72Research Article Receiv ed: 22 May 2012 Revis ed: 10 June 2012 Accepted: 11 Jun e 2012 Pub lishe d onl ine in Wile y Onli ne Libr ary : 20 July 201 2 (wileyonlinelibrary.com) DOI 10.1002/jctb.3892 Low mol ecu lar we ight liq ui d me di a development for Lactobacilli producing bacteriocins Myrto -Pana giota Zacharof∗ and Robert W. Lovitt Abstract BACKGRO UND: Contemp orarypurificat ion techn iquesof Lactobacillibacteriocinsinclu de chemic al precipitationand separ ation through solvents to obtain highly potent semi-purified bacteriocins. These methods are laborious and bacteriocin yields are low. To address thi s problem a set of new, effi cie nt, cos t eff ect ive media,was created,containin g low mol ecularweight nutrient sources (LMWM). Using these media future separation and concentration of the desired metabolic products, using ultra- and nano-filtr ati on from the cul tur ed bro th was pos sib le. RESULTS : The LMWM were made throug h serial filtra tion (filte rs varyin g in pore size 30 kDa, 4 kDa and 1 kDa MWCO) of a modified optimum liquid medium for Lactobacilli growth. The developed media were tested for bacteriocin production and biomass growth, using three known bacteriocin-producing Lactobacilli strains, Lactobacillu s caseiNCIMB 11970, Lactobacillus plantarum NCIMB 8014, Lactobacillu s lactis NCIMB 8586. All were successfully grown ( µmax 0.16 t o 0.18 h −1 ) on the LMWM and produced a si gni ficant amount of bact er iocinsin the range 110 to 130 IU mL −1 . CONCLUSIONS: LMWM do support Lactobacilli growth and bacteriocin production, establishing an alternative to the current produ ctionnutrien t media . The uptak e of thenutrientsourcesis facil itate d as nitro gensources,whichwere prima rilyresponsi ble for growth , wer e sup por ted in les s comple x for ms. c 2012 Soc iet y of Chemic al Indust ry Keywords: Lactobacilli; bacteriocins; low molecular weight medium; yield; filtration INTRODUCTION Sin ce the industrialis ati on of foo d pr odu cti on, foo d saf et y has been an issue of gr eat impor tan ce. Naturally occ ur ring food deteri oration and spoilage due to microbial agen ts has been the main source of hardsh ip in tod ay’ s food indust ry. Numerous pre ser vationmetho ds have been used to pre ven t food poisoning and contamination. These include thermal treatment (pasteurization, heating sterilisation), pH and water activity re- duction (acidification, dehydration) and addition of preservatives (antibiotics, orga nic compoun ds such as prop ionate, sorba te, benz oate, lacta te, and acetate). Regardle ss of thei r prov en succ ess and effectiveness, there is an increasing demand for naturally developed, non-artificial, biologically safe products providing the consumers with high health benefits. 1,5 Currently lactic acid bacteria and especially Lactobacilli have attracted great attention, due to the production of antimicrobial peptide compounds namely bacteriocins. 2 Lactobacilli are widely applied in the food industry as natural acidifiers. Their potential use as bacteriocidal agents would constitute a great commercial bene fit. The use of Lactobacilli- prod uced bact eriocins,is gene rally considered safe (GRAS, Grade One). Most Lactobacilli bacteriocins are small (<10 kDa) cationic, heat-stable, amphiphilic and mem- branepermea biliz ing pept ides . Many of these bacterio cins appe ar to exhibit relatively little adsorption specificity and have greater antibacterial activity at lower pH values (below 5). By means that their adsorption to the cell surface of Gram positive ( +) bacte- ria, eitherto the producing species or to the target strains, is pH dependent. Lactobacilli bacteriocins have been proven to be a highly effective natural barrier against microbial agents causing food poisoning and spoilage. 5,6 Antimicro bial activ ity of bacte rioc ins is dire cted princ ipall y aga inst oth er Gr am pos iti ve ( +) ba cteria. The ma jori ty of Lact obac illibacteri ocinshas beenshownto beeffecti vewhen used in sufficient amounts, towards a wide spectrum of Gram positive (+) bacteria, including Listeria and other species of Lactobacilli. However, a bacteriocin alone induced in a food product is not likely to ensure complete safety; in the case of Gram negative (−) bacteria this has been apparent. Then the use of bacteriocins has to be combined with other technologies that are able to disrupt the cellular membrane so that bacteriocins can kill the pathogenicbacteria. 7,8 Severalother bacteriocins fromLactobacilli have been identified throughout the last decade where research on their production and purification techniques has been highly intensive, due to the growing need for replacement of chemical food preservativ es. 10–13 ∗ Correspondence to: Myrto-Panagio ta Zacharof, College of Engineering, Multi- disciplinary Nanotechnology Centre, Swansea University, Swansea, SA2 8PP, UK. E-mail: myrtozacharof1981@yah oo.com College of Engineering, Multidisciplinary Nanotechnology Centre, Swansea Univ ersit y, Swans ea, SA2 8PP, UKJ Chem Technol Biotechnol2013; 88: 72 – 80 www.soci.org c 2012 Society of Chemical Industry
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Recei ved: 22 May 201 2 Revi sed: 10 Jun e 201 2 Accepted: 11 Ju ne 2012 Pu blish ed on lin e i n Wil ey Onl ine Lib rar y: 20 July 20 12
(wileyonlinelibrary.com) DOI 10.1002/jctb.3892
Low molecular weight liquid media
development for Lactobacilli producingbacteriocinsMyrto-Panagiota Zacharof ∗ and Robert W. Lovitt
Abstract
BACKGROUND: Contemporarypurification techniquesof Lactobacilli bacteriocins include chemical precipitation and separationthrough solvents to obtain highly potent semi-purified bacteriocins. These methods are laborious and bacteriocin yields arelow. To address this problem a set of new, efficient, cost effective media,was created, containing low molecularweight nutrientsources (LMWM). Using these media future separation and concentration of the desired metabolic products, using ultra- andnano-filtration from the cultured broth was possible.
RESULTS: The LMWM were made through serial filtration (filters varying in pore size 30 kDa, 4 kDa and 1 kDa MWCO) of amodified optimum liquid medium for Lactobacilli growth. The developed media were tested for bacteriocin production andbiomass growth, using three known bacteriocin-producing Lactobacilli strains, Lactobacillus casei NCIMB 11970, Lactobacillus
plantarum NCIMB 8014, Lactobacillus lactis NCIMB 8586. All were successfully grown (µ max 0.16 to 0.18 h−1) on the LMWM andproduced a significant amount of bacteriocins in the range 110 to 130 IU mL−1.
CONCLUSIONS: LMWM do support Lactobacilli growth and bacteriocin production, establishing an alternative to the currentproductionnutrient media. The uptake of thenutrientsourcesis facilitated as nitrogensources,whichwere primarilyresponsiblefor growth, were supported in less complex forms.c 2012 Society of Chemical Industry
Figure 1. Schematic diagram of the stirred cell (Amicon cell 8200 Manual,
Sterlitech USA) (1) cap, (2) pressure relief valve, (3) pressure tube fittingassembly, (4) top o-ring, provides seal to maintain pressure in the unit,(5) magneticimpeller,provides cross flowconditions, (6) main body of thestirred cell, (7) bottom o-ring provides seal to maintain pressure in theunit and prevent loss of sample, (8) base with permeate outlet, (9) screwin bottom to secure base in the main body, (10) permeate line and (11)retainingstand prevents displacement of cap when pressure is usedin theunit.
and 4 kDa (polysulfone, Microdyn-Nadir Co., Germany) while
nanofiltration 1 kDa (polysulfone, General Electric-Osmonics Co.
USA). The cell unit was pressurized by constant compressed
nitrogen at 200 kPa.
The operatingtemperaturewas controlledto 25 ◦Cusingawater
jacket with water bath (Grant Water bath, UK). The stirred cell unitwas operatedin batch dead-endmode. After eachexperiment,the
components of the unit cell were soaked in an ethanol solution
(50% v/v) for24 h. Themembraneswererinsed with distilledwater
and sterilised with 25% v/v ethanol solution.
Determinationof permeateflux, membraneresistance and cake
resistance were obtained from the standard equations25 used for
evaluating membrane performance; the flux was defined as
J =
Qf
Am
(3)
the transmembrane pressure (P) was defined as
P = TMP =
Pinl + Pout
2
− Ppermeate (4)
The membrane resistance was defined by Darcy’s law as
Rm =P
J× µ(5)
Each membrane was characterised under different pressure
conditions varying between 0 and 400 kPa with the following
1 and 30 nm, but with a higher percentage of proteins of sizebetween 1 and 10 nm when compared with the non-autoclaved
(a) (b)
(c)
Figure 2. Deposition of solids forming a cake on the outer layer of the ultrafiltration (a, 30 kDa) (b, 4 kDa) and nanofiltration (c, 1 kDa) membranes.
wileyonlinelibrary.com/jctb c 2012 Society of Chemical Industry J Chem TechnolBiotechnol 2013; 88: 72–80
Liquid media development for Lactobacilli producing bacteriocins www.soci.org
Figure 3. Size distribution of media particles in a non-autoclaved media: unfiltered (a) and filtered through ultrafiltration (b, 30 kDa) (c, 4 kDa) andnanofiltration (d, 1 kDa) membranes.
Figure 4. Size distributionof media particlesin autoclavedmedia:unfiltered (a) andfiltered through ultrafiltration(b, 30 kDa)(c, 4 kDa)and nanofiltration(d, 1 kDa) membranes.
J Chem Technol Biotechnol 2013; 88: 72–80 c 2012 Society of Chemical Industry wileyonlinelibrary.com/jctb
the target strain, being highly potent, regardless of the fact that
Lactobacilli were grown on different media. These results are
encouraging as they indicate that these media can be used when
upscaling bacteriocin production and purification using filtration
as the separation method, having solved the problem of excess
proteins.
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wileyonlinelibrary com/jctb c 2012 Society of Chemical Industry J Chem TechnolBiotechnol 2013 88 72 80