S1 and S2) The hypothetical proteinLBA0890 (spots 54ndash56 and 124) was identified both in theacidic and alkaline proteome in the overlapping region (pI6-7)

The identified 49 unique proteins found to change inabundance when NCFM was grown on cellobiose weredistributed among 15 functional categories based on theComprehensive Microbial Research (CMR) database (httpcmrjcviorg) (Figure 2) as follows 3 proteins associated withamino acid metabolism 5 with biosynthesis of cofactorsprosthetic groups and carriers 2 with central intermediatemetabolism 2 with cell envelope 1 with cellular processes2 with DNA metabolism 18 with energy metabolism 1 withfatty acid and phospholipid metabolism 4 with hypotheticalproteins 2 with protein synthesis 1 with protein fate 5 withpurines pyrimidines nucleosides and nucleotides 4 withregulatory functions 1 with transport and 8 of unknownfunction

32 Differentially Expressed Proteins of NCFM Grown onCellobiose A major proportion of the proteins that changedin abundance were associated with energy metabolism(Figure 2) including enzymes involved in the glycolysistriosephosphate isomerase (15ndash26-fold spots 59 60 106and 107 some being higher and some lower in their abun-dance) glyceraldehyde 3-phosphate dehydrogenase (15-

25-fold Table 1 spots 36ndash38 70 and 123 some being higherand some lower in their abundance) and phosphoglyceratekinase (16-17-fold lower abundance Table 1 spots 28ndash31)The two identified glycoside hydrolases phospho-120573-glu-cosidase and phospho-120573-galactosidase II were both upreg-ulated Phospho-120573-glucosidase (Table 1 spot 17) increased24-fold whilst two forms of phospho-120573-galactosidase IIwith different pI (Table 1 spots 16 and 18) increased 70-and 74-fold in NCFM grown on cellobiose as comparedto glucose Notably trehalose-6-phosphate hydrolase (seeTable S1 in Supplementary Material available online athttpdxdoiorg1011552015347216 spot 93) catalyzingconversion of trehalose-6-phosphate to glucose and glucose6-phosphate was upregulated by 21-fold Chromosome par-titioning protein (Table S1 spot 48) and a putative oxalyl-CoA carboxylase (Table S1 spot 11) involved in both energymetabolism and central intermediatemetabolism showed 31-and 19-fold lower abundance respectively

Differentially abundant proteins involved in protein syn-thesis including elongation factor Tu (EF-Tu) (Table S1 spots25 and 27) decreased 42ndash65-fold and a putative phosphatestarvation inducible protein (Table S1 spot 72) increased 28-fold Two forms of a putative serine protease (Table S1 spots131 and 132) involved in protein fate showed a 15-16-folddecrease Two isoforms of asparagine synthetase (Table S1spots 33 and 136 and Table S2 spots 120 and 121) involvedin amino acidmetabolism increased 16ndash19-fold while serinehydroxymethyltransferase (Table S1 spot 26) also associatedwith functional categories purines pyrimidines nucleosidesand nucleotides and biosynthesis of cofactors prostheticgroups and carriers decreased 16-fold

All identified proteins associated with purines pyrim-idines nucleosides and nucleotides were downregulatedwhen grown on cellobiose compared to glucoseThey includeribonucleoside triphosphate reductase (Table S1 spots 2ndash5) inosine-51015840-monophosphate dehydrogenase (Table S1 spot35) uracil p-ribotransferase (Table S2 spot 66) and 2101584031015840-cyclic-nucleotide 21015840-phosphodiesterase (Table 1 spot 92)which decreased 15ndash36-fold Differentially expressed pro-teins involved in DNA metabolism were identified as DNAgyrase subunit B (Table S1 spot 6) and single-stranded DNA-binding protein (Table S1 spot 75) which increased 15- and139-fold respectively when NCFMwas grown on cellobiose

The second largest group of proteins with altered abun-dancy was of unknown function (UF) which included threeupregulated hypothetical proteins LBA0466 (34- and 45-fold Table S1 spots 7 and 8) LBA0890 (15ndash20-fold Table S1spots 54ndash56 58 109 and 124) and LBA1769 (20-fold TableS1 spots 77 and 82) Other proteins in the so-called categoryof unknown function that is myosin-cross-reactive antigen(Table S1 spots 9 and 10) oxidoreductase (Table S1 spots50 and 95) tRNA uridine 5-carboxymethylaminomethylmodification enzyme GidA (Table S1 spots 111 112 and 114)and galactose mutarotase related enzyme (Table S1 spot 49)increased 16ndash63-fold Proteins associated with prostheticgroups and carriers that is hypothetical protein LBA1769(Table S1 spots 77 and 82) nicotinate phosphoribosyltrans-ferase (Table S1 spot 23) and NAD synthetase (Table S1 spot42) were upregulated by 17ndash20-fold while aminotransferase


Table 1 Differentially abundant proteins of Lactobacillus acidophilus NCFM grown on cellobiose compared to growth on glucose shownfor selected proteins having a role in carbohydrate metabolism Differential abundance was based on Progenesis SameSpots analyses of 2Dimages (ge 15-fold spot volume ratio change ANOVA 119875 le 005 and false discovery rate 119902 le 005) A Mascot score of ge 80 (119875 le 005) wasused to confirm proteins identified by peptide mass fingerprint and should have a minimum of six matched peptides Proteins are listedaccording to their fold change All identified proteins including information regarding score ANOVA sequence coverage peptide searchand identificationMW and pI are available in supplementary material

Spot number Fold change Accession number Protein name Localizationa Functional roleblowast102 minus78 gi|58337790 Two-component system regulator C RF16 +74 gi|58337043 Phospho-beta-galactosidase II C EM18 +70 gi|58337043 Phospho-beta-galactosidase II C EM92 minus36 gi|58337251 2101584031015840-Cyclic-nucleotide 21015840-phosphodiesterase E PPNN13 +35 gi|58337008 Phosphoglucomutase C EM60 +26 gi|58337021 Triosephosphate isomerase C EM37 minus25 gi|58337019 Glyceraldehyde-3P dehydrogenase C EM17 +24 gi|58337186 Phospho-beta-glucosidase C EM47 +24 gi|58336768 Catabolite control protein A C RF122 +22 gi|58337323 Putative surface layer protein CW CE123 +20 gi|58337019 Glyceraldehyde-3P dehydrogenase C EM99 minus19 gi|58337088 F0F1 ATP synthase subunit alpha C EM59 minus18 gi|58337021 Triosephosphate isomerase C EM29 minus17 gi|58337020 Phosphoglycerate kinase C EM106 minus17 gi|58337021 Triosephosphate isomerase C EM28 minus16 gi|58337020 Phosphoglycerate kinase C EM30 minus16 gi|58337020 Phosphoglycerate kinase C EM31 minus16 gi|58337020 Phosphoglycerate kinase C EM38 minus16 gi|58337019 Glyceraldehyde-3P dehydrogenase C EM41 minus16 gi|58337089 F0F1 ATP synthase subunit gamma C EM36 +15 gi|58337019 Glyceraldehyde-3P dehydrogenase C EM70 minus15 gi|58337019 Glyceraldehyde-3P dehydrogenase C EM107 minus15 gi|58337021 Triosephosphate isomerase C EMaLocalization C cytoplasmic CW cell wall E extracellularbFunctional role CE cell envelope EM energy metabolism PPNN purines pyrimidines nucleosides and nucleotides RF regulatory functionslowastProtein identification by MSMS was confirmed by a Mascot score of ge 40 (119875 le 005) for each peptide

(Table S1 spot 32) was 19-fold downregulated as comparedto growth in glucose Among proteins involved in regula-tory functions a two-component system regulator (Table 1spot 102) was decreased 78-fold while dihydroxyacetonekinase (Table S1 spot 43) transcriptional regulator (TableS1 spot 116) and catabolite control protein A (Table 1 spot47) increased 24ndash42-fold A putative surface layer protein(Table 1 spot 122) and UDP-N-acetylmuramoyl-L-alanyl-d-glutamate synthetase (Table S1 spot 15) both involved withcell envelope were 22- and 15-fold higher respectively com-pared to cultures grownwith glucose Only one protein of thefunctional class involved in transportwas observed to changethat is an ABC transporter ATP-binding protein (Table S1spots 113 and 117) which decreased 16-fold Moreover twoforms of metallo-120573-lactamase superfamily protein (Table S1spots 115 and 125) presumably involved with cellular pro-teases increased and decreased 22- and 20-fold respectivelyA single protein was identified from the fatty acid andphospholipid metabolism category cyclopropane-fatty-acyl-phospholipid synthase (Table S1 spot 34) which showed15-fold decreased abundance

4 Discussion

Combining cellobiose with a probiotic strain of Lactobacillusrhamnosus was previously reported to give a synergisticincrease of this and other lactic acid bacteria in the cecum ofrats [33] but no proteins important for cellobiose utilizationby lactic acid bacteria were identified in that study Thepresent work provides the first insights into the molecularmechanisms of the utilization of cellobiose by the probioticLactobacillus acidophilusNCFM Differential proteome anal-ysis by 2D-DIGE coupled with mass spectrometric proteinidentification highlighted enzymes and proteins importantfor uptake and catabolism of cellobiose in NCFM

41 Proteins Involved in Carbohydrate Utilization and Regu-latory Functions Growth on cellobiose alters abundance ofa large number of proteins of NCFM involved in glycolysissome of which exist in multiple forms This include pro-teins such as phosphoglucomutase found in two forms withdifferent pI (Figure 1(c) spots 12 and 13) glyceraldehyde 3-phosphate dehydrogenase (GADPH) identified in multiple


LBA0884TLBA0725T

Cellobiose

Glycolysis

IIABCLBA0879T

IIC IIC

IIBLBA0877TLBA0876T

IIA

LBA0726P

LBA0874LBA0881P

LBA0885

Cellobiose-6P

Glucose-6P + glucose

(a)

LBA0724 LBA0726LBA0725

LBA0876LBA0874 LBA0875 LBA0877 LBA0878 LBA0879 LBA0880 LBA0881 LBA0882 LBA0883 LBA0884 LBA0885 LBA0886

(b)

Figure 3 (a) Schematic presentation of cellobiose entry and hydrolysis by L acidophilus NCFM Superscript letters T and P indicateupregulation by transcriptomics [19] or increase in abundance by proteomics respectively (LBA0725 PTS II LBA0726 phospho-120573-galactosidase II LBA0874 phospho-120573-galactosidase I LBA0876 PTS IIC LBA0877 PTS IIA LBA0879 PTS IIC LBA0881 phospho-120573-glucosidase LBA0884 PTS IIC LBA0726 phospho-120573-galactosidase II and LBA0885 120573-glucosidase) (b) Schematic presentation of geneclusters encoding glycoside hydrolases (LBA0726 phospho-120573-galactosidase II LBA0874 phospho-120573-galactosidase I LBA0881 phospho-120573-glucosidase and LBA0885 120573-glucosidase) shown as light grey arrows and PTSs (LBA0725 PTS II LBA0876 PTS IIC LBA0877 PTS IIALBA0879 PTS IIC and LBA0884 PTS IIC) shown as dark grey arrows predicted to be cellobiose specific Transcription antiterminator(LBA0724) hypothetical proteins (LBA0878 LBA0880 and LBA0883) and transcriptional regulators (LBA0875 LBA0882 and LBA0886)are shown as white arrows

forms with different pI as well as molecular size (Figures1(c) and 1(d) spots 36ndash38 70 and 123) triosephosphateisomerase (TPI) present in four forms with different pIand molecular size (Figure 1(c) spots 59 60 106 and 107)and phosphoglycerate kinase found in four forms havingdifferent pI (Figure 1(c) spots 28ndash31) Multiple forms ofphosphoglucomutase GADPH TPI and phosphoglyceratekinase with different pI have previously been identified fromNCFM [8] Notably multiple forms of GADPH and TPI werenot affected in in the same way during growth on cellobiose

Growth of NCFM on cellobiose elicited an increase inabundance of catabolite control protein A (CcpA Table 1spot 47) CcpA is a key enzyme in carbon catabolite repres-sion a mechanism that regulates enzymes involved in carbo-hydrate metabolismThus in the presence of preferred sugars(eg glucose) enzymes involved in utilization of less favorablesugars are repressed [34] Carbohydrate phosphotransferasesystems (PTSs) involved in uptake and phosphorylation ofcarbohydrates participate in the carbon catabolite repression[34] When NCFMwas grown on cellobiose notably 24-foldupregulation was seen for phospho-120573-glucosidase (LBA0881Table 1 spot 17) located in a gene cluster with multiple

PTSs predicted to be cellobiose specific (Figure 3) Interest-ingly a 70ndash74-fold increased abundance was observed forphospho-120573-galactosidase II (LBA0726 Table 1 spots 16 and18) encoded in a gene cluster together with another PTS(LBA0725 Figure 3) indicating a role in cellobiose utilization(Figure 3) This PTS LBA0725 has been reported to have62 amino acid sequence identity to a PTS (ORF 1669) ofL gasseri 33323 of which expression was strongly inducedby cellobiose [35] In another low GC ratio Gram-positivebacteria Clostridium acetobutylicum transcriptional analysisdemonstrated that cellobiose induced expression of two PTSsand three glycoside hydrolases [25] Transcriptional analysisof NCFM grown on cellobiose has reported upregulationof several genes within the loci LBA0724ndashLBA0726 andLBA0877ndashLBA0884 [26] Taken together the results of tran-scriptional and proteomics analyses indicate that NCFMcontains more than one operon encoding protein involved inuptake and metabolism of cellobiose

NCFM encodes 9 two-component system regulators(2CSRs) [7] involved inmediating an intracellular response toextracellular stimuli and typically consisting of a membranebound histidine protein kinase (HPK) and a cytoplasmic


response regulator (RR) [36] When NCFM was grownon cellobiose a 2CSR (Table 1 spot 102) was found to bedownregulated by 78-fold It has been reported that a 2CRS isinvolved in acid tolerance and proteolytic activity of NCFM[37] The decrease in abundance of the 2CSR however mayreflect the different growth rates of NCFM on cellobioseand glucose rather than a decrease in acid tolerance orproteolytic activity This could also explain why all theproteins with changed abundance in the functional categoriesof protein synthesis central intermediate metabolism andpurines pyrimidines nucleosides and nucleotides apartfrom a putative phosphate starvation inducible factor wereless abundant in the cellobiose-grownNCFMMoreover twodifferent subunits of the F

0F1ATP synthase (Table 1 spots 41

and 99) responsible for maintaining the proton motive forceat low pH [36] decreased by 16- and 19-fold respectivelywhenNCFMwas grown on cellobioseThese various changesin the protein profile are in agreement with the observedslower growth of NCFM on cellobiose compared to glucose

Several proteins previously described as moonlightingproteins were identified These include EF-Tu GAPDHand TPI with conventional roles in protein synthesis andglycolysis respectively but which also have been identifiedat the cell wall where they are involved in adhesion activitiesof lactic acid bacteria [38ndash41] Although the present studyincludes only differentially expressed cytosolic proteins aputative surface layer protein was also identified and foundto be upregulated (Table 1 spot 122) Frece et al [42] reportedthat removing the S-layer of L acidophilusM92 by enzymaticand chemical treatment resulted in reduced adhesion tomurine epithelial cells and reduced viability Moreover S-layer proteins of Lactobacillus crispatus are able to inhibitthe adhesion of pathogens to HeLa cells [43] In NCFMS-layer protein A was shown to bind to a dendritic cellreceptor and to regulate immune functions of the dendriticcells [44] The putative S-layer protein upregulated in thepresent study has recently been shown to be an S-layerassociated [45] Although a deletion mutant of this S-layerassociated protein did not affect adhesion to intestinal cells areduction of proinflammatory response in murine dendriticcells was observed [45] thus suggesting an effect on the hostimmune systemThe present findings propose that cellobioseis able to affect the probiotic activity of NCFMwith regard tointeractions with the host

5 Conclusions

Exploring the molecular mechanisms of prebiotic-probioticinteractions is important for understanding how prebioticsbenefit a probiotic strain In the current study the lateexponential growth phase differential proteome of L aci-dophilus NCFM (pH 4ndash7 and pH 6ndash11) was explored usingthe disaccharide cellobiose as carbon source Cellobioseconsists of two 120573(1rarr 4) linked glucosyl residues which afterhydrolysis enter the glycolysis Two hydrolases a phospho-120573-glucosidase (LBA0881 Table 1 spot 17) and a phospho-120573-galactosidase II (LBA0726 Table 1 spots 16 and 18) increased24ndash74-fold in abundance suggesting an involvement in thecellobiose metabolism (Figure 3) Results obtained in thepresent study contribute to the understanding of prebioticutilization by probiotic bacteria

List of Abbreviations

2CSR Two-component system regulatorCcpA Catabolite control protein ACHAPS 3-((3-Cholamidopropyl)dimethylammonio)-1-

propansulfonateGAPDH Glyceraldehyde 3-phosphate dehydrogenaseGH Glycoside hydrolaseHED Bis(2-hydroxyethyl) disulphideHPK Histidine protein kinaseHPr Histidine containing phosphocarrier proteinIEF Isoelectric focusingIPG Immobilized pH gradientLABSEM Lactic acid bacteria semisynthetic mediumNCFM Lactobacillus acidophilus NCFMPTS Phosphotransferase systemRR Response regulatorSCFA Short-chain fatty acidTPI Triosephosphate isomerase

Conflict of Interests

Sampo J Lahtinen is employed by DuPont Nutrition andHealth a manufacturer of L acidophilus NCFM The otherauthors have declared no conflict of interests

Authorsrsquo Contribution

The experimental work including data analysis and interpre-tation was done by Gabriella C van Zanten Nadja SpardingandAvishekMajumderThepaperwas drafted byGabriella Cvan Zanten and Avishek Majumder The experimental workwas conceived and designed by Avishek Majumder SampoJ Lahtinen Birte Svensson and Susanne Jacobsen Sampo JLahtinen Birte Svensson and Susanne Jacobsen helped writeand revise the paper All authors have read the paper andapproved the final version

Acknowledgments

This work was funded by the Danish Strategic ResearchCouncilrsquos Program Committee on Health Food and Wel-fare (FoslashSu) to the project ldquoGene discovery and molecu-lar interactions in prebioticsprobiotics systems Focus oncarbohydrate prebioticsrdquo Avishek Majumder is grateful tothe Technical University of Denmark for a Hans ChristianOslashrsted postdoctoral fellowship and Gabriella C van Zantenwishes to acknowledge DuPont Health andNutrition GroupKantvik Finland for funding The Typhoon scanner wasfunded by The Danish Council for Independent ResearchNatural Sciences and the Mass Spectrometer was fundedin part by the Danish Centre for Advanced Food Studies(LMC) Bjarne G Schmidt is thanked for excellent technicalassistance and Morten Ejby and Joakim M Andersen forstimulating discussions

References

[1] FAOWHO Health and Nutritional Properties of Probiotics inFood Including PowderMilk with Live Lactic Acid Bacteria 2001


[2] E K Vouloumanou G CMakris D E Karageorgopoulos andM E Falagas ldquoProbiotics for the prevention of respiratory tractinfections a systematic reviewrdquo International Journal of Antimi-crobial Agents vol 34 no 3 pp 197e1ndash197e10 2009

[3] R Ashraf and N P Shah ldquoImmune system stimulation byprobioticmicroorganismsrdquoCritical Reviews in Food Science andNutrition vol 54 no 7 pp 938ndash956 2014

[4] A P S Hungin C Mulligan B Pot et al ldquoSystematic reviewprobiotics in the management of lower gastrointestinal symp-toms in clinical practicemdashan evidence-based internationalguiderdquo Alimentary Pharmacology and Therapeutics vol 38 no8 pp 864ndash886 2013

[5] S J Allen B Okoko E Martinez G Gregorio and L FDans ldquoProbiotics for treating infectious diarrhoeardquo CochraneDatabase of Systematic Reviews no 11 Article ID CD0030482010

[6] M E Sanders and T R Klaenhammer ldquoInvited reviewThe sci-entific basis of Lactobacillus acidophilusNCFM functionality asa probioticrdquo Journal of Dairy Science vol 84 no 2 pp 319ndash3312001

[7] E Altermann W M Russell M A Azcarate-Peril et al ldquoCom-plete genome sequence of the probiotic lactic acid bacteriumLactobacillus acidophilus NCFMrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 102 no11 pp 3906ndash3912 2005

[8] A Majumder A Sultan R R Jersie-Christensen et al ldquoPro-teome reference map of Lactobacillus acidophilus NCFM andquantitative proteomics towards understanding the prebioticaction of lactitolrdquo Proteomics vol 11 no 17 pp 3470ndash3481 2011

[9] R Barrangou M A Azcarate-Peril T Duong S B ConnersR M Kelly and T R Klaenhammer ldquoGlobal analysis of car-bohydrate utilization by Lactobacillus acidophilus using cDNAmicroarraysrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 103 no 10 pp 3816ndash38212006

[10] J M Andersen R Barrangou M A Hachem et al ldquoTranscrip-tional and functional analysis of galactooligosaccharide uptakeby lacS in Lactobacillus acidophilusrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 108 no43 pp 17785ndash17790 2011

[11] M Roberfroid G R Gibson L Hoyles et al ldquoPrebiotic effectsmetabolic and health benefitsrdquo British Journal of Nutrition vol104 pp S1ndashS63 2010

[12] S Nakamura T Oku and M Ichinose ldquoBioavailability ofcellobiose by tolerance test and breath hydrogen excretion inhumansrdquo Nutrition vol 20 no 11-12 pp 979ndash983 2004

[13] M L Sanz G R Gibson and R A Rastall ldquoInfluence ofdisaccharide structure on prebiotic selectivity in vitrordquo Journalof Agricultural and FoodChemistry vol 53 no 13 pp 5192ndash51992005

[14] G C van Zanten A Knudsen H Roytio et al ldquoThe effect ofselected synbiotics on microbial composition and short-chainfatty acid production in a model system of the human colonrdquoPLoS ONE vol 7 no 10 Article ID e47212 2012

[15] H M Blottiere B Buecher J-P Galmiche and C CherbutldquoMolecular analysis of the effect of short-chain fatty acids onintestinal cell proliferationrdquo Proceedings of the Nutrition Societyvol 62 no 1 pp 101ndash106 2003

[16] S Tedelind F Westberg M Kjerrulf and A Vidal ldquoAnti-inflammatory properties of the short-chain fatty acids acetateand propionate a study with relevance to inflammatory bowel

diseaserdquo World Journal of Gastroenterology vol 13 no 20 pp2826ndash2832 2007

[17] M Comalada E Bailon O DeHaro et al ldquoThe effects of short-chain fatty acids on colon epithelial proliferation and survivaldepend on the cellular phenotyperdquo Journal of Cancer Researchand Clinical Oncology vol 132 no 8 pp 487ndash497 2006

[18] TNishimura A Andoh THashimoto A Kobori T Tsujikawaand Y Fujiyama ldquoCellobiose prevents the development of dex-tran sulfate sodium (DSS)-induced experimental colitisrdquo Jour-nal of Clinical Biochemistry andNutrition vol 46 no 2 pp 105ndash110 2010

[19] G R Gibson and M B Roberfroid ldquoDietary modulation ofthe human colonic microbiota introducing the concept of pre-bioticsrdquo Journal of Nutrition vol 125 no 6 pp 1401ndash1412 1995

[20] N Rayes D Seehofer T Theruvath et al ldquoEffect of enteralnutrition and synbiotics on bacterial infection rates afterpylorus-preserving pancreatoduodenectomy a randomizeddouble-blind trialrdquo Annals of Surgery vol 246 no 1 pp 36ndash412007

[21] S M Riordan N A Skinner C J Mciver et al ldquoSynbiotic-associated improvement in liver function in cirrhotic patientsrelation to changes in circulating cytokine messenger RNA andprotein levelsrdquoMicrobial Ecology in Health and Disease vol 19no 1 pp 7ndash16 2007

[22] A C Ouwehand K Tiihonen M Saarinen H Putaala andN Rautonen ldquoInfluence of a combination of LactobacillusacidophilusNCFMand lactitol on healthy elderly intestinal andimmune parametersrdquo The British Journal of Nutrition vol 101no 3 pp 367ndash375 2009

[23] MBjorklundACOuwehand SD Forssten et al ldquoGutmicro-biota of healthy elderly NSAID users is selectively modifiedwith the administration of Lactobacillus acidophilusNCFM andlactitolrdquo Age vol 34 no 4 pp 987ndash999 2012

[24] G C van Zanten L Krych and R H Roytio ldquoSynbioticLactobacillus acidophilus NCFM and cellobiose does not affecthuman gut bacterial diversity but increases abundance oflactobacilli bifidobacteria and branched-chain fatty acids arandomized double-blinded cross-over trialrdquo FEMSMicrobiol-ogy Ecology 2014

[25] M D Servinsky J T Kiel N F Dupuy and C J SundldquoTranscriptional analysis of differential carbohydrate utilizationby Clostridium acetobutylicumrdquo Microbiology vol 156 part 11pp 3478ndash3491 2010

[26] J M Andersen R Barrangou M A Hachem et al ldquoTran-scriptional analysis of prebiotic uptake and catabolism byLactobacillus acidophilus NCFMrdquo PLoS ONE vol 7 no 9Article ID e44409 2012

[27] DW Kavanaugh J OrsquoCallaghan L F Butto et al ldquoExposure ofBifidobacterium longum subsp infantis tomilk oligosaccharidesincreases adhesion to epithelial cells and induces a substantialtranscriptional responserdquo PLoS ONE vol 8 no 6 Article IDe67224 2013

[28] R Barrangou E Altermann R Hutkins R Cano and T RKlaenhammer ldquoFunctional and comparative genomic analysesof an operon involved in fructooligosaccharide utilization byLactobacillus acidophilusrdquo Proceedings of the National Academyof Sciences of the United States of America vol 100 no 15 pp8957ndash8962 2003

[29] AMajumder L CaiM Ejby et al ldquoTwo-dimensional gel-basedalkaline proteome of the probiotic bacterium Lactobacillusacidophilus NCFMrdquo Proteomics vol 12 no 7 pp 1006ndash10142012


[30] S H Bae A G Harris P G Hains et al ldquoStrategies for theenrichment and identification of basic proteins in proteomeprojectsrdquo Proteomics vol 3 no 5 pp 569ndash579 2003

[31] M Corton G Villuendas J I Botella J L san Millan H FEscobar-Morreale and B Peral ldquoImproved resolution of thehuman adipose tissue proteome at alkaline and wide range pHby the addition of hydroxyethyl disulfiderdquo Proteomics vol 4 no2 pp 438ndash441 2004

[32] G Candiano M Bruschi L Musante et al ldquoBlue silver avery sensitive colloidal Coomassie G-250 staining for proteomeanalysisrdquo Electrophoresis vol 25 no 9 pp 1327ndash1333 2004

[33] M Umeki K Oue M Mori S Mochizuki and K Sakai ldquoFluo-rescent in situ hybridization analysis of cecal microflora in ratssimultaneously administrated Lactobacillus rhamnosus KY-3and cellobioserdquo Food Science and Technology Research vol 11no 2 pp 168ndash170 2005

[34] JDeutscher C Francke andPWPostma ldquoHowphosphotrans-ferase system-related protein phosphorylation regulates carbo-hydrate metabolism in bacteriardquo Microbiology and MolecularBiology Reviews vol 70 no 4 pp 939ndash1031 2006

[35] A L Francl T Thongaram and M J Miller ldquoThe PTStransporters of Lactobacillus gasseri ATCC 33323rdquo BMCMicro-biology vol 10 article 77 2010

[36] P D Cotter and C Hill ldquoSurviving the acid test responses ofgram-positive bacteria to low pHrdquoMicrobiology and MolecularBiology Reviews vol 67 no 3 pp 429ndash453 2003

[37] M A Azcarate-Peril O McAuliffe E Altermann S Lick WM Russell and T R Klaenhammer ldquoMicroarray analysis of atwo-component regulatory system involved in acid resistanceand proteolytic activity in Lactobacillus acidophilusrdquo Appliedand Environmental Microbiology vol 71 no 10 pp 5794ndash58042005

[38] P Kelly P B Maguire M Bennett et al ldquoCorrelation ofprobiotic Lactobacillus salivarius growth phasewith its cell wall-associated proteomerdquo FEMSMicrobiology Letters vol 252 no 1pp 153ndash159 2005

[39] D Granato G E Bergonzelli R D Pridmore L Marvin MRouvet and I E Corthesy-Theulaz ldquoCell surface-associatedelongation factor Tu mediates the attachment of Lactobacillusjohnsonii NCC533 (La1) to human intestinal cells and mucinsrdquoInfection and Immunity vol 72 no 4 pp 2160ndash2169 2004

[40] K Ramiah C A van Reenen and L M T Dicks ldquoSurface-bound proteins of Lactobacillus plantarum 423 that contributeto adhesion of Caco-2 cells and their role in competitive exclu-sion and displacement of Clostridium sporogenes and Entero-coccus faecalisrdquo Research in Microbiology vol 159 no 6 pp470ndash475 2008

[41] H C Beck S MMadsen J Glenting et al ldquoProteomic analysisof cell surface-associated proteins from probiotic LactobacillusplantarumrdquoFEMSMicrobiology Letters vol 297 no 1 pp 61ndash662009

[42] J Frece B Kos I K Svetec Z Zgaga V Mrsa and JSuskovic ldquoImportance of S-layer proteins in probiotic activity ofLactobacillus acidophilusM92rdquo Journal of AppliedMicrobiologyvol 98 no 2 pp 285ndash292 2005

[43] X Chen J Xu J Shuai J Chen Z Zhang and W FangldquoThe S-layer proteins of Lactobacillus crispatus strain ZJ001 isresponsible for competitive exclusion against Escherichia coliO157H7 and Salmonella typhimuriumrdquo International Journal ofFood Microbiology vol 115 no 3 pp 307ndash312 2007

[44] S R Konstantinov H Smidt W M De Vos et al ldquoS layer pro-tein A of Lactobacillus acidophilus NCFM regulates immature

dendritic cell and T cell functionsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 105 no49 pp 19474ndash19479 2008

[45] B Johnson K Selle S OFlaherty Y J Goh and T Klaenham-mer ldquoIdentification of extracellular surface-layer associatedproteins in Lactobacillus acidophilusNCFMrdquoMicrobiology vol159 part 11 pp 2269ndash2282 2013

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology

Research ArticleThe Differential Proteome of the ProbioticLactobacillus acidophilus NCFM Grown on thePotential Prebiotic Cellobiose Shows Upregulation ofTwo 120573-Glycoside Hydrolases

Gabriella C van Zanten12 Nadja Sparding2 Avishek Majumder2

Sampo J Lahtinen3 Birte Svensson2 and Susanne Jacobsen2

1Department of Food Science Faculty of Science University of Copenhagen Rolighedsvej 26 1958 Frederiksberg C Denmark2Enzyme and Protein Chemistry Department of Systems Biology Technical University of DenmarkSoslashltofts Plads Building 224 2800 Kongens Lyngb Denmark3DuPont Nutrition and Health Sokeritehtaantie 20 02460 Kantvik Finland

Correspondence should be addressed to Birte Svensson bisbiodtudk

Received 16 July 2014 Revised 22 September 2014 Accepted 23 September 2014

Academic Editor Borja Sanchez

Copyright copy 2015 Gabriella C van Zanten et alThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Probiotics prebiotics and combinations thereof that is synbiotics are known to exert beneficial health effects in humans howeverinteractions between pro- and prebiotics remain poorly understood at the molecular level The present study describes changes inabundance of different proteins of the probiotic bacterium Lactobacillus acidophilusNCFM (NCFM) when grown on the potentialprebiotic cellobiose as compared to glucose Cytosolic cell extract proteomes after harvest at late exponential phase of NCFMgrownon cellobiose or glucose were analyzed by two dimensional difference gel electrophoresis (2D-DIGE) in the acidic (pH 4ndash7) andthe alkaline (pH 6ndash11) regions showing a total of 136 spots to change in abundance Proteins were identified by MS or MSMSfrom 81 of these spots representing 49 unique proteins and either increasing 15ndash139-fold or decreasing 15ndash78-fold in relativeabundance Many of these proteins were associated with energy metabolism including the cellobiose related glycoside hydrolasesphospho-120573-glucosidase (LBA0881) and phospho-120573-galactosidase II (LBA0726) The data provide insight into the utilization of thecandidate prebiotic cellobiose by the probiotic bacterium NCFM Several of the upregulated or downregulated identified proteinsassociated with utilization of cellobiose indicate the presence of carbon catabolite repression and regulation of enzymes involvedin carbohydrate metabolism

Dedicated to Susanne Jacobsen who sadly has passed away

1 Background

Probiotics are defined as ldquolive microorganisms that whenadministered in adequate amounts confer a health benefit onthe hostrdquo [1] Reported beneficial effects include preventionand reduced severity of respiratory infections [2]modulationof immune responses [3] diminished symptoms of irritablebowel disease [4] and a remedy against antibiotic-associated

[4] and infectious diarrhea [5] Lactobacillus acidophilusNCFM (NCFM) is a low GC ratio (384) Gram-positivelactic acid bacterium with well-documented probiotic effects[6] NCFMhas a genome of 20Mbwith 1864 predicted openreading frames (ORFs) including different genes and geneloci involved in carbohydrate metabolism [7] Furthermorea large number of identified proteins are dedicated to energymetabolism [8] Noticeably cDNA microarray analyses of

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 347216 9 pageshttpdxdoiorg1011552015347216






2 Methods









4 7

97

10

pH(k

Da)

(a)

6pH

11

(kD

a)

97

10

(b)

4 7pH

(kD

a)

97

10

(c)

6pH

11

(kD

a)

97

10

(d)





3 Results



AA 58BCPC 96

CE 38

CIM 38

CP 19DM 38

EM 346

FP 19HP 77

PS 38

PF 19

PPNN 96

RF 77

TR 19

UF 154













4 Discussion




LBA0884TLBA0725T

Cellobiose

Glycolysis

IIABCLBA0879T

IIC IIC

IIBLBA0877TLBA0876T

IIA

LBA0726P

LBA0874LBA0881P

LBA0885

Cellobiose-6P


(a)



(b)











5 Conclusions









Acknowledgments


References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






2 Methods









4 7

97

10

pH(k

Da)

(a)

6pH

11

(kD

a)

97

10

(b)

4 7pH

(kD

a)

97

10

(c)

6pH

11

(kD

a)

97

10

(d)





3 Results



AA 58BCPC 96

CE 38

CIM 38

CP 19DM 38

EM 346

FP 19HP 77

PS 38

PF 19

PPNN 96

RF 77

TR 19

UF 154













4 Discussion




LBA0884TLBA0725T

Cellobiose

Glycolysis

IIABCLBA0879T

IIC IIC

IIBLBA0877TLBA0876T

IIA

LBA0726P

LBA0874LBA0881P

LBA0885

Cellobiose-6P


(a)



(b)











5 Conclusions









Acknowledgments


References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


4 7

97

10

pH(k

Da)

(a)

6pH

11

(kD

a)

97

10

(b)

4 7pH

(kD

a)

97

10

(c)

6pH

11

(kD

a)

97

10

(d)





3 Results



AA 58BCPC 96

CE 38

CIM 38

CP 19DM 38

EM 346

FP 19HP 77

PS 38

PF 19

PPNN 96

RF 77

TR 19

UF 154













4 Discussion




LBA0884TLBA0725T

Cellobiose

Glycolysis

IIABCLBA0879T

IIC IIC

IIBLBA0877TLBA0876T

IIA

LBA0726P

LBA0874LBA0881P

LBA0885

Cellobiose-6P


(a)



(b)











5 Conclusions









Acknowledgments


References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


AA 58BCPC 96

CE 38

CIM 38

CP 19DM 38

EM 346

FP 19HP 77

PS 38

PF 19

PPNN 96

RF 77

TR 19

UF 154













4 Discussion




LBA0884TLBA0725T

Cellobiose

Glycolysis

IIABCLBA0879T

IIC IIC

IIBLBA0877TLBA0876T

IIA

LBA0726P

LBA0874LBA0881P

LBA0885

Cellobiose-6P


(a)



(b)











5 Conclusions









Acknowledgments


References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





4 Discussion




LBA0884TLBA0725T

Cellobiose

Glycolysis

IIABCLBA0879T

IIC IIC

IIBLBA0877TLBA0876T

IIA

LBA0726P

LBA0874LBA0881P

LBA0885

Cellobiose-6P


(a)



(b)











5 Conclusions









Acknowledgments


References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


LBA0884TLBA0725T

Cellobiose

Glycolysis

IIABCLBA0879T

IIC IIC

IIBLBA0877TLBA0876T

IIA

LBA0726P

LBA0874LBA0881P

LBA0885

Cellobiose-6P


(a)



(b)











5 Conclusions









Acknowledgments


References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






5 Conclusions









Acknowledgments


References

























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

the differential proteome of the probiotic …...ncfm(ncfm)isalowgcratio(38.4%),gram-positive...

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