b. linens (temperature and nacl)

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Appl Microbiol Biotechnol (2003) 62:163167DOI 10.1007/s00253-003-1292-9

O R I G I N A L P A P E R

A. S. Motta A. Brandelli

Influence of growth conditions on bacteriocin productionby Brevibacterium linens

Received: 3 September 2002 / Revised: 13 February 2003 / Accepted: 21 February 2003 / Published online: 2 April 2003 Springer-Verlag 2003

Abstract The influence of temperature, NaCl concentra-tion and cheese whey media on growth of Brevibacteriumlinens ATCC 9175 and production of bacteriocin-like

antimicrobial activity was studied. Bacteriocin productionand activity were higher at 25C than at 30C. Nosignificant growth or production of bacteriocins wasobserved at 37C. When bacteriocin production wasinvestigated in media containing different concentrationsof NaCl, increased activity was observed in mediacontaining 40 or 80 g l1, but not 120 g l1 NaCl. Theaddition of NaCl resulted in a significant increase inspecific production rates of bacteriocin-like activity.Antimicrobial activity was also observed by cultivationof B. linens at 25C in cheese whey media.

Introduction

Bacteriocins are ribosomally synthesized antimicrobialpeptides that are not lethal to producer cells. Thesepeptides are generally active against species closelyrelated to the producer microorganisms. The bacteriocinsof Gram-positive bacteria have been examined extensive-ly since they show lethal activity against important foodpathogens such as Listeria monocytogenes and Clostrid-ium botulinum (Jack et al. 1995). Although manybacteriocins have been studied in recent years, mostresearch has been carried out on lactic acid bacteria(McAuliffe et al. 2001), and very little is known aboutbacteriocins produced by coryneform bacteria (Motta andBrandelli 2002).

Some coryneform bacteria isolated from surface-ripened cheese have been shown to produce inhibitorysubstances active against L. monocytogenes (Valds-

Stauber et al. 1991). Particularly, certain strains ofBrevibacterium linens have been identified as producersof antilisterial bacteriocins (Maisnier-Patin and Richard

1995; Motta and Brandelli 2002)Studies on factors affecting the production of bacte-riocins are relatively scarce excepting for those on nisin,which is the most studied bacteriocin (De Vuyst 1995).Nevertheless, the effect of media composition on theproduction of pediocin AcH (Biswas et al. 1991),amylovorin L471 (De Vuyst et al. 1996) and enterocin1146 (Parente and Ricciardi 1994) has been described.Furthermore, pH and temperature are also very importantfor the production of bacteriocins by lactic acid bacteria(Vignolo et al. 1995; Krier et al. 1998).

In the present work, we investigate the influence oftemperature and NaCl concentration on the growth of B.

linens and bacteriocin production. Production of bacteri-ocins by B. linens grown in cheese whey media was alsostudied.

Materials and methods

Bacterial strains and media

B. linens ATCC 9175 was used as the producer strain. Trypticasesoy broth (TSB, Difco) with 20% (v/v) glycerol was used formaintenance of the strain at a temperature of 20C. Strains used asindicator microorganisms were B. linens ATCC 9172, Corynebac-terium fimi NCTC 7547 and L. monocytogenes ATCC 7644.Indicator strains were laboratory stocks and were kept frozen in

media containing 20% (v/v) glycerol at 20C.

Detection of antimicrobial activity

The antimicrobial activity of bacteriocins was monitored asdescribed previously (Motta and Brandelli 2002). Aliquots of20ml cell-free culture supernatant fluid were applied to discs onagar plates which had been previously inoculated with 0.3 ml ofeach indicator strain suspension in 8.75 g l1 NaCl. Plates wereincubated at the optimal temperature of the test organism. Thebacteriocin titre was determined by the serial twofold dilutionmethod previously described by Mayr-Harting et al. (1972).Activity was defined as the reciprocal of the dilution after the

A. S. Motta A. Brandelli ())Departamento de Cincia de Alimentos, ICTA,Universidade Federal do Rio Grande do Sul,Av. Bento Gonalves 9500, 91501-970 Porto Alegre, Brazile-mail: [email protected]: +55-51-3316-7048


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last serial dilution giving a zone of inhibition and expressed asactivity units (AU) per millilitre.

Cell concentration

The cell concentration was estimated by measuring absorbance in aspectrophotometer at 600 nm, and relating the readings to biomassdry weight with a calibration curve. An absorbance of 1.0 wasequivalent to 0.15 g l1 dry cells.

Effect of growth conditions of B. linens on bacteriocin production

B. linens ATCC 9175 was grown in 100 ml of TSB-modifiedmedium (10 g l1 trypticase peptone, 3 g l1 bacto-peptone, 2.5 g l1

yeast extract, 5 g l1 glucose, 2.5 g l1 K2HPO4, 0 . 2 g l1

MgSO4.7H2O) (Rattray et al. 1995) in a rotary shaker at 125 cy-cles min1 for the desired time. The culture supernatants were usedin the antimicrobial activity assay against B. linens ATCC 9172 andL. monocytogenes ATCC 7644. All experiments were carried out intriplicate.

The influence of temperature on growth and production ofbacteriocin was studied at 25, 30 and 37C.

Bacteriocin production was investigated by culture of B. linensATCC 9175 at 25C in TSB-modified medium containing 40, 80 or

120 g l1

NaCl.To investigate the bacteriocin production on cheese whey, thebasic media was made up with 70 g l 1 of reconstituted sweetcheese whey powder (composition: 71% lactose, 11.1% solubleprotein, 0.7% fat, 3% humidity, 7.2% ash; Eleg Laticnios SA,Lageado, Brazil). B. linens ATCC 9175 was grown at 25C. Toavoid protein precipitation during the sterilization process (121C,15 min), cheese whey proteins were hydrolyzed with a commercialprotease (Alcalase 2.4 L, 1 ml l1, Novo Nordisk) at 55C for 3 h.

The maximal specific growth rate (mmax) was calculated fromthe equation:

m dX=Xdt

where X is the biomass concentration in g l1 cell dry mass and t isthe time (hours).

Specific production rates (qp), defined as the amount of activity

produced per gram of cell dry mass and per hour (AU g1

h1

) werecalculated during growth from the relation:

qp dP=Xdt

where P is the activity expressed in AU l1 and X is the biomassconcentration in g l1 (Sinclair and Cantero 1990).

Extraction of bacteriocin from producer cells

The effect of different NaCl concentrations on physical desorptionof the bacteriocin was evaluated essentially as described elsewhere(Yang et al. 1992). The producer strain was grown in TSB-modifiedmedium to reach the early stationary phase. The cells were washedwith 5 mM sodium phosphate (pH 6.5), then suspended in this samebuffer containing 40, 80 or 120 g l1 NaCl, and mixed with a

magnetic stirrer for 1 h at 4C. Cell suspensions were centrifuged at20,000 g for 20 min and the supernatants were evaluated forbacteriocin activity.

Results

Bacteriocin production

B. linens ATCC 9175 was cultivated in TSB-modifiedmedium at 25C in rotary shaker. Maximum antibacterialactivity was observed starting at 24 h. Bacteriocin activity

was observed against B. linens ATCC 9172, L. monocy-togenes ATCC 7644 and C. fimi NCTC 7547 (Table 1)and it was stable for up to 6 months storage at 4C.

Effect of temperature on growthand bacteriocin production

B. linens ATCC 9175 grew well at 25C and 30C. The

growth curves at these temperatures were similar(Fig. 1A). The stationary growth phase was reached at24 h and 32 h at 25C and 30C, respectively. Nosignificant growth or bacteriocin activity was observedduring cultivation of B. linens at 37C (not shown).

Bacteriocin activity was observed from 24 h ofcultivation at 25C, and the antibacterial activity waspresent until the end of incubation (Fig. 1B). When B.linens was cultivated at 30C, the bacteriocin productionstarted at 32 h, but activity against L. monocytogenes andB. linens was lost after 48 h and 64 h, respectively. Forboth temperatures, 25C and 30C, maximum activitiescorresponded to the maximum biomass (Xmax).

The kinetic constants, obtained by testing the effect oftemperature on growth and production of antibacterialactivity by B. linens ATCC 9175 during batch cultivation,are shown in Table 2.

The qp were maximum at 24 h and 32 h at 25C and30C, respectively (Fig. 1C).

Table 1 Antibacterial activity produced by Brevibacterium linensATCC 9175. Activity of a 24-h culture supernatant of B. linensgrown in TSB at 25C. Values are the means of three determina-tions

Indicator strain Temperature(C)

Activity(AU ml1)

B. linens ATCC 9172 25 300Corynebacterium fimi NCTC 7547 37 300Listeria monocytogenes ATCC 7644 37 400

Table 2 Kinetic constants for growth and bacteriocin productionduring cultivation of B. linens ATCC 9175. Xmax is the maximumbiomass concentration in g l1 cell dry mass; mmax is the maximalspecific growth rate; Amax is the maximum activity (values are themeans of three determinations); YP/X is the yield product/biomoss

Growth condition Xmax

(gl1)

max

(h1)

Amax

(AUml1)

YP/X

(AU 103 g1)

25C 0.300 0.095 400 1.3330C 0.283 0.093 400 1.4140 g l1 NaCl 0.314 0.047 600 1.9180 g l1 NaCl 0.308 0.055 550 1.79

120 g l1 NaCl 0.278 0.025 400 1.44Whey 0.320 0.014 400 1.25

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Effect of NaCl concentration on growthand bacteriocin production

When the growth and production of bacteriocins by B.linens ATCC 9175 were evaluated in TSB-modifiedmedium containing 40 g l1 NaCl, an increase in thelength of the exponential growth phase was observed,reaching the stationary phase at 40 h (Fig. 2A). Inexperiments with TSB-modified medium containing 80 gl-1 or 120 g l1 NaCl, an increase in the lag growth phasewas observed (Fig. 2A).

The production and activity of bacteriocins wasobserved after 16 h of cultivation in TSB-modified mediacontaining either 40 g l-1 or 80 g l1 NaCl, (Fig. 2B).

Maximum activity coincided with Xmax, then decreased toabout 400 AU ml1. The production of bacteriocins inmedia containing 120 g l1 NaCl reached maximum

values after 32 h (Fig. 2B). Antilisterial activity was notobserved when cultivation was performed with increasedNaCl concentration.

The addition of NaCl to TSB resulted in the decreaseofmmax values, but not Xmax values (Table 2). Maximumactivity in media containing either 40 g l-1 or 80 g l1

NaCl was higher than that observed in TSB-modifiedmedium (Table 2). The effect of NaCl on the physicaldesorption of bacteriocins was tested, but no activity wasreleased from B. linens cells (not shown).

Fig. 2AC Effect of NaCl on bacteriocin production and activityby B. linens ATCC 9175. B. linens was cultivated at 25C in TSB-modified broth containing 40 g l1 (l), 80 g l1 (n) and 120 g l1

(s) NaCl. A Bacterial growth. B Bacteriocin activity. C Specificproduction rate. Each point represents the mean of three indepen-dent experiments

Fig. 1AC Effect of growth temperature on bacteriocin productionand activity by Brevibacterium linens ATCC 9175. B. linens wascultivated at 25C (l) and 30C (n) in TSB-modified broth. ABacterial growth. B. Bacteriocin activity. C Specific productionrate. Each point represents the mean of three independentexperiments

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The qp were maximum at 16 h for both 40 g l-1 and80 g l1 NaCl, and at 32 h in media containing 120 g l 1

NaCl (Fig. 2C).

Bacteriocin production in cheese whey media

Cheese whey was used to growth B. linens ATCC 9175and to evaluate the production and activity of bacteri-ocins. The growth of B. linens was observed to start theexponential growth phase after 16 h and reach thestationary phase at 72 h of cultivation (Fig. 3). Bacterio-cin production was observed after 32 h, but activity waslost after 40 h. The qp was maximum at 32 h (Fig. 3).

Discussion

A bacteriocin was produced by B. linens ATCC 9175under different growth conditions. It was active against B.linens ATCC 9172, L. monocytogenes ATCC 7644 and C.fimi NCTC 7547. The bacteriocin was effective against L.monocytogenes; this is considered to be a very importantproperty in food safety. In addition, this bacteriocin wasvery resistant to storage at cooling or freezing tempera-tures, a characteristic that is relevant to food use.

Bacteriocin production by other B. linens strains hasbeen described, but there are no previous reports

comparing different growth conditions. Linenscin OC2was produced by cultivation of B. linens OC2 at 27C inTSBYE medium for up to 72 h (Maisnier-Patin andRichard 1995; Siswanto et al. 1996; Boucabeille et al.1997); these conditions were considered satisfactory forthe production of the bacteriocin, but other conditionswere not investigated. Production of linocin M18 (Valds-Stauber and Scherer 1994) and linencin A (Kato et al.1991) were achieved by cultivation in TSB medium at30C. Temperature had little effect on the final biomassof the producer strain and the volume of bacteriocin

produced. Maximum activities coincided with Xmax, andmaximum qp were observed in the exponential phase.Since the bacteriocin was produced during the exponen-tial growth phase only, it can be considered to be aprimary metabolite, similar to bacteriocins produced byEnterococcus faecium (Parente and Ricciardi 1994) andLactobacillus amylovorus (De Vuyst et al. 1996).

The loss of bacteriocin activity during cultivation at

30C may be associated with proteolysis, since differentB. linens strains produce extracellular proteases showingtemperature optima ranging from 25C to 55C (Rattrayand Fox 1999). Production of bacterial proteases is ofteninduced by the presence of complex organic nitrogen suchas proteins (casein, gelatin) and is suppressed by rapidlymetabolizable nitrogen sources (Beg et al. 2002). Anincrease in protease production by B. linens when corngluten or egg albumin are added to the growth mediumhas been described (Zemanovic and Skrka 1987).Similarly, peptides from cheese whey could stimulateprotease production.

Since B. linens is a halotolerant microorganism and

sometimes has its growth stimulated by elevated amountsof NaCl (Rattray and Fox 1999), it is relevant to study thebacteriocin production in different NaCl concentrations.The addition of NaCl influenced growth and bacteriocinproduction by B. linens. A very significant increase(nearly 200-fold) in the qp was observed by increasing theNaCl concentration at the start of the exponential growthphase, indicating a primary metabolite behaviour. Theincreased activity observed with high salt was not a resultof physical desorption from the producer cells, such asthat observed by the cationic antimicrobial peptidesproduced by lactic acid bacteria (Yang et al. 1992). Thisagrees with the fact that B. linens bacteriocins are often

secreted as large aggregates (Rattray and Fox 1999).However, L. monocytogenes was not inhibited when thebacteriocin was produced in the presence of increasedamounts of NaCl, indicating that the salt concentration insome cheeses must be unfavourable for production ofantilisterial activity by B. linens ATCC 9175.

The production of bacteriocins is normally performedin complex growth media. Although these media promoteabundant growth and relatively high bacteriocin levels,their expensive cost make them unsuitable for large-scaleproduction. Furthermore, some medium components (e.g.large amounts of proteins, which are not totally consumedby the producer strains at the end of fermentation) mayinterfere with the subsequent bacteriocin purification(Barefoot and Klaenhammer 1984).

It seems more economical to use some of the wastefrom the food industry as the raw material for the basis ofthe culture media. Whey is generated in large amounts bythe cheese industry and its use as culture medium forproduction of nisin and pediocin (Goulhen et al. 1999;Guerra and Pastrana 2001) and lactocin 705 (Vignolo etal. 1995) has been studied. The broad-spectrum bacterio-cin lacticin was produced in demineralized whey. Thefermentate was spray dried and the resulting powderexhibited inhibitory activity against L. monocytogenes

Fig. 3 Production of bacteriocins by B. linens ATCC 9175 incheese whey media. B. linens was cultivated in cheese whey at25C and monitored for growth (l), production of antibacterialactivity (n) and specific production rate (s). Each point representsthe mean of three independent experiments

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Scott A and Staphylococcus aureus (Morgan et al. 1999).The development of a bacteriocin powder from wheycould be applied to a number of food systems; this powderwould have applications in all foods where whey powderis an existing ingredient.

Production of a bacteriocin was observed duringcultivation of B. linens ATCC 9175 in cheese wheymedium. Although stability of the bacteriocin was lower

in whey medium than in TSB, the use of whey inbacteriocin production merits further study as an alterna-tive use of this by-product.

References

Barefoot SF, Klaenhammer TR (1984) Detection and activity oflacticin B, a bacteriocin produced by Lactobacillus acidophilus.Appl Environ Microbiol 45:18081815

Beg QK, Saxena RK, Gupta R (2002) De-repression and subse-quent induction of protease synthesis by Bacillus mejavensisunder fed-batch operations. Process Biochem 37:11031109

Biswas SR, Ray P, Johnson MC, Ray B (1991) Influence of growthconditions on the production of a bacteriocin, pediocin AcH, by

Pediococcus acidilactici H. Appl Environ Microbiol 57:12651267

Boubabeille C, Mengin-Lecreulx D, Henckes G, Somonet JM, vanHeijenoort J (1997) Antibacterial and hemolytic activities oflinenscin OC2, a hydrophobic substance produced by Brevi-bacterium linens OC2. FEMS Microbiol Lett 153:295301

De Vuyst L (1995) Nutritional factors affecting nisin production byLactococcus lactis subsp. lactis NIZO 22186 in a syntheticmedium. J Appl Bacteriol 78:2833

De Vuyst L, Callewaert R, Crabb K (1996) Primary metabolitekinetics of bacteriocin biosynthesis by Lactobacillus amylo-vorus and evidence for stimulation of bacteriocin productionunder unfavourable growth conditions. Microbiology 142: 817827

Goulhen F, Meghrous J, Lacroix C (1999) Production of nisin Z/pediocin mixture by pH-controlled mixed-strain batch cultures

in supplemented whey permeate. J Appl Microbiol 86:399406Guerra NP, Pastrana L (2001) Enhanced nisin and pediocin

production on whey supplements with different nitrogensources. Biotechnol Lett 23:609612

Jack RW, Tagg JR, Ray B (1995) Bacteriocins of gram-positivebacteria. Microbiol Rev 59:171200

Kato F, Eguchi Y, Nakano M, Oshima T, Murata A (1991)Purification and characterization of Linecin A, a bacteriocin ofBrevibacterium linens. Agric Biol Chem 55:161166

Krier F, Revol-Junelles AM, Germain P (1998) Influence oftemperature and pH on production of two bacteriocins by

Leuconostoc mesenteroides subsp. mesenteroides FR52 duringbatch fermentation. Appl Microbiol Biotechnol 50:359363

Maisnier-Patin S, Richard J (1995) Activity and purification ofLinenscin OC2, an antibacterial substance produced by Brev-ibacterium linens OC2, an orange cheese coryneform bacteri-um. Appl Environ Microbiol 61:18471852

Mayr-Harting A, Hedjes AJ, Berkeley CW (1972) Methods forstudying bacteriocins. In: Norris JB, Ribbons D (eds) Methodsin microbiology, vol 7. Academic, New York, pp 315412

McAuliffe O, Ross RP, Hill C (2001) Lantibiotics: structure,

biosynthesis and mode of action. FEMS Microbiol Rev 25:285308

Morgan SM, Galvin M, Kelly J, Ross RP, Hill C (1999)Development of a lacticin 3147-enriched whey powder withinhibitory activity against foodborne pathogens. J Food Protect62:10111016

Motta AS, Brandelli A (2002) Characterization of an antibacterialpeptide produced by Brevibacterium linens. J Appl Microbiol92:6371

Parente E, Ricciardi A (1994) Influence of pH on the production ofenterocin 1146 during batch fermentation. Lett Appl Microbiol19:1215

Rattray FP, Fox PF (1999) Aspects of enzymology and biochemicalproperties ofBrevibacterium linens relevant to cheese ripening:a review. J Dairy Sci 82:891909

Rattray FP, Bockelmann W, Fox PF (1995) Purification and

characterization of an extracellular proteinase from Brevibac-terium linens ATCC 9174. Appl Environ Microbiol 61:34543456

Sinclair CG, Cantero D (1990) Fermentation modelling. In: McNeilB, Harvey LM (eds) Fermentations, a pratical approach. IRL,Oxford, pp. 65113

Siswanto HP, Gratadoux JJ, Richard J (1996) Potentiel inhibiteurde la souche de Brevibacterium linens productrice de lalinenscine OC2, vis--vis des listeria et de Staphylococcusaureus. Lait 76:501512

Valds-Stauber N, Gtz H, Busse M (1991) Antagonistic effect ofcoryneform bacteria from red smear cheese against Listeriaspecies. Int J Food Microbiol 13:119130

Valds-Stauber N, Scherer S (1994) Isolation and characterizationof Linocin M18, a bacteriocin produced by Brevibacteriumlinens M18. Appl Environ Microbiol 60:38093814

Vignolo GM, Kairuz MN, Ruiz Holgado AAP, Oliver G (1995)Influence of growth conditions on the production of lactocin705, a bacteriocin produced by Lactobacillus casei CRL 705. JAppl Bacteriol 78:5-10

Yang R, Johnson MC, Ray B (1992) Novel method to extract largeamounts of bacteriocins from lactic acid bacteria. Appl EnvironMicrobiol 58:33553359

Zemanovic J, Skrka B (1987) Culture media for extracellularproteinases production by Brevibacterium linens. In: NeihsselOM, van der Meer RR, Luyben KCAM (eds) Proceedings ofthe 4th European Congress on Biotechnology, vol 3. Elsevier,Amsterdam, pp 541544

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