Research Article A Sequential Statistical Approach towards an …downloads.hindawi.com/journals/tswj/2014/396304.pdf · 2019-07-31 · Research Article A Sequential Statistical Approach

Research ArticleA Sequential Statistical Approach towards an OptimizedProduction of a Broad Spectrum Bacteriocin Substance froma Soil Bacterium Bacillus sp YAS 1 Strain

Amira M Embaby1 Yasmin Heshmat1 Ahmed Hussein1 and Heba S Marey23

1Department of Biotechnology Institute of Graduate Studies and Research University of Alexandria 163 Horreya AvenuePO Box 832 Chatby 21526 Egypt2Department of Environmental Studies Institute of Graduate Studies and Research University of AlexandriaPO Box 832 Chatby 21526 Egypt3University of Alberta Department of Civil and Environmental Engineering Edmonton AB Canada

Correspondence should be addressed to Amira M Embaby amira mohamed 2000yahoocom

Received 15 July 2014 Revised 7 November 2014 Accepted 10 November 2014 Published 29 December 2014

Academic Editor Jean-Marc Sabatier

Copyright copy 2014 Amira M Embaby et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Bacteriocins ribosomally synthesized antimicrobial peptides display potential applications in agriculture medicine and industryThe present study highlights integral statistical optimization and partial characterization of a bacteriocin substance from a soilbacterium taxonomically affiliated as Bacillus sp YAS 1 after biochemical and molecular identifications A sequential statisticalapproach (Plackett-Burman and Box-Behnken) was employed to optimize bacteriocin (BAC YAS 1) production Using optimallevels of three key determinants (yeast extract (048 (wv) incubation time (62 hrs) and agitation speed (207 rpm)) inpeptone yeast beef based production medium resulted in 16-fold enhancement in BAC YAS 1 level (470AUmL arbitrary unitsagainst Erwinia amylovora) BAC YAS 1 showed activity over a wide range of pH (1ndash13) and temperature (45ndash80∘C) A widespectrum antimicrobial activity of BAC YAS 1 against the human pathogens (Clostridium perfringens Staphylococcus epidermidisCampylobacter jejuni Enterobacter aerogenes Enterococcus sp Proteus sp Klebsiella sp and Salmonella typhimurium) the plantpathogen (E amylovora) and the food spoiler (Listeria innocua) was demonstrated On top and above BAC YAS 1 showedno antimicrobial activity towards lactic acid bacteria (Lactobacillus bulgaricus L casei L lactis and L reuteri) Promisingcharacteristics of BAC YAS 1 prompt its commercialization for efficient utilization in several industries

1 Introduction

Ribosomally synthesized antimicrobial peptides (AMPs) areproduced by prokaryotes (eg Gram-negative bacteria andGram-positive bacteria) and eukaryotes (eg plant and awide variety of animals both invertebrates and vertebrates) Inunicellular microorganisms those AMPs produced by bacte-ria are designated as bacteriocins [1ndash4] The present researchpaper focuses on AMPs of bacterial origin (bacteriocins)

Bacteriocins can antagonize the growth of closely relatedspecies (narrow spectrum) or across genera (broad spec-trum) Their usage as potential weapons against human

animalplant pathogens and food spoilermicroorganisms hasattracted the scientific attention in recent years [1 5ndash8]

Classification of bacteriocins is not an easy task becausecontinuous alterations in this classification exist These alter-ations are mainly derived from heterogeneity in bacteriocinspertaining to their structures amino acid sequences andmechanisms of action Although bacteriocins are a hetero-gonous group they could be categorized into two mainclasses class I (lanthionines with unusual amino acids)and class II (non-lanthionine containing unmodified bac-teriocins) Lantibiotics (lanthionines containing posttransla-tionally modified bacteriocins) contain unusual amino acids

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 396304 16 pageshttpdxdoiorg1011552014396304

2 The Scientific World Journal

such as lanthionine (Lan) methyl lanthionine (MeLan)dehydroalanine (Dha) dehydrobutyrine (Dhb) or D-alanine(D-Ala) whilst non-lanthionines containing bacteriocins donot undergo extensive posttranslationalmodifications So fara confined number of bacteriocins have been studied [8ndash11]

Diverse mechanisms describing the bacteriocin mode ofaction were outlined but the cell membrane still remainsthe main target of these AMPs The net positive charges ofbacteriocins stimulate preferential binding of bacteriocins tothe negatively charged bacterial cell membranes resulting increation of multiple pores in the cell membranes [12ndash14]In the context of bacteriocins as an adequate alternative toantibiotics against multidrug resistant pathogens along witha continuous urgent need for developing new antimicro-bial agents bacteriocins represent a promising inexhaustiblesource of new antibiotics [15 16]

The objective of the present study is to isolate a bacte-riocin producing bacterium with a wide spectrum antimi-crobial property The aim is extended to optimize the pro-duction of the produced bacteriocin through a sequentialstatistical approach To the best of our knowledge thereexists a plethora of literature concerning characterizationof bacteriocins but there exists a few reports pertaining tohighlighting key determinants controlling the production ofbacteriocins by their producers

2 Materials and Methods

21 Chemicals Majority of bacteriological media used inthis study were purchased from Oxoid Co UK unlessotherwise stated Proteinase K and Dream Taq Green MasterMix 2X were purchased from Thermo Scientific Co USAIsopropanol acetone acetonitrile toluene methyl alcoholand ethyl alcohol were purchased from Sigma Co USALysozyme and agarose were purchased from Bio Basic IncCanada DNA ladder (100 bp DNA Ladder H3 RTU (Ready-to-Use) was purchased from GeneDirex Inc Taiwan Uni-versal primers specific for 16S rDNA gene were synthesizedby Bioneer Co Korea

22 Indicator Strains Erwinia amylovora pear fire blightpathogen and lactic acid bacterial strains (Lactobacillusbulgaricus L casei L lactis and L reuteri) were obtainedfrom the bacterial culture collection of the Department ofPlant Pathology and the Department of Dairy Productsrespectively Faculty of Agriculture University of AlexandriaEgypt The human pathogens indicator strains used in thisstudy were Salmonella typhimurium NCTC 12023ATCC14028 Clostridium perfringens NCTC 8237ATCC 13124Staphylococcus aureus NCTC 12981ATCC 25923 S epider-midis NCTC 13360ATCC 12228 Escherichia coli NCTC12241ATCC 25922 Shigella boydii NCTCATCC 9207Campylobacter jejuni NCTC 11322ATCC 29428 Enterococ-cus faecalis NCTC 12697ATCC29212 Enterobacter aero-genes NCTC10006ATCC13043 Candida albicans NCPF3179ATCC10231 and Vibrio parahaemolyticus NCTC 3178Other human clinical pathogens such asProteus spKlebsiellasp Enterobacter sp Citrobacter sp Enterococcus sp S

typhi Pseudomonas aeruginosa Acinetobacter sp E coliand S paratyphi were obtained from the Department ofMicrobiology Faculty of Medicine University of AlexandriaEgypt Candida tropicalis was obtained from the Departmentof Microbiology Medical Research Institute University ofAlexandria Egypt The food spoiler indicator strain wasListeria innocua NCTC 11288ATCC 33090 Other environ-mental microbial strains included B cereus B licheniformisSHG2B licheniformis SHG6B licheniformis SHG10Bacillussubtilis NCTC 10400ATCC 6633 B subtilis AS1 B subtilisB subtilis EMBLAKE Bacillus sp Ash2 P aeruginosa strainEGYII Aspergillus brasiliensis NCPF2275ATCC16404 andSaccharomyces cerevisiae NCPF 3178 All indicator bacterialstrains were preserved in 15 glycerol stocks at minus80∘CHowever fungal and yeast strains were preserved in agarslopes at 4∘C

23 Media Peptone yeast beef medium (PYB in gL 10 gpeptone 5 g yeast extract and 3 g beef extract) was used asa bacteriocin core production medium E amylovora wasgrown on PYB supplemented with 2 sucrose The indicatorstrains (S typhimurium S paratyphi E coli P aeruginosaKlebsiella sp Enterobacter sp Citrobacter sp and Proteussp) were grown on BHI (Brain Heart Infusion mediumOxoid CM 1135) All strains of lactic acid bacteria weregrown on MRS (De Man Rogosa Sharpe Oxoid CM0361)C albicans and C tropicalis were grown on SAB DextroseAgar medium (Sabouraud Dextrose Agar Oxoid CM004)S aureus and S epidermidis were grown on MSA (Manni-tol Salt Agar Oxoid CM 0085) C perfringens was grownon TSC (Tryptose Sulphite Cycloserine Agar LAB M194)A brasiliensis and S cerevisiae were grown on DichloranGlycerol medium (HIMEDIA M1129-500G) L innocua wasgrown Brilliance Listeria Agar Base (Oxoid CM1080) Allstrains belonging to the genus Bacillus were grown on PYBmedium V parahaemolyticus was grown on thiosulphatecitrate bile salts sucrose agar (TCBS medium LAB M96) Efaecalis was grown on KF Streptococcus Agar Base (OxoidCM0701) Sporulation medium (1XSG) [17] in gL 6 g beefextract 10 g peptone 2 g KCl 1 g glucose plus 1mM CaCl

2

1 mMFeSO4 1 mMMnCl

2 and 2mMMgSO

4) was used dur-

ing the course of studying the most promising isolate underthe electron microscope MYP (Mannitol egg yolkphenolred CM0929 Oxoid Co) medium consisted of 10 g meatextract 10 g peptone 10 g NaCl 10 g mannitol 0025 g perliter plus supplements of polymyxin and egg yolk suspension[18] Sheep blood agar was obtained from the Department ofMicrobiology Faculty of Medicine University of AlexandriaEgypt

24 Isolation of Bacteriocin Producing Bacteria Differentsamples including soils leaves and fruits were collected fromdifferent sites in Borg El-Arab in Alexandria city Egypt Thecandidate sites were naturally contaminated with the pearfirelight pathogen E amylovora One gram of each sampleseparately was used to inoculate 20mL of PYB broth in250mL Erlenmeyer flask for enrichment The inoculatedbroth was incubated for an overnight at 37∘C with agitation

The Scientific World Journal 3

speed of 150 rpm (New Brunswick Incubator Shaker USA)Serial dilutions were prepared from each enriched cultureOne hundred microliters of an appropriate dilution fromeach culture was selected to be spread on the surface ofPYB agar plates Next day raised single colonies were puri-fied by streaking technique Bacteriocin capability of eachbacterial isolate was checked via tooth picking techniqueas a primary screening Briefly PYB agar was inoculatedwith 1 of an overnight culture of the indicator strain Eamylovora Then the inoculated agar was poured into platesAfter solidification the agar plates were stabbed using steriletooth picks carrying the previously isolated single colonies toassess their efficiency for bacteriocin production After thatthese stabbed inoculated agar plates were incubated at 37∘Cfor an overnight (JSGI-100T Incubator Korea) Halo zonesof the growth of the indicator strain around the stabbingpoints were taken as a tentative indicative for bacteriocinproduction Bacterial isolates having antimicrobial effectagainst E amylovora were further subjected to a secondaryscreening via well cut diffusion assay as mentioned belowin Section 29 The most potent bacteriocin producer wasselected as a good candidate for a further detailed study inorder to integrally optimize and partially characterize theproduced bacteriocin

25 Identification of the Bacteriocin Producing Isolate

251Morphological and Biochemical Identification Themostpotent bacteriocin producing isolate was identified morpho-logically and biochemically according to Bergeyrsquos manual ofdeterminative bacteriology [19] Cell morphological featuresof the bacteriocin producer were studied under light micro-scope and electronmicroscope (transmission electronmicro-scope (TEM) and scanning electron microscope (SEM))

252 Molecular Identification Amplification of 16S rDNAgene was carried out to identify the bacteriocin producerisolate on a molecular level Genomic DNA of the bac-teriocin producer was extracted according to a procedurereported previously by Sambrook et al [20] The geneencoding 16S rDNA was amplified from the genomic DNAby PCR using previously described universal primers F8-27 (51015840-AGAGTTTGATCCTGGCTCAG-31015840) and R1510-1492(51015840-GGTTACCTTGTTACGACTT-31015840) of Escherichia coli 16SrDNA gene [21] The reaction mixture (50 120583L) contained 3 120583L(30 ng genomic DNA) 25120583L of Dream Taq Green MasterMix 2X (Thermo Scientific Co USA) 15 120583L (15 pmol) ofeach forward and reverse primer and 19 120583L of nuclease freewaterThermocycler primus 25 (peQlab Erlangen Germany)was used in this study PCR conditions consisted of an initialdenaturation at 95∘C for 5min 30 cycles each cycle consistedof denaturation at 94∘C for 30 s annealing at 55∘C for 45 sextension at 72∘C for 15min and a final extension at 72∘C for10min The amplified PCR product was analyzed via agarosegel electrophoresis After that the PCR product was purifiedusing Wizard SV Gel and PCR Clean-Up kit PromegaCo USA Then the purified PCR product was sequenceddirectly as sequencing templates using the abovementioned

primers Sequencing was carried out at Eurofin DNA CoLuxembourg The obtained nucleotide sequence was editedand analyzed by Geneious R8 software A partial nucleotidesequence (1331 bases) was used to search the internationalnucleotide databases (eg GenBank EMBL DDBJ etc)through BLASTN (Basic Local Alignment Search Tool) ofNCBI (National Center for Biotechnology Information) todetermine the relative phylogenetic position of the bacterialisolate of inquiry Moreover the phylogenetic tree depictingthe phylogenetic affiliation of the isolate of inquiry wasconstructed via Geneious R8 software

26 Inoculum Preparation An overnight single colony of themost potent bacteriocin producer from PYB agar plate waspicked to inoculate 20mL of PYB broth in 250mL Erlen-meyer flask The inoculated broth was incubated for 18 hrsat 37∘C with agitation speed at 150 rpm (New BrunswickIncubator Shaker USA) One mL of this culture (80 times103 CFUmL) was used to inoculate the bacteriocin produc-tion medium unless otherwise stated

27 Optimizing Bacteriocin Productivity The most potentbacteriocin producing bacterial isolate was selected toconduct an integral statistical optimization study for theproduced bacteriocin A sequential statistical approach(Plackett-Burman design followed by response surfacemethodology) was employed in this study in order to opti-mize the production of bacteriocin

271 Plackett-Burman Design (PBD) PBD is a two-factorialdesign created by Plackett and Burman [22] It allows study-ing of119873-factors through at least119873+1 experiments assumingthat the interactions among the studied factors are negligible(ie the effect of other factors is cancelled when that of aparticular factor is considered)Here the effect of nine factors(eg incubation time pH temperature glycerol glucosestarch agitation speed inoculums size and yeast extract) onbacteriocin production was studied in twelve experimentaltrials Each factor was examined in two levels designated asminus1 (low level) and +1 (high level)The design relies on the factthat each factor is studied in a low level in a set of (119873 + 1)2trials and in a high level in the other set (119873 + 1)2 trialsAll trials were carried out in triplicate in 250mL Erlenmeyerflasks with aworking volume of 100mL brothThe averages ofthree readings were taken as the experimental response valuePBD is based on a first order polynomial equation

119884 = 120573119900 + Σ120573119894 119909119894 (1)

where 119884 is the response 120573o is the model intercept 119909119894 isthe independent variable (factor) and 120573i is the independentvariable estimate

272 Response Surface Methodology (RSM) Response sur-face methodology approach in the form of Box-Behnkendesign (BBD) [23] was applied in this study to determinethe optimal level of each key determinant that was identifiedby PBD All forms of interactions among the independent


variables are shown in this design Each factorwas deliberatedin three levels denoted as minus1 (low level) 0 (center level) and+1 (high level) through fifteen experimental runs All trialswere conducted in triplicate in 250mL Erlenmeyer flaskswith a working volume of 100mL broth The averages ofthree readings were considered as the response experimentalvalues Box-Behnken design (BBD) is based on a secondorder polynomial equation

119884 = 120573119900 + 120573119899119883119899 + Σ120573119899119899119883119899 sdot 119883119899 + Σ120573119899119898119883119899 sdot 119883119898 (2)

where 119884 is the response 120573o is the model intercept 120573n is thelinear estimate 120573nn is the quadratic estimate and 120573nm is theinteraction estimate

28 Statistical Analysis of Data PBD and BBD were cre-ated by Minitab version 150 Multiple regressions canon-ical analysis and three-dimensional surface plots wereperformed via RSM package (R development Core team2009) available from the Comprehensive R Archive Network(httpCRANR-projectorgpackage=rsm) [24]

29 Bacteriocin Bioassay and Its Inhibition Spectrum Theantimicrobial effect of bacteriocin against the abovemen-tioned indicator strains was estimated usingwell cut diffusionassay Briefly 100mL agar medium specific for each indicatorstrain (bacteria or yeast) separately was seeded with 1mLof an overnight grown culture For fungal strains 1mL offungal spore suspension was used to inoculate 100mL agarmedium specific for fungi After that the seededmediumwaspoured into plates After solidification wells were created inplates One hundred microliters of the bacteriocin producingculture was centrifuged at 8000 rpm for 3min (Mikro 200Hettich Germany) Each well was injected with 100 120583L of thecell-free supernatant containing the crude bacteriocin Theplates were incubated at 37∘C for 1ndash3 days The activity ofthe examined bacteriocin was expressed in arbitrary absoluteunits (AU) One AU is defined as inhibition zone diameterin mm produced by the tested bacteriocin on the bacterialindicator lawn [25] AUmL = inhibition diameter zone inmmsdot1000volume of cell-free supernatant in 120583L Here thebacterial indicator lawn involved in assessment of bacteriocinactivity was E amylovora

210 Partial Characterization of Bacteriocin The effect ofdifferent pH(s) and temperatures on the activity of theproduced bacteriocin was examined Moreover the influenceof some organic solvents (eg acetone acetonitrile toluenemethyl alcohol and ethyl alcohol) on the activity of thetested bacteriocin was also studied Furthermore the impactof proteinase K on bacteriocin activity was examined toprove the proteinaceous nature of the claimed bacteriocinsubstance

211 Monitoring the Production of Bacteriocin Productionof bacteriocin was monitored along with the growth ofthe producer isolate Briefly 100mL of BYP broth wasinoculated with 05mL of an overnight grown culture The

newly inoculated broth was incubated at 37∘C with agitationspeed of 200 rpm (New Brunswick Incubator Shaker USA)One mL samples were withdrawn from the culture eachhour tomeasure the bacterial growth spectrophotometricallyat 420 nm (UV-Vis Spectronic helios spectrophotometerThermo Scientific Co USA)Then the cell-free supernatantsobtained via centrifugation at 7000 rpm for 3min (Mikro 200Hettich Germany) were kept at 4∘C until their usage in wellcut diffusion assay to determine the bacteriocin activity asmentioned above in Section 29

3 Results

31 Bacillus sp YAS 1 Strain In the course of a screeningprogramme for bacteriocin producers eighty-seven samples(22 of fruits (F) 15 of leaves (L) and 50 of soils (S))from different sites naturally infected with the pear fireblight disease were collected to isolate bacteriocin producingbacteria Preliminary screening implicated only six bacterialisolates nominated as F2 L18 S4 S9 S25 and S33 provingtheir capabilities for bacteriocin production through toothpicking technique andwell diffusion assay versus the pear fireblight pathogen E amylovora as shown in Table 1 The largestzone of inhibition (228 plusmn 25) was achieved upon usingthe antimicrobial agent of the isolate S9 The most potentproducer (S9) was selected to conduct the current studyMor-phological and biochemical identifications of the bacteriocinproducer unraveled that this isolate was assigned to Bacillussp (Table 2 and Figure 1) Furthermore this identificationwas verified via alignment of 16S rDNAsequence of the isolateS9 using BLASTN algorithm Results of BLASTN demon-strated that the query sequence has 99 identity and 100query coverage with the 16S rDNA of the bacterium recordedin the GenBank under the accession number HQ6009941Phylogenetic tree illustrated in Figure 2 inferred the degreeof relatedness between 16S rDNA sequence of the isolateS9 with other closely related 16S rDNA sequences depositedin the international nucleotide sequences databases (egGenBank EMBL DDBJ etc) The biochemical taxonomyfor the bacterial isolate (S9) was in a good agreement withthe molecular taxonomy Based on these data our bacterialisolate (S9) was nominated as Bacillus sp YAS 1 strainAdditionally the nucleotide sequence of 16S rDNA gene ofBacillus sp YAS 1 strain was submitted in the GenBank andwas given an accession number KM111594

32 Optimizing of Bacteriocin (BAC YAS 1) Production Thebacteriocin produced from Bacillus sp YAS 1 strain wasnominated as BAC YAS 1 Its production was optimizedthrough a two-step optimization procedure

321 PBD Results The matrix of PBD and the experimentalvalues versus the predicted values of the process outcomewere displayed in Table 3(a) whilst coded real values ofthe independent variables and results of linear multipleregression were illustrated in Table 3(b) The 119865 and 119875 valuesof the model were 5758 and 0017 respectively as elucidatedfromANOVAresults Significance of themodel was indicated


Table 1 Sensitivity of E amylovora to antimicrobial agents produced by six bacterial isolates

Bacterial isolateScreening method F2 L18 S4 S9 S25 S33Toothpick technique (mm)a 130 70 77 143 40 173Well cut diffusion method (mmbc

plusmn SEd) 63 plusmn 117 Nil 193 plusmn 18 228 plusmn 25 77 plusmn 18 156 plusmn 188abmm = diameter of inhibition zone cmean of three readings dstandard error

(a)133120583m

046120583m

(a)

(b)

(b)

(c)

(c)

(d)

(d)

(e)

(e)

Figure 1 Electron micrographs ((a) SEM and (b)ndash(d) TEM) and (e) light micrograph of Bacillus sp YAS 1 strain

gb|KJ9441051| Bacillus sp M

|gb|KM0198551| Bacillus cere

|gb|KM0198331| Bacillus cere98

100






|gb|KJ9441141| Bacillus sp762

737

|KJ5344961| Bacillus cere|gb

|gb|KJ5344961| Bacillus cere725







|gb|KM1115941| Bacillus sp955 gb|HQ6009941| Bacillus sp I

955|gb|

|gb|

JN9751011| Bacillus sp B955

533KJ9441431| Bacillus sp

908 |gb|KJ9441421| Bacillus sp

|gb|KJ9441421| Bacillus sp

40E minus 4

4

u

u

u

u

u

u

u

u

u

u

u

u

u

u

u

L

L

L

L

Y

D

7

Figure 2 Neighbor-joining tree showing the phylogenetic relationship between 16S rDNA sequence of the candidate bacterial isolate S9(taxonomically nominated as Bacillus sp YAS 1 strain) and other 16S rDNA sequences belonging to closely related bacteria Phylogenetic treewas constructed via Geneious R8 software Numbers on branch nodes represent bootstrap values (1000 resamplings)


Table 2 Morphological and biochemical identification of S9 bacte-riocin producing bacterial isolate

Test ResultCell shape RodsGram stain +veSpore formation +veGrowth on MYP Non-mannitol utilizerMotility +veGrowth at pH 6ndash8 +veGrowth at 50∘C +veGrowth at 75 NaCl minusveStarch hydrolysis +veCasein hydrolysis +veTween 20 hydrolysis minusveNitrate reduction +veCatalase +veOxidase +veUrease minusveIndole production minusveCitrate utilization minusveVoges-Proskauer (VP) +veMethyl red (MR) minusveGelatin liquefaction +veHemolysis on sheep blood agar 120573-HemolyticLecithinase on MYP +vePenicillin resistance +veOxidative fermentation (OF)

Glucose fermentation +ve (yellowblue ONF)lowast

Galactose fermentation +ve (yellowblue ONF)Sucrose fermentation +ve (yellowyellow OF)lowastlowast

lowastONF oxidativenon-fermentativelowastlowastOF oxidativefermentative

by this 119865 value while the model 119875 value reflects that thechance is only 17 that this model 119865 value could occur dueto noise Significance of coefficients was evaluated by lookingat 119875 value of the estimate As a rule of thumb significanceof an estimate is inversely proportional to its 119875 value anddirectly proportional to its 119905-test [26] Based on data derivedfrom linear multiple regression there exist five independentvariables (incubation time agitation speed glycerol starchand yeast extract) provoking significant consequences onBAC YAS 1 productivity Only three (119883

1 incubation time

119883

5 agitation speed and 119883

9 yeast extract) out of the five

independent variables imposed positive significance on theprocess outcome whilst the other two remaining indepen-dent variables (119883

6 glycerol and119883

7 starch) showed dramatic

negative significant impact Pareto chart was applied hereas an informative tool to display the significance conse-quences of the nine investigated independent variables on theprocess outcome in a descending order according to their119875 values (Figure 3) After exclusion of the non-significantmodel terms a first order polynomial equation in terms ofcoded values was established to explain the linear relation

0

5

10

15

20

25

30

Effec

t (

)

X1

(incu

batio

n tim

e)

X5

(rpm

)

X7

(sta

rch)

X9

(yea

st ex

trac

t)

X6

(gly

cero

l)

X3

(pH

)

X4

(incu

batio

n te

mpe

ratu

re)

X8

(inoc

ulum

size

)

X2

(glu

cose

)

Figure 3 Pareto chart in a descending layout for Plackett-Burmanparameter estimates of nine tested independent variables

between the independent variables and the process outcomeas follows

119884 = 3358 + 5416119883

1+ 3417119883

5

minus 175119883

6minus 2416119883

7+ 225119883

9

(3)

Independent variables demonstrating non-significantimpact on the process outcome were either used in themedium at their lowest values (119883

3 pH 119883

4 incubation

temperature and 1198838 inoculums size) or excluded from the

medium (1198832 glucose) in the next experiments On the

contrary independent variables (1198836 glycerol and119883

7 starch)

presenting dramatic negative significant effects were omittedfrom the production medium in the next experimentsHowever incubation time (119883

1) agitation speed (119883

5) and

yeast extract (1198839) were identified as three key determinants

controlling BAC YAS 1 productivity and were further sub-jected to detailed analysis through RSM approach

322 RSM Results RSM is an empirical mathematical toolused in optimization processes purposes in order to locatethe optimal level of a set of controllable factors along with themaximal level of the process outcome Here Box-Behnkendesign was applied The design matrix and the experimentalversus the predicted levels of BAC YAS 1 were shown inTable 4(a) Coded real values of the tested independentvariables and results of multiple non-linear regression weredisplayed in Table 4(b) ANOVA results inferred that themodel 119865 value and 119875 value were 999 and 001 respectivelyThese values implied the significance of the model and thechance (1) that this 119865 value could occur due to noise Modelaptness is evidenced by the value of 1198772 (095) that conferredthe good agreement between the experimental and thepredicted values of the response A second order polynomialequation was fixed in terms of coded values to explain all


Table 3 (a) PBD with coded levels of nine independent variables and experimental versus predicted levels of BAC YAS 1 (b) Real-codedvalues of independent variables and regression analysis for BAC YAS 1 optimization by PBD

(a)

Trial number 119883

1

119883

2

119883

3

119883

4

119883

5

119883

6

119883

7

119883

8

119883

9

119884 (AUmL CFS)lowast

Expa Predb

1 minus1 minus1 1 1 1 minus1 1 1 minus1 290 296672 minus1 minus1 minus1 minus1 minus1 minus1 minus1 minus1 minus1 250 248333 minus1 1 1 1 minus1 1 1 minus1 1 220 218334 minus1 1 1 minus1 1 minus1 minus1 minus1 1 340 341675 1 minus1 minus1 minus1 1 1 1 minus1 1 380 386676 1 1 minus1 1 1 minus1 1 minus1 minus1 420 413337 1 minus1 1 minus1 minus1 minus1 1 1 1 360 353338 minus1 minus1 minus1 1 1 1 minus1 1 1 390 383339 1 minus1 1 1 minus1 1 minus1 minus1 minus1 320 3216710 1 1 minus1 1 minus1 minus1 minus1 1 1 460 4666711 1 1 1 minus1 1 1 minus1 1 minus1 400 3983312 minus1 1 minus1 minus1 minus1 1 1 1 minus1 200 20167aExperimental values and bpredicted values [119883

1 incubation time 119883

2 glucose 119883

3 pH 119883

4 incubation temperature 119883

5 agitation speed 119883

6 glycerol 119883

7

starch1198838 inoculum size and119883

9 yeast extract] lowastAUmL sample arbitrary units as a measure for bacteriocin activity = diameter of inhibition zone in mm

1000volume of CFS in 120583LlowastCFS cell-free supernatant

(b)

Independent variable Low level High level Main effect 119861-coefficient 119875 value 119905-value confidenceminus1 +1

Incubation time (X1 hrs) 24 48 10834 5417 0004lowast 1576482 996lowast

Glucose (1198832

wv) 1 2 833 4167 0349 1212678 651pH (119883

3

) 65 75 minus2833 minus14167 00541 minus412311 9459Incubation temperature (119883

4

∘C) 28 30 2833 14167 00541 4123106 9459Agitation speed (X5 rpm) 120 200 6833 34167 00099lowast 9943961 9901lowast

Glycerol (X6 wv) 0 1 minus3500 minus175 00363lowast minus509325 9637lowast

Starch (X7 wv) 0 1 minus4833 minus24167 00196lowast minus703353 9804lowast

Inoculum size (1198838

vv) 10 12 2833 14167 0054095 4123106 9459Yeast extract (X9 gL) 3 5 4500 22500 00225lowast 6548462 9775lowastlowastSignificant 119875 value lt005 1198772 = 099 adjusted 1198772 = 098 and 119875 value for the model = 0017

possible interactions among the tested independent variablesthat could impose an effect on the response

119884 = 4433 + 25119883

1+ 3125119883

5minus 875119883

9minus 2166119883

1sdot 119883

1

minus 9416119883

5sdot 119883

5minus 13417119883

9119883

9minus 325119883

1sdot 119883

5

+ 425119883

1sdot 119883

9+ 5119883

5sdot 119883

9

(4)

Anchored in results of multiple non-linear regression onlytwo model terms displayed significant effect (119875 lt 005) onBACYAS 1 productivityThere exist quadratic effects imposedby agitation speed and yeast extract on the process outcome

To attain the optimized conditions canonical analysiswasperformed Determining the overall of the response shapeand the nature of the stationary point whether it ismaximumsaddle orminimum could be achieved via canonical analysisEigenvalues and eigenvectors in the matrix of second orderare used to characterize the response shape Eigenvectors areused to determine the directions of principle orientation for

the surface However signs and magnitude of eigenvaluesindicate surface shape in these directions Meyer 1976 statedtwo rules concerning the concept of eigenvalues and theirmathematical designations [27] Upward and downwardcurvatures are proved by positive and negative eigenvaluesrespectively (1st rule) The larger an eigenvalue is in itsabsolute value the more pronounced is the curvature of theresponse in the associated direction (2nd rule) Our modelindicated that it has the eigenvalues (120582

1= minus144 120582

5= minus976

and 1205829= minus1381) Based on 1st rule of Meyer the present

negative eigenvalues conferred that the stationary point ismaximum The two largest eigenvalues in their absolutevalues (120582

5= 976 and 120582

9= 1381) reflected the pronounced

curvature of the response in the directions of the independentvariables (119883

5and 119883

9) This finding authenticated to a great

extent the results of regression analysis that stated that1198835and

119883

9showed the highest significant effect in quadratic forms

on the level of BAC YAS 1 The predicted stationary point asderived from canonical analysis along with differentiation of


Table 4 (a) BBDwith coded levels of three independent variables and experimental versus predicted BACYAS 1 levels (b) Real-coded valuesof independent variables and regression analysis for BAC YAS 1 optimization by BBD

(a)

Trial number 119883

1

119883

5

119883

9


Expa Predb

1 minus1 minus1 0 220 238752 1 minus1 0 400 366253 minus1 1 0 320 353754 1 1 0 370 351255 minus1 0 minus1 300 313756 1 0 minus1 210 211257 minus1 0 1 280 278758 1 0 1 360 346259 0 minus1 minus1 220 1875010 0 1 minus1 200 1800011 0 minus1 1 240 2600012 0 1 1 200 2325013 0 0 0 430 4433314 0 0 0 450 4433315 0 0 0 450 44333aExperimental values and bpredicted values1198831 incubation time119883

5 agitation speed and119883

9 yeast extract

lowastAUmL sample arbitrary units as a measure for bacteriocin activity = diameter of inhibition zone in mm 1000volume of CFS in 120583LlowastCFS cell-free supernatant

(b)

Variable Low level Middle level High level Model term Main effect 119861-coefficient 119905-value 119875 value confidenceminus1 0 +1

Incubation time(1198831

hrs) 24 48 72 119883

1

5000 25 165144 0159558 840

Agitation speed(1198835

rpm) 80 200 320 119883

5

6250 3125 007506 0943073 569

Yeast extract(1198839

gL) 2 5 8 119883

9

minus1750 minus875 112598 0311295 6887

119883

1

sdot 119883

1

minus4334 minus2167 minus088395 0417196 5828119883

5

sdot 119883

5

minus18834 minus9417 minus384178 0012102lowast 9879lowast

119883

9

sdot 119883

9


119883

1

sdot 119883

5


1

sdot 119883

9

8500 4250 180470 0130962 8690119883

5

sdot 119883

9

minus1000 minus500 minus021232 0840243 1598lowastSignificant 119875 value lt005 1198772 = 095 adjusted 1198772 = 085 119875 value for the model = 001

(4) was found to be at coded values 1198831= 059 119883

5= 006

and 1198839= minus0059 to achieve a predicted level of BAC YAS

1 of 470AUmL This predicted stationary point is obviouslylocated inside the model domain (model constrains)

Three-dimensional surface plots were depicted to furtherexplore the nature of the response at the stationary point asshown in Figures 4(a) 4(b) and 4(c) Normally the three-dimensional surface plots are based on the model keepingone independent variable constant at its optimal level wherechanging the other two independent variables within thedesign constrains

Figure 4(a) illustrated the response of the dependentvariable (BACYAS 1) for the optimal level of the independentvariable yeast extract The maximal predicted level of BACYAS 1 (470AUmL) was noticed at levels of 62 hrs and207 rpm Supportively the three-dimensional surface plotdepicted in Figure 4(b) revealed that the maximal point ofBAC YAS 1 at the optimal level for agitation speed could bereached at 62 hrs and 048 (wv) yeast extract Likewisethese predicted levels of both dependent and the independentvariables were further proved by the three-dimensional sur-face plot illustrated in Figure 4(c)


BAC

YAS 1

activ

ity (A

Um

L)

X5 (agitation speed)

X1(incubation tim

e)

480

420

360

300

240

12

12

08

08

04

04

minus04minus04

minus08minus08

minus12minus12

gt4142849

lt4142849

lt3908564

lt3674279

lt3439995

lt320571

lt2971425

lt273714

lt2502855

lt226857

lt2034285

minus29802E minus 8minus29802E minus 8

(a)

BAC

YAS1

activ

ity (A

Um

L)

X9 (yeast extract)

X1(incubation tim

e)minus29802E minus 8

minus29802E minus 8

480

420

360

300

240

12

12

08

08

04

04

minus04minus04

minus08minus08

minus12minus12

gt4193656

lt4193656

lt395429

lt3714925

lt3475559

lt3236194

lt2996828

lt2757462

lt2518097

lt2278731

lt2039366

(b)

BAC

YAS 1

activ

ity (A

Um

L)

480

420

360

300

240

12

12

08

08

0404

minus04minus04

minus08minus08

minus12minus12


X9 (yeast extract)

X5(agitation speed)

gt4185689

lt4185689

lt394712

lt3708551

lt3469982

lt3231413

lt2992845

lt2754276

lt2515707

lt2277138

lt2038569

(c)

Figure 4 (a) Three-dimensional surface plot for the dependent variable BAC YAS 1 antimicrobial activity versus the independent variablesincubation time and agitation speed (b) Three-dimensional surface plot for the dependent variable BAC YAS 1 antimicrobial activity versusthe independent variables incubation time and yeast extract (c) Three-dimensional surface plot for the dependent variable BAC YAS 1antimicrobial activity versus the independent variables agitation speed and yeast extract


A

B

Figure 5 Antimicrobial activity of BACYAS 1 on E amylovora uponusing non-optimized conditions (A) and optimized conditions (B)

0

50

100

150

200

250

300

350

0010

0100

1000

10000

0 1 2 3 4 5 6 7 8 9 10

BAC

YAS

1 ac

tivity

(AU

mL)

Bact

eria

l gro

wth

Ab

at 420

nm

Incubation time (hrs)

Figure 6 The BAC YAS 1 production time course vs the growth ofBacillus sp YAS 1 strain on bacteriocin optimized medium Symbol998771 bacterial growth Symbol e BAC YAS 1 activity (AUmL) uponusing the indicator strain E amylovora

323 Model Validation In order to determine model ade-quacy the predicted levels of incubation time agitationspeed and yeast extract were fully considered in the labora-toryThe experiment was run in triplicate Data indicated thatthe maximal obtained level of BAC YAS 1 was 470 (AUmL)The obtained data conferred the high precision of model100 (ie good agreement between the predicted and theexperimental levels of BAC YAS 1) Furthermore by the endof the optimization plan the level of BACYAS 1was enhanced16-fold that was obtained upon using the non-optimizedmedium as shown in Figure 5

33 BAC YAS 1 Production Time Course versus BacterialGrowth Production of BAC YAS 1 during the growth ofBacillus sp YAS 1 strain was monitored Figure 6 demon-strated that the first onset of BAC YAS 1 was observed afterfive hrs of the bacterial growth (ie late exponential phase)

Table 5 Effect of temperature on BAC YAS 1 antimicrobial activity

Temperature (∘C) Residual activitylowast

Control (BAC YAS 1 without treatment) 10045∘C 10060∘C 10075∘C 8380∘C 3685∘C 00lowastResidual activity = AU after BAC YAS 1 treatmentAU before BAC YAS 1treatmentTreatment 15min exposure to each indicated temperature

Increased levels of BAC YAS 1 were recorded during thestationary phase of bacterial growth

34 Partial Characterization of BACYAS 1 Theantimicrobialactivity of the crude BAC YAS 1 was completely lost aftertreatment with proteinase K (data not shown) Total inacti-vation of the antimicrobial activity by proteinase K greatlyaffirms the proteinaceous nature of BAC YAS 1 On the otherhand BACYAS 1 couldmaintain its full antimicrobial activityover a wide range of pH (3ndash12) A dramatic decrease inBAC YAS 1 antimicrobial activity was noticed only at theextreme acidic or alkaline pH(s) (1 2 and 13) Additionallypresent data revealed a good stability of BAC YAS 1 over awide range of pH (1ndash13) along five hrs of incubation at theindicated pH(s) Pertaining to the effect of temperature onantimicrobial activity of BAC YAS 1 (Table 5) full activity ofBAC YAS 1 was detected after 15min of exposure at 60∘Cwhilst BAC YAS 1 could preserve 83 of its activity at 75∘Cafter 15min of heat exposure A dramatic decrease in BACYAS 1 activity was observed after exposure to temperaturesgreater than 75∘CComplete loss of activity was noted at 85∘CConcerning the effect of some organic solvents onBACYAS 1none of the tested organic solvents stated an inhibitory effecton the BAC YAS 1 activity

35 Antimicrobial Spectrum of BACYAS 1 BACYAS 1 offeredantimicrobial activity against some Gram-negative humanpathogens (eg S typhimurium E aerogenes Proteus spKlebsiella sp Enterococcus sp and C jejuni) Gram-positivehuman pathogens (eg C perfringens and S epidermidis)a food spoilage bacterium (eg L innocua) and a plantpathogen bacterium (E amylovora) as well while the growthof other human pathogens (S aureus S typhi S paratyphiand S boydii) had not been affected by BAC YAS 1 antimicro-bial activity Among the examined indicator strains the mostsensitive strains to BAC YAS 1 could be listed in the followingorder regarding the inhibition zone diameter E amylovoraL innocua Klebsiella sp and S epidermidis Additionallyno antimicrobial activity of BAC YAS 1 could be detectedagainst E coli and E faecalis Regarding the influence ofBAC YAS 1 on some environmental bacterial strains thegrowth of only two strains belonging to the genus Bacillus(B licheniformis SHG2 and B subtilis AS1) was inhibited byBAC YAS1 Conversely none of the tested fungal and yeast


strains were influenced by BAC YAS 1 antimicrobial activitySurprisingly none of the tested lactic acid bacterial strains(Lactobacillus bulgaricus L casei L lactis and L reuteri)were affected by BAC YAS 1 antimicrobial activity as shownin Table 6

4 Discussion

Although the literature reported the production of bacteri-ocins from bacteria belonging to different genera and speciessuch as lactic acid bacteria Bacillus spp Escherichia coli andVibrio spp a few reports exist concerning the key determi-nants controlling the levels of the produced bacteriocins fromthese bacteria Additionally the bottleneck for bacteriocinproduction frommost bacterial strains is the low yield In thiscontext optimizing BAC YAS 1 production is the first step inthe agenda of large scale production Here the productionof BAC YAS 1 was affected by neither soluble starch norinitial pH of the production medium The present finding isinconsistent with other findings of Zhong et al [28] Pal et al[29] andKaur et al [30] where the production of bacteriocinfromB cereusXH25Weissella paramesenteroidesDFR-8 andPediococcus acidilactici BA28 respectively was significantlyinfluenced by soluble starch and initial pH of the productionmedium However present finding concerning the impactof yeast extract on BAC YAS1 was in good agreement withthose of Zhong et al and Meera and Devi who stated thatyeast extract did exert significant effect on the productivityof a bacteriocin-like substance from B cereus XH25 andprobiotic lactic acid bacteria respectively [28 31] Pertainingto the influence of glycerol on BAC YAS 1 productivity itdid not exhibit any significant consequence The presentfinding was in discordance with those of Yi et al [32] andKamoun et al [33] where addition of glycerol resulted in anincrease in the productivity of bacteriocin from Lactobacillusparacasei J23 and Bacthuricin F4 (bacteriocin) produced byB thuringiensis subsp kurstaki strain respectively Glucosewas one of the noninfluential factors exerted on productivityof BAC YAS 1 whilst glucose exerted a significant impact onthe productivity bacteriocin fromWparamesenteroidesDFR-8 [29] Bacillus sp GU057 [34] and Bacthuricin F4 (bac-teriocin) produced by B thuringiensis subsp kurstaki strain[33] With respect to optimal temperature for bacteriocinproduction there exists a discrepancy in optimal temperatureto produce bacteriocin from various bacteria [35ndash37] Theobserved discrepancy in key determinants controlling theproductivity of various bacteriocins produced by differentbacteria greatly addresses the indispensable need for carryingout optimization step for each newly isolated bacteriocinproducer Alleviation of the overall cost for a bioprocesscommercialization is considered one of the great challengesNonetheless the overall cost of a bioprocess is usuallyconstricted to the high cost of productionmedium BACYAS1 production medium is somehow simple and cost effectiveupon comparison with other media required for bacteriocinsproduction from other bacterial sources (eg MRS mediumplus other supplements required for bacteriocin productionfrom lactic acid bacteria) Therefore the cost encountered in

BAC YAS1 production will alleviate This in turn gives BACYAS 1 productivity from Bacillus sp YAS 1 a privilege overproductivity of other bacteriocins fromother bacteriaHencethe overall cost for BAC YAS 1 production will reduce Fromanother side the timeline for BAC YAS 1 production fromBacillus sp YAS1 strain is 62 hrs which is to some extent not aprolonged time Subsequently this will create a rapid processupon scaling up the production of BAC YAS1

Determining the onset of the genes encoding for bac-teriocin is one of the prerequisite data in the agenda ofbacteriocin production on an industrial scale Thus BACYAS 1 production was monitored along with the growthof its producer strain Bacillus sp YAS 1 in the bacteriocinproductionmediumThe onset of BACYAS 1 production wasrecorded to be at the late exponential phase and increasedduring the stationary phase of growth reaching its maximumafter 62 hrs of cultivation This finding was in disagreementwith other reported findings [29 38ndash41] For instance theproduction of bacteriocin (Bac14) by B subtilis 14B startedafter a 24-h-lag phase and then increased exponentially andreached its maximum within 96 h cultivation [38] BAC YAS1 production was not associated with the growth of Bacillussp YAS 1 strain because the maximum yield of BAC YAS 1was achieved during the stationary phaseThe present findingis partially in agreement with that of cerein from B cereuswhere cerein could be traced at the beginning of stationaryphase [1 39] On the other hand the present finding wascontradicted by Pal et al and Banerjee et al who affirmedthat the bacteriocin ofW paramesenteroides DFR-8 [29] andthat of L brevis FPTLB3 [40] were growth-dependent sincemajority of the bacteriocin was traced during the exponentialphase of growth Activity of cerein 7 was detected in culturesof B cereus Bc7 near the end of exponential phase andreached its maximum at the beginning of stationary phase[41] It seems that the onset of bacteriocins encoding genesvaries widely not only among distally related species andgenera but also among strains belonging to the same specieswhich necessitates the indispensable need to explore the exacttime at which each bacteriocin encoding gene turns on ineach newly isolated producer strain

To gain more insights into BAC YAS 1 partial character-ization was performed as mentioned above Bacteriocins bydefinitions are sensitive to the action of proteolytic enzymesSensitivity of BAC YAS 1 to the action of proteinase Kconfers its proteinaceous nature and agrees well with thereported sensitivity of other bacteriocins to proteinase K[26 40 42 43] In contrast the temperature stability profilefor various bacteriocins [39 42 44] was somehow compa-rable to that of BAC YAS 1 For instance the temperaturestability profile (45∘Cndash75∘C) for cerein 8A and cerein ofB cereus [1 5] was in partial agreement with that of thepresent finding Conversely Bac14B of B subtilis [38] exertedcomplete higher thermostability over a broad range (30∘Cndash100∘C) for two hrs when compared to that of BAC YAS1 over a broad range (45∘Cndash80∘C) for 15min Moreoverbacteriocin from Propionibacterium thoeniit was found tobe heat labile [45] Regarding optimal pH and pH stabilityof BAC YAS 1 present findings were somehow consistentwith some reported bacteriocins derived from either closely


Table6Sensitivityof

someind

icator

strainstoBA

CYA

S1

Indicatorstrain

BACYA

S1a

ctivity

(inhibitio

nzone

diam

eterm

mplusmnSE

)lowastGram

nature

Bacteria

Acinetobactersp(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty

ofMedicineUniversity

ofAlexand

riaE

gypt)

000

Gram-negative

Bacilluscereus(environm

entalstrainiso

latedfro

msoilEg

ypt)

000

Gram-positive

Blicheniform

isSH

G2(enviro

nmentalstrainiso

latedfro

msoilEg

ypt)

13plusmn17

0Gram-positive

Blicheniform

isSH

G6(enviro

nmentalstrainiso

latedfro

msoilEg

ypt)

000

Gram-positive

Blicheniform

isSH

G10

(enviro

nmentalstrainiso

lated

from

soilEg

ypt)

000

Gram-positive

BsubtilisE

MBL

AKE

(enviro

nmentalstrainiso

lated

from

El-M

ahmou

diaL

akeEg

ypt)

000

Gram-positive

BsubtilisA

S1(enviro

nmentalstrainiso

lated

from

soilEg

ypt)

1025plusmn048

Gram-positive

BsubtilisN

CTC1040

0AT

CC6633

000

Gram-positive

Bsubtilis(environm

entalstrainiso

latedfro

msoilEg

ypt)

000

Gram-positive

BacillusspAs

h2(enviro

nmentalstrainiso

lated

from

soilEg

ypt)

000

Gram-positive

Campyloba

cter

jejuni

NCT

C11322AT

CC29

428

1400plusmn041

Gram-negative

Clostridium

perfrin

gens

NCT

C82

37ATC

C13124

1550plusmn15

Gram-positive

Citro

bacte

rsp(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Enteroba

cter

aerogenesN

CTC1

0006

ATC

C130

431550plusmn046

Gram-negative

Enterobacter

sp(hu

man

clinicalisolateIDepartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Enterobacter

sp(hu

man

clinicalisolateIID

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Enterococcus

sp(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

1375plusmn035

Gram-negative

Ecoli(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

EcoliNCT

C12241A

TCC25922

000

Gram-negative

Enterococcus

faecalisNCT

C12697AT

CC29212

000

Gram-negative

Eam

ylovora(D

epartm

ento

fPlant

Patholog

yFaculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

4100plusmn043

Gram-negative

Klebsie

llasp(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

295plusmn04

Gram-negative

Lactobacillus

bulga

ricus

(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Lcasei(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Llactis(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Lreuteri(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Liste

riainno

cuaNCT

C1128

8AT

CC3309

03775plusmn049

Gram-positive

Pseudomonas

aeruginosa

strain

EGYII

000

Gram-negative

Paeruginosa

(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Proteussp

(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

1400plusmn03

Gram-negative

Salm

onellatyph

imurium

NCT

C1202

3AT

CC

1402

81725plusmn049

Gram-negative

Styphi(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Sparatyphi(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

ShigellaboydiiAT

CC9207

000

Gram-negative

Staphylococcus

aureus

NCT

C12981A

TCC25923

000

Gram-positive

Stap

hylococcus

epidermidisNCT

C1336

0AT

CC1222

82375plusmn075

Gram-positive


Table6Con

tinued

Indicatorstrain

BACYA

S1a

ctivity

(inhibitio

nzone

diam

eterm

mplusmnSE

)lowastGram

nature

Vibrioparahaem

olyticu

sNCT

C3178

000

Gram-negative

Fung

iand

yeast

Aspergillus

brasiliensis

NCP

F2275ATC

C1640

4000

Cand

idaalbicans

NCP

F3179ATC

C10231

000

Ctro

picalis

(hum

anclinicalisolatefro

mdiabeticfootD

epartm

ento

fMicrobiolog

yMedicalRe

search

Institu

teU

niversity

ofAlexand

riaE

gypt)

000

Saccharomycescerevisia

eNCP

F3178

000

lowast

Meanof

four

readings

with

stand

arderror(SE

)


related bacterial genera or other bacteriocins from otherdistally related bacterial genera andor species [1 31 39 4245] For example Naclerio et al [39] stated that cerein of Bcereus exhibited pH stability profile (3ndash12) quite similar tothat of BACYAS 1 Nevertheless Kayalvizhi andGunasekaranreported a pH stability profile (3ndash10) of bacteriocin from BlicheniformisMKU3 [42] Bacteriocins ofB subtilis 14B and Lbrevis FPTLB3 demonstrated lower pH stability range (3ndash8)[38 40] This reported divergence in thermostability and pHstability broadened by bacteriocins from different bacteria ismainly attributed to their amino acids structures that nec-essarily impose a strong influence on their physicochemicalpropertiesThis highlights the substantial necessity to unravelthe amino acid structure of the BAC YAS 1 under studyFurther characterization of BAC YAS 1 implied its ability toretain the full activity in presence of some tested organicsolvents (eg chloroform acetone ethyl alcohol acetonitrileand methyl alcohol) Sensitivity of bacteriocin to organicsolvents differs among different bacteriocins from differentbacteria [1 38 39 42] This retained activity in presence ofthe abovementioned organic solvent may give a hint aboutthe hydrophobic nature of BAC YAS 1 Additionally retainedactivity of BAC YAS 1 under a wide range of pH could beattributed to the type of amino acid residues of BAC YAS1 Consequently these two findings profoundly address theindispensable need to study the amino acid composition ofBAC YAS 1 in the future

Bacteriocins by definitions are antimicrobial agents andthose that demonstrate wide spectrum antimicrobial activityagainst both Gram-negative and Gram-positive bacteria areof a prime importance as chemotherapeutic and prophy-lactic agents BAC YAS 1 demonstrated a broad spectrumantimicrobial activity against bothGram-negative andGram-positive bacteria as mentioned above The antimicrobialspectrum activity of BAC YAS 1 exhibited a similar patternto that obtained by bacteriocins from the lactic acid bacteria(L plantarum and L fermentum) where their bacteriocinsexerted a profound antimicrobial effect against some Gram-positive bacteria (eg S aureus and Streptococcus pneumo-niae) and Gram-negative bacteria (eg E coli P aeruginosaKlebsiella and Proteus) [46] In contrast the BAC YAS 1antimicrobial spectrum did not agree with other bacteriocins[41 47] Bacteriocins of B cereus Bc7 and lactic acid bacteria(eg L lactis and P acidilactici) inhibited a broad spectrumof Gram-positive bacteria including food-borne pathogensbut not any of the tested Gram-negative bacteria [41 47]In this context cerein of B cereus displayed antimicrobialeffect against only closely related Bacillus spp [1] On topand above the tested lactic acid bacterial species have notbeen influenced by the antimicrobial activity of BAC YAS1 This finding in turn gives BAC YAS 1 a privilege overother bacteriocins reported in the literature and underpins itspotential usage as a food biopreservative without killing thebeneficial lactic acid bacteria included in foodstuffs

5 Conclusion

In a nutshell employment of optimization strategies in bio-processes particularly for bacteriocin industry is considered

to be an essential step It aims not only to minimize the costencountered in medium compositions but also to maximizethe yield as well By the end of sequentially statistical opti-mization procedure we could end up with appreciable levelsof the bacteriocin (470AUmL) after 62 hrs of the growth ofBacillus sp YAS 1 strain under optimized conditions againstE amylovora Conclusively BAC YAS 1 of Bacillus sp YAS1 strain is a promising bacteriocin that could offer numer-ous biotechnological applications in wide variety of fieldsagricultural medical and industrial ones as a biocontrolagent against the fire blight plant pathogen E amylovoraas an antibiotic agent against the human pathogens (egC perfringens S epidermidis S typhimurium E aerogenesC jejuni Enterococcus sp Proteus sp and Klebsiella sp)and as a food biopreservative against the food spoiler Linnocua On top and above absence of BAC YAS 1 inhibitoryeffect towards the beneficial lactic acid bacteria along withits potent inhibitory effect against the food spoiler L innocuadonates BACYAS 1 a privilege over other bacteriocins derivedfrom other bacteria

The present data exceedingly prompt applying directedevolution methodologies in order to improve the character-istics of the promising broad spectrum antimicrobial activityof BAC YAS 1 in the future work In this context improvingthermostability of the BAC YAS 1 over a wide range of tem-perature degrees along with prolonged exposure times is anissue of a prime importance if commercial industrialization ofBAC YAS 1 is being intended in the future Rational proteindesign one of the currently applied methodologies could bea powerful tool to achieve this task that in turn necessitatesthe indispensable need to purify and study the crystallinestructure of BAC YAS 1

Conflict of Interests

The authors declare that there is no conflict of interests

Authorsrsquo Contribution

Amira M Embaby put the design of most laboratory exper-iments especially those concerned with statistical modelingand identified the producer bacterial strain via molecularapproach She was responsible for writing-revision of thewhole paper statistical analysis of data scientific interpreta-tion and data discussion Yasmin Heshmat carried out mostof the laboratory experiments Ahmed Hussein participatedin data analysis results interpretation paper revision andlanguage editing Heba S Marey performed statistical anal-ysis of RSM experiment especially canonical analysis

References

[1] P A Risoslashen P Roslashnning I K Hegna and A-B KolstoslashldquoCharacterization of a broad range antimicrobial substancefrom Bacillus cereusrdquo Journal of Applied Microbiology vol 96no 4 pp 648ndash655 2004

[2] Y Sang and F Blecha ldquoAntimicrobial peptides and bacteriocinsalternatives to traditional antibioticsrdquo Animal Health ResearchReviews vol 9 no 2 pp 227ndash235 2008


[3] J Nissen-Meyer and I F Nes ldquoRibosomally synthesized antimi-crobial peptides their function structure biogenesis andmechanism of actionrdquoArchives of Microbiology vol 167 no 2-3pp 67ndash77 1997

[4] M Papagianni ldquoRibosomally synthesized peptides with antimi-crobial properties biosynthesis structure function and appli-cationsrdquo Biotechnology Advances vol 21 no 6 pp 465ndash4992003

[5] D Bizani A P M Dominguez and A Brandelli ldquoPurificationand partial chemical characterization of the antimicrobialpeptide cerein 8Ardquo Letters in Applied Microbiology vol 41 no3 pp 269ndash273 2005

[6] R Lappe A S Motta V SantrsquoAnna and A Brandelli ldquoInhibi-tion of Salmonella Enteritidis by cerein 8A EDTA and sodiumlactaterdquo International Journal of Food Microbiology vol 135 no3 pp 312ndash316 2009

[7] J C Oscariz I Lasa and A G Pisabarro ldquoDetection and char-acterization of cerein 7 a new bacteriocin produced by Bacilluscereus with a broad spectrum of activityrdquo FEMS MicrobiologyLetters vol 178 no 2 pp 337ndash341 1999

[8] A Ansari A Aman N N Siddiqui S Iqbal and S AUl Qader ldquoBacteriocin (BAC-IB17) screening isolation andproduction fromBacillus subtilisKIBGE IB-17rdquoPakistan Journalof Pharmaceutical Sciences vol 25 no 1 pp 195ndash201 2012

[9] J L Smith Structural and functional characterization of thelantibiotic mutacin 1140 [PhD thesis] University Of FloridaGainesville Fla USA 2002

[10] D Cotter C Hill and R P Ross ldquoBacterial lantibioticsstrategies to improve therapeutic potentialrdquo Current Protein ampPeptide Science vol 6 no 1 pp 61ndash75 2005

[11] J Nissen-Meyer P Rogne C Oppergard H S Haugen andP E Kristiansen ldquoStructure-function relationships of the non-lanthionine-containing peptide (class II) bacteriocins producedby gram-positive bacteriardquo Current Pharmaceutical Biotechnol-ogy vol 10 no 1 pp 19ndash37 2009

[12] M-T Lee F-Y Chen and H W Huang ldquoEnergetics of poreformation induced bymembrane active peptidesrdquoBiochemistryvol 43 no 12 pp 3590ndash3599 2004

[13] M R Yeaman and N Y Yount ldquoMechanisms of antimicrobialpeptide action and resistancerdquoPharmacological Reviews vol 55no 1 pp 27ndash55 2003

[14] R Mani S D Cady M Tang A J Waring R I Lehrer and MHong ldquoMembrane-dependent oligomeric structure and poreformation of a 120573-hairpin antimicrobial peptide in lipid bilayersfrom solid-state NMRrdquo Proceedings of the National Academy ofSciences of the United States of America vol 103 no 44 pp16242ndash16247 2006

[15] M Al-Mahrous S K Sandiford J R Tagg and M UptonldquoPurification and characterization of a novel delta-lysin variantthat inhibits Staphylococcus aureus and has limited hemolyticactivityrdquo Peptides vol 31 no 9 pp 1661ndash1668 2010

[16] S S Gauri S M Mandal B R Pati and S Dey ldquoPurificationand structural characterization of a novel antibacterial peptidefrom Bellamya bengalensis activity against ampicillin and chlo-ramphenicol resistant Staphylococcus epidermidisrdquo Peptides vol32 no 4 pp 691ndash696 2011

[17] T J Leighton and R H Doi ldquoThe stability of messengerribonucleic acid during sporulation in Bacillus subtilisrdquo TheJournal of Biological Chemistry vol 246 no 10 pp 3189ndash31951971

[18] M Fricker R Reissbrodt and M Ehling-Schulz ldquoEvaluationof standard and new chromogenic selective plating media for

isolation and identification of Bacillus cereusrdquo InternationalJournal of Food Microbiology vol 121 no 1 pp 27ndash34 2008

[19] Bergys Bergys Manual of Determinative Bacteriology 1990[20] J Sambrook E F Frisch and T ManiatisMolecular Cloning A

LaboratoryManual Cold SpringHarbor Laboratory Press NewYork NY USA 2nd edition 1989

[21] P A Eden T M Schmidt R P Blakemore and N R PaceldquoPhylogenetic analysis ofAquaspirillummagnetotacticum usingpolymerase chain reaction-amplified 16S rRNA-specific DNArdquoInternational Journal of Systematic Bacteriology vol 41 no 2pp 324ndash325 1991

[22] R L Plackett and J P Burnam ldquoThe design of optimummultifactorial experimentsrdquo Biometrika vol 33 pp 305ndash3251946

[23] G E P Box and D W Behnken ldquoSome new three level designsfor the study of quantitative variablesrdquo Technometrics vol 2 pp455ndash475 1960

[24] RDevelopment Core Team R A Language and Environment forStatistical Computing R Foundation for Statistical ComputingVienna Austria 2009 httpwwwR-projectorg

[25] P Iyapparaj T Maruthiah R Ramasubburayan et alldquoOptimization of bacteriocin production by Lactobacillussp MSU3IR against shrimp bacterial pathogensrdquo AquaticBiosystems vol 9 no 12 pp 2ndash10 2013

[26] C M Douglas Design and Analysis Experiments JohnWiley ampSons Tucson Ariz USA 5th edition 2001

[27] R H Myers Response Surface Methodology Edwards BrothersAnn Arbor Mich USA 1976

[28] X T Zhong Q Zhang K Lin et al ldquoOptimization ofbacteriocin-like substance production by Bacillus cereus XH25using response surface methodologyrdquo Research Journal ofBiotechnology vol 8 no 8 pp 42ndash48 2003

[29] A Pal K V Ramana and A S Bawa ldquoSimplification andoptimization of deMan Rogosa Sharpe (MRS) medium forenhanced production of bacteriocin by Weissella paramesen-teroidesDFR-8rdquo Journal of Food Science and Technology vol 47no 3 pp 258ndash265 2010

[30] B Kaur N Garg and A Sachdev ldquoOptimization of bacteriocinproduction in Pediococcus acidilactici BA28 using responsesurface methodologyrdquo Asian Journal of Pharmaceutical andClinical Research vol 6 no 1 pp 192ndash195 2013

[31] N S Meera and M C Devi ldquoPartial characterization and opti-mization of parameters for Bacteriocin production by ProbioticLactic acid bacteriardquo Journal of Microbiology and BiotechnologyResearch vol 2 no 2 pp 357ndash365 2012

[32] H Yi X Han Y Yang et al ldquoEffect of exogenous factorson bacteriocin production from Lactobacillus paracasei J23 byusing a resting cell systemrdquo International Journal of MolecularSciences vol 14 no 12 pp 24355ndash24365 2013

[33] F Kamoun N Zouari I Saadaoui and S Jaoua ldquoImprovementof Bacillus thuringiensis bacteriocin production through cultureconditions optimizationrdquo Preparative Biochemistry and Biotech-nology vol 39 no 4 pp 400ndash412 2009

[34] A AminM Ayaz KhanM Ehsanullah U Haroon SMuham-mad Farooq Azam and A Hameed ldquoProduction of peptideantibiotics by Bacillus SP GU 057 indigenously isolated fromsaline soilrdquo Brazilian Journal of Microbiology vol 43 no 4 pp1340ndash1346 2012

[35] I Javed M I Ali B Ahmed P B Ghumro A Hameed and SAhmed ldquoOptimization and partial purification of bacteriocinsfrom Enterococcus spp Indigenous to Pakistanrdquo Food Biotech-nology vol 25 no 2 pp 130ndash139 2011


[36] S Usmiati and T Marwati ldquoSelection and optimization processof bacteriocin production from Lactobacillus sprdquo IndonesianJournal of Agriculture vol 2 no 2 pp 82ndash92 2009

[37] M V Leal-Sanchez R Jimenez-Dıaz A Maldonado-BarraganA Garrido-Fernandez and J L Ruiz-Barba ldquoOptimization ofbacteriocin production by batch fermentation of Lactobacillusplantarum LPCO10rdquo Applied and Environmental Microbiologyvol 68 no 9 pp 4465ndash4471 2002

[38] I Hammami A Rhouma B Jaouadi A Rebai and X NesmeldquoOptimization and biochemical characterization of a bacte-riocin from a newly isolated Bacillus subtilis strain 14B forbiocontrol of Agrobacterium spp strainsrdquo Letters in AppliedMicrobiology vol 48 no 2 pp 253ndash260 2009

[39] GNaclerio E RiccaM Sacco andM de Felice ldquoAntimicrobialactivity of a newly identified bacteriocin of Bacillus cereusrdquoApplied and Environmental Microbiology vol 59 no 12 pp4313ndash4316 1993

[40] S P Banerjee KCDora and SChowdhury ldquoDetection partialpurification and characterization of bacteriocin produced byLactobacillus brevis FPTLB3 isolated from freshwater fishrdquoJournal of Food Science and Technology vol 50 no 1 pp 17ndash252013

[41] J C Oscariz and A G Pisabarro ldquoCharacterization andmechanism of action of cerein 7 a bacteriocin produced byBacillus cereus Bc7rdquo Journal of Applied Microbiology vol 89 no2 pp 361ndash369 2000

[42] N Kayalvizhi and P Gunasekaran ldquoProduction and charac-terization of a low-molecular-weight bacteriocin from BacilluslicheniformisMKU3rdquoLetters inAppliedMicrobiology vol 47 no6 pp 600ndash607 2008

[43] S Sebei T Zendo A Boudabous J Nakayama and KSonomoto ldquoCharacterization N-terminal sequencing and clas-sification of cerein MRX1 a novel bacteriocin purified froma newly isolated bacterium Bacillus cereus MRX1rdquo Journal ofApplied Microbiology vol 103 no 5 pp 1621ndash1631 2007

[44] D R Bhatta and B P Kapadnis ldquoProduction optimizationand characterization of bioactive compound against Salmonellabacillus subtilis KBB isolated from Nepalrdquo Scientific World vol8 no 8 pp 19ndash29 2010

[45] W J Lyon and B A Glatz ldquoPartial purification and characteri-zation of a bacteriocin produced by Propionibacterium thoeniirdquoApplied and Environmental Microbiology vol 57 no 3 pp 701ndash706 1991

[46] S Saranya and N Heashenpagam ldquoPurification and charac-terization of bacteriocin produced by different Lactobacillusspecies isolated from fermented foodsrdquo International Journal ofMicrobiology Research vol 5 no 1 pp 341ndash348 2013

[47] M Millette C Dupont D Archambault and M LacroixldquoPartial characterization of bacteriocins produced by humanLactococcus lactis and Pediococccus acidilactici isolatesrdquo Journalof Applied Microbiology vol 102 no 1 pp 274ndash282 2007

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


such as lanthionine (Lan) methyl lanthionine (MeLan)dehydroalanine (Dha) dehydrobutyrine (Dhb) or D-alanine(D-Ala) whilst non-lanthionines containing bacteriocins donot undergo extensive posttranslationalmodifications So fara confined number of bacteriocins have been studied [8ndash11]

Diverse mechanisms describing the bacteriocin mode ofaction were outlined but the cell membrane still remainsthe main target of these AMPs The net positive charges ofbacteriocins stimulate preferential binding of bacteriocins tothe negatively charged bacterial cell membranes resulting increation of multiple pores in the cell membranes [12ndash14]In the context of bacteriocins as an adequate alternative toantibiotics against multidrug resistant pathogens along witha continuous urgent need for developing new antimicro-bial agents bacteriocins represent a promising inexhaustiblesource of new antibiotics [15 16]

The objective of the present study is to isolate a bacte-riocin producing bacterium with a wide spectrum antimi-crobial property The aim is extended to optimize the pro-duction of the produced bacteriocin through a sequentialstatistical approach To the best of our knowledge thereexists a plethora of literature concerning characterizationof bacteriocins but there exists a few reports pertaining tohighlighting key determinants controlling the production ofbacteriocins by their producers

2 Materials and Methods

21 Chemicals Majority of bacteriological media used inthis study were purchased from Oxoid Co UK unlessotherwise stated Proteinase K and Dream Taq Green MasterMix 2X were purchased from Thermo Scientific Co USAIsopropanol acetone acetonitrile toluene methyl alcoholand ethyl alcohol were purchased from Sigma Co USALysozyme and agarose were purchased from Bio Basic IncCanada DNA ladder (100 bp DNA Ladder H3 RTU (Ready-to-Use) was purchased from GeneDirex Inc Taiwan Uni-versal primers specific for 16S rDNA gene were synthesizedby Bioneer Co Korea

22 Indicator Strains Erwinia amylovora pear fire blightpathogen and lactic acid bacterial strains (Lactobacillusbulgaricus L casei L lactis and L reuteri) were obtainedfrom the bacterial culture collection of the Department ofPlant Pathology and the Department of Dairy Productsrespectively Faculty of Agriculture University of AlexandriaEgypt The human pathogens indicator strains used in thisstudy were Salmonella typhimurium NCTC 12023ATCC14028 Clostridium perfringens NCTC 8237ATCC 13124Staphylococcus aureus NCTC 12981ATCC 25923 S epider-midis NCTC 13360ATCC 12228 Escherichia coli NCTC12241ATCC 25922 Shigella boydii NCTCATCC 9207Campylobacter jejuni NCTC 11322ATCC 29428 Enterococ-cus faecalis NCTC 12697ATCC29212 Enterobacter aero-genes NCTC10006ATCC13043 Candida albicans NCPF3179ATCC10231 and Vibrio parahaemolyticus NCTC 3178Other human clinical pathogens such asProteus spKlebsiellasp Enterobacter sp Citrobacter sp Enterococcus sp S

typhi Pseudomonas aeruginosa Acinetobacter sp E coliand S paratyphi were obtained from the Department ofMicrobiology Faculty of Medicine University of AlexandriaEgypt Candida tropicalis was obtained from the Departmentof Microbiology Medical Research Institute University ofAlexandria Egypt The food spoiler indicator strain wasListeria innocua NCTC 11288ATCC 33090 Other environ-mental microbial strains included B cereus B licheniformisSHG2B licheniformis SHG6B licheniformis SHG10Bacillussubtilis NCTC 10400ATCC 6633 B subtilis AS1 B subtilisB subtilis EMBLAKE Bacillus sp Ash2 P aeruginosa strainEGYII Aspergillus brasiliensis NCPF2275ATCC16404 andSaccharomyces cerevisiae NCPF 3178 All indicator bacterialstrains were preserved in 15 glycerol stocks at minus80∘CHowever fungal and yeast strains were preserved in agarslopes at 4∘C

23 Media Peptone yeast beef medium (PYB in gL 10 gpeptone 5 g yeast extract and 3 g beef extract) was used asa bacteriocin core production medium E amylovora wasgrown on PYB supplemented with 2 sucrose The indicatorstrains (S typhimurium S paratyphi E coli P aeruginosaKlebsiella sp Enterobacter sp Citrobacter sp and Proteussp) were grown on BHI (Brain Heart Infusion mediumOxoid CM 1135) All strains of lactic acid bacteria weregrown on MRS (De Man Rogosa Sharpe Oxoid CM0361)C albicans and C tropicalis were grown on SAB DextroseAgar medium (Sabouraud Dextrose Agar Oxoid CM004)S aureus and S epidermidis were grown on MSA (Manni-tol Salt Agar Oxoid CM 0085) C perfringens was grownon TSC (Tryptose Sulphite Cycloserine Agar LAB M194)A brasiliensis and S cerevisiae were grown on DichloranGlycerol medium (HIMEDIA M1129-500G) L innocua wasgrown Brilliance Listeria Agar Base (Oxoid CM1080) Allstrains belonging to the genus Bacillus were grown on PYBmedium V parahaemolyticus was grown on thiosulphatecitrate bile salts sucrose agar (TCBS medium LAB M96) Efaecalis was grown on KF Streptococcus Agar Base (OxoidCM0701) Sporulation medium (1XSG) [17] in gL 6 g beefextract 10 g peptone 2 g KCl 1 g glucose plus 1mM CaCl

2

1 mMFeSO4 1 mMMnCl

2 and 2mMMgSO

4) was used dur-

ing the course of studying the most promising isolate underthe electron microscope MYP (Mannitol egg yolkphenolred CM0929 Oxoid Co) medium consisted of 10 g meatextract 10 g peptone 10 g NaCl 10 g mannitol 0025 g perliter plus supplements of polymyxin and egg yolk suspension[18] Sheep blood agar was obtained from the Department ofMicrobiology Faculty of Medicine University of AlexandriaEgypt

24 Isolation of Bacteriocin Producing Bacteria Differentsamples including soils leaves and fruits were collected fromdifferent sites in Borg El-Arab in Alexandria city Egypt Thecandidate sites were naturally contaminated with the pearfirelight pathogen E amylovora One gram of each sampleseparately was used to inoculate 20mL of PYB broth in250mL Erlenmeyer flask for enrichment The inoculatedbroth was incubated for an overnight at 37∘C with agitation










119884 = 120573119900 + Σ120573119894 119909119894 (1)





119884 = 120573119900 + 120573119899119883119899 + Σ120573119899119899119883119899 sdot 119883119899 + Σ120573119899119898119883119899 sdot 119883119898 (2)







3 Results








(a)133120583m

046120583m

(a)

(b)

(b)

(c)

(c)

(d)

(d)

(e)

(e)





100







737










955|gb|

|gb|





40E minus 4

4

u

u

u

u

u

u

u

u

u

u

u

u

u

u

u

L

L

L

L

Y

D

7









1 incubation time

119883







0

5

10

15

20

25

30

Effec

t (

)

X1

(incu

batio

n tim

e)

X5

(rpm

)

X7

(sta

rch)

X9

(yea

st ex

trac

t)

X6

(gly

cero

l)

X3

(pH

)

X4

(incu

batio

n te

mpe

ratu

re)

X8

(inoc

ulum

size

)

X2

(glu

cose

)



119884 = 3358 + 5416119883

1+ 3417119883

5

minus 175119883

6minus 2416119883

7+ 225119883

9

(3)


3 pH 119883

4 incubation




7 starch)



5) and






(a)

Trial number 119883

1

119883

2

119883

3

119883

4

119883

5

119883

6

119883

7

119883

8

119883

9


Expa Predb



2 glucose 119883

3 pH 119883



6 glycerol 119883

7




(b)



Glucose (1198832

wv) 1 2 833 4167 0349 1212678 651pH (119883

3


4







119884 = 4433 + 25119883

1+ 3125119883

5minus 875119883

9minus 2166119883

1sdot 119883

1

minus 9416119883

5sdot 119883

5minus 13417119883

9119883

9minus 325119883

1sdot 119883

5

+ 425119883

1sdot 119883

9+ 5119883

5sdot 119883

9

(4)




1= minus144 120582

5= minus976



5= 976 and 120582



5and 119883



119883





(a)

Trial number 119883

1

119883

5

119883

9


Expa Predb



9 yeast extract


(b)



hrs) 24 48 72 119883

1

5000 25 165144 0159558 840


rpm) 80 200 320 119883

5

6250 3125 007506 0943073 569


gL) 2 5 8 119883

9

minus1750 minus875 112598 0311295 6887

119883

1

sdot 119883

1


5

sdot 119883

5


119883

9

sdot 119883

9


119883

1

sdot 119883

5


1

sdot 119883

9

8500 4250 180470 0130962 8690119883

5

sdot 119883

9



5= 006






BAC

YAS 1

activ

ity (A

Um

L)


X1(incubation tim

e)

480

420

360

300

240

12

12

08

08

04

04

minus04minus04

minus08minus08

minus12minus12

gt4142849

lt4142849

lt3908564

lt3674279

lt3439995

lt320571

lt2971425

lt273714

lt2502855

lt226857

lt2034285


(a)

BAC

YAS1

activ

ity (A

Um

L)

X9 (yeast extract)

X1(incubation tim


minus29802E minus 8

480

420

360

300

240

12

12

08

08

04

04

minus04minus04

minus08minus08

minus12minus12

gt4193656

lt4193656

lt395429

lt3714925

lt3475559

lt3236194

lt2996828

lt2757462

lt2518097

lt2278731

lt2039366

(b)

BAC

YAS 1

activ

ity (A

Um

L)

480

420

360

300

240

12

12

08

08

0404

minus04minus04

minus08minus08

minus12minus12


X9 (yeast extract)

X5(agitation speed)

gt4185689

lt4185689

lt394712

lt3708551

lt3469982

lt3231413

lt2992845

lt2754276

lt2515707

lt2277138

lt2038569

(c)



A

B


0

50

100

150

200

250

300

350

0010

0100

1000

10000

0 1 2 3 4 5 6 7 8 9 10

BAC

YAS

1 ac

tivity

(AU

mL)

Bact

eria

l gro

wth

Ab

at 420

nm













4 Discussion






Table6Sensitivityof

someind

icator

strainstoBA

CYA

S1

Indicatorstrain

BACYA

S1a

ctivity

(inhibitio

nzone

diam

eterm

mplusmnSE

)lowastGram

nature

Bacteria

Acinetobactersp(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative


entalstrainiso

latedfro

msoilEg

ypt)

000

Gram-positive

Blicheniform

isSH

G2(enviro

nmentalstrainiso

latedfro

msoilEg

ypt)

13plusmn17

0Gram-positive

Blicheniform

isSH

G6(enviro

nmentalstrainiso

latedfro

msoilEg

ypt)

000

Gram-positive

Blicheniform

isSH

G10

(enviro

nmentalstrainiso

lated

from

soilEg

ypt)

000

Gram-positive

BsubtilisE

MBL

AKE

(enviro

nmentalstrainiso

lated

from

El-M

ahmou

diaL

akeEg

ypt)

000

Gram-positive

BsubtilisA

S1(enviro

nmentalstrainiso

lated

from

soilEg

ypt)

1025plusmn048

Gram-positive

BsubtilisN

CTC1040

0AT

CC6633

000

Gram-positive

Bsubtilis(environm

entalstrainiso

latedfro

msoilEg

ypt)

000

Gram-positive

BacillusspAs

h2(enviro

nmentalstrainiso

lated

from

soilEg

ypt)

000

Gram-positive

Campyloba

cter

jejuni

NCT

C11322AT

CC29

428

1400plusmn041

Gram-negative

Clostridium

perfrin

gens

NCT

C82

37ATC

C13124

1550plusmn15

Gram-positive

Citro

bacte

rsp(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Enteroba

cter

aerogenesN

CTC1

0006

ATC

C130

431550plusmn046

Gram-negative

Enterobacter

sp(hu

man


ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Enterobacter

sp(hu

man

clinicalisolateIID

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Enterococcus

sp(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

1375plusmn035

Gram-negative

Ecoli(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

EcoliNCT

C12241A

TCC25922

000

Gram-negative

Enterococcus

faecalisNCT

C12697AT

CC29212

000

Gram-negative

Eam

ylovora(D

epartm

ento

fPlant

Patholog

yFaculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

4100plusmn043

Gram-negative

Klebsie

llasp(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

295plusmn04

Gram-negative

Lactobacillus

bulga

ricus

(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Lcasei(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Llactis(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Lreuteri(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Liste

riainno

cuaNCT

C1128

8AT

CC3309

03775plusmn049

Gram-positive

Pseudomonas

aeruginosa

strain

EGYII

000

Gram-negative

Paeruginosa

(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Proteussp

(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

1400plusmn03

Gram-negative

Salm

onellatyph

imurium

NCT

C1202

3AT

CC

1402

81725plusmn049

Gram-negative

Styphi(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Sparatyphi(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

ShigellaboydiiAT

CC9207

000

Gram-negative

Staphylococcus

aureus

NCT

C12981A

TCC25923

000

Gram-positive

Stap

hylococcus

epidermidisNCT

C1336

0AT

CC1222

82375plusmn075

Gram-positive


Table6Con

tinued

Indicatorstrain

BACYA

S1a

ctivity

(inhibitio

nzone

diam

eterm

mplusmnSE

)lowastGram

nature

Vibrioparahaem

olyticu

sNCT

C3178

000

Gram-negative

Fung

iand

yeast

Aspergillus

brasiliensis

NCP

F2275ATC

C1640

4000

Cand

idaalbicans

NCP

F3179ATC

C10231

000

Ctro

picalis

(hum


mdiabeticfootD

epartm

ento

fMicrobiolog

yMedicalRe

search

Institu

teU

niversity

ofAlexand

riaE

gypt)

000


eNCP

F3178

000

lowast

Meanof

four

readings

with

stand

arderror(SE

)




5 Conclusion








References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










119884 = 120573119900 + Σ120573119894 119909119894 (1)





119884 = 120573119900 + 120573119899119883119899 + Σ120573119899119899119883119899 sdot 119883119899 + Σ120573119899119898119883119899 sdot 119883119898 (2)







3 Results








(a)133120583m

046120583m

(a)

(b)

(b)

(c)

(c)

(d)

(d)

(e)

(e)





100







737










955|gb|

|gb|





40E minus 4

4

u

u

u

u

u

u

u

u

u

u

u

u

u

u

u

L

L

L

L

Y

D

7









1 incubation time

119883







0

5

10

15

20

25

30

Effec

t (

)

X1

(incu

batio

n tim

e)

X5

(rpm

)

X7

(sta

rch)

X9

(yea

st ex

trac

t)

X6

(gly

cero

l)

X3

(pH

)

X4

(incu

batio

n te

mpe

ratu

re)

X8

(inoc

ulum

size

)

X2

(glu

cose

)



119884 = 3358 + 5416119883

1+ 3417119883

5

minus 175119883

6minus 2416119883

7+ 225119883

9

(3)


3 pH 119883

4 incubation




7 starch)



5) and






(a)

Trial number 119883

1

119883

2

119883

3

119883

4

119883

5

119883

6

119883

7

119883

8

119883

9


Expa Predb



2 glucose 119883

3 pH 119883



6 glycerol 119883

7




(b)



Glucose (1198832

wv) 1 2 833 4167 0349 1212678 651pH (119883

3


4







119884 = 4433 + 25119883

1+ 3125119883

5minus 875119883

9minus 2166119883

1sdot 119883

1

minus 9416119883

5sdot 119883

5minus 13417119883

9119883

9minus 325119883

1sdot 119883

5

+ 425119883

1sdot 119883

9+ 5119883

5sdot 119883

9

(4)




1= minus144 120582

5= minus976



5= 976 and 120582



5and 119883



119883





(a)

Trial number 119883

1

119883

5

119883

9


Expa Predb



9 yeast extract


(b)



hrs) 24 48 72 119883

1

5000 25 165144 0159558 840


rpm) 80 200 320 119883

5

6250 3125 007506 0943073 569


gL) 2 5 8 119883

9

minus1750 minus875 112598 0311295 6887

119883

1

sdot 119883

1


5

sdot 119883

5


119883

9

sdot 119883

9


119883

1

sdot 119883

5


1

sdot 119883

9

8500 4250 180470 0130962 8690119883

5

sdot 119883

9



5= 006






BAC

YAS 1

activ

ity (A

Um

L)


X1(incubation tim

e)

480

420

360

300

240

12

12

08

08

04

04

minus04minus04

minus08minus08

minus12minus12

gt4142849

lt4142849

lt3908564

lt3674279

lt3439995

lt320571

lt2971425

lt273714

lt2502855

lt226857

lt2034285


(a)

BAC

YAS1

activ

ity (A

Um

L)

X9 (yeast extract)

X1(incubation tim


minus29802E minus 8

480

420

360

300

240

12

12

08

08

04

04

minus04minus04

minus08minus08

minus12minus12

gt4193656

lt4193656

lt395429

lt3714925

lt3475559

lt3236194

lt2996828

lt2757462

lt2518097

lt2278731

lt2039366

(b)

BAC

YAS 1

activ

ity (A

Um

L)

480

420

360

300

240

12

12

08

08

0404

minus04minus04

minus08minus08

minus12minus12


X9 (yeast extract)

X5(agitation speed)

gt4185689

lt4185689

lt394712

lt3708551

lt3469982

lt3231413

lt2992845

lt2754276

lt2515707

lt2277138

lt2038569

(c)



A

B


0

50

100

150

200

250

300

350

0010

0100

1000

10000

0 1 2 3 4 5 6 7 8 9 10

BAC

YAS

1 ac

tivity

(AU

mL)

Bact

eria

l gro

wth

Ab

at 420

nm













4 Discussion






Table6Sensitivityof

someind

icator

strainstoBA

CYA

S1

Indicatorstrain

BACYA

S1a

ctivity

(inhibitio

nzone

diam

eterm

mplusmnSE

)lowastGram

nature

Bacteria

Acinetobactersp(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative


entalstrainiso

latedfro

msoilEg

ypt)

000

Gram-positive

Blicheniform

isSH

G2(enviro

nmentalstrainiso

latedfro

msoilEg

ypt)

13plusmn17

0Gram-positive

Blicheniform

isSH

G6(enviro

nmentalstrainiso

latedfro

msoilEg

ypt)

000

Gram-positive

Blicheniform

isSH

G10

(enviro

nmentalstrainiso

lated

from

soilEg

ypt)

000

Gram-positive

BsubtilisE

MBL

AKE

(enviro

nmentalstrainiso

lated

from

El-M

ahmou

diaL

akeEg

ypt)

000

Gram-positive

BsubtilisA

S1(enviro

nmentalstrainiso

lated

from

soilEg

ypt)

1025plusmn048

Gram-positive

BsubtilisN

CTC1040

0AT

CC6633

000

Gram-positive

Bsubtilis(environm

entalstrainiso

latedfro

msoilEg

ypt)

000

Gram-positive

BacillusspAs

h2(enviro

nmentalstrainiso

lated

from

soilEg

ypt)

000

Gram-positive

Campyloba

cter

jejuni

NCT

C11322AT

CC29

428

1400plusmn041

Gram-negative

Clostridium

perfrin

gens

NCT

C82

37ATC

C13124

1550plusmn15

Gram-positive

Citro

bacte

rsp(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Enteroba

cter

aerogenesN

CTC1

0006

ATC

C130

431550plusmn046

Gram-negative

Enterobacter

sp(hu

man


ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Enterobacter

sp(hu

man

clinicalisolateIID

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Enterococcus

sp(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

1375plusmn035

Gram-negative

Ecoli(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

EcoliNCT

C12241A

TCC25922

000

Gram-negative

Enterococcus

faecalisNCT

C12697AT

CC29212

000

Gram-negative

Eam

ylovora(D

epartm

ento

fPlant

Patholog

yFaculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

4100plusmn043

Gram-negative

Klebsie

llasp(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

295plusmn04

Gram-negative

Lactobacillus

bulga

ricus

(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Lcasei(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Llactis(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Lreuteri(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Liste

riainno

cuaNCT

C1128

8AT

CC3309

03775plusmn049

Gram-positive

Pseudomonas

aeruginosa

strain

EGYII

000

Gram-negative

Paeruginosa

(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Proteussp

(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

1400plusmn03

Gram-negative

Salm

onellatyph

imurium

NCT

C1202

3AT

CC

1402

81725plusmn049

Gram-negative

Styphi(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Sparatyphi(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

ShigellaboydiiAT

CC9207

000

Gram-negative

Staphylococcus

aureus

NCT

C12981A

TCC25923

000

Gram-positive

Stap

hylococcus

epidermidisNCT

C1336

0AT

CC1222

82375plusmn075

Gram-positive


Table6Con

tinued

Indicatorstrain

BACYA

S1a

ctivity

(inhibitio

nzone

diam

eterm

mplusmnSE

)lowastGram

nature

Vibrioparahaem

olyticu

sNCT

C3178

000

Gram-negative

Fung

iand

yeast

Aspergillus

brasiliensis

NCP

F2275ATC

C1640

4000

Cand

idaalbicans

NCP

F3179ATC

C10231

000

Ctro

picalis

(hum


mdiabeticfootD

epartm

ento

fMicrobiolog

yMedicalRe

search

Institu

teU

niversity

ofAlexand

riaE

gypt)

000


eNCP

F3178

000

lowast

Meanof

four

readings

with

stand

arderror(SE

)




5 Conclusion








References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



119884 = 120573119900 + 120573119899119883119899 + Σ120573119899119899119883119899 sdot 119883119899 + Σ120573119899119898119883119899 sdot 119883119898 (2)







3 Results








(a)133120583m

046120583m

(a)

(b)

(b)

(c)

(c)

(d)

(d)

(e)

(e)





100







737










955|gb|

|gb|





40E minus 4

4

u

u

u

u

u

u

u

u

u

u

u

u

u

u

u

L

L

L

L

Y

D

7









1 incubation time

119883







0

5

10

15

20

25

30

Effec

t (

)

X1

(incu

batio

n tim

e)

X5

(rpm

)

X7

(sta

rch)

X9

(yea

st ex

trac

t)

X6

(gly

cero

l)

X3

(pH

)

X4

(incu

batio

n te

mpe

ratu

re)

X8

(inoc

ulum

size

)

X2

(glu

cose

)



119884 = 3358 + 5416119883

1+ 3417119883

5

minus 175119883

6minus 2416119883

7+ 225119883

9

(3)


3 pH 119883

4 incubation




7 starch)



5) and






(a)

Trial number 119883

1

119883

2

119883

3

119883

4

119883

5

119883

6

119883

7

119883

8

119883

9


Expa Predb



2 glucose 119883

3 pH 119883



6 glycerol 119883

7




(b)



Glucose (1198832

wv) 1 2 833 4167 0349 1212678 651pH (119883

3


4







119884 = 4433 + 25119883

1+ 3125119883

5minus 875119883

9minus 2166119883

1sdot 119883

1

minus 9416119883

5sdot 119883

5minus 13417119883

9119883

9minus 325119883

1sdot 119883

5

+ 425119883

1sdot 119883

9+ 5119883

5sdot 119883

9

(4)




1= minus144 120582

5= minus976



5= 976 and 120582



5and 119883



119883





(a)

Trial number 119883

1

119883

5

119883

9


Expa Predb



9 yeast extract


(b)



hrs) 24 48 72 119883

1

5000 25 165144 0159558 840


rpm) 80 200 320 119883

5

6250 3125 007506 0943073 569


gL) 2 5 8 119883

9

minus1750 minus875 112598 0311295 6887

119883

1

sdot 119883

1


5

sdot 119883

5


119883

9

sdot 119883

9


119883

1

sdot 119883

5


1

sdot 119883

9

8500 4250 180470 0130962 8690119883

5

sdot 119883

9



5= 006






BAC

YAS 1

activ

ity (A

Um

L)


X1(incubation tim

e)

480

420

360

300

240

12

12

08

08

04

04

minus04minus04

minus08minus08

minus12minus12

gt4142849

lt4142849

lt3908564

lt3674279

lt3439995

lt320571

lt2971425

lt273714

lt2502855

lt226857

lt2034285


(a)

BAC

YAS1

activ

ity (A

Um

L)

X9 (yeast extract)

X1(incubation tim


minus29802E minus 8

480

420

360

300

240

12

12

08

08

04

04

minus04minus04

minus08minus08

minus12minus12

gt4193656

lt4193656

lt395429

lt3714925

lt3475559

lt3236194

lt2996828

lt2757462

lt2518097

lt2278731

lt2039366

(b)

BAC

YAS 1

activ

ity (A

Um

L)

480

420

360

300

240

12

12

08

08

0404

minus04minus04

minus08minus08

minus12minus12


X9 (yeast extract)

X5(agitation speed)

gt4185689

lt4185689

lt394712

lt3708551

lt3469982

lt3231413

lt2992845

lt2754276

lt2515707

lt2277138

lt2038569

(c)



A

B


0

50

100

150

200

250

300

350

0010

0100

1000

10000

0 1 2 3 4 5 6 7 8 9 10

BAC

YAS

1 ac

tivity

(AU

mL)

Bact

eria

l gro

wth

Ab

at 420

nm













4 Discussion






Table6Sensitivityof

someind

icator

strainstoBA

CYA

S1

Indicatorstrain

BACYA

S1a

ctivity

(inhibitio

nzone

diam

eterm

mplusmnSE

)lowastGram

nature

Bacteria

Acinetobactersp(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative


entalstrainiso

latedfro

msoilEg

ypt)

000

Gram-positive

Blicheniform

isSH

G2(enviro

nmentalstrainiso

latedfro

msoilEg

ypt)

13plusmn17

0Gram-positive

Blicheniform

isSH

G6(enviro

nmentalstrainiso

latedfro

msoilEg

ypt)

000

Gram-positive

Blicheniform

isSH

G10

(enviro

nmentalstrainiso

lated

from

soilEg

ypt)

000

Gram-positive

BsubtilisE

MBL

AKE

(enviro

nmentalstrainiso

lated

from

El-M

ahmou

diaL

akeEg

ypt)

000

Gram-positive

BsubtilisA

S1(enviro

nmentalstrainiso

lated

from

soilEg

ypt)

1025plusmn048

Gram-positive

BsubtilisN

CTC1040

0AT

CC6633

000

Gram-positive

Bsubtilis(environm

entalstrainiso

latedfro

msoilEg

ypt)

000

Gram-positive

BacillusspAs

h2(enviro

nmentalstrainiso

lated

from

soilEg

ypt)

000

Gram-positive

Campyloba

cter

jejuni

NCT

C11322AT

CC29

428

1400plusmn041

Gram-negative

Clostridium

perfrin

gens

NCT

C82

37ATC

C13124

1550plusmn15

Gram-positive

Citro

bacte

rsp(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Enteroba

cter

aerogenesN

CTC1

0006

ATC

C130

431550plusmn046

Gram-negative

Enterobacter

sp(hu

man


ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Enterobacter

sp(hu

man

clinicalisolateIID

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Enterococcus

sp(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

1375plusmn035

Gram-negative

Ecoli(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

EcoliNCT

C12241A

TCC25922

000

Gram-negative

Enterococcus

faecalisNCT

C12697AT

CC29212

000

Gram-negative

Eam

ylovora(D

epartm

ento

fPlant

Patholog

yFaculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

4100plusmn043

Gram-negative

Klebsie

llasp(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

295plusmn04

Gram-negative

Lactobacillus

bulga

ricus

(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Lcasei(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Llactis(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Lreuteri(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Liste

riainno

cuaNCT

C1128

8AT

CC3309

03775plusmn049

Gram-positive

Pseudomonas

aeruginosa

strain

EGYII

000

Gram-negative

Paeruginosa

(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Proteussp

(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

1400plusmn03

Gram-negative

Salm

onellatyph

imurium

NCT

C1202

3AT

CC

1402

81725plusmn049

Gram-negative

Styphi(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Sparatyphi(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

ShigellaboydiiAT

CC9207

000

Gram-negative

Staphylococcus

aureus

NCT

C12981A

TCC25923

000

Gram-positive

Stap

hylococcus

epidermidisNCT

C1336

0AT

CC1222

82375plusmn075

Gram-positive


Table6Con

tinued

Indicatorstrain

BACYA

S1a

ctivity

(inhibitio

nzone

diam

eterm

mplusmnSE

)lowastGram

nature

Vibrioparahaem

olyticu

sNCT

C3178

000

Gram-negative

Fung

iand

yeast

Aspergillus

brasiliensis

NCP

F2275ATC

C1640

4000

Cand

idaalbicans

NCP

F3179ATC

C10231

000

Ctro

picalis

(hum


mdiabeticfootD

epartm

ento

fMicrobiolog

yMedicalRe

search

Institu

teU

niversity

ofAlexand

riaE

gypt)

000


eNCP

F3178

000

lowast

Meanof

four

readings

with

stand

arderror(SE

)




5 Conclusion








References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





(a)133120583m

046120583m

(a)

(b)

(b)

(c)

(c)

(d)

(d)

(e)

(e)





100







737










955|gb|

|gb|





40E minus 4

4

u

u

u

u

u

u

u

u

u

u

u

u

u

u

u

L

L

L

L

Y

D

7









1 incubation time

119883







0

5

10

15

20

25

30

Effec

t (

)

X1

(incu

batio

n tim

e)

X5

(rpm

)

X7

(sta

rch)

X9

(yea

st ex

trac

t)

X6

(gly

cero

l)

X3

(pH

)

X4

(incu

batio

n te

mpe

ratu

re)

X8

(inoc

ulum

size

)

X2

(glu

cose

)



119884 = 3358 + 5416119883

1+ 3417119883

5

minus 175119883

6minus 2416119883

7+ 225119883

9

(3)


3 pH 119883

4 incubation




7 starch)



5) and






(a)

Trial number 119883

1

119883

2

119883

3

119883

4

119883

5

119883

6

119883

7

119883

8

119883

9


Expa Predb



2 glucose 119883

3 pH 119883



6 glycerol 119883

7




(b)



Glucose (1198832

wv) 1 2 833 4167 0349 1212678 651pH (119883

3


4







119884 = 4433 + 25119883

1+ 3125119883

5minus 875119883

9minus 2166119883

1sdot 119883

1

minus 9416119883

5sdot 119883

5minus 13417119883

9119883

9minus 325119883

1sdot 119883

5

+ 425119883

1sdot 119883

9+ 5119883

5sdot 119883

9

(4)




1= minus144 120582

5= minus976



5= 976 and 120582



5and 119883



119883





(a)

Trial number 119883

1

119883

5

119883

9


Expa Predb



9 yeast extract


(b)



hrs) 24 48 72 119883

1

5000 25 165144 0159558 840


rpm) 80 200 320 119883

5

6250 3125 007506 0943073 569


gL) 2 5 8 119883

9

minus1750 minus875 112598 0311295 6887

119883

1

sdot 119883

1


5

sdot 119883

5


119883

9

sdot 119883

9


119883

1

sdot 119883

5


1

sdot 119883

9

8500 4250 180470 0130962 8690119883

5

sdot 119883

9



5= 006






BAC

YAS 1

activ

ity (A

Um

L)


X1(incubation tim

e)

480

420

360

300

240

12

12

08

08

04

04

minus04minus04

minus08minus08

minus12minus12

gt4142849

lt4142849

lt3908564

lt3674279

lt3439995

lt320571

lt2971425

lt273714

lt2502855

lt226857

lt2034285


(a)

BAC

YAS1

activ

ity (A

Um

L)

X9 (yeast extract)

X1(incubation tim


minus29802E minus 8

480

420

360

300

240

12

12

08

08

04

04

minus04minus04

minus08minus08

minus12minus12

gt4193656

lt4193656

lt395429

lt3714925

lt3475559

lt3236194

lt2996828

lt2757462

lt2518097

lt2278731

lt2039366

(b)

BAC

YAS 1

activ

ity (A

Um

L)

480

420

360

300

240

12

12

08

08

0404

minus04minus04

minus08minus08

minus12minus12


X9 (yeast extract)

X5(agitation speed)

gt4185689

lt4185689

lt394712

lt3708551

lt3469982

lt3231413

lt2992845

lt2754276

lt2515707

lt2277138

lt2038569

(c)



A

B


0

50

100

150

200

250

300

350

0010

0100

1000

10000

0 1 2 3 4 5 6 7 8 9 10

BAC

YAS

1 ac

tivity

(AU

mL)

Bact

eria

l gro

wth

Ab

at 420

nm













4 Discussion






Table6Sensitivityof

someind

icator

strainstoBA

CYA

S1

Indicatorstrain

BACYA

S1a

ctivity

(inhibitio

nzone

diam

eterm

mplusmnSE

)lowastGram

nature

Bacteria

Acinetobactersp(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative


entalstrainiso

latedfro

msoilEg

ypt)

000

Gram-positive

Blicheniform

isSH

G2(enviro

nmentalstrainiso

latedfro

msoilEg

ypt)

13plusmn17

0Gram-positive

Blicheniform

isSH

G6(enviro

nmentalstrainiso

latedfro

msoilEg

ypt)

000

Gram-positive

Blicheniform

isSH

G10

(enviro

nmentalstrainiso

lated

from

soilEg

ypt)

000

Gram-positive

BsubtilisE

MBL

AKE

(enviro

nmentalstrainiso

lated

from

El-M

ahmou

diaL

akeEg

ypt)

000

Gram-positive

BsubtilisA

S1(enviro

nmentalstrainiso

lated

from

soilEg

ypt)

1025plusmn048

Gram-positive

BsubtilisN

CTC1040

0AT

CC6633

000

Gram-positive

Bsubtilis(environm

entalstrainiso

latedfro

msoilEg

ypt)

000

Gram-positive

BacillusspAs

h2(enviro

nmentalstrainiso

lated

from

soilEg

ypt)

000

Gram-positive

Campyloba

cter

jejuni

NCT

C11322AT

CC29

428

1400plusmn041

Gram-negative

Clostridium

perfrin

gens

NCT

C82

37ATC

C13124

1550plusmn15

Gram-positive

Citro

bacte

rsp(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Enteroba

cter

aerogenesN

CTC1

0006

ATC

C130

431550plusmn046

Gram-negative

Enterobacter

sp(hu

man


ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Enterobacter

sp(hu

man

clinicalisolateIID

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Enterococcus

sp(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

1375plusmn035

Gram-negative

Ecoli(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

EcoliNCT

C12241A

TCC25922

000

Gram-negative

Enterococcus

faecalisNCT

C12697AT

CC29212

000

Gram-negative

Eam

ylovora(D

epartm

ento

fPlant

Patholog

yFaculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

4100plusmn043

Gram-negative

Klebsie

llasp(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

295plusmn04

Gram-negative

Lactobacillus

bulga

ricus

(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Lcasei(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Llactis(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Lreuteri(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Liste

riainno

cuaNCT

C1128

8AT

CC3309

03775plusmn049

Gram-positive

Pseudomonas

aeruginosa

strain

EGYII

000

Gram-negative

Paeruginosa

(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Proteussp

(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

1400plusmn03

Gram-negative

Salm

onellatyph

imurium

NCT

C1202

3AT

CC

1402

81725plusmn049

Gram-negative

Styphi(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Sparatyphi(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

ShigellaboydiiAT

CC9207

000

Gram-negative

Staphylococcus

aureus

NCT

C12981A

TCC25923

000

Gram-positive

Stap

hylococcus

epidermidisNCT

C1336

0AT

CC1222

82375plusmn075

Gram-positive


Table6Con

tinued

Indicatorstrain

BACYA

S1a

ctivity

(inhibitio

nzone

diam

eterm

mplusmnSE

)lowastGram

nature

Vibrioparahaem

olyticu

sNCT

C3178

000

Gram-negative

Fung

iand

yeast

Aspergillus

brasiliensis

NCP

F2275ATC

C1640

4000

Cand

idaalbicans

NCP

F3179ATC

C10231

000

Ctro

picalis

(hum


mdiabeticfootD

epartm

ento

fMicrobiolog

yMedicalRe

search

Institu

teU

niversity

ofAlexand

riaE

gypt)

000


eNCP

F3178

000

lowast

Meanof

four

readings

with

stand

arderror(SE

)




5 Conclusion








References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








1 incubation time

119883







0

5

10

15

20

25

30

Effec

t (

)

X1

(incu

batio

n tim

e)

X5

(rpm

)

X7

(sta

rch)

X9

(yea

st ex

trac

t)

X6

(gly

cero

l)

X3

(pH

)

X4

(incu

batio

n te

mpe

ratu

re)

X8

(inoc

ulum

size

)

X2

(glu

cose

)



119884 = 3358 + 5416119883

1+ 3417119883

5

minus 175119883

6minus 2416119883

7+ 225119883

9

(3)


3 pH 119883

4 incubation




7 starch)



5) and






(a)

Trial number 119883

1

119883

2

119883

3

119883

4

119883

5

119883

6

119883

7

119883

8

119883

9


Expa Predb



2 glucose 119883

3 pH 119883



6 glycerol 119883

7




(b)



Glucose (1198832

wv) 1 2 833 4167 0349 1212678 651pH (119883

3


4







119884 = 4433 + 25119883

1+ 3125119883

5minus 875119883

9minus 2166119883

1sdot 119883

1

minus 9416119883

5sdot 119883

5minus 13417119883

9119883

9minus 325119883

1sdot 119883

5

+ 425119883

1sdot 119883

9+ 5119883

5sdot 119883

9

(4)




1= minus144 120582

5= minus976



5= 976 and 120582



5and 119883



119883





(a)

Trial number 119883

1

119883

5

119883

9


Expa Predb



9 yeast extract


(b)



hrs) 24 48 72 119883

1

5000 25 165144 0159558 840


rpm) 80 200 320 119883

5

6250 3125 007506 0943073 569


gL) 2 5 8 119883

9

minus1750 minus875 112598 0311295 6887

119883

1

sdot 119883

1


5

sdot 119883

5


119883

9

sdot 119883

9


119883

1

sdot 119883

5


1

sdot 119883

9

8500 4250 180470 0130962 8690119883

5

sdot 119883

9



5= 006






BAC

YAS 1

activ

ity (A

Um

L)


X1(incubation tim

e)

480

420

360

300

240

12

12

08

08

04

04

minus04minus04

minus08minus08

minus12minus12

gt4142849

lt4142849

lt3908564

lt3674279

lt3439995

lt320571

lt2971425

lt273714

lt2502855

lt226857

lt2034285


(a)

BAC

YAS1

activ

ity (A

Um

L)

X9 (yeast extract)

X1(incubation tim


minus29802E minus 8

480

420

360

300

240

12

12

08

08

04

04

minus04minus04

minus08minus08

minus12minus12

gt4193656

lt4193656

lt395429

lt3714925

lt3475559

lt3236194

lt2996828

lt2757462

lt2518097

lt2278731

lt2039366

(b)

BAC

YAS 1

activ

ity (A

Um

L)

480

420

360

300

240

12

12

08

08

0404

minus04minus04

minus08minus08

minus12minus12


X9 (yeast extract)

X5(agitation speed)

gt4185689

lt4185689

lt394712

lt3708551

lt3469982

lt3231413

lt2992845

lt2754276

lt2515707

lt2277138

lt2038569

(c)



A

B


0

50

100

150

200

250

300

350

0010

0100

1000

10000

0 1 2 3 4 5 6 7 8 9 10

BAC

YAS

1 ac

tivity

(AU

mL)

Bact

eria

l gro

wth

Ab

at 420

nm













4 Discussion






Table6Sensitivityof

someind

icator

strainstoBA

CYA

S1

Indicatorstrain

BACYA

S1a

ctivity

(inhibitio

nzone

diam

eterm

mplusmnSE

)lowastGram

nature

Bacteria

Acinetobactersp(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative


entalstrainiso

latedfro

msoilEg

ypt)

000

Gram-positive

Blicheniform

isSH

G2(enviro

nmentalstrainiso

latedfro

msoilEg

ypt)

13plusmn17

0Gram-positive

Blicheniform

isSH

G6(enviro

nmentalstrainiso

latedfro

msoilEg

ypt)

000

Gram-positive

Blicheniform

isSH

G10

(enviro

nmentalstrainiso

lated

from

soilEg

ypt)

000

Gram-positive

BsubtilisE

MBL

AKE

(enviro

nmentalstrainiso

lated

from

El-M

ahmou

diaL

akeEg

ypt)

000

Gram-positive

BsubtilisA

S1(enviro

nmentalstrainiso

lated

from

soilEg

ypt)

1025plusmn048

Gram-positive

BsubtilisN

CTC1040

0AT

CC6633

000

Gram-positive

Bsubtilis(environm

entalstrainiso

latedfro

msoilEg

ypt)

000

Gram-positive

BacillusspAs

h2(enviro

nmentalstrainiso

lated

from

soilEg

ypt)

000

Gram-positive

Campyloba

cter

jejuni

NCT

C11322AT

CC29

428

1400plusmn041

Gram-negative

Clostridium

perfrin

gens

NCT

C82

37ATC

C13124

1550plusmn15

Gram-positive

Citro

bacte

rsp(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Enteroba

cter

aerogenesN

CTC1

0006

ATC

C130

431550plusmn046

Gram-negative

Enterobacter

sp(hu

man


ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Enterobacter

sp(hu

man

clinicalisolateIID

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Enterococcus

sp(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

1375plusmn035

Gram-negative

Ecoli(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

EcoliNCT

C12241A

TCC25922

000

Gram-negative

Enterococcus

faecalisNCT

C12697AT

CC29212

000

Gram-negative

Eam

ylovora(D

epartm

ento

fPlant

Patholog

yFaculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

4100plusmn043

Gram-negative

Klebsie

llasp(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

295plusmn04

Gram-negative

Lactobacillus

bulga

ricus

(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Lcasei(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Llactis(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Lreuteri(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Liste

riainno

cuaNCT

C1128

8AT

CC3309

03775plusmn049

Gram-positive

Pseudomonas

aeruginosa

strain

EGYII

000

Gram-negative

Paeruginosa

(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Proteussp

(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

1400plusmn03

Gram-negative

Salm

onellatyph

imurium

NCT

C1202

3AT

CC

1402

81725plusmn049

Gram-negative

Styphi(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Sparatyphi(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

ShigellaboydiiAT

CC9207

000

Gram-negative

Staphylococcus

aureus

NCT

C12981A

TCC25923

000

Gram-positive

Stap

hylococcus

epidermidisNCT

C1336

0AT

CC1222

82375plusmn075

Gram-positive


Table6Con

tinued

Indicatorstrain

BACYA

S1a

ctivity

(inhibitio

nzone

diam

eterm

mplusmnSE

)lowastGram

nature

Vibrioparahaem

olyticu

sNCT

C3178

000

Gram-negative

Fung

iand

yeast

Aspergillus

brasiliensis

NCP

F2275ATC

C1640

4000

Cand

idaalbicans

NCP

F3179ATC

C10231

000

Ctro

picalis

(hum


mdiabeticfootD

epartm

ento

fMicrobiolog

yMedicalRe

search

Institu

teU

niversity

ofAlexand

riaE

gypt)

000


eNCP

F3178

000

lowast

Meanof

four

readings

with

stand

arderror(SE

)




5 Conclusion








References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



(a)

Trial number 119883

1

119883

2

119883

3

119883

4

119883

5

119883

6

119883

7

119883

8

119883

9


Expa Predb



2 glucose 119883

3 pH 119883



6 glycerol 119883

7




(b)



Glucose (1198832

wv) 1 2 833 4167 0349 1212678 651pH (119883

3


4







119884 = 4433 + 25119883

1+ 3125119883

5minus 875119883

9minus 2166119883

1sdot 119883

1

minus 9416119883

5sdot 119883

5minus 13417119883

9119883

9minus 325119883

1sdot 119883

5

+ 425119883

1sdot 119883

9+ 5119883

5sdot 119883

9

(4)




1= minus144 120582

5= minus976



5= 976 and 120582



5and 119883



119883





(a)

Trial number 119883

1

119883

5

119883

9


Expa Predb



9 yeast extract


(b)



hrs) 24 48 72 119883

1

5000 25 165144 0159558 840


rpm) 80 200 320 119883

5

6250 3125 007506 0943073 569


gL) 2 5 8 119883

9

minus1750 minus875 112598 0311295 6887

119883

1

sdot 119883

1


5

sdot 119883

5


119883

9

sdot 119883

9


119883

1

sdot 119883

5


1

sdot 119883

9

8500 4250 180470 0130962 8690119883

5

sdot 119883

9



5= 006






BAC

YAS 1

activ

ity (A

Um

L)


X1(incubation tim

e)

480

420

360

300

240

12

12

08

08

04

04

minus04minus04

minus08minus08

minus12minus12

gt4142849

lt4142849

lt3908564

lt3674279

lt3439995

lt320571

lt2971425

lt273714

lt2502855

lt226857

lt2034285


(a)

BAC

YAS1

activ

ity (A

Um

L)

X9 (yeast extract)

X1(incubation tim


minus29802E minus 8

480

420

360

300

240

12

12

08

08

04

04

minus04minus04

minus08minus08

minus12minus12

gt4193656

lt4193656

lt395429

lt3714925

lt3475559

lt3236194

lt2996828

lt2757462

lt2518097

lt2278731

lt2039366

(b)

BAC

YAS 1

activ

ity (A

Um

L)

480

420

360

300

240

12

12

08

08

0404

minus04minus04

minus08minus08

minus12minus12


X9 (yeast extract)

X5(agitation speed)

gt4185689

lt4185689

lt394712

lt3708551

lt3469982

lt3231413

lt2992845

lt2754276

lt2515707

lt2277138

lt2038569

(c)



A

B


0

50

100

150

200

250

300

350

0010

0100

1000

10000

0 1 2 3 4 5 6 7 8 9 10

BAC

YAS

1 ac

tivity

(AU

mL)

Bact

eria

l gro

wth

Ab

at 420

nm













4 Discussion






Table6Sensitivityof

someind

icator

strainstoBA

CYA

S1

Indicatorstrain

BACYA

S1a

ctivity

(inhibitio

nzone

diam

eterm

mplusmnSE

)lowastGram

nature

Bacteria

Acinetobactersp(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative


entalstrainiso

latedfro

msoilEg

ypt)

000

Gram-positive

Blicheniform

isSH

G2(enviro

nmentalstrainiso

latedfro

msoilEg

ypt)

13plusmn17

0Gram-positive

Blicheniform

isSH

G6(enviro

nmentalstrainiso

latedfro

msoilEg

ypt)

000

Gram-positive

Blicheniform

isSH

G10

(enviro

nmentalstrainiso

lated

from

soilEg

ypt)

000

Gram-positive

BsubtilisE

MBL

AKE

(enviro

nmentalstrainiso

lated

from

El-M

ahmou

diaL

akeEg

ypt)

000

Gram-positive

BsubtilisA

S1(enviro

nmentalstrainiso

lated

from

soilEg

ypt)

1025plusmn048

Gram-positive

BsubtilisN

CTC1040

0AT

CC6633

000

Gram-positive

Bsubtilis(environm

entalstrainiso

latedfro

msoilEg

ypt)

000

Gram-positive

BacillusspAs

h2(enviro

nmentalstrainiso

lated

from

soilEg

ypt)

000

Gram-positive

Campyloba

cter

jejuni

NCT

C11322AT

CC29

428

1400plusmn041

Gram-negative

Clostridium

perfrin

gens

NCT

C82

37ATC

C13124

1550plusmn15

Gram-positive

Citro

bacte

rsp(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Enteroba

cter

aerogenesN

CTC1

0006

ATC

C130

431550plusmn046

Gram-negative

Enterobacter

sp(hu

man


ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Enterobacter

sp(hu

man

clinicalisolateIID

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Enterococcus

sp(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

1375plusmn035

Gram-negative

Ecoli(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

EcoliNCT

C12241A

TCC25922

000

Gram-negative

Enterococcus

faecalisNCT

C12697AT

CC29212

000

Gram-negative

Eam

ylovora(D

epartm

ento

fPlant

Patholog

yFaculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

4100plusmn043

Gram-negative

Klebsie

llasp(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

295plusmn04

Gram-negative

Lactobacillus

bulga

ricus

(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Lcasei(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Llactis(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Lreuteri(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Liste

riainno

cuaNCT

C1128

8AT

CC3309

03775plusmn049

Gram-positive

Pseudomonas

aeruginosa

strain

EGYII

000

Gram-negative

Paeruginosa

(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Proteussp

(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

1400plusmn03

Gram-negative

Salm

onellatyph

imurium

NCT

C1202

3AT

CC

1402

81725plusmn049

Gram-negative

Styphi(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Sparatyphi(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

ShigellaboydiiAT

CC9207

000

Gram-negative

Staphylococcus

aureus

NCT

C12981A

TCC25923

000

Gram-positive

Stap

hylococcus

epidermidisNCT

C1336

0AT

CC1222

82375plusmn075

Gram-positive


Table6Con

tinued

Indicatorstrain

BACYA

S1a

ctivity

(inhibitio

nzone

diam

eterm

mplusmnSE

)lowastGram

nature

Vibrioparahaem

olyticu

sNCT

C3178

000

Gram-negative

Fung

iand

yeast

Aspergillus

brasiliensis

NCP

F2275ATC

C1640

4000

Cand

idaalbicans

NCP

F3179ATC

C10231

000

Ctro

picalis

(hum


mdiabeticfootD

epartm

ento

fMicrobiolog

yMedicalRe

search

Institu

teU

niversity

ofAlexand

riaE

gypt)

000


eNCP

F3178

000

lowast

Meanof

four

readings

with

stand

arderror(SE

)




5 Conclusion








References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



(a)

Trial number 119883

1

119883

5

119883

9


Expa Predb



9 yeast extract


(b)



hrs) 24 48 72 119883

1

5000 25 165144 0159558 840


rpm) 80 200 320 119883

5

6250 3125 007506 0943073 569


gL) 2 5 8 119883

9

minus1750 minus875 112598 0311295 6887

119883

1

sdot 119883

1


5

sdot 119883

5


119883

9

sdot 119883

9


119883

1

sdot 119883

5


1

sdot 119883

9

8500 4250 180470 0130962 8690119883

5

sdot 119883

9



5= 006






BAC

YAS 1

activ

ity (A

Um

L)


X1(incubation tim

e)

480

420

360

300

240

12

12

08

08

04

04

minus04minus04

minus08minus08

minus12minus12

gt4142849

lt4142849

lt3908564

lt3674279

lt3439995

lt320571

lt2971425

lt273714

lt2502855

lt226857

lt2034285


(a)

BAC

YAS1

activ

ity (A

Um

L)

X9 (yeast extract)

X1(incubation tim


minus29802E minus 8

480

420

360

300

240

12

12

08

08

04

04

minus04minus04

minus08minus08

minus12minus12

gt4193656

lt4193656

lt395429

lt3714925

lt3475559

lt3236194

lt2996828

lt2757462

lt2518097

lt2278731

lt2039366

(b)

BAC

YAS 1

activ

ity (A

Um

L)

480

420

360

300

240

12

12

08

08

0404

minus04minus04

minus08minus08

minus12minus12


X9 (yeast extract)

X5(agitation speed)

gt4185689

lt4185689

lt394712

lt3708551

lt3469982

lt3231413

lt2992845

lt2754276

lt2515707

lt2277138

lt2038569

(c)



A

B


0

50

100

150

200

250

300

350

0010

0100

1000

10000

0 1 2 3 4 5 6 7 8 9 10

BAC

YAS

1 ac

tivity

(AU

mL)

Bact

eria

l gro

wth

Ab

at 420

nm













4 Discussion






Table6Sensitivityof

someind

icator

strainstoBA

CYA

S1

Indicatorstrain

BACYA

S1a

ctivity

(inhibitio

nzone

diam

eterm

mplusmnSE

)lowastGram

nature

Bacteria

Acinetobactersp(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative


entalstrainiso

latedfro

msoilEg

ypt)

000

Gram-positive

Blicheniform

isSH

G2(enviro

nmentalstrainiso

latedfro

msoilEg

ypt)

13plusmn17

0Gram-positive

Blicheniform

isSH

G6(enviro

nmentalstrainiso

latedfro

msoilEg

ypt)

000

Gram-positive

Blicheniform

isSH

G10

(enviro

nmentalstrainiso

lated

from

soilEg

ypt)

000

Gram-positive

BsubtilisE

MBL

AKE

(enviro

nmentalstrainiso

lated

from

El-M

ahmou

diaL

akeEg

ypt)

000

Gram-positive

BsubtilisA

S1(enviro

nmentalstrainiso

lated

from

soilEg

ypt)

1025plusmn048

Gram-positive

BsubtilisN

CTC1040

0AT

CC6633

000

Gram-positive

Bsubtilis(environm

entalstrainiso

latedfro

msoilEg

ypt)

000

Gram-positive

BacillusspAs

h2(enviro

nmentalstrainiso

lated

from

soilEg

ypt)

000

Gram-positive

Campyloba

cter

jejuni

NCT

C11322AT

CC29

428

1400plusmn041

Gram-negative

Clostridium

perfrin

gens

NCT

C82

37ATC

C13124

1550plusmn15

Gram-positive

Citro

bacte

rsp(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Enteroba

cter

aerogenesN

CTC1

0006

ATC

C130

431550plusmn046

Gram-negative

Enterobacter

sp(hu

man


ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Enterobacter

sp(hu

man

clinicalisolateIID

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Enterococcus

sp(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

1375plusmn035

Gram-negative

Ecoli(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

EcoliNCT

C12241A

TCC25922

000

Gram-negative

Enterococcus

faecalisNCT

C12697AT

CC29212

000

Gram-negative

Eam

ylovora(D

epartm

ento

fPlant

Patholog

yFaculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

4100plusmn043

Gram-negative

Klebsie

llasp(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

295plusmn04

Gram-negative

Lactobacillus

bulga

ricus

(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Lcasei(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Llactis(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Lreuteri(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Liste

riainno

cuaNCT

C1128

8AT

CC3309

03775plusmn049

Gram-positive

Pseudomonas

aeruginosa

strain

EGYII

000

Gram-negative

Paeruginosa

(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Proteussp

(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

1400plusmn03

Gram-negative

Salm

onellatyph

imurium

NCT

C1202

3AT

CC

1402

81725plusmn049

Gram-negative

Styphi(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Sparatyphi(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

ShigellaboydiiAT

CC9207

000

Gram-negative

Staphylococcus

aureus

NCT

C12981A

TCC25923

000

Gram-positive

Stap

hylococcus

epidermidisNCT

C1336

0AT

CC1222

82375plusmn075

Gram-positive


Table6Con

tinued

Indicatorstrain

BACYA

S1a

ctivity

(inhibitio

nzone

diam

eterm

mplusmnSE

)lowastGram

nature

Vibrioparahaem

olyticu

sNCT

C3178

000

Gram-negative

Fung

iand

yeast

Aspergillus

brasiliensis

NCP

F2275ATC

C1640

4000

Cand

idaalbicans

NCP

F3179ATC

C10231

000

Ctro

picalis

(hum


mdiabeticfootD

epartm

ento

fMicrobiolog

yMedicalRe

search

Institu

teU

niversity

ofAlexand

riaE

gypt)

000


eNCP

F3178

000

lowast

Meanof

four

readings

with

stand

arderror(SE

)




5 Conclusion








References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


BAC

YAS 1

activ

ity (A

Um

L)


X1(incubation tim

e)

480

420

360

300

240

12

12

08

08

04

04

minus04minus04

minus08minus08

minus12minus12

gt4142849

lt4142849

lt3908564

lt3674279

lt3439995

lt320571

lt2971425

lt273714

lt2502855

lt226857

lt2034285


(a)

BAC

YAS1

activ

ity (A

Um

L)

X9 (yeast extract)

X1(incubation tim


minus29802E minus 8

480

420

360

300

240

12

12

08

08

04

04

minus04minus04

minus08minus08

minus12minus12

gt4193656

lt4193656

lt395429

lt3714925

lt3475559

lt3236194

lt2996828

lt2757462

lt2518097

lt2278731

lt2039366

(b)

BAC

YAS 1

activ

ity (A

Um

L)

480

420

360

300

240

12

12

08

08

0404

minus04minus04

minus08minus08

minus12minus12


X9 (yeast extract)

X5(agitation speed)

gt4185689

lt4185689

lt394712

lt3708551

lt3469982

lt3231413

lt2992845

lt2754276

lt2515707

lt2277138

lt2038569

(c)



A

B


0

50

100

150

200

250

300

350

0010

0100

1000

10000

0 1 2 3 4 5 6 7 8 9 10

BAC

YAS

1 ac

tivity

(AU

mL)

Bact

eria

l gro

wth

Ab

at 420

nm













4 Discussion






Table6Sensitivityof

someind

icator

strainstoBA

CYA

S1

Indicatorstrain

BACYA

S1a

ctivity

(inhibitio

nzone

diam

eterm

mplusmnSE

)lowastGram

nature

Bacteria

Acinetobactersp(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative


entalstrainiso

latedfro

msoilEg

ypt)

000

Gram-positive

Blicheniform

isSH

G2(enviro

nmentalstrainiso

latedfro

msoilEg

ypt)

13plusmn17

0Gram-positive

Blicheniform

isSH

G6(enviro

nmentalstrainiso

latedfro

msoilEg

ypt)

000

Gram-positive

Blicheniform

isSH

G10

(enviro

nmentalstrainiso

lated

from

soilEg

ypt)

000

Gram-positive

BsubtilisE

MBL

AKE

(enviro

nmentalstrainiso

lated

from

El-M

ahmou

diaL

akeEg

ypt)

000

Gram-positive

BsubtilisA

S1(enviro

nmentalstrainiso

lated

from

soilEg

ypt)

1025plusmn048

Gram-positive

BsubtilisN

CTC1040

0AT

CC6633

000

Gram-positive

Bsubtilis(environm

entalstrainiso

latedfro

msoilEg

ypt)

000

Gram-positive

BacillusspAs

h2(enviro

nmentalstrainiso

lated

from

soilEg

ypt)

000

Gram-positive

Campyloba

cter

jejuni

NCT

C11322AT

CC29

428

1400plusmn041

Gram-negative

Clostridium

perfrin

gens

NCT

C82

37ATC

C13124

1550plusmn15

Gram-positive

Citro

bacte

rsp(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Enteroba

cter

aerogenesN

CTC1

0006

ATC

C130

431550plusmn046

Gram-negative

Enterobacter

sp(hu

man


ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Enterobacter

sp(hu

man

clinicalisolateIID

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Enterococcus

sp(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

1375plusmn035

Gram-negative

Ecoli(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

EcoliNCT

C12241A

TCC25922

000

Gram-negative

Enterococcus

faecalisNCT

C12697AT

CC29212

000

Gram-negative

Eam

ylovora(D

epartm

ento

fPlant

Patholog

yFaculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

4100plusmn043

Gram-negative

Klebsie

llasp(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

295plusmn04

Gram-negative

Lactobacillus

bulga

ricus

(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Lcasei(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Llactis(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Lreuteri(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Liste

riainno

cuaNCT

C1128

8AT

CC3309

03775plusmn049

Gram-positive

Pseudomonas

aeruginosa

strain

EGYII

000

Gram-negative

Paeruginosa

(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Proteussp

(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

1400plusmn03

Gram-negative

Salm

onellatyph

imurium

NCT

C1202

3AT

CC

1402

81725plusmn049

Gram-negative

Styphi(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Sparatyphi(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

ShigellaboydiiAT

CC9207

000

Gram-negative

Staphylococcus

aureus

NCT

C12981A

TCC25923

000

Gram-positive

Stap

hylococcus

epidermidisNCT

C1336

0AT

CC1222

82375plusmn075

Gram-positive


Table6Con

tinued

Indicatorstrain

BACYA

S1a

ctivity

(inhibitio

nzone

diam

eterm

mplusmnSE

)lowastGram

nature

Vibrioparahaem

olyticu

sNCT

C3178

000

Gram-negative

Fung

iand

yeast

Aspergillus

brasiliensis

NCP

F2275ATC

C1640

4000

Cand

idaalbicans

NCP

F3179ATC

C10231

000

Ctro

picalis

(hum


mdiabeticfootD

epartm

ento

fMicrobiolog

yMedicalRe

search

Institu

teU

niversity

ofAlexand

riaE

gypt)

000


eNCP

F3178

000

lowast

Meanof

four

readings

with

stand

arderror(SE

)




5 Conclusion








References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


A

B


0

50

100

150

200

250

300

350

0010

0100

1000

10000

0 1 2 3 4 5 6 7 8 9 10

BAC

YAS

1 ac

tivity

(AU

mL)

Bact

eria

l gro

wth

Ab

at 420

nm













4 Discussion






Table6Sensitivityof

someind

icator

strainstoBA

CYA

S1

Indicatorstrain

BACYA

S1a

ctivity

(inhibitio

nzone

diam

eterm

mplusmnSE

)lowastGram

nature

Bacteria

Acinetobactersp(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative


entalstrainiso

latedfro

msoilEg

ypt)

000

Gram-positive

Blicheniform

isSH

G2(enviro

nmentalstrainiso

latedfro

msoilEg

ypt)

13plusmn17

0Gram-positive

Blicheniform

isSH

G6(enviro

nmentalstrainiso

latedfro

msoilEg

ypt)

000

Gram-positive

Blicheniform

isSH

G10

(enviro

nmentalstrainiso

lated

from

soilEg

ypt)

000

Gram-positive

BsubtilisE

MBL

AKE

(enviro

nmentalstrainiso

lated

from

El-M

ahmou

diaL

akeEg

ypt)

000

Gram-positive

BsubtilisA

S1(enviro

nmentalstrainiso

lated

from

soilEg

ypt)

1025plusmn048

Gram-positive

BsubtilisN

CTC1040

0AT

CC6633

000

Gram-positive

Bsubtilis(environm

entalstrainiso

latedfro

msoilEg

ypt)

000

Gram-positive

BacillusspAs

h2(enviro

nmentalstrainiso

lated

from

soilEg

ypt)

000

Gram-positive

Campyloba

cter

jejuni

NCT

C11322AT

CC29

428

1400plusmn041

Gram-negative

Clostridium

perfrin

gens

NCT

C82

37ATC

C13124

1550plusmn15

Gram-positive

Citro

bacte

rsp(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Enteroba

cter

aerogenesN

CTC1

0006

ATC

C130

431550plusmn046

Gram-negative

Enterobacter

sp(hu

man


ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Enterobacter

sp(hu

man

clinicalisolateIID

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Enterococcus

sp(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

1375plusmn035

Gram-negative

Ecoli(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

EcoliNCT

C12241A

TCC25922

000

Gram-negative

Enterococcus

faecalisNCT

C12697AT

CC29212

000

Gram-negative

Eam

ylovora(D

epartm

ento

fPlant

Patholog

yFaculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

4100plusmn043

Gram-negative

Klebsie

llasp(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

295plusmn04

Gram-negative

Lactobacillus

bulga

ricus

(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Lcasei(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Llactis(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Lreuteri(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Liste

riainno

cuaNCT

C1128

8AT

CC3309

03775plusmn049

Gram-positive

Pseudomonas

aeruginosa

strain

EGYII

000

Gram-negative

Paeruginosa

(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Proteussp

(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

1400plusmn03

Gram-negative

Salm

onellatyph

imurium

NCT

C1202

3AT

CC

1402

81725plusmn049

Gram-negative

Styphi(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Sparatyphi(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

ShigellaboydiiAT

CC9207

000

Gram-negative

Staphylococcus

aureus

NCT

C12981A

TCC25923

000

Gram-positive

Stap

hylococcus

epidermidisNCT

C1336

0AT

CC1222

82375plusmn075

Gram-positive


Table6Con

tinued

Indicatorstrain

BACYA

S1a

ctivity

(inhibitio

nzone

diam

eterm

mplusmnSE

)lowastGram

nature

Vibrioparahaem

olyticu

sNCT

C3178

000

Gram-negative

Fung

iand

yeast

Aspergillus

brasiliensis

NCP

F2275ATC

C1640

4000

Cand

idaalbicans

NCP

F3179ATC

C10231

000

Ctro

picalis

(hum


mdiabeticfootD

epartm

ento

fMicrobiolog

yMedicalRe

search

Institu

teU

niversity

ofAlexand

riaE

gypt)

000


eNCP

F3178

000

lowast

Meanof

four

readings

with

stand

arderror(SE

)




5 Conclusion








References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



4 Discussion






Table6Sensitivityof

someind

icator

strainstoBA

CYA

S1

Indicatorstrain

BACYA

S1a

ctivity

(inhibitio

nzone

diam

eterm

mplusmnSE

)lowastGram

nature

Bacteria

Acinetobactersp(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative


entalstrainiso

latedfro

msoilEg

ypt)

000

Gram-positive

Blicheniform

isSH

G2(enviro

nmentalstrainiso

latedfro

msoilEg

ypt)

13plusmn17

0Gram-positive

Blicheniform

isSH

G6(enviro

nmentalstrainiso

latedfro

msoilEg

ypt)

000

Gram-positive

Blicheniform

isSH

G10

(enviro

nmentalstrainiso

lated

from

soilEg

ypt)

000

Gram-positive

BsubtilisE

MBL

AKE

(enviro

nmentalstrainiso

lated

from

El-M

ahmou

diaL

akeEg

ypt)

000

Gram-positive

BsubtilisA

S1(enviro

nmentalstrainiso

lated

from

soilEg

ypt)

1025plusmn048

Gram-positive

BsubtilisN

CTC1040

0AT

CC6633

000

Gram-positive

Bsubtilis(environm

entalstrainiso

latedfro

msoilEg

ypt)

000

Gram-positive

BacillusspAs

h2(enviro

nmentalstrainiso

lated

from

soilEg

ypt)

000

Gram-positive

Campyloba

cter

jejuni

NCT

C11322AT

CC29

428

1400plusmn041

Gram-negative

Clostridium

perfrin

gens

NCT

C82

37ATC

C13124

1550plusmn15

Gram-positive

Citro

bacte

rsp(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Enteroba

cter

aerogenesN

CTC1

0006

ATC

C130

431550plusmn046

Gram-negative

Enterobacter

sp(hu

man


ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Enterobacter

sp(hu

man

clinicalisolateIID

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Enterococcus

sp(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

1375plusmn035

Gram-negative

Ecoli(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

EcoliNCT

C12241A

TCC25922

000

Gram-negative

Enterococcus

faecalisNCT

C12697AT

CC29212

000

Gram-negative

Eam

ylovora(D

epartm

ento

fPlant

Patholog

yFaculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

4100plusmn043

Gram-negative

Klebsie

llasp(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

295plusmn04

Gram-negative

Lactobacillus

bulga

ricus

(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Lcasei(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Llactis(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Lreuteri(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Liste

riainno

cuaNCT

C1128

8AT

CC3309

03775plusmn049

Gram-positive

Pseudomonas

aeruginosa

strain

EGYII

000

Gram-negative

Paeruginosa

(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Proteussp

(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

1400plusmn03

Gram-negative

Salm

onellatyph

imurium

NCT

C1202

3AT

CC

1402

81725plusmn049

Gram-negative

Styphi(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Sparatyphi(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

ShigellaboydiiAT

CC9207

000

Gram-negative

Staphylococcus

aureus

NCT

C12981A

TCC25923

000

Gram-positive

Stap

hylococcus

epidermidisNCT

C1336

0AT

CC1222

82375plusmn075

Gram-positive


Table6Con

tinued

Indicatorstrain

BACYA

S1a

ctivity

(inhibitio

nzone

diam

eterm

mplusmnSE

)lowastGram

nature

Vibrioparahaem

olyticu

sNCT

C3178

000

Gram-negative

Fung

iand

yeast

Aspergillus

brasiliensis

NCP

F2275ATC

C1640

4000

Cand

idaalbicans

NCP

F3179ATC

C10231

000

Ctro

picalis

(hum


mdiabeticfootD

epartm

ento

fMicrobiolog

yMedicalRe

search

Institu

teU

niversity

ofAlexand

riaE

gypt)

000


eNCP

F3178

000

lowast

Meanof

four

readings

with

stand

arderror(SE

)




5 Conclusion








References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table6Sensitivityof

someind

icator

strainstoBA

CYA

S1

Indicatorstrain

BACYA

S1a

ctivity

(inhibitio

nzone

diam

eterm

mplusmnSE

)lowastGram

nature

Bacteria

Acinetobactersp(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative


entalstrainiso

latedfro

msoilEg

ypt)

000

Gram-positive

Blicheniform

isSH

G2(enviro

nmentalstrainiso

latedfro

msoilEg

ypt)

13plusmn17

0Gram-positive

Blicheniform

isSH

G6(enviro

nmentalstrainiso

latedfro

msoilEg

ypt)

000

Gram-positive

Blicheniform

isSH

G10

(enviro

nmentalstrainiso

lated

from

soilEg

ypt)

000

Gram-positive

BsubtilisE

MBL

AKE

(enviro

nmentalstrainiso

lated

from

El-M

ahmou

diaL

akeEg

ypt)

000

Gram-positive

BsubtilisA

S1(enviro

nmentalstrainiso

lated

from

soilEg

ypt)

1025plusmn048

Gram-positive

BsubtilisN

CTC1040

0AT

CC6633

000

Gram-positive

Bsubtilis(environm

entalstrainiso

latedfro

msoilEg

ypt)

000

Gram-positive

BacillusspAs

h2(enviro

nmentalstrainiso

lated

from

soilEg

ypt)

000

Gram-positive

Campyloba

cter

jejuni

NCT

C11322AT

CC29

428

1400plusmn041

Gram-negative

Clostridium

perfrin

gens

NCT

C82

37ATC

C13124

1550plusmn15

Gram-positive

Citro

bacte

rsp(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Enteroba

cter

aerogenesN

CTC1

0006

ATC

C130

431550plusmn046

Gram-negative

Enterobacter

sp(hu

man


ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Enterobacter

sp(hu

man

clinicalisolateIID

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Enterococcus

sp(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

1375plusmn035

Gram-negative

Ecoli(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

EcoliNCT

C12241A

TCC25922

000

Gram-negative

Enterococcus

faecalisNCT

C12697AT

CC29212

000

Gram-negative

Eam

ylovora(D

epartm

ento

fPlant

Patholog

yFaculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

4100plusmn043

Gram-negative

Klebsie

llasp(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

295plusmn04

Gram-negative

Lactobacillus

bulga

ricus

(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Lcasei(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Llactis(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Lreuteri(dairy

prod

uctisolateDepartm

ento

fDairy

Prod

ucts

Faculty

ofAgricultureU

niversity

ofAlexand

riaE

gypt)

000

Gram-positive

Liste

riainno

cuaNCT

C1128

8AT

CC3309

03775plusmn049

Gram-positive

Pseudomonas

aeruginosa

strain

EGYII

000

Gram-negative

Paeruginosa

(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Proteussp

(hum

anclinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

1400plusmn03

Gram-negative

Salm

onellatyph

imurium

NCT

C1202

3AT

CC

1402

81725plusmn049

Gram-negative

Styphi(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

Sparatyphi(hu

man

clinicalisolateD

epartm

ento

fMicrobiolog

yFaculty


ofAlexand

riaE

gypt)

000

Gram-negative

ShigellaboydiiAT

CC9207

000

Gram-negative

Staphylococcus

aureus

NCT

C12981A

TCC25923

000

Gram-positive

Stap

hylococcus

epidermidisNCT

C1336

0AT

CC1222

82375plusmn075

Gram-positive


Table6Con

tinued

Indicatorstrain

BACYA

S1a

ctivity

(inhibitio

nzone

diam

eterm

mplusmnSE

)lowastGram

nature

Vibrioparahaem

olyticu

sNCT

C3178

000

Gram-negative

Fung

iand

yeast

Aspergillus

brasiliensis

NCP

F2275ATC

C1640

4000

Cand

idaalbicans

NCP

F3179ATC

C10231

000

Ctro

picalis

(hum


mdiabeticfootD

epartm

ento

fMicrobiolog

yMedicalRe

search

Institu

teU

niversity

ofAlexand

riaE

gypt)

000


eNCP

F3178

000

lowast

Meanof

four

readings

with

stand

arderror(SE

)




5 Conclusion








References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table6Con

tinued

Indicatorstrain

BACYA

S1a

ctivity

(inhibitio

nzone

diam

eterm

mplusmnSE

)lowastGram

nature

Vibrioparahaem

olyticu

sNCT

C3178

000

Gram-negative

Fung

iand

yeast

Aspergillus

brasiliensis

NCP

F2275ATC

C1640

4000

Cand

idaalbicans

NCP

F3179ATC

C10231

000

Ctro

picalis

(hum


mdiabeticfootD

epartm

ento

fMicrobiolog

yMedicalRe

search

Institu

teU

niversity

ofAlexand

riaE

gypt)

000


eNCP

F3178

000

lowast

Meanof

four

readings

with

stand

arderror(SE

)




5 Conclusion








References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




5 Conclusion








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





















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article A Sequential Statistical Approach towards an …downloads.hindawi.com/journals/tswj/2014/396304.pdf · 2019-07-31 · Research Article A Sequential Statistical Approach