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Research ArticleSimultaneous Production of Biosurfactants and Bacteriocins byProbiotic Lactobacillus casei MRTL3

Deepansh Sharma and Baljeet Singh Saharan

Microbial Resource Technology Laboratory Department ofMicrobiology Kurukshetra University Kurukshetra Haryana 136 119 India

Correspondence should be addressed to Baljeet Singh Saharan baljeetkukgmailcom

Received 31 July 2013 Accepted 13 November 2013 Published 29 January 2014

Academic Editor Todd R Callaway

Copyright copy 2014 D Sharma and B Singh Saharan This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Lactic acid bacteria (LAB) are ubiquitous and well-known commensal bacteria in the human and animal microflora LAB areextensively studied and used in a variety of industrial and food fermentations They are widely used for humans and animals asadjuvants probiotic formulation and dietary supplements and in other food fermentation applications In the present investigationLAB were isolated from raw milk samples collected from local dairy farms of Haryana India Further the isolates were screenedfor simultaneous production of biosurfactants and bacteriocins Biosurfactant produced was found to be a mixture of lipid andsugar similar to glycolipids The bacteriocin obtained was found to be heat stable (5min at 100∘C) Further DNA of the strain wasextracted and amplified by the 16S rRNA sequencing using universal primers The isolate Lactobacillus caseiMRTL3 was found tobe a potent biosurfactant and bacteriocin producer It seems to have huge potential for food industry as a biopreservative andorfood ingredient

1 Introduction

Probiotics are living microbial preparations that have benefi-cial effects on the well-being of the host when administeredin adequate amount [1] A range of beneficial effects havebeen reported for probiotics including improvement indigestion [2] antidiarrheal property [3] and prevention offood-borne pathogens [4] Food-borne pathogens such asListeria monocytogenes L innocua Pseudomonas aeruginosaSalmonella typhi Staphylococcus aureus S epidermidis andBacillus cereus not only are harmful to human health but alsolead to spoilage of foodstuff Although chemical preservativesefficiently inhibit the growth of food-borne pathogens theiruse in food industry is still under scrutiny as some of themhave been reported to be unsafe to human health and forthe environment LAB produce a large number of antimi-crobial compounds such as organic acids H

2O2 diacetyl

enzymes bacteriocins and biosurfactants which are effectiveagainst food spoilage and pathogenic bacteria Amongst theseantimicrobial metabolites bacteriocins have been found as apotentially safe class of biopreservatives For example nisin

a bacteriocin produced by Lactococcus lactis has been usedsince last decade to extend the shelf life of preserved food [56]Thebacteriocins are latent biopreservatives particularly inmeat packaged food and dairy products [7] A large numberof reports indicate the potential use of bacteriocins in thecontrol of important gastric pathogens specially Salmonellasp [8]Campylobacter jejuni Lmonocytogenes [9] and E coliO157H7 [10]

Microbial biosurfactants are amphiphilic metaboliteswith a pronounced surface activity with a broad rangeof chemical structures (such as glycolipids lipopeptidespolysaccharide-protein complexes phospholipids fattyacids and neutral lipids) with several advantages overchemical surfactants that is low toxicity biodegradableand effective at different ranges of temperature and pH[11 12] They are being used for industrial applicationsin the pharmaceuticals biomedical and food processingindustries [11 13 14] This paper deals with the study ofthe biotechnological potential of strain L casei MRTL3The selective potential of the strain for bacteriocins andbiosurfactants productions has been described

Hindawi Publishing CorporationInternational Journal of MicrobiologyVolume 2014 Article ID 698713 7 pageshttpdxdoiorg1011552014698713

2 International Journal of Microbiology

2 Materials and Methods

21 Isolation of Lactic Acid Bacteria Various strains of LABwere isolated from raw milk by enrichment in 100mL ofsterile minimal medium (MM) with 2 paraffin as carbonsource The suspension was incubated at 37∘C for 48 h inincubator cum shaker (NSW India) and it was subculturedonMRSmedium [15] Further all these isolated cultures weresubjected to series of physiological biochemical and species-specific standard identification tests [16] The isolates werestored at minus20∘C in MRS broth containing 20 (vv) glycerolstock as master stock until they were further usedThe isolateMRTL3 was chosen for further studies

22 Biosurfactants and Bacteriocins Extraction The isolatewas grown in MRS broth at 37∘C for 72 hThe cell free super-natant (CFS) was adjusted to pH 65 with 5M HCl heatedat 100∘C for 3min and centrifuged at 10000 g for 15minat 10∘C for the recovery of bacteriocins On the otherhand biomass was washed twice with demineralized watercentrifuged (10000 g 15min 10∘C) resuspended in a volumeof phosphate buffer saline (PBS pH 70) incubated for 2 h atroom temperature and centrifuged at (10000 g 15min 10∘C)to take the PBS extract free of biomass

23 Biosurfactants Production Biosurfactants produced bythe isolateMRTL3were determined bymeasuring the surfacetension (ST) of the culture supernatant in case of the excretedbiosurfactants and of the PBS extracts in the case of thecell-bound biosurfactants For the recovery of the cell-bound biosurfactants fermentationmediumwas centrifuged(10000 g 15min 10∘C) to recover the cells that were washedtwice with demineralized water and resuspended in PBS (pH70) incubated for 2 h at room temperature and centrifuged(10000 g 15min 10∘C) ST of sample was measured by theRing method [17] using a Tensiometer (Lauda Germany)equipped with a 19 cm De Nouy platinum ring at roomtemperature About 10mL sample was withdrawn every 12 hand surface tension was measured

24 Bacteriocins Production The pH of the samples wasadjusted to 65with 5MNaOH heated at 100∘C for 3min andcentrifuged (10000 g for 15min at 10∘C)The presence of bac-teriocins in the extracts was qualitatively determined by agarwell diffusion assay method against Staphylococcus aureus(ATCC 6538P) S epidermidis (ATCC 12228) Shigella flexneri(ATCC 9199) Salmonella typhi (MTCC 733) Pseudomonasaeruginosa (ATCC 15442) Bacillus cereus (ATCC 11770)Listeria monocytogenes (MTCC 657) and L innocua (ATCC33090) grown at 37∘C in brain heart infusion (BHI HimediaIndia) The antibacterial activity in CFS was determined bywell diffusion method [17] with slight modification CFSof overnight (16ndash18 h) culture of L casei MRTL3 grown inMRS broth at 37∘C was obtained by centrifugation (10000 g15min 4∘C) and the pH was adjusted to 65 To avoidproteolytic degradation of the bacteriocin CFS was boiledfor 3min Soft nutrient agar (08 wv) was allowed to cooldown in sterile Petri dish after addition of test organism

culture grown up to the early stationary phase Wells weremade in the lawn of hardened soft agar and aliquots of50 120583L of supernatant were poured in the wells After 24 h ofincubation at the optimal growth temperature of indicatorstrain a clear zone of inhibition of at least 2mm in diameteraround cut wells was recorded as positive [18] Additionallythe antibacterial activity of the biosurfactants produced wasalso determined by the same procedure

25 Colony PCR (16S rRNA Gene Amplification) ColonyPCR of the isolate was performed according to the Sheuet al [19] The optimized colony PCR reaction mixturecontained 1X PCR amplification buffer (20mM (NH

4)2SO4

725mM TrisHCl 01 Tween-20 and pH 90) 25mMMgCl

2 200120583M each deoxynucleotide triphosphate 25 120583M

each primer (27f 51015840-AGAGTTTGATCMTGGCTCAG-31015840 and1385r 31015840-AATTCAAATTTAATTTCTTTCC-51015840) and 125UDNA polymerase in 50 120583L PCR reaction mixture Colonies(approximately 1mm in diameter) were picked up with asterilized toothpick and directly transferred to the PCR tubesas DNA templates The thermal cycle programme run on athermocycler PCR system (Eppendorf Germany) consistedof one cycle of 94∘C for 10min 51∘C for 2min 72∘C for 2minand 35 cycles of 94∘C for 20 s 57∘C for 45 s (decreased by 1 sper cycle) and 72∘C for 1min and then incubation at 72∘Cfor 5min and a final incubation at 4∘C

26 Detection of PCR Products PCR-amplified DNA frag-ments were observed by Agarose gel electrophoresis in 13Agarose gel in TAE buffer (004M Tris acetate 002M aceticacid and 0001M EDTA) containing 1 gmL of SYBR greenBriefly 10 microlitres of each amplification mixture andthe molecular mass marker were subjected to Agarose gelelectrophoresis and SYBR green stainingThe amplifiedDNAfragments were visualized by UV illumination

27 Sequencing and Analysis of 16S rRNA Gene The ABIPrism Big Dye Terminator Cycle Sequencing Ready ReactionKit (Applied Biosystems) was used for the sequencing ofthe PCR product A combination of universal primers waschosen to sequence the gene sequence Samples were run onan ABI PRISM 3730XLDNAAnalyzer (Applied Biosystems)Each alignment was checked manually corrected and thenanalyzed using the UPGMA method [19] Phylogenetic treewas constructed using the MEGA 5 (Molecular EvolutionaryGenetics Analysis) software [20 21]

28 Product Characterization

281Thin Layer Chromatography Samples were dissolved inethyl acetate and applied to dried precoated silica TLC plates(Merck India) Briefly biosurfactant molecules were priorextracted as follows Aliquot (4mL) of CFS was extractedtwice with ethyl acetate (Rankem India) 1 125 by vigorousvortexing for 2min Then upper phase was extracted twotimes with ethyl acetate and the ethyl acetate was evaporatedat room temperature [22 23] The extracted product wascharacterized by using analytical TLC carried out on Silica

International Journal of Microbiology 3

Table 1 Testing of MRTL3 for biosurfactant production by various methods

Strain(s) Surface tension Emulsification index HemolysisCFSlowast PBS extractlowastlowast Kerosene Diesel

MRTL3 468 plusmn 015 408 plusmn 020 58 plusmn 015 57 plusmn 020 ++lowastSurface tension of MRS is 530mNm lowastlowastsurface tension of PBS is 724mNm

(a) (b) (c)

Figure 1 Biosurfactant and bacteriocin production (a) Hemolysis on blood agar plates (b) Emulsification of kerosene and diesel oil byculture supernatant (c) Bacteriocin activity against Listeria innocua

gel plates Briefly one mL aliquot of each crude biosur-factant sample was concentrated resuspended in 5 120583L ofethyl acetate and separated on a precoated silica gel plate(Merck India) using chloroformmethanolglacial acetic acid(65 15 2 vv) as developing solvent system with differentpost-color-developing reagents The sugar moieties werestained with anisaldehyde whereas the fatty acid moietieswere stained with ammoniummolybdatecerium sulfate [2425]

282 Purification of Biosurfactant Crude biosurfactant resi-due was partially purified in silica gel (60ndash120 mesh) col-umn eluted with gradient of chloroform and methanol ran-ging from 20 1 to 2 1 (vv) The fractions were pooled afterTLC analysis and solvents were evaporated Once dried thebiosurfactant was stored at 20∘C

283 Partial Purification of Bacteriocins CFS from 05 L ofovernight culture of isolateMRTL3was prepared as describedearlier (Section 24) Ammonium sulfate was gently added tothe supernatant maintained at 4∘C to obtain 60 saturationand themixturewas stirred for 4 h at 4∘CAfter centrifugationfor 1 h at 10000 g at 4∘C the resulting pellet was resuspendedin 30mL of 25mM ammonium acetate buffer (pH 65) untilfurther use

284 Fourier Transform Infrared Spectroscopy (FTIR) Mo-lecular characterization was performed with a crude biosur-factant sample which was dialyzed against demineralizedwater at 4∘C in a dialysis membrane (molecular weightcutoff 10000 kDa Himedia India) and then freeze-driedCharacterization was carried out by FTIR analysis of the

biosurfactant by scanning it in the range of 4000ndash400 cmminus1at a resolution of 4 cmminus1 (Model-ABB and MB-3000)

285 NMR Spectroscopy The purified biosurfactant was dis-solved in deuterated chloroform and 1H analysis was carriedout using a Bruker 300 spectrometer The biosurfactant wasdissolved in deuterated chloroform (50mgmLminus1) and thespectrum was recorded 1H chemical shifts were expressed inppm relative to the solvent shift as chemical standard

29 Statistical Analysis All the values were recorded intriplicates and were subjected to an analysis of standarddeviation by using SPSS (v 16) statistical software package

3 Results and Discussion

31 Screening Assays for Biosurfactant Production Initiallythe isolate was screened for its ability to produce biosurfac-tant and bacteriocin using various qualitative and quantita-tivemethods As the strainwas spot inoculated over the bloodagar plate it resulted in a significant zone of clearance aroundthe colony confirming the production of biosurfactants Incase of drop collapsing test the flattened drops of CFSover the oil coated surface also indicated the presence ofbiosurfactant A clear halo zone (lt3 cm2) was observed inthe oil displacement test and had significant emulsificationactivity against kerosene oil (Table 1 Figure 1)

32 Biosurfactant and Bacteriocin Production In currentstudy Lactobacillus caseiMRTL3 was tested for biosurfactantand bacteriocin production using different qualitative andquantitative methods (Figure 1) Lactobacillus casei MRTL3was found positive for cell-bound and excreted biosurfactant


Table 2 Surface tension values (mNm) of the crude biosurfactant produced by MRTL3

Strain(s) Surface tension (mNm)lowast

0 h 12 h 24 h 36 h 48 h 60 h 72 hMRTL3 531 plusmn 005 408 plusmn 015 407 plusmn 026 409 plusmn 010 412 plusmn 010 411 plusmn 017 414 plusmn 005

lowastMRS surface tension was 530mNm

Table 3 Chemical shift assignment of biosurfactant in 1H NMR

Assignment Chemical shift (ppm)CH3ndash(CH2)119899ndash 0918ndash(CH2)119899ndash 1274ndashCH2OH 36621-H (sugar) 4384

and extracellular bacteriocin production The crude bacteri-ocin and biosurfactant isolated from strain Lactobacillus caseiMRTL3 showed significant antimicrobial activity against abroad range of pathogens including gram positive (Staphy-lococcus aureus ATCC 6538P S epidermidis ATCC 12228Bacillus cereus ATCC 11770 Listeria monocytogenes MTCC657 and L innocuaATCC 33090) and gram negative bacteria(Shigella flexneri ATCC 9199 Salmonella typhi MTCC 733and Pseudomonas aeruginosa ATCC 15442) Several biosur-factants and bacteriocins that exhibit antimicrobial activityhave been previously described [12] but their simultaneousproduction has not been reported earlier Gudina et al [26]have suggested various approaches for successful recoveryof biosurfactant and bacteriocin under different pH con-ditions using Lactococcus lactis and they recovered boththe product separately Antibacterial properties of biosur-factants and bacteriocins isolated from Lactobacillus caseiMRTL3 against pathogenic bacteria were found similar tothose obtained with the crude biosurfactant recovered fromStreptococcus thermophiluswith various concentrations (25 to100mgmLminus1) The surface tension of the production mediawas reduced to 408mNm from its initial value of 53mNm(Table 2) This decrease of surface tension confirmed theproduction of biosurfactants by the isolate and its accu-mulation within the media Such a reduction in surfacetension during the logarithmic and stationary phase hasalready been reported for various biosurfactant producingmicroorganisms [27 28]

Decrease in surface tension of the culture broth wasobserved for all the strains after 72 h of incubation althoughthose reductions varied markedly from 9 to 14mNm whencompared to the surface tension of MRS broth (530mNm)The ability of a biosurfactant to bring down the surfacetension of water from 724 to 408mNm is considered tobe a good characteristic of a potent surface active agent Thebiosurfactant production was found to be growth associatedas a parallel relationship was observed between biomassproduction and the decrease in culture broth surface ten-sion Similar results have been reported by Gudina et al[26] while using different lactobacilli for the production ofbiosurfactants under optimal conditions The lowest value ofsurface tension was obtained at the end of the shake flask

MRTL3 MRS RL

Figure 2 Anisaldehyde staining of BS TLC plate

4000 3750 3500 3250 3000 2750 2500 2250 2000 1750 1500 1250

105

975

90

825

75

675

T(

) 3310

2924

2854

1736

1651

1458

1404 1319 1227

1119

1057

980

903

841

802

710

656

(1cm)

Figure 3 FTIR absorption spectra of partially purified biosurfactantproduced by strain MRTL3

experiment The strain MRTL3 showed the highest excretedbiosurfactant production rate with a reduction in the culturebroth surface tension from 530 to 408mNm The growthand biomass were restricted due to high yield of lactic acidproduction during the shake flask experiments The culturesupernatant responded positively to oil collapse method andemulsification capacity and reduction of surface tension wasobserved

33 TLC Analysis Separated compounds were detected onthe plate as a single pink spot (Figure 2) and dark bluespots for fatty acids It confirmed the presence of glycolipidbiosurfactant

34 Molecular Characterization of the Isolate MRTL3 Using16S rRNA Sequencing Amplified DNA fragments were


0018

5360

5281

5258

5235

5211

5187

5065

5034

5010

4985

4933

4897

4880

4767

4758

4714

4574

4407

4384

4361

4338

4258

4080

4037

3985

3979

3880

3853

3745

3727

3662

3555

60 55 50 45 40 35 30

096

171

100

35 30 25 20 15 10 05(ppm)

041

139

058

030

012

DS-3

(ppm)

2425

2399

2373

2341

2315

2196

2173

2160

2070

2049

2036

1957

1931

1907

1889

1782

1774

1739

1719

1656

1629

1607

1583

1526

1507

1484

1459

1435

1274

1179

1138

1116

1089

1066

1030

1000

0978

0953

0918

0910

0898

0876

2706

Figure 4 1H NMR spectrum of biosurfactant produced by Lactobacillus caseiMRTL3

Table 4 Antibacterial activity shown by bacteriocin and biosurfactant against various test organisms

Indicator organisms Origin Bacteriocinlowast Biosurfactantlowast

Pseudomonas aeruginosa ATCC 15442 +++ +++Salmonella typhi MTCC 733 +++ +++Shigella flexneri ATCC 9199 +++ +++Staphylococcus aureus ATCC 6538P ++++ ++++Staphylococcus epidermidis ATCC 12228 ++++ +++Listeria monocytogenes MTCC 657 ++++ ++++Listeria innocua ATCC 33090 ++++ ++++Bacillus cereus ATCC 11770 ++++ ++++lowastExcellent gt8mm (++++) good lt8mm (+++) and fair lt5mm (++)

sequenced using Sanger Dideoxy method [29] Forward andreverse sequence of isolate was joined using DNA Baserv 353 software and finally was identified as Lactobacilluscasei The 16S rRNA gene sequence obtained from theisolate was compared with other bacterial sequences by usingNCBI mega BLAST (httpblastncbinlmnihgovBlastcgi)for their pairwise identities Isolate has shown 99 identitywith Lactobacillus casei ATCC 334 with maximum querycoverage of 98 Further Consensus sequences were alignedand compared with the available database in NCBI The 16S

rRNA sequence of the isolated bacterium was submitted inthe Genbank (NCBI) with an accession number KC568563

35 FTIR Spectrometry Analysis Themolecular compositionof the partially purified biosurfactant was determined byFTIR spectroscopy (Figure 3) which reveals the presence ofcarbohydrate and lipid combination The most significantbands were located 1736 cmminus1 (for the C=O ester bond)and 1057 cmminus1 (polysaccharides) 3310 cmminus1 confirming OHstretching and 1400ndash1460 cmminus1 for C=H stretching which


Lactobacillus pantheris strain LMG 21017 16S ribosomal RNA complete sequenceLactobacillus camelliae strain MCH3-1 16S ribosomal RNA complete sequence

Lactobacillus manihotivorans strain OND 32 16S ribosomal

Lactobacillus rhamnosus strain JCM 1136 16S ribosomal RNA complete s Lactobacillus zeae strain RIA 482 16S ribosomal RNA partial sequenceLactobacillus casei subsp casei ATCC 393 strain ATCC 393 16S ribosom

Lactobacillus paracasei subsp paracasei strain R094 16S ribosomal R Lactobacillus casei strain MRTL3 16S ribosomal RNA g

Lactobacillus paracasei subsp tolerans strain NBRC 15906 16S ribosom

Lactobacillus casei ATCC 334 strain ATCC 334 16S ribosomal RNA

0008

Lactobacillus rhamnosus GG strain GG (ATCC 53103) ribosomal RNA

Figure 5 Phylogenetic relatedness of Lactobacillus caseiMRTL3 using UPGMA tree

confirmed the presence of glycolipid moieties The com-pound showed the CndashH stretching vibrations in the transmit-tance range 2924 cmminus1 indicating the aliphatic chain

36 NMR Analysis The data of 1H-NMR spectrum of com-pound (Table 3 Figure 4) indicate that the compound wasa glycolipid Proton NMR analysis confirmed the presenceof lipid and sugar moiety in the biosurfactant The presenceof the methyl esters in the structure of biosurfactant can berelated to an increase of its hydrophobicity and consequentlythis leads to the increment of their surfactant powers andantifungal and hemolytic activities

37 Antibacterial Property of Bacteriocin and BiosurfactantThe antibacterial activity of bacteriocin and biosurfactantsproduced by the isolate L casei MRTL3 was tested against 8pathogenic culturesThe results show that the culture exhibitsantibacterial activity against these food-borne pathogens(Table 4)

38 Phylogenetic Relationship The phylogenetic tree wasconstructed using MEGA 505 software to determine evolu-tionary relationships of the isolate (Figure 5)

4 Conclusion

In the present study isolate (Lactobacillus casei MRTL3)possessing simultaneous bacteriocin and biosurfactant-producing ability was identified and characterizedLactobacillus casei MRTL3 shows remarkable ability todecrease surface tension and also shows inhibitory effectagainst various food-borne pathogens Further the structuralcharacteristics were determined using TLC FTIR and 1HNMR spectroscopy which confirmed that the compound is aglycolipid

Thus the result obtained suggests that the isolate L caseiMRTL3 is a potent lactic acid bacterium as a biosurfactantand bacteriocin producer and possibly can be used as thesource of biopreservatives (bacteriocin andor biosurfactant)in the food processing sector being a suitable alternative tochemicalconventional preservatives

Conflict of Interests

The authors declare that there is no conflict of interests

Acknowledgment

The grant obtained from University Grant Commission(UGC India) in the form of major research project (sanc-tioned to Dr Baljeet Singh Saharan) is fully acknowledged

References

[1] P R Marteau ldquoProbiotics in clinical conditionsrdquo ClinicalReviews in Allergy and Immunology vol 22 no 3 pp 255ndash2732002

[2] ldquoReport of a Joint FAOWHO Expert Consultation on Evalua-tion of Health and Nutritional Properties of Probiotics in FoodIncluding Powder Milk with Live Lactic Acid Bacteriardquo FAOWHO 2012

[3] J Saavedra ldquoProbiotics and infectious diarrheardquoAmerican Jour-nal of Gastroenterology vol 95 no 1 pp S16ndashS18 2000

[4] P M Matricardi F Rosmini V Panetta L Ferrigno and SBonini ldquoHay fever and asthma in relation to markers of infec-tion in the United Statesrdquo Journal of Allergy and Clinical Immu-nology vol 110 no 3 pp 381ndash387 2002

[5] L H Deegan P D Cotter C Hill and P Ross ldquoBacteriocinsbiological tools for bio-preservation and shelf-life extensionrdquoInternational Dairy Journal vol 16 no 9 pp 1058ndash1071 2006

[6] B Collins M Caitriona P D Guinane and C H Cot-ter ldquoAssessing the contributions of the LiaS histidine kinaseto the innate resistance of Listeria monocytogenes to nisincephalosporins and disinfectantsrdquo Applied and EnvironmentalMicrobiology vol 78 no 8 pp 2923ndash2929 2012

[7] S Mills C Stanton C Hill and R P Ross ldquoNew developmentsand applications of bacteriocins and peptides in foodsrdquo AnnualReview of Food Science and Technology vol 2 pp 299ndash329 2011

[8] P G Casey G E Gardiner G Casey et al ldquoA five-strain pro-biotic combination reduces pathogen shedding and alleviatesdisease signs in pigs challengedwith Salmonella enterica serovarTyphimuriumrdquo Applied and Environmental Microbiology vol73 no 6 pp 1858ndash1863 2007

[9] AAllende F A Tomas-Barberan andM I Gil ldquoMinimal proc-essing for healthy traditional foodsrdquo Trends in Food Science andTechnology vol 17 no 9 pp 513ndash519 2006


[10] M M Brashears M L Galyean G H Loneragan J E Mannand K Killinger-Mann ldquoPrevalence of Escherichia coliO157H7and performance by beef feedlot cattle given Lactobacillusdirect-fed microbialsrdquo Journal of Food Protection vol 66 no5 pp 748ndash754 2003

[11] B S Saharan R K Sahu and D Sharma ldquoA Review on biosur-factants fermentation current developments and perspectivesrdquoGenetic Engineering and Biotechnology Journal vol 2011 14pages 2011

[12] L Rodrigues I M Banat J Teixeira and R Oliveira ldquoBio-surfactants potential applications in medicinerdquo Journal ofAntimicrobial Chemotherapy vol 57 no 4 pp 609ndash618 2006

[13] M Henkel M M Mueller J H Kuegler et al ldquoRhamnolipidsas biosurfactants from renewable resources concepts for next-generation rhamnolipid productionrdquo Process Biochemistry vol47 pp 1207ndash1219 2012

[14] M M Mueller J H Kuegler M Henkel et al ldquoRhamnolipids-next generation surfactantsrdquo Journal of Biotechnology vol 162pp 366ndash380 2012

[15] M Rogosa and M E Sharpe ldquoSpecies differentiation of humanvaginal lactobacillirdquo Journal of GeneralMicrobiology vol 23 pp197ndash201 1960

[16] J G Cappuccino and N Sherman Microbiology A LaboratoryManual Pearson San Francisco Calif USA 10th edition 2013

[17] S H Kim E J Lim S O Lee J D Lee and T H Lee ldquoPurifi-cation and characterization of biosurfactants fromNocardia spL-417rdquo Biotechnology and Applied Biochemistry vol 31 no 3 pp249ndash253 2000

[18] U Schillinger and F K Lucke ldquoAntibacterial activity of Lac-tobacillus sake isolated from meatrdquo Applied and EnvironmentalMicrobiology vol 55 no 8 pp 1901ndash1906 1989

[19] D-S Sheu Y-T Wang and C-Y Lee ldquoRapid detection ofpolyhydroxyalkanoate-accumulating bacteria isolated from theenvironment by colony PCRrdquo Microbiology vol 146 no 8 pp2019ndash2025 2000

[20] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987

[21] S Kumar K Tamura andMNeiManual forMEGAMolecularEvolutionary Genetics Analysis Software Pennsylvania StateUniversity University Park Pa USA 1993

[22] C Syldatk S Lang UMatulovic and FWagner ldquoProduction offour interfacial active rhamnolipids from n-alkanes or glycerolby resting cells of Pseudomonas species DSM 2874rdquo Zeitschriftfur Naturforschung C vol 40 no 1-2 pp 61ndash67 1985

[23] T Schenk I Schuphan and B Schmidt ldquoHigh-performanceliquid chromatographic determination of the rhamnolipidsproduced by Pseudomonas aeruginosardquo Journal of Chromatog-raphy A vol 693 no 1 pp 7ndash13 1995

[24] B Hormann M M Muller C Syldatk and R HausmannldquoRhamnolipid production by Burkholderia plantarii DSM9509Trdquo European Journal of Lipid Science and Technology vol112 no 6 pp 674ndash680 2010

[25] B Anandaraj and P Thivakaran ldquoIsolation and production ofbiosurfactant producing organism from oil spilled soilrdquo Journalof Biosciences Technology vol 1 no 3 pp 120ndash126 2010

[26] E Z Gudina J A Teixeira and L R Rodrigues ldquoBiosurfactant-producing lactobacilli screening production profiles andeffect of medium compositionrdquo Applied and Environmental SoilScience vol 2011 Article ID 201254 9 pages 2011

[27] S-C Lee S-J Lee S-H Kim et al ldquoCharacterization of newbiosurfactant produced by Klebsiella sp Y6-1 isolated fromwaste soybean oilrdquo Bioresource Technology vol 99 no 7 pp2288ndash2292 2008

[28] N Rodrıguez J M Salgado S Cortes and J M DomınguezldquoAlternatives for biosurfactants and bacteriocins extractionfrom Lactococcus lactis cultures produced under different pHconditionsrdquo Letters in Applied Microbiology vol 51 no 2 pp226ndash233 2010

[29] M Hattori and Y Sakaki ldquoDideoxy sequencing method usingdenatured plasmid templatesrdquo Analytical Biochemistry vol 152no 2 pp 232ndash238 1986

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2 Materials and Methods

21 Isolation of Lactic Acid Bacteria Various strains of LABwere isolated from raw milk by enrichment in 100mL ofsterile minimal medium (MM) with 2 paraffin as carbonsource The suspension was incubated at 37∘C for 48 h inincubator cum shaker (NSW India) and it was subculturedonMRSmedium [15] Further all these isolated cultures weresubjected to series of physiological biochemical and species-specific standard identification tests [16] The isolates werestored at minus20∘C in MRS broth containing 20 (vv) glycerolstock as master stock until they were further usedThe isolateMRTL3 was chosen for further studies

22 Biosurfactants and Bacteriocins Extraction The isolatewas grown in MRS broth at 37∘C for 72 hThe cell free super-natant (CFS) was adjusted to pH 65 with 5M HCl heatedat 100∘C for 3min and centrifuged at 10000 g for 15minat 10∘C for the recovery of bacteriocins On the otherhand biomass was washed twice with demineralized watercentrifuged (10000 g 15min 10∘C) resuspended in a volumeof phosphate buffer saline (PBS pH 70) incubated for 2 h atroom temperature and centrifuged at (10000 g 15min 10∘C)to take the PBS extract free of biomass

23 Biosurfactants Production Biosurfactants produced bythe isolateMRTL3were determined bymeasuring the surfacetension (ST) of the culture supernatant in case of the excretedbiosurfactants and of the PBS extracts in the case of thecell-bound biosurfactants For the recovery of the cell-bound biosurfactants fermentationmediumwas centrifuged(10000 g 15min 10∘C) to recover the cells that were washedtwice with demineralized water and resuspended in PBS (pH70) incubated for 2 h at room temperature and centrifuged(10000 g 15min 10∘C) ST of sample was measured by theRing method [17] using a Tensiometer (Lauda Germany)equipped with a 19 cm De Nouy platinum ring at roomtemperature About 10mL sample was withdrawn every 12 hand surface tension was measured

24 Bacteriocins Production The pH of the samples wasadjusted to 65with 5MNaOH heated at 100∘C for 3min andcentrifuged (10000 g for 15min at 10∘C)The presence of bac-teriocins in the extracts was qualitatively determined by agarwell diffusion assay method against Staphylococcus aureus(ATCC 6538P) S epidermidis (ATCC 12228) Shigella flexneri(ATCC 9199) Salmonella typhi (MTCC 733) Pseudomonasaeruginosa (ATCC 15442) Bacillus cereus (ATCC 11770)Listeria monocytogenes (MTCC 657) and L innocua (ATCC33090) grown at 37∘C in brain heart infusion (BHI HimediaIndia) The antibacterial activity in CFS was determined bywell diffusion method [17] with slight modification CFSof overnight (16ndash18 h) culture of L casei MRTL3 grown inMRS broth at 37∘C was obtained by centrifugation (10000 g15min 4∘C) and the pH was adjusted to 65 To avoidproteolytic degradation of the bacteriocin CFS was boiledfor 3min Soft nutrient agar (08 wv) was allowed to cooldown in sterile Petri dish after addition of test organism

culture grown up to the early stationary phase Wells weremade in the lawn of hardened soft agar and aliquots of50 120583L of supernatant were poured in the wells After 24 h ofincubation at the optimal growth temperature of indicatorstrain a clear zone of inhibition of at least 2mm in diameteraround cut wells was recorded as positive [18] Additionallythe antibacterial activity of the biosurfactants produced wasalso determined by the same procedure

25 Colony PCR (16S rRNA Gene Amplification) ColonyPCR of the isolate was performed according to the Sheuet al [19] The optimized colony PCR reaction mixturecontained 1X PCR amplification buffer (20mM (NH

4)2SO4

725mM TrisHCl 01 Tween-20 and pH 90) 25mMMgCl

2 200120583M each deoxynucleotide triphosphate 25 120583M

each primer (27f 51015840-AGAGTTTGATCMTGGCTCAG-31015840 and1385r 31015840-AATTCAAATTTAATTTCTTTCC-51015840) and 125UDNA polymerase in 50 120583L PCR reaction mixture Colonies(approximately 1mm in diameter) were picked up with asterilized toothpick and directly transferred to the PCR tubesas DNA templates The thermal cycle programme run on athermocycler PCR system (Eppendorf Germany) consistedof one cycle of 94∘C for 10min 51∘C for 2min 72∘C for 2minand 35 cycles of 94∘C for 20 s 57∘C for 45 s (decreased by 1 sper cycle) and 72∘C for 1min and then incubation at 72∘Cfor 5min and a final incubation at 4∘C

26 Detection of PCR Products PCR-amplified DNA frag-ments were observed by Agarose gel electrophoresis in 13Agarose gel in TAE buffer (004M Tris acetate 002M aceticacid and 0001M EDTA) containing 1 gmL of SYBR greenBriefly 10 microlitres of each amplification mixture andthe molecular mass marker were subjected to Agarose gelelectrophoresis and SYBR green stainingThe amplifiedDNAfragments were visualized by UV illumination

27 Sequencing and Analysis of 16S rRNA Gene The ABIPrism Big Dye Terminator Cycle Sequencing Ready ReactionKit (Applied Biosystems) was used for the sequencing ofthe PCR product A combination of universal primers waschosen to sequence the gene sequence Samples were run onan ABI PRISM 3730XLDNAAnalyzer (Applied Biosystems)Each alignment was checked manually corrected and thenanalyzed using the UPGMA method [19] Phylogenetic treewas constructed using the MEGA 5 (Molecular EvolutionaryGenetics Analysis) software [20 21]

28 Product Characterization

281Thin Layer Chromatography Samples were dissolved inethyl acetate and applied to dried precoated silica TLC plates(Merck India) Briefly biosurfactant molecules were priorextracted as follows Aliquot (4mL) of CFS was extractedtwice with ethyl acetate (Rankem India) 1 125 by vigorousvortexing for 2min Then upper phase was extracted twotimes with ethyl acetate and the ethyl acetate was evaporatedat room temperature [22 23] The extracted product wascharacterized by using analytical TLC carried out on Silica





(a) (b) (c)





















MRTL3 MRS RL


4000 3750 3500 3250 3000 2750 2500 2250 2000 1750 1500 1250

105

975

90

825

75

675

T(

) 3310

2924

2854

1736

1651

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(ppm)

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1089

1066

1030

1000

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0953

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0910

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0876

2706















0008







4 Conclusion





Acknowledgment


References






































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





(a) (b) (c)





















MRTL3 MRS RL


4000 3750 3500 3250 3000 2750 2500 2250 2000 1750 1500 1250

105

975

90

825

75

675

T(

) 3310

2924

2854

1736

1651

1458

1404 1319 1227

1119

1057

980

903

841

802

710

656

(1cm)






0018

5360

5281

5258

5235

5211

5187

5065

5034

5010

4985

4933

4897

4880

4767

4758

4714

4574

4407

4384

4361

4338

4258

4080

4037

3985

3979

3880

3853

3745

3727

3662

3555

60 55 50 45 40 35 30

096

171

100

35 30 25 20 15 10 05(ppm)

041

139

058

030

012

DS-3

(ppm)

2425

2399

2373

2341

2315

2196

2173

2160

2070

2049

2036

1957

1931

1907

1889

1782

1774

1739

1719

1656

1629

1607

1583

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1507

1484

1459

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1179

1138

1116

1089

1066

1030

1000

0978

0953

0918

0910

0898

0876

2706















0008







4 Conclusion





Acknowledgment


References






































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










MRTL3 MRS RL


4000 3750 3500 3250 3000 2750 2500 2250 2000 1750 1500 1250

105

975

90

825

75

675

T(

) 3310

2924

2854

1736

1651

1458

1404 1319 1227

1119

1057

980

903

841

802

710

656

(1cm)






0018

5360

5281

5258

5235

5211

5187

5065

5034

5010

4985

4933

4897

4880

4767

4758

4714

4574

4407

4384

4361

4338

4258

4080

4037

3985

3979

3880

3853

3745

3727

3662

3555

60 55 50 45 40 35 30

096

171

100

35 30 25 20 15 10 05(ppm)

041

139

058

030

012

DS-3

(ppm)

2425

2399

2373

2341

2315

2196

2173

2160

2070

2049

2036

1957

1931

1907

1889

1782

1774

1739

1719

1656

1629

1607

1583

1526

1507

1484

1459

1435

1274

1179

1138

1116

1089

1066

1030

1000

0978

0953

0918

0910

0898

0876

2706















0008







4 Conclusion





Acknowledgment


References






































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0018

5360

5281

5258

5235

5211

5187

5065

5034

5010

4985

4933

4897

4880

4767

4758

4714

4574

4407

4384

4361

4338

4258

4080

4037

3985

3979

3880

3853

3745

3727

3662

3555

60 55 50 45 40 35 30

096

171

100

35 30 25 20 15 10 05(ppm)

041

139

058

030

012

DS-3

(ppm)

2425

2399

2373

2341

2315

2196

2173

2160

2070

2049

2036

1957

1931

1907

1889

1782

1774

1739

1719

1656

1629

1607

1583

1526

1507

1484

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1179

1138

1116

1089

1066

1030

1000

0978

0953

0918

0910

0898

0876

2706















0008







4 Conclusion





Acknowledgment


References






































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








0008







4 Conclusion





Acknowledgment


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

Documents

Research Article Simultaneous Production of Biosurfactants ...downloads.hindawi.com/journals/ijmicro/2014/698713.pdf · Research Article Simultaneous Production of Biosurfactants