Probiotics and prebiotics in the elderly

In vitro investigations of the e?ect of probiotics and prebiotics on

selected human intestinal pathogens

Laura J.Fooks *,Glenn R.Gibson

Food Microbial Sciences Unit,School of Food Biosciences,The University of Reading,Whiteknights,Reading RG66BZ UK

Received 27July 2001;received in revised form 19October 2001;accepted 19October 2001

First published online 10December 2001

Abstract

This study investigated the effects of selected probiotic microorganisms,in combination with prebiotics,on certain human intestinal food-borne pathogens.Probiotics grown with different carbohydrate sources were observed to inhibit growth of Escherichia coli ,Campylobacter jejuni and Salmonella enteritidis ,with the extent of inhibition varying according to the carbohydrate source provided.Prebiotics identified as being preferentially utilised by the probiotics tested were oligofructose (FOS),inulin,xylo-oligosaccharide (XOS),and mixtures of inulin:FOS (80:20w/w)and FOS:XOS (50:50w/w).Two of the probiotics,Lactobacillus plantarum and Bifidobacterium bifidum were selected for further co-culture experiments.Each was combined with the selected prebiotics,and was observed to inhibit pathogen growth strongly.Results suggested that acetate and lactate were directly conferring antagonistic action,which was not necessarily related to a lowering of culture pH.?2002Federation of European Microbiological Societies.Published by Elsevier Science B.V.All rights reserved.

Keywords:Oligosaccharides;Lactobacillus plantarum ;Bi￠dobacterium bi￠dum ;Escherichia coli ;Campylobacter jejuni ;Salmonella enteritidis

1.Introduction

The large bowel harbours a nutritionally and physiolog-ically diverse range of bacteria,promoting normal intesti-nal function,and o?ering the host protection against in-fections [1].Disruption of the colonic ￡ora,due to pathogens [2,3],dietary antigens [1]or other harmful sub-stances [4^6]can,however,lead to intestinal https://www.360docs.net/doc/179923372.html,ctobacillus and Bi￠dobacterium ,which have a long and safe history in the manufacture of dairy products,are traditionally included in probiotic products to help protect against such e?ects [7].Both are thought to prevent the adherence,establishment,replication and/or virulence of speci￠c enteropathogens [8].A number of mechanisms have been proposed:decreasing the luminal pH via the production of volatile short chain fatty acids (SCFA);rendering speci￠c nutrients unavailable to pathogens;and/or producing speci￠c inhibitory compounds such as bacteriocins [9].In the normal intestinal ￡ora these mech-

anisms are essential to the component bacterial popula-tions,as a means of gaining advantage over competing bacteria.

An ability to compete for limiting nutrients is perhaps the most important factor that determines the composition of the gut ￡ora,with species that are unable to compete being e?ectively eliminated from the system.One of the primary objectives of this research was to identify a syn-biotic,that is,a combination of a probiotic and prebiotic [10],which could ultimately be used for fermentation ex-periments and be an e?ective antimicrobial agent against common enteropathogens.The range of carbohydrates tested included non-prebiotic controls such as lactitol,starch and dextran,and prebiotics,short and long chain fructo-oligosaccharides,xylo-oligosaccharide (XOS)and lactulose.

In this study,a range of probiotics,were tested using disc/spot assays for their ability to inhibit the growth of some common enteropathogens:Campylobacter jejuni ,Escherichia coli and Salmonella enteritidis .Each probiotic strain tested was combined with a range of prebiotic sour-ces in an attempt to move towards identifying synbiotic combinations.Subsequently,two of the probiotic strains,Lactobacillus plantarum and Bi￠dobacterium bi￠dum ,com-bined with preferred candidate prebiotics,were examined

w w w.f e m s -m i c r o b i o l o g y.o r g

in co-culture for their ability to inhibit growth of the en-teropathogens.The mechanism underlying any antimicro-bial activity was addressed by monitoring changes of pH in the culture medium and levels of SCFA at speci￠c time intervals,when samples were removed for enumeration of probiotic and pathogen strains.

2.Materials and methods

2.1.Growth substrates

Oligofructose(FOS)and inulin extracted from chicory root were supplied by Orafti Ra¤nerie(Tienen,Belgium). Inulin was94%pure with fructose(V1%),glucose(V1%) and sucrose(V5%)as impurities.FOS was98%pure with fructose(0.9%),glucose(60.1%)and sucrose(0.9%)as impurities.XOS35P was supplied by Suntory Ltd.(To-kyo,Japan).The XOS was obtained by enzymatically treating xylan derived from corncobs.The￠nal product contained35%w/w oligosaccharides[11].Growth media and supplements were obtained from Oxoid Ltd.,Basing-stoke,Hampshire,UK,unless otherwise stated.All other carbohydrates and chemicals were obtained from Sigma Chemicals Ltd.,Poole,Dorset,UK.Carbohydrates used were FOS,inulin,inulin:FOS mixture(80:20,w/w),XOS, FOS:XOS mixture(50:50,w/w),lactulose,lactitol,starch and dextran.

2.2.Bacterial strains

Probiotic strains tested were L.plantarum0407and Lac-tobacillus pentosus905(St.Ivel European Food,Swindon, UK),Lactobacillus reuteri SD2112(Biogaia),Lactobacillus acidophilus La5and B.bi￠dum Bb12(Chr.Hansen,Den-mark).Enteropathogens used were E.coli NCIMB9517 (National Collections of Industrial and Marine Bacteria Ltd.),C.jejuni ATCC11351(American Type Culture Col-lection)and S.enteritidis var.danysz NCTC4444(Na-tional Collection of Type Cultures).

2.3.Bacterial growth conditions

Probiotic strains were grown in basal medium(30ml) containing,in g l31,peptone,2.0;yeast extract,2.0;NaCl, 0.1;K2HPO4,0.04;KH2PO4,0.04;CaCl2W6H20,0.01; MgSO4W7H20,0.01;NaHCO3,2.0;Tween80,2ml;hemin (dissolved in three drops1M NaOH),0.05;vitamin K1, 10W l;cysteine^HCl,0.5;bile salts,0.5with various car-bohydrate sources(1%w/v)anaerobically in an anaerobic cabinet(Don Whitley,Yorkshire,UK;10:10:80, H2:CO2:N2).E.coli and S.enteritidis were grown aerobi-cally in Mueller^Hinton broth.C.jejuni was grown in Brucella broth(Difco Laboratories,Detroit,MI,USA) supplemented with Campylobacter growth supplement (Oxoid)under microaerobic conditions,achieved using a variable atmosphere incubator(VAIN;Don Whitley, Yorkshire,UK;4:4:10:82,H2:O2:CO2:N2).

2.4.Disc assay technique

Tryptone soya agar(TSA;2%w/v;Oxoid Ltd.,Basing-stoke,UK)(15ml),prepared and autoclaved according to manufacturer's instructions,was poured into sterile Petri dishes and allowed to set.Overnight cultures(109cfu ml31)of each probiotic were centrifuged at14000U g for 10min at43C.Sterile￠lter paper discs(Whatman No.2, 5mm)were soaked(10W l)in one of the culture fractions and laid upon the agar surface.Three fractions were tested.Probiotic cells were prepared by removing super-natant and washing the cells twice with phosphate bu?er (pH7.4;1M)before re-suspension in200W l phosphate bu?er.Supernatant consisted of cell-free extract following centrifugation at14000U g for10min.The pH of the supernatant from each probiotic culture was recorded.Ab-sence of cells was con￠rmed by serially diluting and plat-ing the extract;no growth was observed.Neutralised supernatant was prepared by neutralising to pH7using 1M NaOH.

Controls used were basal medium,and acetate and lac-tate solutions(10mmol l31,pH4.5).Four discs were placed on each plate,one test and three controls.Tryptic soy soft agar(10ml;0.75%)inoculated with V108cells (0.5ml)of the indicator organism(E.coli,S.enteritidis)in the late exponential phase was then poured over the sur-face.The plates were incubated anaerobically at373C for up to48h.The extent of inhibition was assessed after incubation by measuring the diameter of the clear zone surrounding each disc.Plates were prepared in triplicate, i.e.three per fraction,for each carbohydrate.The exper-imental set-up was repeated in triplicate.

2.5.Overlay assay technique

The disc assay technique was modi￠ed for testing with C.jejuni since reliable results were not obtained with the above technique,as its growth was restricted to the base of the inoculated agar layer.Consequently,a disc laying on the top agar surface was ine?ective and10W l of culture fraction was therefore spotted onto2%(w/v)TSA,al-lowed to evaporate and overlain with0.75%(w/v)agar inoculated with108cells of C.jejuni.

2.6.Growth curves^probiotic strains

MRS broth(10ml)was inoculated with one cryogenic bead of each probiotic strain and incubated overnight under anaerobic conditions(oxygen-free nitrogen)at 373C.Basal medium(50ml)contained in a50-ml batch culture fermenter was pre-reduced overnight and0.5g(1% w/v)of the selected carbohydrate added.1ml of each overnight culture was then inoculated,in duplicate,into

L.J.Fooks,G.R.Gibson/FEMS Microbiology Ecology39(2002)67^75 68

50ml carbohydrate-containing basal medium and cultures were incubated under the same conditions.The optical density at 650nm of each culture was determined at hourly intervals for up to 24h.This was repeated four times and mean values plotted.Subsequently,speci￠c growth rates of the probiotics in each carbohydrate me-dium were calculated.Exponential phase growth was de-rived from growth plots of the probiotics (not shown).

2.7.Growth curves ^pathogenic strains:carbohydrate

e?ect

Mueller^Hinton broth (10ml)was inoculated with one cryogenic bead of either E.coli or S.enteritidis and incu-bated overnight under aerobic conditions at 373C.Brucel-la broth (50ml)supplemented with Campylobacter growth supplement was inoculated with one cryogenic bead of C.jejuni and incubated overnight under microaerobic con-ditions at 373C.Basal medium (50ml),contained in a 50-ml batch culture fermenter,was pre-reduced overnight and 0.5g (1%w/v)of the selected carbohydrate added.1ml of each overnight culture (109^1010cells ml 31)was then inoculated,in triplicate,into 50ml carbohydrate-con-taining basal medium and cultures were incubated under the same conditions for each organism.1ml aliquots were removed at hourly intervals and used to prepare a dilution series,which was plated onto a suitable agar medium (Oxoid Manual);MacConkey No.3(E.coli ),Brilliant green (S.enteritidis )or Campylobacter blood-free speci￠c (CBFS)+CCDA supplement (C.jejuni ).Plates were incu-bated for up to 48h,when colonies were enumerated for each time point.

2.8.Growth curves ^pathogenic strains:pH e?ect

To assess survival of each pathogenic strain at di?erent initial culture pH values,overnight cultures were prepared as previously described.Mueller^Hinton broth (50ml)and Brucella supplemented broth (50ml)were prepared,and the pH adjusted using 4M HCl to give a range of initial pH values from 1to 7.These were incubated overnight under aerobic (E.coli and S.enteritidis )or microaerobic (C.jejuni )atmospheric conditions.1ml of each overnight culture was then inoculated,in triplicate,into 50ml of pH-adjusted medium (Mueller^Hinton for E.coli and S.enteritidis and Brucella for C.jejuni )and these were incubated under the same conditions for each organism.1ml aliquots were removed at hourly intervals and used to prepare a dilution series,which was plated onto a suitable agar medium;MacConkey No.3(E.coli ),Brilliant green (S.enteritidis )or CBFS+CCDA supplement (C.jejuni ).Plates were incubated for up to 48h,when colonies were enumerated.

T a b l e 1P l a t e a s s a y i n h i b i t i o n o f E .c o l i ,C .j e j u n i a n d S .e n t e r i t i d i s b y s e l e c t e d p r o b i o t i c m i c r o o r g a n i s m s g r o w n i n b r o t h c u l t u r e w i t h a r a n g e o f c a r b o h y d r a t e s o u r c e s

L .p l a n t a r u m 0407

L .p e n t o s u s 905

L .a c i d o p h i l u s L a 5

L .r e u t e r i S D 2112

B .b i ￠d u m B b 12

C e l l s

S /N

N .S /N

C e l l s S /N

N .S /N

C e l l s

S /N

N .S /N

C e l l s

S /N

N .S /N

C e l l s

S /N

N .S /N

E .c o l i

F O S 2.0(1.4)8.0*(1.4)2.5(0.7)3.5(2.1)

5.0(0.0)4.0(1.4)^4.5(0.7)0.5(0.5)^^^0.5(0.0)1

6.0*(1.4)

7.5(0.7)I n u l i n 4.5(2.1)7.5*(3.5)5.0(1.4)^12.0*(0.0)2.5(0.7)

4.5(0.7)4.0(0.0)2.5(0.7)^0.5(0.5)4.0(0.0)

4.0(0.0)

5.5(0.7)1.5(0.7)X O S 5.5(0.7)9.5(0.7)0.5(0.7)^9.5*(0.7)^2.0(1.4)

5.5(0.7)0.5(0.5)

2.5(0.7)6.0(1.4)^^11.5*(0.7)^F O S :X O S 2.5(0.7)5.5(0.7)

3.5(3.5)0.5(0.5)

5.5*(2.1)2.0(0.0)^1.0(0.0)^1.5(0.7)

2.0(0.0)0.5(0.5)2.5(2.1)12.5*(

3.5)5.5(0.7)C .j e j u n i F O S 5.0(0.0)10.5(0.7)8.5(0.7)^8.0*(0.0)2.5(0.7)3.5(0.7)

4.5(2.1)1.5(2.1)

^5.5(0.7)

5.0(0.0)

8.5(0.7)13.0*(1.4)9.5(0.7)F O S :X O S 4.5(0.7)9.0(0.0)7.0(0.0)3.5(0.7)7.0(0.0)4.5(0.7)^5.5(0.7)^^^^5.5(0.7)15.5*(0.7)12.5(0.7)S .e n t e r i t i d i s F O S 4.0(0.0)6.5(0.7)2.5(0.7)2.5(0.7)3.5(0.7)3.0(1.4)3.5(0.7)6.0(1.4)3.0(0.0)1.0(0.7)2.5(0.7)0.5(0.0)6.0(0.0)6.0(1.4)2.5(1.4)I n u l i n 6.0(0.0)^1.0(0.0)3.0(1.4)^1.5(0.7)

4.5(0.7)4.0(1.4)1.5(1.4)

0.5(0.0)

6.0(1.4)1.5(0.7)1.5(0.7)

5.5(0.7)2.5(0.7)I n u l i n :F O S 3.0(0.0)

6.0(1.4)2.5(0.7)3.0(0.0)^^2.5(0.7)5.5(0.7)^^6.5(2.1)4.0(2.8)^4.5(0.7)2.0(0.0)X O S ^6.5*(3.5)3.0(1.4)

7.0(2.8)3.0(0.0)2.5(0.7)2.0(1.4)4.0(0.0)1.5(0.7)

2.5(0.7)7.5(0.7)4.5(0.7)^^^F O S :X O S 4.0

(0.0)

9.0*(2.8)

1.0(0.0)4.5

(0.7)

4.0(1.4)

3.0

(1.4)

0.5

(0.3)

3.5(0.7)

2.0

(1.4)

6.0

(1.4)

4.0

(1.4)

2.0

(0.0)

10.0*(4.2)

1.0(0.0)

C u l t u r e s w e r e f r a c t i o n e d i n t o c e l l s ,s u p e r n a t a n t (S /N )a n d n e u t r a l i s e d (p H 7.0)s u p e r n a t a n t (N .S /N ).R e s u l t s a r e d i a m e t e r o f t h e i n h i b i t i o n z o n e (m m )s u r r o u n d i n g t h e d i s c .R e s u l t s a r e c o r r e c t e d f o r t h e d i s c s i z e (5m m )f o r E .c o l i a n d S .e n t e r i t i d i s .V a l u e s i n p a r e n t h e s e s a r e S .E .M .o f n i n e d e t e r m i n a t i o n s .A s t e r i s k s d e n o t e s i g n i ￠c a n t l y g r e a t e r i n h i b i t o r y e ?e c t o f s u p e r n a t a n t c o m p a r e d t o c e l l s o r n e u t r a l -i s e d s u p e r n a t a n t .

L.J.Fooks,G.R.Gibson /FEMS Microbiology Ecology 39(2002)67^7569

2.9.Co-culture experiments

On the basis of disc/spot assay results,two of the pro-biotic strains,L.plantarum 0407and B.bi￠dum Bb12,were selected for further study because of their greater ability to inhibit pathogenic organisms.Overnight cultures (109cells ml 31)of each probiotic strain and each entero-pathogenic strain were prepared.MRS broth (10ml)was inoculated with 0.01g of lyophilised powder of each pro-biotic strain.The cultures were incubated overnight under anaerobic conditions (10:10:80,H 2:CO 2:N 2)at 373C.Mueller^Hinton broth (10ml)was inoculated with one cryogenic bead of either E.coli or S.enteritidis ,and in-cubated overnight under aerobic conditions at 373C.Bru-cella broth (50ml)supplemented with Campylobacter growth supplement (Oxoid)was inoculated with one cryo-genic bead of C.jejuni ,and incubated overnight under microaerobic conditions at 373C.Basal medium (50ml)was pre-reduced overnight and 0.5g (1%w/v)of the se-lected carbohydrate added.Carbohydrates used were:starch,FOS P95,XOS 35P,mixtures of inulin:FOS (80:20w/w)and FOS:XOS (50:50w/w).1ml of each overnight culture (V 108^109cells)was then inoculated,in triplicate,into 50ml carbohydrate-containing basal me-dium and cultures were incubated under anaerobic condi-tions.Agar plates (Oxoid;Beerens,1990)for the enumer-ation of each organism were prepared as appropriate.Growth media used were:Rogosa (L.plantarum ),Beerens (B.bi￠dum ),MacConkey No.3(E.coli ),CBFS+CCDA supplement (C.jejuni )and Brilliant green (S.enteritidis ).Samples were removed at 0,3,6,9and 24h.1ml was used to prepare a dilution series,which was then plated,in triplicate,onto the appropriate agar.For example,if L.plantarum 0407and E.coli 9517were co-cultured,the sample was plated onto Rogosa and MacConkey agar.Plates were incubated for up to 48h,under appropriate atmospheric conditions (L.plantarum and B.bi￠dum :an-aerobically,E.coli and S.enteritidis :aerobically,C.jejuni :microaerobically)and colonies enumerated.The pH of the culture medium was monitored throughout the fermenta-tion period.Each experiment was repeated in triplicate.A further 1ml sample was also removed for the analysis of SCFAs (lactate and acetate),according to the method de-scribed by Parham and Gibson [12].

2.10.Statistical analyses

Data were statistically analysed using pairwise Student's t -test to evaluate signi￠cance of inhibition observed (disc/spot assays)or changes in bacterial numbers (co-culture experiments).3.Results 3.1.Plate assays

Basal medium gave no inhibition of the enteropatho-gens tested.Acetate and lactate solutions gave inhibition zones of 10mm (t1.8mm)and 7mm (t0.6mm)https://www.360docs.net/doc/179923372.html,e of lactulose,lactitol,starch and dextran as carbohydrate sources was generally ine?ective in inducing any inhibitory e?ects from the probiotic strains.

L.plantarum 0407and L.pentosus 905,combined with FOS,inulin,XOS,and mixtures of inulin:FOS and FOS:XOS,were e?ective in inhibiting growth of E.coli and S.enteritidis (Table 1).The cell supernatant (cell-free ex-tract)consistently conferred a signi￠cantly greater inhibi-tory e?ect than either the cells (P 60.01)or neutralised supernatant (P 60.05)fractions.FOS and FOS:XOS were the only carbohydrate sources where inhibition of C.jejuni was observed with L.plantarum ,whilst

with

Fig.1.Growth rates (h 31)of probiotic and enteropathogenic bacteria utilising various carbohydrate sources.In,Inulin;In:FOS,mixture 80:20w/w;FOS:XOS,mixture 50:50w/w

Table 2

pH values of supernatant fraction of the overnight cultures of probiotics grown with various carbohydrate sources Probiotic strain FOS In In:FOS XOS FOS:XOS Lactulose Lactitol Starch Dextran L.plantarum 0407 4.29(t0.05) 4.52(t0.04) 5.00(t0.03) 4.92(t0.02) 4.78(t0.03) 5.98(t0.04) 6.55(t0.05) 6.56(t0.04) 6.73(t0.07)L.pentosus 905 4.35(t0.08) 4.77(t0.05) 5.02(t0.03) 4.83(t0.09) 4.72(t0.05) 6.14(t0.05) 6.39(t0.04) 6.54(t0.06) 6.84(t0.03)L.acidophilus La5 4.31(t0.06) 5.10(t0.02) 5.38(t0.04) 5.15(t0.03) 4.63(t0.07) 6.27(t0.04) 6.09(t0.05) 6.33(t0.08) 6.41(t0.05)L.reuteri SD2112 4.54(t0.03) 5.26(t0.04) 5.24(t0.08) 5.85(t0.06) 4.89(t0.05) 6.23(t0.07) 6.86(t0.02) 6.78(t0.04) 6.67(t0.04)B.

bi￠dum Bb12

4.27

(t0.02)

4.49

(t0.06)

5.72

(t0.06)

4.47

(t0.03)

4.17

(t0.03)

6.08

(t0.03)

6.11

(t0.04)

6.05

(t0.01)

6.18

(t0.03)

Values in parentheses are average tS.E.M.of triplicate determinations.In:Inulin;In:FOS,mixture 80:20w/w;FOS:XOS,mixture 50:50w/w.

L.J.Fooks,G.R.Gibson /FEMS Microbiology Ecology 39(2002)67^75

L.pentosus ,the most e?ective carbohydrate sources were FOS,inulin,XOS and mixtures thereof.Inhibition was signi￠cant for FOS (P 60.01)and FOS:XOS (P 60.05).The improved antimicrobial e¤cacy of the probiotic with these carbohydrate sources was repeated with L.acidophi-lus La5and L.reuteri SD2112although the magnitude of inhibition was less than that of the other two lactobacilli.In contrast,B.bi￠dum Bb12was most e?ective,combina-tions with FOS,inulin,XOS and related mixtures impart-ing high inhibition levels against all three enteropathogens.Again,the cell-free fraction of the Bb12culture was most e?ective at inhibiting pathogen growth,which was signi￠-cant compared to the other cell fractions (P 60.05).

The pH of the cell-free extract was recorded for each probiotic strain grown with the various carbohydrates (Table 2).pH was approximately 7.0prior to inoculation.In general,the largest decrease in pH was observed when FOS and the mixture of FOS:XOS (50:50)was used as the

carbohydrate source,with the variation being strain-de-pendent.

3.2.Probiotic growth curves

Highest growth rates of the lactobacilli were obtained in media that contained glucose as the carbon and energy source.For the bi￠dobacterial strain,the mixture of FOS:XOS gave optimal growth.L.plantarum 0407and B.bi￠dum Bb12generally grew faster than the other strains,regardless of the carbohydrate source used.In general,the oligosaccharides FOS,XOS or their mixtures were fermented preferably to all other carbohydrate sour-ces (Fig.1).

3.3.Enteropathogen growth curves

Each of the three enteropathogens was tested for

its

Fig.2.Survival of (a)E.coli ,(b)C.jejuni and (c)S.enteritidis at di?erent initial culture pH values.Samples were removed at hourly intervals and enumerated using on selected agars.Data are averaged (tS.E.M.)from triplicate determinations.

L.J.Fooks,G.R.Gibson /FEMS Microbiology Ecology 39(2002)67^7571

ability to grow with various carbohydrate sources (Fig.1).E.coli grew well on glucose.S.enteritidis also utilised glucose e?ectively,although growth rate was approxi-mately half that observed for E.coli .C.jejuni failed to grow on any of the carbohydrates.The e?ect of pH on growth of the pathogens was also investigated.Initial me-dium pH of 3or below completely inhibited growth of E.coli (Fig.2a)and of C.jejuni (Fig.2b)and S.enteritidis (Fig.2c)at initial pH values of 4and below.3.4.Co-culture investigation

Both L.plantarum 0407(Table 3)and B.bi￠dum Bb12(Table 4)survived in all culture conditions tested such that,generally,their numbers after 24h fermentation were maintained.B.bi￠dum numbers were enhanced by 1-log value in the presence of FOS and up to 2logs when a FOS:XOS (50:50)mixture was used as the carbo-hydrate source.An increase in probiotic numbers after 24h fermentation was observed when both L.plantarum and B.bi￠dum were grown with C.jejuni ,again regardless of the carbohydrate source used.

L.plantarum combined with FOS was the most e?ective at inhibiting pathogen growth (Table 3).A signi￠cant,6-log (P 60.01)decrease in E.coli numbers was observed when FOS was used,whilst after the same time period, C.jejuni and S.enteritidis were undetectable (P 60.001).B.bi￠dum combined with FOS:XOS proved an e?ective synbiotic combination (Table 4).C.jejuni and S.enteritidis were decreased to below detectable levels (P 60.001),whilst a 2-log decrease in E.coli numbers was ob-served.

Changes in pH during the 24-h fermentation were ob-served but were not signi￠cant for either L.plantarum 0407(Table 3)or B.bi￠dum Bb12(Table 4).No de￠nitive correlation could be made between the observed decrease in pH and the extent of inhibition.Culture pH decrease was generally observed after fermentation for 3h,yet a lowering of pathogen numbers was not observed until fer-mentation for 9^24h.Furthermore,when B.bi￠dum Bb12was co-cultured with E.coli in the presence of XOS,a pH decrease was observed,even though there was no evidence of antimicrobial activity.

With L.plantarum ,the ￠nal concentration of lactate varied depending on the carbohydrate source provided,ranging from 12^17mM with starch and inulin:FOS,to 20^25mM with FOS and FOS:XOS (Table 3).For B.bi￠dum ,acetate concentrations were consistently higher than levels of lactate by the end of the fermentation period (Table 4).Both FOS and FOS:XOS gave rise to higher levels of acetate,reaching approximately 20^30mM 24h after inoculation.

Table 3

Inhibition of enteropathogens by probiotic L.plantarum 0407in co-culture experiments

Carbohydrate source

Change in numbers,pH or concentration (0-24h)Starch

FOS

Inulin:FOS

FOS:XOS

XOS

E.coli

Probiotic numbers +1.62U 109(t1.33U 109)+2.14U 109(t2.00U 109)+1.35U 1010(t2.83U 109)+1.40U 108(t2.02U 107)+9.40U 108(t2.11U 109)Pathogen numbers +4.50U 109(t4.33U 108)38.25U 108**(t2.83U 107)+3.27U 109(t1.31U 109)32.37U 107(t6.66U 107)32.25U 108(t1.98U 107)Culture pH +0.03(t0.04)31.15(t0.06)31.02(t0.03)31.00(t0.24)30.94(t0.22)[Acetate]mM +2.32(t0.33)+2.24(t0.18)+2.35(t0.47)+1.23(t0.04)+1.20(t0.09)[Lactate]mM +16.64(t2.12)

+25.38(t0.55)

+9.77(t2.02)

+23.06(t0.51)

+17.62(t0.75)

C.jejuni

Probiotic numbers +9.98U 1012(t0.00)+1.00U 1013(t0.00)

+1.00U 1013(t0.00)

+1.00U 1013(t0.00)+1.00U 1013(t0.00)Pathogen numbers 31.29U 107(t1.78U 105)32.09U 109***(t0.00)31.74U 109(t0.00)31.30U 109(t0.00)31.89U 109(t0.00)Culture pH 30.41(t.021)32.20(t0.06)31.99(t0.14)32.33(t0.25)32.13(t0.18)[Acetate]mM +2.73(t0.33)+2.26(t0.13)+2.27(t0.11)+1.87(t0.30)+1.92(t0.05)[Lactate]mM +17.39(t2.86)+20.17(t0.41)+11.37(t1.12)+21.27(t0.89)+20.68(t1.29)S.enteritidis

Probiotic numbers +1.21U 1010(t7.36U 109)+2.15U 1010(t1.70U 1010)+2.20U 1010(t1.48U 1010)+1.76U 108(t5.65U 107)+6.10U 109(t9.19U 108)Pathogen numbers +7.42U 1010(t8.08U 109)37.25U 1010***(t0.00)

38.08U 108(t2.09U 102)32.68U 107(t1.04U 107)37.15U 106(t2.26U 106)Culture pH 30.35(t0.04)+1.79(t0.04)31.55(t0.04)31.87(t0.08)31.43(t0.03)[Acetate]mM +2.58(t0.43)+0.94(t0.10)+1.52(t0.22)+2.04(t0.16)+1.81(t0.05)[Lactate]mM

+11.55(t5.36)

+20.89(t0.70)

+11.06(t1.62)

+20.74(t0.38)

+13.46(t0.33)

Data presented are changes in parameter between 0and 24h inoculation.Probiotic and enteropathogen numbers were enumerated at various time points after inoculation.Results are cfu ml 31(tS.E.M.)averaged from six determinations.For culture pH and acetate and lactate concentrations (mM),1-ml samples were removed at speci￠ed intervals and parameters determined.Results are average (tS.E.M.)of triplicate determinations.+and 3denote an increase or decrease respectively of the described parameter.A signi￠cant decrease of pathogen numbers from baseline (0h fermentation)is denoted;**P 60.01,***P 60.001.

L.J.Fooks,G.R.Gibson /FEMS Microbiology Ecology 39(2002)67^75

4.Discussion

All of the probiotic strains tested using plate assays o?ered some inhibition of each of the pathogenic strains, E.coli,S.enteritidis and C.jejuni.The extent of inhibition was dependent on the probiotic strain,such that L.plan-tarum0407and B.bi￠dum Bb12tended to inhibit patho-gen growth to a greater extent than that observed for the other strains included,particularly with E.coli.These re-sults,in this respect,compound the￠ndings of a number of investigations,which used similar pure culture in vitro methodologies to establish the antimicrobial potential of lactobacilli and bi￠dobacteria.Jacobsen et al.[13]used the spot assay to examine the capabilities of47strains of Lactobacillus spp.to inhibit a range of pathogenic organ-isms,including E.coli and Salmonella typhimurium. Twenty eight of the strains tested inhibited E.coli growth, whilst12of the47strains tested inhibited growth of S.typhimurium.A plethora of information surrounds the proposed probiotic activity of L.reuteri[14],primarily ascribed to production of an antimicrobial protein called reuterin[15,16],whose synthesis requires glycerol in the growth medium[17].This was omitted from media used in the current investigation,since it is not known how much glycerol would be present in the gut,to become available for utilisation by L.reuteri,in the production of reuterin[14].Six B.bi￠dum strains,tested using well di?usion assays,inhibited growth of E.coli and to a lesser extent,Salmonella typhosa[18,19].Gibson and Wang[20] reported the strain-dependent ability of eight di?erent spe-cies of bi￠dobacteria,including B.bi￠dum,to inhibit the growth of a range of pathogenic bacteria,including E.coli, a Salmonella spp.,and a Campylobacter spp.

The antimicrobial potential exhibited by each of the probiotics used here appeared to depend on the carbohy-drate source used.FOS,inulin,XOS,and mixtures of FOS:XOS(50:50w/w)and inulin:FOS(80:20w/w)all caused greater inhibition than lactulose,lactitol,starch and dextran,perhaps suggesting a structure-to-function relationship in terms of the prebiotic used.The type of bond linking the component monomers,in view of speci￠c cleavage enzymes being required for fermentation of the carbohydrate,may e?ect fermentation rate,and thereby determine the speed at which potential inhibitory metabol-ic end products are released.Chain length of the carbohy-drate is also likely to be a contributory factor,since long chain oligosaccharides,with multiple branching,require more enzymatic hydrolysis by the organisms before its complete fermentation.

The major metabolic end products of lactobacilli and bi￠dobacterial fermentations are acetate and lactate[21], leading to a decrease in the culture pH.In both plate assay and co-culture experiments,the pH of the cell-free extract was lower when FOS,inulin and XOS,and mixtures there-

Table4

Inhibition of enteropathogens by probiotic B.bi￠dum Bb12in co-culture experiments

Change in numbers,pH or concentration(0^24h)

Carbohydrate source

Starch FOS Inulin:FOS FOS:XOS XOS

E.coli Probiotic

numbers +1.69U107

(t5.99U107)

+1.89U109

(t1.20U109)

+5.27U107

(t3.09U107)

+1.26U109

(t2.18U108)

+2.02U108

(t6.78U107)

Pathogen numbers 32.56U108

(t2.61U108)

31.92U107

(t6.46U106)

34.80U107

(t4.89U107)

31.25U106

(t6.13U105)

36.68U107

(t4.08U107)

Culture pH+0.16(t0.13)31.46(t0.09)31.02(t0.52)30.77(t0.12)30.97(t0.21) [Acetate]mM+14.02(t0.57)+28.39(t1.32)+22.70(t1.07)+14.63(t0.47)+11.15(t1.18) [Lactate]mM+9.05(t1.34)+12.64(t0.72)+14.52(t0.53)+29.11(t2.38)+18.29(t1.40)

C.jejuni Probiotic

numbers +1.00U1013

(t1.11U105)

+1.00U1013(t0.00)+1.00U1013(t0.00)+1.00U1013(t0.00)+1.00U1013(t0.00)

Pathogen numbers 31.26U108

(t4.92U107)

32.29U108**

(t1.24U109)

32.38U108**(t0.00)32.77U108***(t0.00)32.07U108

(t5.03U108)

Culture pH30.49(t0.73)32.20(t0.18)32.06(t0.19)32.43(t0.26)31.97(t0.51) [Acetate]mM+10.82(t1.85)+25.82(t0.97)+21.71(t2.05)+26.19(t1.04)+15.19(t2.49) [Lactate]mM+10.93(t0.92)+12.80(t1.92)+14.23(t2.00)+16.50(t1.28)+11.01(t1.27)

S.enteritidis Probiotic

numbers +3.68U108(t0.00)+1.42U109

(t3.91U108)

+8.84U107

(t5.95U107)

+8.75U108

(t2.07U108)

+1.94U107

(t9.00U106)

Pathogen numbers 31.66U108

(t1.11U105)

31.36U109(t0.00)33.19U107

(t1.99U107)

31.70U109***(t0.00)33.05U108

(t2.10U108)

Culture pH+0.09(t0.16)31.59(t0.08)31.38(t0.64)31.37(t0.13)30.96(t0.12)

[Acetate]mM+10.65(t1.25)+21.55(t1.97)+22.98(t1.23)+26.26(t3.03)+12.81(t3.98)

[Lactate]mM+11.40(t1.50)+13.08(t0.25)+17.12(t2.25)+17.45(t3.20)+11.49(t1.62)

Data presented are changes in parameter between0and24h inoculation.Probiotic and enteropathogen numbers were enumerated at various time points after inoculation.Results are cfu ml31(tS.E.M.)averaged from six determinations.For culture pH and acetate and lactate concentrations (mM),1-ml samples were removed at speci￠ed intervals and parameters determined.Results are average(tS.E.M.)of triplicate determinations.+and 3denote an increase or decrease respectively of the described parameter.A signi￠cant decrease of pathogen numbers from baseline(0h fermentation) is denoted;**P60.01,***P60.001.

L.J.Fooks,G.R.Gibson/FEMS Microbiology Ecology39(2002)67^7573

of were provided as the carbohydrate sources.The extent of inhibition with these prebiotic carbohydrates was often correspondingly increased,suggesting that a possible mechanism of antimicrobial action may be attributable to the low culture pH.However,since some results contra-dicted this￠nding,it cannot be de￠nitively stated that low pH acts as the principal inhibitory parameter.The en-hanced antimicrobial e?ect of FOS and FOS:XOS may be attributed to the action of the synbiotic since numbers of the pathogen decreased in co-cultures,but not in mono-cultures of the pathogen.Con￠rmatory evidence of the inability of E.coli,C.jejuni and S.enteritidis to withstand an acidic environment was obtained(Fig.2)where these bacteria failed to proliferate at the pH environment (pH95.0)normally induced by bi￠dobacterial and lacto-bacilli fermentation.Similar￠ndings have been observed for B.bi￠dum1452[19],an L.plantarum strain[22]and a bi￠dobacterial strain B.longum[23],where a decrease in pH during incubation time increased the antagonistic properties against E.coli,Staphylococcus aureus,Klebsiella pneumoniae,and a number of clostridia and bacteroides species,including Clostridium perfringens and Bacteroides fragilis.

The toxicity of fermentation acids at a low pH has been traditionally explained by the transmembrane￡ux of un-dissociated acids,dissociation of the acids in the more alkaline cytoplasm,and metabolic uncoupling[24].How-ever,Russell and Diez-Gonzalez[25]suggested that the mechanistic explanation lies with the pH gradient-medi-ated anion accumulation within the bacterial cytoplasm. Fermentation acid dissociation in the more alkaline inte-rior causes an accumulation of the anionic species,and this accumulation is dependent on a pH gradient across the membrane.Where culture pH of the cell-free extract decreased to pH4.17,when short chain carbohydrates had been utilised,this would theoretically induce a consider-able pH gradient across the pathogenic organism mem-brane,thus allowing accumulation of the fermentation acids in the cytoplasm.Furthermore,since the cell-free extract promoted the greatest bactericidal e?ect,it can be assumed that the fermentation acids accumulated in this fraction.

In conclusion,this study has shown that lactobacilli and bi￠dobacteria species can inhibit some important patho-genic species.This antagonism was in￡uenced by the car-bohydrate provided in vitro.The inhibitory mechanism underlying the e?ect has been initially addressed,and a strong case has been presented for the production of SCFA as the underlying mechanism of inhibition of enter-opathogens.

Acknowledgements

L.J.F.was a recipient of a PhD studentship from St. Ivel European Food,Wootton Bassett,UK.References

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