Antimicrobial Activity and Chemical Composition of Kpètè...

Research ArticleAntimicrobial Activity and ChemicalComposition of (Kpegravetegrave-Kpegravetegrave) A Starter ofBenin Traditional Beer Tchoukoutou

Christine Nrsquotcha1 Haziz Sina1 Adeacutechola Pierre Polycarpe Kayodeacute2

Joachim D Gbenou3 and Lamine Baba-Moussa1

1Laboratoire de Biologie et de Typage Moleculaire en Microbiologie Universite drsquoAbomey-Calavi 05 BP 1604 Cotonou Benin2Laboratoire de Valorisation et de Gestion de la Qualite de Bioingredient Alimentaire Universite drsquoAbomey-Calavi01 BP 526 Cotonou Benin3Laboratoire de Pharmacognosie et des Huiles Essentielles FSS Universite drsquoAbomey-Calavi 01 BP 4521 Cotonou Benin

Correspondence should be addressed to Lamine Baba-Moussa laminesaidyahoofr

Received 3 August 2016 Accepted 6 February 2017 Published 6 March 2017

Academic Editor Gail B Mahady

Copyright copy 2017 Christine Nrsquotcha et alThis is an open access article distributed under theCreativeCommonsAttributionLicensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The aim of this study was to investigate the antibacterial effect of the crude starter ldquokpete-kpeterdquo and lactic acid bacteria usedduring the production of ldquotchoukoutourdquo To achieve this a total of 11 lactic acid bacteria and 40 starter samples were collectedfrom four communesThe samples were tested on 29 gram + and minus strains by disk diffusion methodTheminimum inhibitory andbactericidal concentrations of starter and lactic acid bacteria were determined by conventional methods Organic acids sugar andvolatile compounds were determined using the HPLC method The ldquokpete-kpeterdquo displays a high antibacterial activity against thetested strains The most sensitive strain was S epidermidis (125mm) whereas the resistance strain was Proteus mirabilis (8mm)All the tested ferment has not any inhibitory effect on Enterococcus faecalis The lactic acid bacteria isolates of Parakou showed thehighest (1748mm) antibacterial activity whereas the smallest diameter was obtained with the ferment collected from Boukoumbe(980mm)The startersrsquo chemical screening revealed the presence of tannins anthocyanin flavonoids triterpenes steroids reducingcompounds and mucilage O-glycosides These compounds are probably the source of recorded inhibition effect The lactic acidbacteria of the ldquokpete-kpeterdquo could be used to develop a food ingredient with probiotic property

1 Introduction

Major scientific advances have been done in several areas ofhealth sciences for many years mainly in the development ofantimicrobial agents Moreover pathogenic species such asEscherichia coli Salmonella sppVibrio anguillarumV algin-olyticus Aeromonas hydrophila A salmonicida and Yersiniaruckeri were isolated from gastrointestinal tract (stomachjejunum and caecum) samples of poultry rabbit fish andpork [1] However humans and animals deaths due tomicrobial infections (acute respiratory infections hospitalacquired diarrhea cancer andmalaria) are reported [2] Oneof the main reasons of this situation can be the increasingantibiotic resistance observed over decades Indeed theappearance of antibacterial resistance considerably reduced

the efficacy of infections treatments So pharmaceuticalindustries find difficult to follow the resistance rate andthen develop new effective antimicrobial agents against resis-tant pathogens [3] Thus many multiresistant bacteria arereported in food and clinical samples collected in Benin[4] Cote drsquoIvoire [5ndash8] and other countries in sub-SaharanAfrica [9]

To face this public health problem it is necessary to inves-tigate new method that can help to strengthen conventionalantibiotics through the investigation of new antimicrobialmolecules Thus several paths can be explored includingindigenous knowledge through traditional medicine In spiteof its lack of scientific certification the traditional medicineis known to be efficient to cure several diseases [10] Amongthe traditional knowledge the use of fermented beverage

HindawiBioMed Research InternationalVolume 2017 Article ID 6582038 10 pageshttpsdoiorg10115520176582038

2 BioMed Research International

(particularly their lactic bacteria content) to overcome somebacterial infections [11 12] and food conservation [13] iscurrent So the use of lactic acid bacteria could be consideredas an alternative to control resistant pathogenic bacterialstrains

Several studies shows that some lactic acid bacteriasuch as Lactobacillus isolated from fermented foods haveantimicrobial activity [14 15] Up to date the principalmicroorganisms reported to have probiotic properties arebacteria (Lactobacillus Bifidobacterium PropionibacteriumEnterococcus etc) and yeast (Saccharomyces Candida etc)[16]Thesemicroorganisms are even used for the formulationof new probiotic products [17]

In Benin the starter of the most popular traditionalsorghum beer named ldquotchoukoutourdquo is reported to be usedto cure several infections such as stomach pains and diarrhea[12] However a part of the preliminary study conducted in2012 by Kayode et al [18] showed that there is no scientificevidence to support the traditional use of the tchoukoutoursquosstarter named ldquokpete-kpeterdquo in infection treatments Thusthe aim of the present study was to investigate the antimi-crobial activity of starter ldquokpete-kpeterdquo and it isolated lacticacid bacteria not only on some reference microorganismsrsquostrains but also on some food and clinical isolated pathogenicbacteria strains

2 Material and Methods

21 Kpete-Kpete Sample Collection The starters used for thestudywere the ones used in the fermentation of the traditionalbeer ldquotchoukoutourdquo and locally called ldquokpete-kpeterdquo Sampleswere collected in four communes selected from the twodepartments (Atacora and Borgou) displaying the highestproduction and consumption level of the ldquotchoukoutourdquoThe 4 communes are Natitingou (10∘1810158402410158401015840N 1∘2210158403010158401015840E)Boukoumbe (10∘1810158402410158401015840N 1∘2210158403010158401015840E) Tanguieta (10∘3710158401110158401015840N1∘1510158405210158401015840E) the department of Atacora (North-West Benin)and Parakou (9∘2110158400010158401015840N 2∘3710158400010158401015840E) in the department ofBorgou (Northern-East) In each commune 10 samples oftchoukoutoursquos starter were collected for laboratory analysisThus a total of 40 samples were collected and tested for thisstudy

22 Tested Strains The stains used for the antimicrobialtest were composed of 12 references strains (Staphylococcusaureus ATCC 29213 S epidermidis T22695 Proteus mirabilisA24974 P vulgaris A25015Micrococcus luteus Streptococcusoralis Enterococcus faecalis ATCC 29212 Escherichia coliATCC25922E coliO157H7ATTC700728 Salmonella typhiR 30951401 Candida albicans MHMR and PseudomonasaeruginosaATCC 27853) 15 clinical and food isolated methi-cillin resistance S aureus (MRSA) 2 extended-spectrumbeta-lactamase E coli strains and 11 lactic acid bacteriaisolated previously from ldquotchoukoutourdquo [12]

23 In Vitro Determination of ldquoKpete-Kpeterdquo AntimicrobialActivity The antimicrobial activity of the collected starterswas evaluated by disk diffusion method [19] Briefly 100 120583Lof 106 CFUmL microbial suspension was used to inoculate

Mueller-Hinton agar by flooding (Bio-Rad France) [20]Sterile paper discs impregnated with 30120583L of startersrsquo super-natant (20mgmL) were then deposited on the mediumFor each test the experiment is duplicated and a negativecontrol was performed using sterile distilled water Plateswere then left for 15ndash30min at room temperature before beingincubated at 37∘C for 24 to 48 h Diameters of inhibitionzones were then measured using a sliding graduated scaleafter incubation times (24 h and 48 h)

24 Antibacterial Activity of Lactic Acid Bacteria Strains

241 Bacteriocins Extraction The method describe bySavadogo et al [21] was used to extract the bacteriocinproduced by the lactic acid bacteria Briefly each lactic acidbacteria strains were previously mixed in 1000mL of MRSbroth (pH 70) For the bacteriocin extraction a cell-free solu-tion was obtained by centrifuging (10000 rpm for 20minat 4∘C) the culture Thus the obtained cell-free solutionwas precipitated with ammonium sulphate (40 saturation)for 2 h at 4∘C Cells were harvested by centrifugation at20000 rpm for an hour at 4∘C and resuspended in 005Mpotassium phosphate buffer (25mL pH 70) for further diskdiffusion assay

242 Determination of Bacteriocin Activity The bacteriocinactivity was determined by disk diffusion assay [22] For thisassay the aliquot of each bacteriocin (50 120583L) was used toimpregnate disk Once impregnated the disks were lodgedon Mueller-Hinton agar dishes containing 5 sdot 105 CFUmLof tested pathogenic strains (E coli O157 H7 ATCC 700728Salmonella typhi R 30951401 E coli S aureus ATCC 29213and 2 clinical isolated MRSA) The dishes were then incu-bated at 30∘C or 37∘C for 24 h Diameters of inhibition zoneswere then measured using a sliding graduated scale [23]

25 Sensitivity of Antagonistic Substances of Lactic AcidBacteria to Enzymes The sensitivity of antimicrobial sub-stances to enzymes was tested according to the methodpreviously described by Savadogo et al [21] Briefly the cell-free supernatants of 13 lactic acid bacteria isolates that showedantimicrobial activity against reference microorganism werecollected after centrifugation (7500timesg for 10min at 4∘C)ThepHof the supernatants was adjusted to 6with 10NNaOHandtreated with 02mgmL of specific enzymes (Sigma-AldrichBrasil Sao Paulo Brazil) The used enzymes were lipase(86Umg in 005M Tris hydrochloride pH 80 and 001MCaCl2) chymotrypsin (47Umg in 005MTris hydrochloridepH 80 and 001M CaCl2) type x trypsin (15000Umg in02M citrate pH 60) pepsin (32 UmL in 02M citrate pH60) and catalase (20Umg in 10mM potassium phosphatepH 70)The samples of bacteriocins (500 120583L) were incubatedwith the appropriate enzymes (500120583L) for 60min at 37∘C or25∘C (for trypsin chymotrypsin and catalase) The negativecontrols contain supernatant solutions and 01M sodiumphosphate buffer without enzymes

26 Determination of the Startersrsquo Minimum Inhibitory Con-centrations (MIC) The minimum inhibitory concentrations

BioMed Research International 3

of starter were determined by macrodilution method [24]Concentrations tested were 10mgmL 5mgmL 25mgmL125mgmL 0625mgmL 03125mgmL 015625mgmL007812mgmL and 003906mgmL Culture medium with-out starters and without microorganisms was used as con-trols Thus after gently mix tubes were incubated at 37∘Cfor 18ndash24 h and growth was indicated by turbidityThe lowestconcentration of the starter at which the tested microorgan-ism does not demonstrate visible growth was considered asminimum inhibitory concentrations

27 Determination of Lactic Acid Bacteriarsquos MinimumInhibitory Concentrations (MIC) against E coli ATCC25922For the determination of the MIC the crude supernatantobtained after centrifugation (12000119892 for 10min) of the lacticacid bacteria precultures was consecutively 2-ratio factordiluted up to 512 Antibacterial activities were subsequentlycarried out with the different concentration of extract ofbacteriocinThus the lowest dilution which showed a positiveantibacterial activity against the tested reference strain wasconsidered as the minimum inhibitory concentration

28 Determination of the Startersrsquo Minimum BactericidalConcentration (MBC) The minimum bactericidal concen-tration of the tested microorganisms was determined bysubculturingmethodThus the content of each test tube usedin the minimum inhibitory concentration assay that did notshow microorganism growth after incubation was streakedon a solid nutrient agar plate and then incubated at 37∘Cfor 24 h The starterrsquos lowest concentration without bacterialgrowth was identified and taken as Minimum BactericidalConcentration [25]

29 Determination of the Startersrsquo Contents in Organic AcidsSugar and Volatile Compounds Organic acids sugar andvolatile compounds were determined using the high per-formance liquid chromatography (HPLC) method [26] Anamount (25mg) of each fermented starter samples wasmixed with 1mL of 5mM H2SO4 in screw-capped tubesThe mixtures were centrifuged (12000119892 for 10min) and thesupernatants filtered with 045 120583m filter The separation oforganic acids and the volatile compounds was achievedwith an Aminex HPX-87H HPLC column (Bio-Rad LabsRichmond Calif USA) at 45∘C using 5mM H2SO4 as amobile phase and the externally calibrated with standardsolutions The organic acids and volatile compound wereidentified and quantified by comparison of their retentiontimes with these standard acids

The content of a compound (mg100 g of dry matter) =(SRe times 119865 times 10000)(CRs times PE times MS) SRe is the responsesurface of the sugar in the sample 119865 is the dilution factorCRs is the standard response factor and PE peak areaconcentration (mgmL)

210 Startersrsquo Chemical Screening Chemical screening of theldquokpete-kpeterdquo was carried out using themethod of Houghtonand Raman [27]

E co

0

S ty

Psa

e

S a

u

S ep

Pr m

i

M l

u

S o

r

E co

En f

a

Pr v

u

C a

l

0

5

10

15

References strains

Inhi

bito

ry d

iam

eter

(mm

)

24 H48H

Figure 1 Medium inhibitory diameter of tchoukoutoursquos starter onreference strains after 24 h and 48 h E co 0 Escherichia coli O157S ty Salmonella typhimurium Ps ae Pseudomonas aeruginosa Sau Staphylococcus aureus S ep Staphylococcus epidermidis Pr miProteusmirabilisM luMicrococcus luteus S or Streptococcus oralisE co Escherichia coli En fa Enterococcus faecalis ATCC 29212 Prvu Proteus vulgaris C al Candida albicans

211 Data Analysis Statistical analysis of antibacterial datawas done with Excel 2010 and Graph Pad Prism 5 Thechemical compounds data were analyzed using SAS (SASInstitute Cary NC USA) to determine the significant dif-ference between the various treatments The mean differencewas determined by Student Newman-Keuls test (119901 lt 005)For normal distributions the rate of organic acids sugars andvolatile compound obtained was processed with 2 arcsin radic119899[28] where 119899 is the actual value

3 Results

31 Inhibition of Reference Strains by the Starter ldquoKpete-Kpeterdquo Tested The collected ldquokpete-kpeterdquo samples showedan antibacterial effect on some reference strainsThemajorityof the collected ferment in the four localities exhibitedantibacterial effect on 9167 (1112) of the reference strainsThe only one tested reference strain that was not inhibitedby the starter was Enterococcus faecalis Figure 1 shows thecompilation of inhibition diameter measured after 24 and 48hours of incubation Considering the sensitive strains themean inhibitory diameter zones vary from 8mm (Proteusmirabilis) to 1250mm (Staphylococcus epidermidis) Our datashows that there was not a significant variation betweendiameter recorded at 24 h and those of 48 h (119901 gt 005)

32 Antimicrobial Activity of ldquoKpete-Kpeterdquo on Some Path-ogenic Bacteria The antibacterial activities of ldquokpete-kpeterdquocollected from four towns on some pathogenic food andclinical isolated bacteria are compiled on Figure 2 Thuswe observed that clinical isolated MRSA are sensitive tothe tested ldquokpete-kpeterdquo independently from their collectionarea (Figure 2(a)) This sensitivity was higher with the


Tan Nat Bou ParTown

0

5

10

15In

hibi

tory

dia

met

er (m

m)

24 H48H

(a) Clinical isolated MRSA

Tan Nat Bou Par0

5

10

Town

Inhi

bito

ry d

iam

eter

(mm

)

24 H48H

(b) Food isolated MRSA

Tan Nat Bou Par

Clinical isolated MRSAFood isolated MRSA

Town

0

5

10

Inhi

bito

ry d

iam

eter

(mm

) lowastlowastlowastlowast

(c) Food isolated MRSA versus clinical MRSA

Tan Nat Bou Par0

5

10

15

Town

Inhi

bito

ry d

iam

eter

(mm

)

24 H48H

(d) Clinical isolated ESBL EC

Figure 2 Mean inhibition zonesrsquo diameter of starter on some isolated pathogenic strains after incubation (24 and 48 h) Tan Tanguieta NatNatitingou Bou Boukoumbe Par Parakou MRSA Methicillin Resistant S aureus and ESBL EC extended-spectrum beta-lactamases Ecoli lowast 119901 lt 005

starter collected at Parakou (1140mm) and less with thosefrom Tanguieta (1075mm) However considering the startercollection area the recorded difference from a starter toanother is not significant (119901 gt 005) Our data shows thatthe food isolated MRSA are more sensitive to ldquokpete-kpeterdquocollected at Parakou (989mm) (Figure 2(b)) The recordedinhibition diameters of ldquokpete-kpeterdquo on pathogenic bacteriadid not vary over the incubation time Globally the clinicalMRSA are more sensitive to ldquokpete-kpeterdquo than food isolatedMRSA (119901 lt 005) (Figure 2(c))

The clinical isolated extended-spectrum beta-lactamasesE coli tested strains are sensitive to ldquokpete-kpeterdquo indepen-dently from their origin (Figure 2(d)) The inhibitory diam-eter zones of the starter tested vary slightly from the starterscollected at Boukoumbe (1050mm) to those of Natitingou(12mm) from a starter to another but the difference is notstatistically significant (119901 gt 005) The recorded diametersdid not vary over the time

33 Antibacterial Activity of Lactic Acid Bacteria Isolates onPathogenic Strains Theantibacterial activity of the lactic acidbacteria isolates showed that all pathogenic strains tested(66) are sensitive to the isolates independently from theircollection area (Figure 3) It was observed that the inhibition

diameters zones vary according not only to the pathogensstrains but also to the lactic acid bacteria isolates The resultsalso showed that there was no significant difference betweenthe isolates inhibition diameters in the time (119901 gt 005) Ecoli (1675mm) and Salmonella typhi are the most sensitive(1543mm) strains to lactic acid bacteria isolates The lowerdiameters were obtained with clinical isolated MRSA strains(1318mm) and E coli O157 strain (12mm)

34 Sensitivity of Antagonistic Substances to Enzymes Theantibacterial compounds identified have no effect on threeproteolytic enzymes (chymotrypsin trypsin and pepsin)indicating that the inhibitory compounds are protein ageneral characteristic of bacteriocin (Table 1) There was notinhibition zone of the bacteriocin extracts in presence ofvarious proteolytic enzymes However in the presence ofamylase lipase and catalase we noticed a zone of inhibition

35 Minimum Inhibitory and Bacterial Concentrations ofStarter on Some References and Pathogenic Strains TheMICand MBC vary depending on the types of strains andstarter (Table 2) However there was no significant differencebetween these different concentrations So the MIC variesfrom 03125mgmL (clinical MRSA with starter collected


Table 1 The inhibitory diameters recorded on bacterial culture after enzymatic hydrolysis

LAB strain Reference strains Chymotrypsin Trypsin Pepsin Lipase Amylase CatalaseLAB 1 Candida albicans 0mm 0mm 0mm 8mm 8mm 10mmLAB 1 Enterococcus faecalis 0mm 0mm 0mm 9mm 9mm 8mmLAB 2 Escherichia coli O 157 0mm 0mm 0mm 6mm 5mm 9mmLAB 2 Escherichia coli 0mm 0mm 0mm 7mm 8mm 9mmLAB 3 Micrococcus luteus 0mm 0mm 0mm 0mm 0mm 11mmLAB 4 Proteus mirabilis 0mm 0mm 0mm 8mm 6mm 10mmLAB 6 Proteus vulgaris 0mm 0mm 0mm 7mm 0mm 10mmLAB 7 Pseudomonas aeruginosa 0mm 0mm 0mm 8mm 7mm 10mmLAB 8 Salmonella typhimurium 0mm 0mm 0mm 6mm 5mm 7mmLAB 10 Staphylococcus aureus 0mm 0mm 0mm 8mm 7mm 9mmLAB 10 S epidermidis 0mm 0mm 0mm 6mm 6mm 8mmLAB 10 Streptococcus oralis 0mm 0mm 0mm 6mm 0mm 9mm

from Tanguieta) to 5mgmL (Table 2) whereas the MBCvaried from 125mgmL (S epidermidis and clinical MRSAwith the starter of Boukoumbe) to 20mgmL These resultsshowed that the starter can have bacteriostatic and bacterici-dal effect on reference strains andMRSA Indeed the ratio ofthe two parameters indicates bactericidal effect of the starterson references strains (Pseudomonas aeruginosa Salmonellatyphi E coli ATCC 25922 S epidermidis Proteus mirabilisProteus vulgaris Streptococcus oralis and Candida albicans)and clinical isolated MRSA (Table 2)

36 Effect of Lactic Acid Bacterial against E coli ATCC25922Table 3 shows inhibition diameters of lactic acid bacteriaagainst E coli ATCC 25922 strain The different dilutionsof supernatant obtained from lactic acid bacteria culturersquosshowed inhibition diameters ranging from 6mm to 17mmup to a dilution factor of 256 above which no activity isobserved This dilution factor (256) represents thus the CMIof the assumed bacteriocin produced by lactic acid bacteriaHowever it is noticed that the inhibition power decreaseswhen the dilution of the supernatant increases

37 Production of Organic Acid Sugar and Volatile Com-pounds in Starter of ldquoTchoukoutourdquo Table 4 shows the con-centration of organic acid sugar and volatile compoundsin a starter of tchoukoutou In this study the organic acidsproduced and detectedwere lactic and propionic acids Lacticacid was the main organic produced in the starter culture(119901 lt 005) The lactic acid content in starter of tchouk-outou varied between 049mg100 g to 072mg100 g whereasthe propionic acid content varied between 012mg100 g to030mg100 g

The principal volatile compound detected and identifiedin tchoukoutoursquos starter was ethanol Thus the level ofthis alcohol varied between 146mg100 g and 178mg100 gThere was a significant difference (119901 lt 001) betweenthe ethanol concentrations of starter of tchoukoutou fromdifferent regions

The sugars detected in starter were raffinose maltoseglucose and fructose Raffinose was the main sugar pro-duced in starter with concentrations ranging from 027 to

035mg100 g and the fructose was the less produced (013ndash015mg100 g) There was no significant difference betweenraffinose and fructose levels (119901 gt 005) but there was asignificant difference (119901 lt 001) in the levels of maltose andglucose in the starter

38 Chemical Composition of the Tchoukoutoursquos Starter Thechemical screening of starter samples showed that the mainchemical compounds were polyphenolic compounds (tan-nins catechic tannins flavonoids anthocyanin and leucoan-thocyanes) mucilage reducing compounds triterpenoidand steroids (Table 5) Other compounds such as alkaloidsquinone derivatives saponosides coumarin and anthracenederivatives are absent in the starter

4 Discussion

The ldquokpete-kpeterdquo and the associated lactic acid bacteriahave a remarkable antimicrobial potential on the majorityof tested microorganisms (Figures 1ndash3) Thus it appearthat many pathogenic strains such as Candida albicansSalmonella typhi E coli and S aureus were inhibited by thestarter ldquokpete-kpeterdquo (Figure 1) These results confirm thosereported by Silva de et al [29] in their study on antimicrobialactivity of strains involved in the fermentation of ldquokefirrdquoThisactivity is very interesting and can explain the large inhibi-tion proportion of spoilage germs responsible for poisoningduring fermentation process To support this some lactic acidbacteria strains isolated from many African fermented foodsare reported to produce antimicrobial substances againstseveral spoilagemicroorganisms [30] So the inhibition effectmay be due to the action of lactic bacteria contained inthe tested starter Indeed several studies suggested that theantimicrobial effect of the lactic acid bacteria isolated fromfermented paste of maize is mainly due to the effect of pHH2O2 or bacteriocin [31]

Among the pathogenicmicroorganisms tested onlyEnte-rococcus faecalis ATCC 29212 was resistant to the testedldquokpete-kpeterdquo Meanwhile this microorganism is reported tobe sensitive to the lactic acid bacteria isolated in BurkinaFaso from milk [21] This difference may be explained by


Table2Com

pilationof

thetestedsta

rtersrsquominim

uminhibitory

andbactericidalconcentrationon

somer

eferencesstrains

Pathogenicstrains

Tang

uieta

Natitingou

Bouk

oumbe

Parakou

MIC

(mgmL)

MBC

(mgm)MBC

MIC

MIC

(mgmL)

MBC

(mgm)MBC

MIC

MIC

(mgmL)

MBC

(mgm)MBC

MIC

MIC

(mgmL)

MBC

(mgmL)

MBC

MIC

Escherich

iacoliO157

125

119862gt20

mdash5

119862gt20

mdash25

104

25

104

Pseudomonas

aeruginosa

062

1016

5119862gt20

mdash25

52lowast

25

104

Salm

onellatyphi

125

2016

55

1lowast5

102lowast

25

208

Staphylococcus

aureus

062

2032

5119862gt20

mdash25

119862gt20

mdash12

520

16Proteusm

irabilis

25

104

5119862gt20

mdash12

55

412

525

2lowastE

coliAT

CC25922

25

52lowast

520

425

104

125

108

Sepidermidis

062

125

2lowast5

119862gt20

mdash25

52lowast

25

104

Proteusv

ulgaris

125

54

510

2lowast25

52lowast

25

208

Streptococcuso

ralis

125

54

5119862gt20

mdash5

102lowast

25

119862gt20

mdashCa

ndidaalbicans

25

52lowast

125

2016

25

52lowast

25

208

ESBL

Ecoli

125

54

25

208

25

208

25

52lowast

Clinicaliso

latedMRS

A25

52lowast

25

104

0312

516

062

125

2lowastFo

odiso

latedMRS

A12

55

412

55

412

55

4062

25

4MRS

Am

ethicillinresis

tanceS

aureusES

BLEC

extended-spectrum

beta-la

ctam

asesEcoliMICm

inim

uminhibitory

concentrationMBC

minim

umbactericidalconcentrationwithlowastbacteric

idaleffects

and

with

outlowast

bacterio

static

effects


Table 3 Effect of lactic acid bacterial supernatant dilution against E coli ATCC25922

Dilutions1 2 4 8 16 32 64 128 256 512

Inhibition diameter (mm) 17 17 15 15 12 11 10 8 6 0

Table 4 Organic acids sugars contents and volatile compound (mg100 g) of the traditional beer tchoukoutoursquos starter (mean plusmn standarderror)

Commune Raffinose Maltose Glucose Fructose Lactic acid Propionic acid Acetic acid EthanolBoukoumbe 027 plusmn 002alowast 023 plusmn 001a 022 plusmn 003a 014 plusmn 001a 049 plusmn 003c 012 plusmn 001c 033 plusmn 011b 146 plusmn 008b

Natitingou 035 plusmn 004a 025 plusmn 001a 016 plusmn 003b 015 plusmn 001a 063 plusmn 004ba 020 plusmn 002b 063 plusmn 004ba 206 plusmn 015a

Parakou 025 plusmn 003a 016 plusmn 002b 013 plusmn 001b 014 plusmn 002a 063 plusmn 004bc 063 plusmn 004ba 063 plusmn 004ba 135 plusmn 014b

Tanguieta 035 plusmn 004a 024 plusmn 001a 010 plusmn 001b 013 plusmn 001a 072 plusmn 003a 030 plusmn 014a 094 plusmn 012a 063 plusmn 004ba

Probabilite 006 00002 00023 084 00007 lt00001 0008 0001lowastData with the same letters are not significantly different (119901 gt 005)

Table 5 Chemical content of the tchoukoutoursquos starter

Chemical groups Subgroups Test resultsAlkaloids minus

Polyphenolic compound

Tannins ++Catechic tannins ++Gallic tannins minus

Flavonoids ++ flavoneAnthocyanin minus

Leucoanthocyanes ++Quinone derivatives minus

Saponosides minus

Triterpenoids ++Steroids ++Cardenolides minus

Cyanogenic compound minus

Mucilage +++Coumarins minus

Reducing compound +++

Anthracene derivatives

Free anthracene minus

Combine anthracene-O-Glycosides minus

-C-Glycosides minus

+++ great presence ++ fair presence minus absence

the fact that the active substance (bacteriocins) producedby lactic acid bacteria of the milk is not the same as thoseproduced by lactic acid bacteria isolated from the ldquokpete-kpeterdquo In addition it could be also due to lactic acid bacteriastrains implicated and their culture conditions because theantibacterial effect may vary depending not only on the lacticacid bacteria strain but also on the cultures conditionsrdquo [32]

Concerning the results of antimicrobial activities on Ecoli E coli O157 and S aureus recorded in this study theyare similar to those of by Savadogo et al [21] in their study onantimicrobial activities of lactic acid bacteria strains isolatedfrom Burkina Fasorsquos fermented milk Indeed in their study

they show that lactic acid bacteria exhibited antimicrobialactivity on pathogenic strains such as S aureus and E coli

Both ldquokpete-kpeterdquo and lactic acid bacteria showed avery interesting activity against methicillin resistant S aureusisolated from food and clinical (Figures 2 and 3) Thus thetested starter and lactic acid bacteria have an inhibitoryeffect on pathogenic bacteria as previously reported forbacteria isolated from traditional fermented Algerian milk[33] The lactic acid bacteria are previously reported tobe able to eliminate about 99 of multiresistant bacteriasuch as methicillin resistant S aureus [34] In addition ourdata shows that the antimicrobial activity was completely


E co 0 S ty E co S au c MRSA 0

5

10

15

20

Strains

Inhi

bito

ry d

iam

eter

(mm

)

24 H48H

Figure 3 Lactic acid bacteria isolatesrsquo antimicrobial activity onsome pathogenic strains after 24 h and 48 h of incubation E co 0E coliO157 H7 ATCC 700728 S ty Salmonella typhi R 30951401 Eco E coli ATCC 25922 S au S aureus ATCC 29213 and c MRSAclinical isolated MRSA

inhibited by the proteolytic enzymes action (Table 1) So thebiologically active fraction of the inhibitory substance mayhave a protein natureThough the nature of the bacteriocin isvery important to determine it is more interesting to confirmthese results in vitro as done by other authors [35 36]

According to our data the in vitro test shows the smallestminimum inhibitory concentration (031mgmL) with themethicillin resistant S aureus strains in comparison to thevalue (0625mgmL) of some reference strains (Pseudomonasaeruginosa S aureus and S epidermidis) (Table 2) Thus theldquokpete-kpeterdquo can have an inhibiting effect at low dose on thepathogenic strains such as methicillin resistant S aureus

The ratio between the CMB and CMI shows that thestarter can have bactericidal or bacteriostatic effect depend-ing on the tested strains (Table 2) However there is lessbactericidal effect with the reference strains (Pseudomonasaeruginosa Salmonella typhi E coli ATCC 25922 S epi-dermidis Proteus vulgaris Streptococcus oralis and Candidaalbicans) and clinical isolated methicillin resistant S aureusThese results are not similar to those found by Dramaneet al [37] when they showed that the extract of Erythrinasenegalensis has bactericidal effect on several pathogenicreferences strains (Candida albicans Enterococcus faecalisS epidermidis S aureus Proteus mirabilis E coli andPseudomonas aeruginosa) The difference of the bactericidalcapacity could be explained not only by the fact that theauthors used plants extracts instead of a fermented food butalso by the fact that the active substance of each product actsdifferently on the strains

The analysis of the starterrsquos composition by HPLCrevealed the presence of the organic acids in particular thelactic and acetic acid (Table 4) These compounds may thenbe mainly responsible for the kpete-kpetersquos antimicrobialactivity Indeed the organic acids were indexed in theantimicrobial activity of lactic acid bacteria isolates fromGhanaian fermented maize called ldquokenkeyrdquo [38] In addi-tion the chemical screening of the starter showed that it

contains several compounds such as steroids polyphenoliccompounds (tannins flavonoids and anthocyans) mucilageand reducing compounds (Table 5) The presence of tanninsand flavonoids in the starter suggests that those ldquokpete-kpeterdquomay have some biological and pharmaceutical (antidiarrheicantibacterial antiviral and anticarcinogenic) properties [3940] So these compounds in the starter can explain itsantimicrobial activity in addition to the organic acid effectIndeed these secondary metabolites have been recognized asthe base of medicinal plantsrsquo antibacterial properties [41ndash44]The results of this work give us the probable origin of goodantimicrobial activity of the starter and lactic acid bacteriaobserved in this study Apart from the antibacterial effectof those starters the presence of polyphenolic compoundsindicates that it may have other biological activities such asantioxidant activity

5 Conclusion

Tchoukoutou starter named ldquokpete-kpeterdquo prepared inBenin displayed antimicrobial activity These properties areexpressed not only by the production of the bacteriocin(protein substance) but also by the chemical compoundscontained in the startersThus the lactic acid bacteria presentin the starters are able to produce lactic acid acetic acidethanol peptides (bacteriocins) and other biologically activecompounds which increase the starter capacity to inhibit orkill pathogenic strains So these results provide a scientificsupport to the traditional use of the ldquokpete-kpeterdquo in treat-ment of bacterial infections such as diarrhea and dysenteryThe ldquokpete-kpeterdquo can therefore be used for the elaboration ofa probiotic food ingredient for human and animals in orderto effectively control resistant microorganisms This startercan be also used to control food and clinical multiresistantbacteria

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

The authors thank the University of Abomey-Calavi forfinancial support through the BioZoo project The authorsalso thank the producers of traditional beers for their collabo-ration and themembers of Laboratory of Pharmacognosy andEssential Oil Faculty of Science and Techniques (Universityof Abomey-Calavi)

References

[1] A O Adejuwon E O Agbaje and N Idika ldquoAntifungal andantibacterial activities of aqueous and methanolic root extractsof Carica papaya linn (Caricaceae)rdquo International ResearchJournal of Microbiology vol 2 no 8 pp 270ndash277 2011

[2] OMS Strategie Mondiale OMS pour la Maitrise de la Resistanceaux Antibiotiques Organisation Mondiale de la Sante 2001httpwwwwhointantimicrobial-resistanceexecsumfpdf


[3] R D Rolfe ldquoThe role of probiotic cultures in the control ofgastrointestinal healthrdquo Journal of Nutrition vol 130 no 2 ppS396ndashS402 2000

[4] H Sina F Baba-Moussa T A Ahoyo et al ldquoAntibioticsusceptibility and toxins production of staphylococcus aureusisolated from clinical samples from beninrdquo African Journal ofMicrobiology Research vol 5 no 18 pp 2797ndash2803 2011

[5] P Attien H Sina W Moussaoui et al ldquoPrevalence and antibi-otic resistance of Staphylococcus strains isolated from meatproducts sold in Abidjan streets (Ivory Coast)rdquo African Journalof Microbiology Research vol 7 no 26 pp 3285ndash3293 2013

[6] C Akoua-Koffi N Guessennd V Gbonon H Faye-Kette andM Dosso ldquoMethicillin-resistance of Staphylococcus aureus inAbidjan (1998ndash2001) a new hospital problemrdquo Medecine etMaladies Infectieuses vol 34 no 3 pp 132ndash136 2004

[7] M Benbachir S Benredjeb C S Boye et al ldquoTwo-year surveil-lance of antibiotic resistance in Streptococcus pneumoniae infour African citiesrdquo Antimicrobial Agents and Chemotherapyvol 45 no 2 pp 627ndash629 2001

[8] A Kacou-NrsquoDouba S A Bouzid K N Guessennd A AKouassi-MrsquoBengue A YH Faye-Kette andMDosso ldquoAntimi-crobial resistance of nasopharyngeal isolates of Streptococcuspneumoniae in healthy carriers report of a study in 5-year-olds in Marcory Abidjan Cote drsquoIvoirerdquo Annals of TropicalPaediatrics vol 21 no 2 pp 149ndash154 2001

[9] K O Akinyemi O Oladapo C E Okwara C C Ibe andK A Fasure ldquoScreening of crude extracts of six medicinalplants used in South-West Nigerian unorthodox medicine foranti-methicillin resistant Staphylococcus aureus activityrdquo BMCcomplementary and alternative medicine vol 5 article 6 2005

[10] HMiollogo-Kone I P Guissou O Nacoulma andA S TraoreldquoComparative study of leaf and stem bark extracts of Parkiabiglobosa against enterobacteriardquoAfrican Journal of TraditionalComplementary andAlternativeMedicines vol 5 no 3 pp 238ndash243 2008

[11] L M T Dickset D A Heunis D A van Staden A BrandK Sutyak Noll and M L Chinkindas ldquoMedical and personalcare application ns of bacteriocins produced by lactic acidbacteriardquo in Prokaryotic Antimicrobial Peptides From Genesto Applications D Drider and S Rebuffat Eds pp 391ndash421Springer Stellenbosch South Africa 2011

[12] C Nrsquotcha A Adeyemi A Kayode et al ldquoIndigenous knowledgeassociated with the production of starters culture used toproduce Beninese opaque sorghum beersrdquo Journal of AppliedBiosciences vol 88 no 1 pp 8223ndash8234 2015

[13] AGalvezHAbriouelN BenOmar andR Lucas ldquoFood appli-cations and regulationrdquo in Prokaryotic Antimicrobial PeptidesFrom Genes to Applications D Drider and S Rebuffat Eds pp253ndash390 Springer Jaen Spain 2011

[14] S Ammor G Tauveron E Dufour and I Chevallier ldquoAntibac-terial activity of lactic acid bacteria against spoilage andpathogenic bacteria isolated from the same meat small-scalefacility 1 Screening and characterization of the antibacterialcompoundsrdquo Food Control vol 17 no 6 pp 454ndash461 2006

[15] M Beausoleil N Fortier S Guenette et al ldquoEffect of afermented milk combining Lactobacillus acidophilus CL1285andLactobacillus casei in the prevention of antibiotic-associateddiarrhea a randomized double-blind placebo-controlled trialrdquoCanadian Journal of Gastroenterology vol 21 no 11 pp 732ndash736 2007

[16] A Mercenier ldquoLactic acid bacteria as vaccinesrdquo in Probiotics ACritical Review G Tannock Ed pp 113ndash128 Horizon ScientificPress Norfolk UK 1999

[17] A Blandino M E Al-Aseeri S S Pandiella D Cantero andC Webb ldquoCereal-based fermented foods and beveragesrdquo FoodResearch International vol 36 no 6 pp 527ndash543 2003

[18] A P P Kayode D C Deh L Baba-Moussa S O Kotchoni andJ D Hounhouigan ldquoStabilization and preservation of probioticproperties of the traditional starter of African opaque sorghumbeersrdquoAfrican Journal of Biotechnology vol 11 no 30 pp 7725ndash7730 2012

[19] K Anani J B Hudson C De Souza et al ldquoInvestigationof medicinal plants of Togo for antiviral and antimicrobialactivitiesrdquo Pharmaceutical Biology vol 38 no 1 pp 40ndash452000

[20] SFM (Societe Francaise de Microbiologie) Recommandationsdu Comite de lrsquoAntibiogramme de la Societe Francaise de Micro-biologie 2008

[21] A Savadogo C A T Ouattara P W Savadogo N Barro AS Ouattara and A S Traore ldquoIdentification of exopolysac-charides-producing lactic acid bacteria from Burkina Fasofermented milk samplesrdquo African Journal of Biotechnology vol3 no 3 pp 189ndash194 2004

[22] J R Tagg and A R McGiven ldquoAssay system for bacteriocinsrdquoApplied Microbiology vol 21 no 5 p 943 1971

[23] M Rammelsberg and F Radler ldquoAntibacterial polypeptides ofLactobacillus speciesrdquo Journal of Applied Bacteriology vol 69no 2 pp 177ndash184 1990

[24] N N Farshori MM Al-Oqail E S Al-Sheddi M A Siddiquiand A Rauf ldquoAntimicrobial potentiality of Polyalthia longifoliaseed oil against multi drug resistant (MDR) strains of bacteriaand fungus of clinical originrdquo African Journal of MicrobiologyResearch vol 7 no 19 pp 1977ndash1982 2013

[25] SMAKawsar EHuq andNNahar ldquoCytotoxicity assessmentof the aerial parts ofMacrotyloma uniflorum linnrdquo InternationalJournal of Pharmacology vol 4 no 4 pp 297ndash300 2008

[26] J A Narvhus K Oslashsteraas T Mutukumira and R K Abraham-sen ldquoProduction of fermented milk using a malty compound-producing strain of Lactococcus lactis subsp lactis biovardiacetylactis isolated from Zimbabwean naturally fermentedmilkrdquo International Journal of Food Microbiology vol 41 no 1pp 73ndash80 1998

[27] P J Houghton and A Raman Laboratory Handbook for theFractionation of Natural Extracts Chapman amp Hall New YorkNY USA 1988

[28] P Dagnelie StatistiqueTheorique et Appliquee Tome2 InferenceStatistique a Une et a Deux Dimensions De Boeck BruxellesBelgium 1998

[29] K R Silva de S A Rodrigues L X Filho and A S LimaldquoActivite antimicrobienne de bouillon fermente avec des grainsde kefirrdquo Kheira vol 67 2012

[30] S K Mbugua and J Njenga ldquoThe antimicrobial activity offermented ujirdquo Ecology of Food and Nutrition vol 28 no 3 pp191ndash198 1992

[31] P Mensah A M Tomkins B S Drasar and T J HarrisonldquoAntimicrobial effect of fermented Ghanaian maize doughrdquoJournal of Applied Bacteriology vol 70 no 3 pp 203ndash210 1991

[32] L JHarrisMADaeschelM E Stiles andTRKlaenhammerldquoAntimicrobial activity of lactic acid bacteria against Listeriamonocytogenesrdquo Journal of Food Protection vol 52 no 6 pp384ndash387 1989


[33] M Abdelbasset and K Djamila ldquoAntimicrobial activity ofautochthonous lactic acid bacteria isolated from Algerian tra-ditional fermented milk Raibrdquo African Journal of Biotechnologyvol 7 no 16 pp 2908ndash2914 2008

[34] B Karska-Wysocki M Bazo andW Smoragiewicz ldquoAntibacte-rial activity of Lactobacillus acidophilus and Lactobacillus caseiagainst methicillin-resistant Staphylococcus aureus (MRSA)rdquoMicrobiological Research vol 165 no 8 pp 674ndash686 2010

[35] Q Shu and H S Gill ldquoA dietary probiotic (Bifidobacteriumlactis HN019) reduces the severity of Escherichia coli O157H7infection in micerdquo Medical Microbiology and Immunology vol189 no 3 pp 147ndash152 2001

[36] C-C Tsai L-F Huang C-C Lin andH-Y Tsen ldquoAntagonisticactivity against Helicobacter pylori infection in vitro by a strainof Enterococcus faecium TM39rdquo International Journal of FoodMicrobiology vol 96 no 1 pp 1ndash12 2004

[37] S Dramane K W Mamidou and K Kagoyire ldquoEvalua-tion des activites antimicrobiennes et anti-radicaux libres dequelques taxons bioactifs de Coterdquo European Journal of Scien-tific Research vol 40 no 2 pp 307ndash317 2010

[38] A Olsen M Halm and M Jakobsen ldquoThe antimicrobialactivity of lactic acid bacteria from fermented maize (kenkey)and their interactions during fermentationrdquo Journal of AppliedBacteriology vol 79 no 5 pp 506ndash512 1995

[39] E Haslam ldquoNatural polyphenols (vegetable tannins) as drugspossible modes of actionrdquo Journal of Natural Products vol 59no 2 pp 205ndash215 1996

[40] P Iserin Encyclopedie des Plantes Medicinales IdentificationPresentations Soins LarousseVUEF Paris France 2001

[41] J A Adejumbi M O Ogundiya A L Kolapo and M BOkunade ldquoPhytochemical composition and in vitro antimicro-bial activity of Anogeissus leiocarpus on some common oralpathogensrdquo Journal of Medicinal Plants Research vol 2 no 8pp 193ndash196 2008

[42] MM Cowan ldquoPlant products as antimicrobial agentsrdquoClinicalMicrobiology Reviews vol 12 no 4 pp 564ndash582 1999

[43] C O EsimoneM U Adikwu O O Ndu P O Udeogaranya OEzeugwu and W Obonga ldquoEffect of Garcinia kola seed extracton the antimicrobial properties of some antibiotics in-vitrordquoJournal Pharmaceutical and Allied Sciences vol 2 pp 114ndash1202003

[44] M Parida P K Dash N K Tripathi et al ldquoJapanese encephali-tis outbreak India 2005rdquo Emerging Infectious Diseases vol 12no 9 pp 1427ndash1430 2006

Submit your manuscripts athttpswwwhindawicom

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


(particularly their lactic bacteria content) to overcome somebacterial infections [11 12] and food conservation [13] iscurrent So the use of lactic acid bacteria could be consideredas an alternative to control resistant pathogenic bacterialstrains

Several studies shows that some lactic acid bacteriasuch as Lactobacillus isolated from fermented foods haveantimicrobial activity [14 15] Up to date the principalmicroorganisms reported to have probiotic properties arebacteria (Lactobacillus Bifidobacterium PropionibacteriumEnterococcus etc) and yeast (Saccharomyces Candida etc)[16]Thesemicroorganisms are even used for the formulationof new probiotic products [17]

In Benin the starter of the most popular traditionalsorghum beer named ldquotchoukoutourdquo is reported to be usedto cure several infections such as stomach pains and diarrhea[12] However a part of the preliminary study conducted in2012 by Kayode et al [18] showed that there is no scientificevidence to support the traditional use of the tchoukoutoursquosstarter named ldquokpete-kpeterdquo in infection treatments Thusthe aim of the present study was to investigate the antimi-crobial activity of starter ldquokpete-kpeterdquo and it isolated lacticacid bacteria not only on some reference microorganismsrsquostrains but also on some food and clinical isolated pathogenicbacteria strains

2 Material and Methods

21 Kpete-Kpete Sample Collection The starters used for thestudywere the ones used in the fermentation of the traditionalbeer ldquotchoukoutourdquo and locally called ldquokpete-kpeterdquo Sampleswere collected in four communes selected from the twodepartments (Atacora and Borgou) displaying the highestproduction and consumption level of the ldquotchoukoutourdquoThe 4 communes are Natitingou (10∘1810158402410158401015840N 1∘2210158403010158401015840E)Boukoumbe (10∘1810158402410158401015840N 1∘2210158403010158401015840E) Tanguieta (10∘3710158401110158401015840N1∘1510158405210158401015840E) the department of Atacora (North-West Benin)and Parakou (9∘2110158400010158401015840N 2∘3710158400010158401015840E) in the department ofBorgou (Northern-East) In each commune 10 samples oftchoukoutoursquos starter were collected for laboratory analysisThus a total of 40 samples were collected and tested for thisstudy

22 Tested Strains The stains used for the antimicrobialtest were composed of 12 references strains (Staphylococcusaureus ATCC 29213 S epidermidis T22695 Proteus mirabilisA24974 P vulgaris A25015Micrococcus luteus Streptococcusoralis Enterococcus faecalis ATCC 29212 Escherichia coliATCC25922E coliO157H7ATTC700728 Salmonella typhiR 30951401 Candida albicans MHMR and PseudomonasaeruginosaATCC 27853) 15 clinical and food isolated methi-cillin resistance S aureus (MRSA) 2 extended-spectrumbeta-lactamase E coli strains and 11 lactic acid bacteriaisolated previously from ldquotchoukoutourdquo [12]

23 In Vitro Determination of ldquoKpete-Kpeterdquo AntimicrobialActivity The antimicrobial activity of the collected starterswas evaluated by disk diffusion method [19] Briefly 100 120583Lof 106 CFUmL microbial suspension was used to inoculate

Mueller-Hinton agar by flooding (Bio-Rad France) [20]Sterile paper discs impregnated with 30120583L of startersrsquo super-natant (20mgmL) were then deposited on the mediumFor each test the experiment is duplicated and a negativecontrol was performed using sterile distilled water Plateswere then left for 15ndash30min at room temperature before beingincubated at 37∘C for 24 to 48 h Diameters of inhibitionzones were then measured using a sliding graduated scaleafter incubation times (24 h and 48 h)

24 Antibacterial Activity of Lactic Acid Bacteria Strains

241 Bacteriocins Extraction The method describe bySavadogo et al [21] was used to extract the bacteriocinproduced by the lactic acid bacteria Briefly each lactic acidbacteria strains were previously mixed in 1000mL of MRSbroth (pH 70) For the bacteriocin extraction a cell-free solu-tion was obtained by centrifuging (10000 rpm for 20minat 4∘C) the culture Thus the obtained cell-free solutionwas precipitated with ammonium sulphate (40 saturation)for 2 h at 4∘C Cells were harvested by centrifugation at20000 rpm for an hour at 4∘C and resuspended in 005Mpotassium phosphate buffer (25mL pH 70) for further diskdiffusion assay

242 Determination of Bacteriocin Activity The bacteriocinactivity was determined by disk diffusion assay [22] For thisassay the aliquot of each bacteriocin (50 120583L) was used toimpregnate disk Once impregnated the disks were lodgedon Mueller-Hinton agar dishes containing 5 sdot 105 CFUmLof tested pathogenic strains (E coli O157 H7 ATCC 700728Salmonella typhi R 30951401 E coli S aureus ATCC 29213and 2 clinical isolated MRSA) The dishes were then incu-bated at 30∘C or 37∘C for 24 h Diameters of inhibition zoneswere then measured using a sliding graduated scale [23]

25 Sensitivity of Antagonistic Substances of Lactic AcidBacteria to Enzymes The sensitivity of antimicrobial sub-stances to enzymes was tested according to the methodpreviously described by Savadogo et al [21] Briefly the cell-free supernatants of 13 lactic acid bacteria isolates that showedantimicrobial activity against reference microorganism werecollected after centrifugation (7500timesg for 10min at 4∘C)ThepHof the supernatants was adjusted to 6with 10NNaOHandtreated with 02mgmL of specific enzymes (Sigma-AldrichBrasil Sao Paulo Brazil) The used enzymes were lipase(86Umg in 005M Tris hydrochloride pH 80 and 001MCaCl2) chymotrypsin (47Umg in 005MTris hydrochloridepH 80 and 001M CaCl2) type x trypsin (15000Umg in02M citrate pH 60) pepsin (32 UmL in 02M citrate pH60) and catalase (20Umg in 10mM potassium phosphatepH 70)The samples of bacteriocins (500 120583L) were incubatedwith the appropriate enzymes (500120583L) for 60min at 37∘C or25∘C (for trypsin chymotrypsin and catalase) The negativecontrols contain supernatant solutions and 01M sodiumphosphate buffer without enzymes

26 Determination of the Startersrsquo Minimum Inhibitory Con-centrations (MIC) The minimum inhibitory concentrations








E co

0

S ty

Psa

e

S a

u

S ep

Pr m

i

M l

u

S o

r

E co

En f

a

Pr v

u

C a

l

0

5

10

15

References strains

Inhi

bito

ry d

iam

eter

(mm

)

24 H48H



3 Results




Tan Nat Bou ParTown

0

5

10

15In

hibi

tory

dia

met

er (m

m)

24 H48H


Tan Nat Bou Par0

5

10

Town

Inhi

bito

ry d

iam

eter

(mm

)

24 H48H


Tan Nat Bou Par


Town

0

5

10

Inhi

bito

ry d

iam

eter

(mm



Tan Nat Bou Par0

5

10

15

Town

Inhi

bito

ry d

iam

eter

(mm

)

24 H48H



















4 Discussion




Table2Com

pilationof

thetestedsta

rtersrsquominim

uminhibitory


somer

eferencesstrains

Pathogenicstrains

Tang

uieta

Natitingou

Bouk

oumbe

Parakou

MIC

(mgmL)

MBC

(mgm)MBC

MIC

MIC

(mgmL)

MBC

(mgm)MBC

MIC

MIC

(mgmL)

MBC

(mgm)MBC

MIC

MIC

(mgmL)

MBC

(mgmL)

MBC

MIC

Escherich

iacoliO157

125

119862gt20

mdash5

119862gt20

mdash25

104

25

104

Pseudomonas

aeruginosa

062

1016

5119862gt20

mdash25

52lowast

25

104

Salm

onellatyphi

125

2016

55

1lowast5

102lowast

25

208

Staphylococcus

aureus

062

2032

5119862gt20

mdash25

119862gt20

mdash12

520

16Proteusm

irabilis

25

104

5119862gt20

mdash12

55

412

525

2lowastE

coliAT

CC25922

25

52lowast

520

425

104

125

108

Sepidermidis

062

125

2lowast5

119862gt20

mdash25

52lowast

25

104

Proteusv

ulgaris

125

54

510

2lowast25

52lowast

25

208

Streptococcuso

ralis

125

54

5119862gt20

mdash5

102lowast

25

119862gt20

mdashCa

ndidaalbicans

25

52lowast

125

2016

25

52lowast

25

208

ESBL

Ecoli

125

54

25

208

25

208

25

52lowast

Clinicaliso

latedMRS

A25

52lowast

25

104

0312

516

062

125

2lowastFo

odiso

latedMRS

A12

55

412

55

412

55

4062

25

4MRS

Am

ethicillinresis

tanceS

aureusES

BLEC

extended-spectrum

beta-la

ctam

asesEcoliMICm

inim

uminhibitory

concentrationMBC

minim


idaleffects

and

with

outlowast

bacterio

static

effects



Dilutions1 2 4 8 16 32 64 128 256 512














Saponosides minus








-C-Glycosides minus








5

10

15

20

Strains

Inhi

bito

ry d

iam

eter

(mm

)

24 H48H







5 Conclusion




Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








E co

0

S ty

Psa

e

S a

u

S ep

Pr m

i

M l

u

S o

r

E co

En f

a

Pr v

u

C a

l

0

5

10

15

References strains

Inhi

bito

ry d

iam

eter

(mm

)

24 H48H



3 Results




Tan Nat Bou ParTown

0

5

10

15In

hibi

tory

dia

met

er (m

m)

24 H48H


Tan Nat Bou Par0

5

10

Town

Inhi

bito

ry d

iam

eter

(mm

)

24 H48H


Tan Nat Bou Par


Town

0

5

10

Inhi

bito

ry d

iam

eter

(mm



Tan Nat Bou Par0

5

10

15

Town

Inhi

bito

ry d

iam

eter

(mm

)

24 H48H



















4 Discussion




Table2Com

pilationof

thetestedsta

rtersrsquominim

uminhibitory


somer

eferencesstrains

Pathogenicstrains

Tang

uieta

Natitingou

Bouk

oumbe

Parakou

MIC

(mgmL)

MBC

(mgm)MBC

MIC

MIC

(mgmL)

MBC

(mgm)MBC

MIC

MIC

(mgmL)

MBC

(mgm)MBC

MIC

MIC

(mgmL)

MBC

(mgmL)

MBC

MIC

Escherich

iacoliO157

125

119862gt20

mdash5

119862gt20

mdash25

104

25

104

Pseudomonas

aeruginosa

062

1016

5119862gt20

mdash25

52lowast

25

104

Salm

onellatyphi

125

2016

55

1lowast5

102lowast

25

208

Staphylococcus

aureus

062

2032

5119862gt20

mdash25

119862gt20

mdash12

520

16Proteusm

irabilis

25

104

5119862gt20

mdash12

55

412

525

2lowastE

coliAT

CC25922

25

52lowast

520

425

104

125

108

Sepidermidis

062

125

2lowast5

119862gt20

mdash25

52lowast

25

104

Proteusv

ulgaris

125

54

510

2lowast25

52lowast

25

208

Streptococcuso

ralis

125

54

5119862gt20

mdash5

102lowast

25

119862gt20

mdashCa

ndidaalbicans

25

52lowast

125

2016

25

52lowast

25

208

ESBL

Ecoli

125

54

25

208

25

208

25

52lowast

Clinicaliso

latedMRS

A25

52lowast

25

104

0312

516

062

125

2lowastFo

odiso

latedMRS

A12

55

412

55

412

55

4062

25

4MRS

Am

ethicillinresis

tanceS

aureusES

BLEC

extended-spectrum

beta-la

ctam

asesEcoliMICm

inim

uminhibitory

concentrationMBC

minim


idaleffects

and

with

outlowast

bacterio

static

effects



Dilutions1 2 4 8 16 32 64 128 256 512














Saponosides minus








-C-Glycosides minus








5

10

15

20

Strains

Inhi

bito

ry d

iam

eter

(mm

)

24 H48H







5 Conclusion




Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Tan Nat Bou ParTown

0

5

10

15In

hibi

tory

dia

met

er (m

m)

24 H48H


Tan Nat Bou Par0

5

10

Town

Inhi

bito

ry d

iam

eter

(mm

)

24 H48H


Tan Nat Bou Par


Town

0

5

10

Inhi

bito

ry d

iam

eter

(mm



Tan Nat Bou Par0

5

10

15

Town

Inhi

bito

ry d

iam

eter

(mm

)

24 H48H



















4 Discussion




Table2Com

pilationof

thetestedsta

rtersrsquominim

uminhibitory


somer

eferencesstrains

Pathogenicstrains

Tang

uieta

Natitingou

Bouk

oumbe

Parakou

MIC

(mgmL)

MBC

(mgm)MBC

MIC

MIC

(mgmL)

MBC

(mgm)MBC

MIC

MIC

(mgmL)

MBC

(mgm)MBC

MIC

MIC

(mgmL)

MBC

(mgmL)

MBC

MIC

Escherich

iacoliO157

125

119862gt20

mdash5

119862gt20

mdash25

104

25

104

Pseudomonas

aeruginosa

062

1016

5119862gt20

mdash25

52lowast

25

104

Salm

onellatyphi

125

2016

55

1lowast5

102lowast

25

208

Staphylococcus

aureus

062

2032

5119862gt20

mdash25

119862gt20

mdash12

520

16Proteusm

irabilis

25

104

5119862gt20

mdash12

55

412

525

2lowastE

coliAT

CC25922

25

52lowast

520

425

104

125

108

Sepidermidis

062

125

2lowast5

119862gt20

mdash25

52lowast

25

104

Proteusv

ulgaris

125

54

510

2lowast25

52lowast

25

208

Streptococcuso

ralis

125

54

5119862gt20

mdash5

102lowast

25

119862gt20

mdashCa

ndidaalbicans

25

52lowast

125

2016

25

52lowast

25

208

ESBL

Ecoli

125

54

25

208

25

208

25

52lowast

Clinicaliso

latedMRS

A25

52lowast

25

104

0312

516

062

125

2lowastFo

odiso

latedMRS

A12

55

412

55

412

55

4062

25

4MRS

Am

ethicillinresis

tanceS

aureusES

BLEC

extended-spectrum

beta-la

ctam

asesEcoliMICm

inim

uminhibitory

concentrationMBC

minim


idaleffects

and

with

outlowast

bacterio

static

effects



Dilutions1 2 4 8 16 32 64 128 256 512














Saponosides minus








-C-Glycosides minus








5

10

15

20

Strains

Inhi

bito

ry d

iam

eter

(mm

)

24 H48H







5 Conclusion




Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











4 Discussion




Table2Com

pilationof

thetestedsta

rtersrsquominim

uminhibitory


somer

eferencesstrains

Pathogenicstrains

Tang

uieta

Natitingou

Bouk

oumbe

Parakou

MIC

(mgmL)

MBC

(mgm)MBC

MIC

MIC

(mgmL)

MBC

(mgm)MBC

MIC

MIC

(mgmL)

MBC

(mgm)MBC

MIC

MIC

(mgmL)

MBC

(mgmL)

MBC

MIC

Escherich

iacoliO157

125

119862gt20

mdash5

119862gt20

mdash25

104

25

104

Pseudomonas

aeruginosa

062

1016

5119862gt20

mdash25

52lowast

25

104

Salm

onellatyphi

125

2016

55

1lowast5

102lowast

25

208

Staphylococcus

aureus

062

2032

5119862gt20

mdash25

119862gt20

mdash12

520

16Proteusm

irabilis

25

104

5119862gt20

mdash12

55

412

525

2lowastE

coliAT

CC25922

25

52lowast

520

425

104

125

108

Sepidermidis

062

125

2lowast5

119862gt20

mdash25

52lowast

25

104

Proteusv

ulgaris

125

54

510

2lowast25

52lowast

25

208

Streptococcuso

ralis

125

54

5119862gt20

mdash5

102lowast

25

119862gt20

mdashCa

ndidaalbicans

25

52lowast

125

2016

25

52lowast

25

208

ESBL

Ecoli

125

54

25

208

25

208

25

52lowast

Clinicaliso

latedMRS

A25

52lowast

25

104

0312

516

062

125

2lowastFo

odiso

latedMRS

A12

55

412

55

412

55

4062

25

4MRS

Am

ethicillinresis

tanceS

aureusES

BLEC

extended-spectrum

beta-la

ctam

asesEcoliMICm

inim

uminhibitory

concentrationMBC

minim


idaleffects

and

with

outlowast

bacterio

static

effects



Dilutions1 2 4 8 16 32 64 128 256 512














Saponosides minus








-C-Glycosides minus








5

10

15

20

Strains

Inhi

bito

ry d

iam

eter

(mm

)

24 H48H







5 Conclusion




Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table2Com

pilationof

thetestedsta

rtersrsquominim

uminhibitory


somer

eferencesstrains

Pathogenicstrains

Tang

uieta

Natitingou

Bouk

oumbe

Parakou

MIC

(mgmL)

MBC

(mgm)MBC

MIC

MIC

(mgmL)

MBC

(mgm)MBC

MIC

MIC

(mgmL)

MBC

(mgm)MBC

MIC

MIC

(mgmL)

MBC

(mgmL)

MBC

MIC

Escherich

iacoliO157

125

119862gt20

mdash5

119862gt20

mdash25

104

25

104

Pseudomonas

aeruginosa

062

1016

5119862gt20

mdash25

52lowast

25

104

Salm

onellatyphi

125

2016

55

1lowast5

102lowast

25

208

Staphylococcus

aureus

062

2032

5119862gt20

mdash25

119862gt20

mdash12

520

16Proteusm

irabilis

25

104

5119862gt20

mdash12

55

412

525

2lowastE

coliAT

CC25922

25

52lowast

520

425

104

125

108

Sepidermidis

062

125

2lowast5

119862gt20

mdash25

52lowast

25

104

Proteusv

ulgaris

125

54

510

2lowast25

52lowast

25

208

Streptococcuso

ralis

125

54

5119862gt20

mdash5

102lowast

25

119862gt20

mdashCa

ndidaalbicans

25

52lowast

125

2016

25

52lowast

25

208

ESBL

Ecoli

125

54

25

208

25

208

25

52lowast

Clinicaliso

latedMRS

A25

52lowast

25

104

0312

516

062

125

2lowastFo

odiso

latedMRS

A12

55

412

55

412

55

4062

25

4MRS

Am

ethicillinresis

tanceS

aureusES

BLEC

extended-spectrum

beta-la

ctam

asesEcoliMICm

inim

uminhibitory

concentrationMBC

minim


idaleffects

and

with

outlowast

bacterio

static

effects



Dilutions1 2 4 8 16 32 64 128 256 512














Saponosides minus








-C-Glycosides minus








5

10

15

20

Strains

Inhi

bito

ry d

iam

eter

(mm

)

24 H48H







5 Conclusion




Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Dilutions1 2 4 8 16 32 64 128 256 512














Saponosides minus








-C-Glycosides minus








5

10

15

20

Strains

Inhi

bito

ry d

iam

eter

(mm

)

24 H48H







5 Conclusion




Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



5

10

15

20

Strains

Inhi

bito

ry d

iam

eter

(mm

)

24 H48H







5 Conclusion




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

Antimicrobial Activity and Chemical Composition of Kpètè...

Documents

Transcript of Antimicrobial Activity and Chemical Composition of Kpètè...