Co esponding au ho : Sh u hi S idha
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S udy o an i-bac e ial ac i i y o bac e iocins isola ed om lac ic acid bac e ia g own
on mille media
Sh u hi S idha *, Padmash ee Kulka ni, Su ya K and Vi elia S
Depa men o Li e Science, Moun Ca mel College Au onomous, Bangalo e, India.
Wo ld Jou nal o Biology Pha macy and Heal h Sciences, 2025, 21(01), 149-154
Publica ion his o y: Recei ed on 28 No embe 2024; e ised on 05 Janua y 2025; accep ed on 07 Janua y 2025
A icle DOI: h ps://doi.o g/10.30574/wjbphs.2025.21.1.0014
Abs ac
Mille s a e g ains ha ha e high nu i i e alues. The cu en s udy aimed a s udying he e ec o p ebio ics on he
p oduc ion o bac e iocins and hei an ibac e ial ac i i y. Bac e iocins we e ex ac ed om p obio ic species o
Lac obacillus plan a um and Lac ocaseibacillus hamnosus g own in mille modi ied medium and MRS medium. Resul s
o an i-mic obial assays o bac e iocin indica e enhanced ac i i y in p esence o p oso mille . Bac e iocin isola ed om
L. hamnosus g owing in p oso modi ied media had a zone o inhibi ion 15 ± 0.35mm agains B. ce eus and 16.8 ±
0.19mm agains E. cloacae. Molecula weigh was de e mined by SDS-PAGE and was ound o be ~ 48 kDa. Thus, i could
be concluded ha p oso mille ac s as an e icien p ebio ic. C ude, unp ocessed ex ac s o mille s we e used in his
s udy and signi ican esul s we e ob ained, sugges ing ha including hem in e e yday die could ende a sou ce o
na u al p ebio ic ha is a o dable and a ailable commonly ac oss he globe.
Keywo ds: P ebio ic; P oso Mille ; Lac obacillus; Bac e iocin; Pa hogen
1. In oduc ion
Bac e iocins a e small size, hea -s able pep ides syn hesised by bac e ia ha a e ac i e agains o he o ganisms and
agains which he p oduce has a de ini e immune mechanism [1]. They a e p oduced by a a ie y o mic oo ganisms
including, G am-posi i e and G am-nega i e bac e ia along wi h some a chaea [2]. Bac e iocins syn hesised by G am-
posi i e bac e ia, commonly lac ic acid bac e ia (LAB), a e di ided in o wo ca ego ies namely: Class I - lan hionine-
con aining bac e iocins/lan ibio ics and Class II - non-lan hionine con aining bac e iocins/lan ibio ics[3]. Acco ding o
he s a is ics p o ided by he Wo ld Heal h O ganiza ion, nea ly 4.2 million indi iduals a e subjec ed o dea h each yea
as a esul o inges ing pa hogenic bac e ia-in ec ed ood[4]. The majo i y o he epo ed oodbo ne diso de ou b eaks
a e known o be caused by common pa hogens such as Salmonella, Campylobac e , Lis e ia monocy ogenes, No o i us,
and Shiga oxin-p oducing Esche ichia coli. O he pa hogens ha a e epo ed o occasionally con ibu e o illnesses a e
Clos idium sp., S aphylococcus au eus, Bacillus ce eus, Ye sinia en e ocoli ica, pa asi es, and some o he s [5].
The mechanism by which bac e iocins kill deadly pa hogens, he a iables ha de e mine he e iciency o bac e iocins
in oods and he physical chemis y o he bac e iocin/pa hogen in e ac ion and a e less known. Such unde s anding is
manda o y o bac e iocins o be used mo e commonly and o hei po ency o be inc eased h ough means o gene ic
enginee ing [6]. Since many bac e iocin p oduce s in his bac e ial g oup a e p obio ics, bac e iocins p oduced by LAB
ha e spa ked a lo o esea ch, pa icula ly in ecen yea s. Since hese mic o-o ganisms a e widely p esen in ou ood,
pa icula ly e men ed oods, hey a e ypically conside ed as sa e o human inges ion [7]. The gas oin es inal ac is
ano he ypical habi a o LAB, whe e hey gene a e complica ed molecula c oss alk wi h he hos and he o he
mic obes [8]. Bac e iocins a e equen ly iewed as weapons, wi h a wide ange o inhibi o y spec a o compe e wi h
o he bac e ia in he same niche. The majo i y o bac e iocins a ge species o gene a ha a e closely ela ed o he
p oduce s; howe e , some can ha e a signi ican ly g ea e spec al ange [9].
Wo ld Jou nal o Biology Pha macy and Heal h Sciences, 2025, 21(01), 149-154
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A ew s udies ha e shown he p esence o p ebio ic oligosaccha ides in g ains ha a e ich in die a y ib es, including ice,
buckwhea and whea [10, 11, 12]. Conside ing he ac ha mille s a e easily a ailable, s ess and hea - esis an c ops, ich in
die a y ib es, hey could be po en ially conside ed as p ebio ics ha enhance g ow h and de elopmen o p obio ics ha can
neu alize pa hogens h ough bac e iocins in he gas o-in es inal ac
2. Ma e ial and me hods
2.1. Sample collec ion
Mille s (namely ba nya d, b own op, ox ail, inge , kodo, li le, pea l, p oso, and so ghum) we e p ocu ed om he local
ma ke in Bengalu u. Cul u es o Lac obacillus plan a um (MTCC 1407), Lac ocaseibacillus hamnosus (MTCC 1408),
Bacillus ce eus (MTCC 1307) and En e obac e cloacae (MTCC 509) we e ob ained om MTCC, Chandiga h, India.
2.2. P epa a ion o modi ied MRS media
Lac obacillus s ains we e g own on s anda d MRS media and modi ied MRS media namely p oso mMRS (PmMRS) and
ox ail mMRS (FmMRS) con aining espec i e mille ex ac s as he sou ce o ca bohyd a e eplacing glucose in MRS
media.
Composi ion o modi ied MRS (mMRS) (g/L): Pep one – 10g; Bee ex ac – 10g; Yeas ex ac – 8g; Ex ac om 20g
mille s; Sodium ace a e ihyd a e – 5g; Polyso ba e – 1g; T i-ammonium ci a e – 2g; Magnesium sulpha e – 0.2g;
Manganese sulpha e – 0.05g; Aga – 15g.
The componen s we e dissol ed in dis illed wa e (1L) and pH was adjus ed o 6.2 and s e ilized by au ocla ing a 121°C
o 15 minu es.
2.3. Bac e iocin ex ac ion
Bac e iocin p oduced by he LAB was ex ac ed ollowing he p o ocol gi en by Zhao [2]. Abou 50 ml o 18h cul u es o
Lac obacillus plan a um and Lac ocaseibacillus hamnosus in MRS, PmMRS and FmMRS b o h we e cen i uge a 8000
pm o 10 minu es a 4°C. The pelle was suspended in 50 mL o espec i e b o hs. The suspension was sonica ed o
20 minu es and cen i uged a 8000 pm o 10 minu es a 4°C. The cell ee ex ac was cen i uged. The supe na an
was collec ed, and equal olume o chlo o o m was added. The mix u e was igo ously shaken and incuba ed o e nigh
a 4°C. The in e acial laye was collec ed and cen i uged a 12,000 pm o 10 minu es a 4°. The pelle was
esuspended in 1mL o phospha e bu e (0.05M; pH-7.0). The esul ing suspension was s o ed a 4°C o u he use.
2.4. An imic obial assay o c ude bac e iocin ex ac .
The an i-mic obial assay was done on Mulle Hin on (MH) aga . B. ce eus and E. cloacae we e he pa hogens used. The
cul u es we e swabbed on MH aga and wells we e made using a 6mm co kbo e . Te acycline (10μg) was used as a
posi i e con ol, and phospha e bu e was aken used as a nega i e con ol. Bac e iocin ex ac s om MRS, PmMRS and
FmMRS we e loaded, and pla es we e incuba ed a 37°C o 24 h s and he zones o inhibi ion we e obse ed.
2.5. De e mina ion o molecula weigh by SDS – PAGE
Molecula weigh s o he c ude bac e iocin ex ac s we e de e mined [2]. To achie e p ope esolu ion sepa a ion o
complex mix u e o p o eins SDS-PAGE is used. The s anda d p o ocol by Laemmli [13] was ollowed. Two gels we e
made, one wi h 5% s acking gel and he o he wi h 12% sepa a ing gel. 25 μl aliquo o ex ac ed bac e iocin was mixed
wi h 25 μl o loading dye and boiled a 100°C o 10 minu es. 40 μl o his mix u e was loaded in he wells along wi h a
ladde . Elec opho esis was ca ied ou a a cons an ol age o 50 milli ol s.
2.6. Analysis o he e ec o pa hogens on he p ebio ic s ains h ough SEM analysis
The mic obial cul u es o L. plan a um and L. hamnosus we e inocula ed in MRS and PmMRS b o hs (5% inoculum
each). A e 24h s, pa hogens we e inocula ed in o p obio ic cul u es in equal p opo ions and incuba ed a 37oC on a
shake incuba o a 100 pm o 24h s. One d op o he cul u e was suspended on a glass slide and was subjec ed o
d ying in a desicca o o 48h s. The p epa a ions we e hen analysed by scanning elec on mic oscopy a IISc.,
Bengalu u.
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3. Resul s and discussion
3.1. An imic obial assay o bac e iocin
An imic obial assay o bac e iocin ex ac s om L. plan a um in he di e en modi ied and s anda d media showed no
zone o inhibi ion. Only posi i e con ol had a consis en zone o inhibi ion 23 ± 2 mm as seen in Pla e 1. Bac e iocin
ex ac s om L. hamnosus g own in PmMRS media had a signi ican zone o inhibi ion 15 ± 5mm agains B. ce eus (Pla e
2 (a)) and 16.8 ± 1 mm agains E. cloacae (Fig 2 (b)). The an imic obial assay was conduc ed in eplica es o 5.
Figu e 1 An i-mic obial assay o L. plan a um bac e iocin ex ac agains (a) B. ce eus and (b) E. cloacae
Figu e 2 Zone o inhibi ion o bac e iocin ex ac p oduced by L. hamnosus g own in PmMRS agains (a) B. ce eus
and (b) E. cloacae
In a s udy conduc ed on L. bulga icus om yoghu , signi ican inhibi ing po en ial has been obse ed agains S. yphi
(16.5 mm), E. coli (15.25 mm), and B. sub ilis (15.32 mm), wi h a maximum o 18.32 mm agains V. chole ae [14].
Lac obacillus also showed e y s ong ac i i y agains Bacillus mycoides wi h he zone o inhibi ion o 15-18mm in
diame e [15]. Simila alues in he ange o 15-16mm has been ob ained in he p esen expe imen as well.
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3.2. De e mina ion o molecula weigh by SDS – PAGE
Two p ominen bands o P07 (L. plan a um on PmMRS) and P08 (L. hamnosus on PmMRS) we e ob ained and he
molecula weigh de e mined was ~ 48 kDa (Fig 3). P oso mille samples showed he p esence o bac e iocin. No bands
we e ob ained om o he samples sugges ing u he pu i ica ion o c ude ex ac s.
Bac e iocins ob ained om L. plan a um isola ed om sou dough ha e ound o be a ound 10 kDa as acco ding o
Zangeneh [16]. The alues o ~ 48 kDa ob ained in he cu en s udy is well in he ange o 2.5 o 100 kDa desc ibed o
bac e iocins o Lac obacillus species [17].
Figu e 3 Bands ob ained on SDS-PAGE gel wi h ex ac ed bac e iocin (indica ed by a ows)
3.3. Analysis o p obio ic pa hogen in e ac ion h ough SEM analysis
Figu e 4a and 4b shows he SEM images o p obio ic bac e ia in PmMRS wi h B. ce eus a wo di e en magni ica ions.
I is e iden om he mic og aph, up u ing o pa hogenic cells could be seen on in e ac ion wi h he p obio ic spp.
Some ex acellula polyme ic subs ances (EPS) a e clea ly seen in Fig 5a and 5b. These may be bac e iocin sec e ed by
p obio ic bac e ia in esponse o he pa hogens g owing in he medium. This shows ha , he p obio ic help in
supp ession o bac e ia, mo e e icien ly in p esence o p ebio ics such as mille . A simila SEM image showing he
enhanced p oduc ion o EPS by p obio ic s ains in he p esence o pa hogens was epo ed by Ma hi anan [18], in hei
in es iga ion.
Figu e 4 (a) and (b): SEM images showing up u ed cell walls o B. ce eus in he p esence o p obio ic sp
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Figu e 5 (a) and (b): SEM image showing EPS sec e ed by pa hogen in esponse o p obio ic bac e iocin
4. Conclusion
An agonis ic ac i i y o he c ude bac e iocin ex ac s agains he selec ed pa hogens sugges s ha he ac i i y
in ensi ied in he p esence o mille s. The molecula weigh s o he ex ac ed bac e iocin we e ound o be ~ 48kDa.
Amongs he wo mille s, p oso mille showed be e p ebio ic p ope ies compa ed o ox ail mille s, hus p o ing as a
supe io p ebio ic g ain. Resul s ob ained in his in i o s udy hin s ha gu mic obio a could be modula ed o h ough
in e en ions like including na u al sou ce o p ebio ics in die . They ac as subs a es o p obio ics and lead o
exploi a i e compe i ion and di ec ly in luence he p oduc ion o bac e iocins ha can ha m he gu pa hogens.
Compliance wi h e hical s anda ds
Acknowledgemen
We a e deeply g a e ul o depa men o Li e sciences o Moun Ca mel College Au onomous o hei unwa e ing
suppo in men o ing us h oughou he esea ch wo k and o IISc o ex ending hei help in speci ic pa s o he wo k.
Disclosu e o con lic o in e es
No con lic o in e es o be disclosed.
Re e ences
[1] Co e PD, Hill C, Ross RP. Bac e iocins: de eloping inna e immuni y o ood. Na Re Mic obiol. 2005;3(10):777–
88.
[2] Zhang B, He L, Wang X, Li H, Han Y. Fox ail mille : nu i ional and ea ing quali y, and p ospec s o gene ic
imp o emen . F on Ag Sci Eng. 2015;2(2):124–33.
[3] Heng NC, Tagg JR. Wha 's in a name? Class dis inc ion o bac e iocins. Na Re Mic obiol. 2006;4(2):160.
[4] F anz CMAP, den Bes en HMW, Böhnlein C, Ga eis M, Zwie e ing MH, Fusco V. Rep in o : Mic obial ood sa e y
in he 21s cen u y: Eme ging challenges and oodbo ne pa hogenic bac e ia. T ends Food Sci Technol.
2019;84:34–7.
[5] Gou ama H. Foodbo ne Pa hogens. In: Demi ci A, Feng H, K ishnamu hy K, edi o s. Food Sa e y Enginee ing.
Food Enginee ing Se ies. Sp inge , Cham; 2020.
[6] Mon ille TJ, Winkowski K, Ludesche RD. Models and mechanisms o bac e iocin ac ion and applica ion. In
Dai y J. 1995;5(8):797–814.
[7] Nes IF, Yoon SS, Diep DB. Ribosomally syn hesized an imic obial pep ides (bac e iocins) in lac ic acid bac e ia: a
e iew. Food Sci Bio echnol. 2007;16(5):675–90.
Wo ld Jou nal o Biology Pha macy and Heal h Sciences, 2025, 21(01), 149-154
154
[8] Pessione E. Lac ic acid bac e ia con ibu ion o gu mic obio a complexi y: ligh s and shadows. F on Cell In ec
Mic obiol. 2012; 2:86.
[9] Dobson A, Co e PD, Ross RP, Hill C. Bac e iocin p oduc ion: a p obio ic ai ? Appl En i on Mic obiol.
2012;78(1):1–6.
[10] D’hoe K, Con e no L, Fa a F, Falony G, Viei a-Sil a S, Ve mei en J, e al. P ebio ic whea b an ac ions induce
speci ic mic obio a changes. F on Mic obiol. 2018; 9:31.
[11] Kyu MT, Da B, Aye SS, Ma suda T. P ebio ic oligosaccha ides p epa ed by enzyma ic deg ada ion o die a y ibe s
in ice g ains. J Nu Sci Vi aminol. 2019;65(Supplemen ): S143–7.
[12] No een S, Rizwan B, Khan M, Fa ooq S. Heal h bene i s o buckwhea (Fagopy um esculen um), po en ial emedy
o diseases, a e o cance : A mini e iew. In ec Diso d D ug Ta ge s. 2021;21(6): e170721189478.
[13] He F. Laemmli-SDS-PAGE. Bio-p o ocol. 2011;1(2): e80.
[14] Ma ia T, Shahzad H, Fa naz M, Tahi a M, Ghazala P, Shazia S, e al. Isola ion and e alua ion o an ibac e ial ac i i y
o bac e iocin p oduced by Lac obacillus bulga icus om yogu . A J Mic obiol Res. 2011;5(22):3842–7.
[15] Mohankuma A, Mu ugala ha N. Cha ac e iza ion and an ibac e ial ac i i y o bac e iocin p oducing
Lac obacillus isola ed om aw ca le milk sample. In J Biol. 2011;3(3):128.
[16] Kho ami S, Khaleghi M. Bac e ios a ic ac i i y and pa ial cha ac e iza ion o he bac e iocin p oduced by L.
plan a um sp. isola ed om adi ional sou dough. Food Sci Nu . 2020;8(11):6023–30.
[17] Gaspa C, Donde s GG, Palmei a-de-Oli ei a R, Quei oz JA, Tomaz C, Ma inez-de-Oli ei a J, e al. Bac e iocin
p oduc ion o he p obio ic Lac obacillus acidophilus KS400. AMB Exp ess. 2018;8(1):153.
[18] Ma hi anan K, Chandi ika JU, Ma himani T, Raja am R, Annadu ai G, Yin H. P oduc ion and unc ionali y o
exopolysaccha ides in bac e ia exposed o a oxic me al en i onmen . Eco oxicol En i on Sa . 2021; 208:111567.
