Professional Documents
Culture Documents
TMP 9 CF5
TMP 9 CF5
TMP 9 CF5
www.elsevier.com/locate/procbio
Abstract
A kinetic model of the production of bacteriocins on MRS medium by Lactococcus lactis subsp. lactis CECT 539 and
Pediococcus acidilactici NRRL B-5627 has been developed. The model is a modification of the Luedeking and Piret expression,
including a term for the influence of pH on both nisin and pediocin production. With this model it was demonstrated that these
bacteriocins are produced as primary metabolites depending on the pH. By modeling the effects of temperature and pH on their
activities, the high heat stability of both bacteriocins was demonstrated, with optimum values at acidic pH and short incubation
times. The initial activity of both nisin and nisaplin was drastically reduced by treatment with trypsin, subtilisin and pepsin, but
50% activity was retained when both antibacterial substances were treated with papain. The inhibitory activity of pediocin almost
totally disappeared after 15 min of treatment with these four proteases. © 2001 Published by Elsevier Science Ltd.
Keywords: Lactic acid bacteria; Nisin; Pediocin; Primary metabolite; Stability; Antibacterial activity
Modelling growth and bacteriocin production en- 2.3. Bacteriocin acti6ity assays
ables the elucidation of underlying metabolic regulatory
mechanisms which could facilitate the optimization of Two methods were employed. For fermentation ki-
production for upscaling. On the other hand, the math- netic purposes, the quantitative bacteriocin activity of
ematical formalization in the experimental planning of L. lactis subsp. lactis CECT 539 (Lc 1.04) and P.
the studies of stability and activity of both nisin and acidilactici NRRL B-5627 (Pc 1.02) was estimated by
pediocin could avoid the difficulty in interpretation and using a photometric assay on culture tubes [26], using
comparison of results. C. piscicola CECT 4020 (Cb 1.01) as target organism.
In this work, a kinetic analysis of both nisin and Each tube contained 2.5 ml of serial dilutions of bacte-
pediocin production, using a modification of the riocin extracts (pH 6.0) and an equal volume of a
Luedeking and Piret model, was developed. A multifac- culture of Cb 1.01, adjusted with sterile buffered MRS
torial experimental design and response surface analysis broth (pH 6.3) to provide an initial absorbance (at 700
was used to determine the effects of pH and tempera- nm) of 0.2. The tubes were incubated for 6 h at 30 °C.
ture on the stability of both bacteriocins and their Growth inhibition was measured spectrophotometri-
sensitivity to the action of proteolytic enzymes. In cally at 700 nm. One bacteriocin unit (BU) was defined
addition, the antibacterial activity of nisin and pediocin as the amount of bacteriocin causing 50% growth inhi-
was compared with bacteriocin-like inhibitory sub- bition (lethal dose 50: LD50 obtained from duplicate
stances from other lactic acid bacteria using hierarchi- samples), compared with a control without
cal clustering analysis. bacteriocins.
For comparative purposes, an agar well-diffusion
method was used [27]. An overnight culture of indicator
2. Materials and methods bacterium was spread on an appropriate agar media
(Rothe agar and Mc Bride agar for strains of Entero-
coccus and Listeria, respectively. MRS agar was used
2.1. Bacterial strains, media and culture conditions for Lactococcus, Lactobacillus, Carnobacterium and
Leuconostoc strains indicators.) Wells (5 mm in diame-
The strains used in this study are listed in Table 1. ter) were cut into plates and 50 ml of culture superna-
Cultures were maintained as frozen stocks at − 40 °C tant fluid of the producing-strains placed into each well.
in Nutrient Broth containing 15% (v/v) of glycerol and Plates were kept for 4 h at 4 °C to allow the diffusion
were propagated twice in appropriate media before use. of the extracts and later they were incubated at 30 °C
All Lactococcus, Lactobacillus, Carnobacterium, Pedio- for 24 h, after which the widths of the clear inhibition
coccus and Leuconostoc strains were grown on MRS zones around the well were measured. A solution of 1
broth. Strains of Enterococcus were grown on Rothe g/l of nisaplin (Aplin and Barrett Ltd., Dorset, UK)
broth and Listeria strains were grown on Mc Bride adjusted to pH 6.0, was used as bacteriocin standard.
agar. The bacteriocin activity was quantified as a relative
All bacteriocin-producing cultures of lactic acid bac- inhibition index (RII) according to the expression:
teria (LAB) were grown in 250 ml Erlenmeyer flasks RII = Ze/(Zn − 1), where Ze and Zn are the mean
containing 50 ml of medium, on a rotary shaker (200 diameters, respectively (obtained from triplicate experi-
rpm) at 30 °C, until the early stationary phase. All ments) of the inhibition zones produced by the extract
cultivations were started with a 2% (v/v) inoculum of a and nisaplin solution.
12-h culture. Samples were taken for biomass, culture
pH, antibacterial activity and analytical determinations, 2.4. Analytical methods
as required.
Samples were taken, at regular intervals, for analysis.
2.2. Extraction of antibacterial acti6ity Growth was monitored by absorbance at 700 nm and
converted to dry cell weight from a standard curve. The
Aliquots from LAB cultures were adjusted to pH 3.5 cells were harvested by centrifugation (12,000× g dur-
with 5 N HCl to avoid the adsorption of molecules of ing 15 min at 4 °C) of culture samples and washed
bacteriocin onto the producer cell surfaces. Thereafter, twice with saline (0.8% NaCl). The culture supernatants
they were heated for 3 min to kill the cells and cen- were used to determine total sugars, phosphorous, ni-
trifuged at 27,200×g for 15 min at 4 °C. The superna- trogen and proteins by methods as described previously
tants containing antibacterial activity (bacteriocin [28,29]. The lactic acid was determined by gas-liquid
extract) were adjusted to pH 6.0 and frozen until chromatography with FI detection [30], using a SU-
further use. PELCO SPB1701 column.
1008 N.P. Guerra, L. Pastrana / Process Biochemistry
2.5. Bacteriocin sensiti6ity to proteolytic enzymes 5 N HCl or NaOH and incubated for 5 and 15 min at
the corresponding temperature, according to the ex-
Solutions of protease in suitable buffers (pepsin: perimental matrix defined by the design used. After
potassium hydrogen phthalate– HCl 0.2 M, pH 2.2; incubation the samples were adjusted to pH 6.0 and
trypsin and subtilisin: TRIS 0.2 M, pH 7.6 and pa- monitored for remaining activity (%RA). Controls
pain: phosphate 0.2 M, pH 7.0) containing 1 enzymic consisted of samples of untreated bacteriocin extracts.
unit (determined according to Murado et al. [29]) were In order to compare the activity of nisin and pe-
mixed with equal volumes of bacteriocin extracts and diocin with other bacteriocins, a cluster analysis was
incubated over 30, 60 and 90 min at each optimum of used. The classification variables used were the in-
temperature for the enzymes: 37 °C for pepsin and hibitory spectrum (defined as the number of inhibited
subtilisin, 25 °C for trypsin and 30 °C for papain. indicator cells) and the potencies of the antibacterial
After the treatment, the samples were adjusted to pH compounds (defined as RII) produced by each pro-
6.0 to determine the remaining antibacterial activity. ducing strain on the indicator organisms. Both vari-
Controls consisted of samples of extracts treated un- ables were standardized by average deviation. The
der the same conditions but without enzymes. Euclidean distance was used as a measure of the simi-
larity index. The average linkage (in the variant of
2.6. Statistical methods unweighted pair-group average) was used as amalga-
mation (linkage) method [32].
A multifactorial composite orthogonal design [31]
based on five levels and two variables was used to
study the combined influence of pH and temperature 3. Results and discussion
on the stability of both nisin and pediocin. The design
consisted of 13 experiments with four (22) factorial 3.1. Nisin and pediocin production on MRS broth
points, four axial points to form a central composite
design with h =1.267 and five centre points for repli- Fig. 1 shows the growth of both L. lactis subsp.
cation. lactis CECT 539 (Lc 1.04) and P. acidilactici NRRL
Bacteriocin extracts from 18 h cultures of Lc 1.04 B-5627 (Pc 1.02) cultures. The last strain produced
and Pc 1.02, were adjusted at different pH values with higher titres of bacteriocin than Lc 1.04. In this
medium, the growth of both strains stopped after 18 h
of cultivation, although nutrients (the carbon, nitrogen
and phosphorous sources) were not totally consumed.
Since rich media (e.g. MRS Broth) are provided with
adequate amounts of vitamins, minerals and amino
acids through the use of yeast and meat extract [33],
the exhaustion of these micronutrients in the medium
could be discarded as explanation for this fact. In
addition, growth of the biomass typically sigmoidal,
did not reflect the characteristic linear shape of limit-
ing situations. Neither bacteriocin autoinhibition nor
the accumulation of by-products with antibacterial ac-
tivity (such as lactic and acetic acids or hydrogen
peroxide) seem to be the causes for the cessation of
growth because the producing strains possess specific
immunity to their own bacteriocin [20,34]. The only
inhibitory by-product synthesized by both strains was
lactic acid (Fig. 1), but the amounts produced (slightly
superior to 4 g/l) were lower than those considered
damaging for these strains, according to Matsusaki et
al. [18].
An acceptable cause of growth inhibition could be
an inadequate final pH reached in the cultures. Ac-
cording to some authors [35–37], one of the growth
rate determining steps in LAB could be nutrient trans-
Fig. 1. Kinetics of growth of Lc 1.04 (open symbols) and Pc 1.02
(closed symbols) on MRS broth. B: biomass; Pr: protein; RS: reduc-
port, which is a function of the pH. Usually, the
ing sugars; BT: bacteriocin; Lact: lactic acid; TP: total phosphorous; optimum pH varies between 6.0 and 6.5 and decreases
TN: total nitrogen. Means of three analytical replications. rapidly at higher or lower values. For this reason, the
N.P. Guerra, L. Pastrana / Process Biochemistry 1009
Bacteriocin samples
Producers Indicators
Cb 1.01 Lb 3.01 Lb 4.05 Lb 5.01 Lb 5.02 Lb 6.02 Lb 6.04 Lb 6.05 Lb 7.01 Lb 8.04 Lb 8.06 Lc 1.05 Lc 1.06 Ln 3.01 Ec 1.01 Ec 1.02 Ec 2.01 Lm 1.01 Lm 1.02
Lb 1.03 + + + − − − − − − − + − − − + + + + −
Lb 2.01 + + − − − − − − − − + − − − − + + + −
Fig. 5. Sensitivity of nisaplin (A), nisin (B) and pediocin (C) to proteases enzymes: pepsin (), subtilisin (
), papain () and trypsin (2).
Experimental data (symbols) and adjustments (continuous lines) according to Eq. (5).
3.5. Comparison of the antibacterial acti6ity of Lc 1.04 monocytogenes (Lm 1.01 and 1.02) strains. Of these, 14
and Pc 1.02 extracts with a set of other bacteriocin-pro- were found to be non-producing strains, while 36 pro-
ducing lactic acid bacteria duced bacteriocin-like inhibitory substances (BLIS)
which to various degrees inhibited the growth of the
Usually the characterization of the bacteriocin activ- indicator organisms. Subsequently, the antibacterial ac-
ity is carried out by determination of the inhibition tivity of these producing strains were compared with
spectrum against a set of target microorganisms (num- those produced by Lc 1.04 and Pc 1.02 using as indica-
ber of sensitive/number of total tested strains) by mea- tors the 14 non-producing strains plus the five target
suring of the diameter of the inhibition zone in agar organisms used in the first step.
plates. In these assays, no standard reference was used Table 7 shows the different inhibitory activity of
and a comparison with other bacteriocins (in the same these bacteriocin producers. Some strains (Lc 1.04, Pc
experimental conditions) is not shown. 1.02, Ln 5.01, Ln 5.03) were inhibitory to almost all the
In this work, the inhibitory spectra of nisin and indicators, while others (Ln 3.07, Lb 3.02, Lb 4.06, Lb
pediocin (expressed as number of inhibited strains, 6.06, Lb 9.01, Pc 1.01, Pc 1.03, Pc 2.01) were active
NIS) and their potencies (expressed as RII) were com- only against four or five strains. These differences in
pared with other extracts containing bacteriocin-like sensitivity and resistance of a strain to the different
inhibitory substances (BLIS) from lactic acid bacteria. bacteriocins are related to the existence of different, but
The study was divided in two steps. Firstly a set of 50 specific, surface receptors for different bacteriocins in
strains of lactic acid bacteria were tested as producers Gram-positive bacterial cells [47].
against Enterococcus (Ec 1.01, 1.02, 2.01) and Listeria The high variability observed for NIS (Table 7) and
Fig. 6. Tree diagram for 38 producer strains. The classification variables used were the inhibitory spectrum (NIS) and the potencies of the
antibacterial compounds (RII).
1014 N.P. Guerra, L. Pastrana / Process Biochemistry
RII makes the comparison difficult between the an- [13] Hirsch A. Growth and nisin production of a strain of Strepto-
coccus lactis. J Gen Microbiol 1951;5:208 – 21.
tibacterial activity of the producing bacteria. Because of
[14] Anderssen EL, Diep DB, Nes IF, Eijsink VGH, Nissen-Meyer J.
this, the data were processed by cluster analysis using Antagonistic activity of Lactobacillus plantarum C11: two new
the inhibitory spectrum and potency of each extract, as two-peptide bacteriocins, plantaricins EF and JK, and the induc-
classification variables. tion factor plantaricin A. Appl Environ Microbiol
The pyramidal representation (Fig. 6) showed that Pc 1998;64:2269 – 72.
[15] De Vuyst L, Vandamme EJ. Influence of the carbon source on
1.02 and Lc 1.04 formed separate clusters, each one
nisin production in Lactococcus lactis subsp lactis batch fermen-
with the highest distance indexes, revealing that nisin tations. J Gen Microbiol 1992;138:571 – 8.
and pediocin are the most potent bacteriocins assayed [16] Parente E, Hill C. A comparison of factors affecting the produc-
and they have the broadest inhibitory spectra. In addi- tion of two bacteriocins from lactic acid bacteria. J Appl Bacte-
tion to this, an independence between lactic acid pro- riol 1992;73:290 – 8.
[17] Daba H, Lacroix C, Huang J, Simard RE. Influence of growth
duction and RII was observed (even the major lactic conditions on production and activity of mesenterocin 5 by a
acid producers: Lb 3.04 and Lb 9.02, did not span the strain of Leuconostoc mesenteroides. Appl Microbiol Biotechnol
broadest inhibitory spectrum). 1993;39:166 – 77.
[18] Matsusaki H, Endo N, Sonomoto K, Ishizaki A. Lantobiotic
nisin Z fermentative production by Lactococcus lactis IO-1:
relationship between production of lantibiotic and lactate and
Acknowledgements cell growth. Appl Microbiol Biotechnol 1996;45:36 – 40.
[19] Cabo ML. Nisina: producción y aplicación a la conservación en
We thank Miguel Anxo Murado (head of the re- pescado refrigerado, PhD Thesis, Universidade de Santiago de
search team of Instituto de Investigacións Mariñas de Compostela, España, 1998.
[20] Kim WS, Hall RJ, Dunn NW. The effect of nisin concentration
Vigo, CSIC) for their aid in the elaboration of this
and nutrient depletion on nisin production of Lactococcus lactis.
work. Nelson P. Guerra was a recipient of ICI and Appl Microbiol Biotechnol 1997;48:449 – 53.
Xunta de Galicia fellowships. [21] Yang R, Ray B. Factors influencing production of bacteriocins
by lactic acid bacteria. Food Microbiol 1994;11:281 – 91.
[22] De Vuyst L, De Poorter G, Vandamme EJ. Nutritional and
metabolic regulation of the nisin fermentation process. Med Fac
References Landbouww Univ Gent 1989;54:1501 – 6.
[23] Liao C, Yousef AE, Richter ER, Chism GW. Pediococcus acidi-
lactici PO2 production in whey permeate and inhibition of
[1] McKay LL, Baldwin KA. Applications for biotechnology: Listeria monocytogenes in foods. J Food Sci 1993;58:430 –4.
present and future improvements in lactic acid bacteria. FEMS [24] Cho HY, Yousef AE, Yang ST. Continuous production of
Microbiol Rev 1990;87:3 –14. pediocin by immobilized Pediococcus acidilactici PO2 in a
[2] Daeschel, MA. Antimicrobial substances from lactic acid bacte- packed-bed bioreactor. Appl Microbiol Biotechnol 1996;45:589 –
ria for use as food preservatives, Food Technol 1989;164 – 167. 94.
[3] Tagg JR, Dajani AD, Wannamaker LW. Bacteriocins of Gram- [25] Yang R, Johnson MC, Ray B. Novel method to extract large
positive bacteria. Bacteriol Rev 1976;40:722 –56. amounts of bacteriocins from lactic acid bacteria. Appl Environ
[4] Barefoot SF, Klaenhammer TR. Detection and activity of Microbiol 1992;58:3355 – 9.
lactacin B, a bacteriocin produced by Lactobacillus acidophilus. [26] Cabo ML, Murado MA, González MP, Pastoriza L. A method
Appl Environ Microbiol 1984;45:1808 –15. for bacteriocin quantification. J Appl Microbiol 1999;87:907 –14.
[5] Delves-Broughton J. Nisin and its use as food preservative. Food [27] Casla D, Requena T, Gómez R. Antimicrobial activity of lactic
Technol 1990;44:100 –17. acid bacteria isolated from goat’s milk and artisanal cheeses:
[6] Klaenhammer TR. Bacteriocins of lactic acid bacteria. Biochimie characteristics of a bacteriocin produced by Lactobacillus cur6a-
1988;70:337 – 49. tus IFPL 105. J Appl Bacteriol 1996;81:35 – 41.
[7] Malik RK, Kumar N, Rao KN, Mathur DK. Bacteriocins —an- [28] González MP, Siso MIG, Murado MA, Pastrana L, Mon-
tibacterial proteins of lactic acid bacteria: a review. Microbiol temayor MI, Miron J. Depuration and valuation of mussel-pro-
Aliments Nutr 1994;12:117 –32. cessing wastes. Characterization of amylolytic postincubates
[8] Ray B. Bacteriocins of starter culture bacteria as food biopreser- from different species grown on an effluent. Biores Technol
vative: an overview. In: Ray B, Daeschel M, editors. Food 1992;42:133 – 40.
Biopreservatives of Microbial Origin. Florida: CRC Press, [29] Murado MA, Siso MIG, González MP, Montemayor MI, Pas-
1992:177 –206 Chapter 8. trana L, Pintado J. Characterization of microbial biomasses and
[9] Hurst A. Biosynthesis of the antibiotic nisin by whole Strepto- amylolytic preparations obtained from mussel processing waste
coccus lactis organisms. J Gen Microbiol 1966;44:209 –20. treatment. Biores Technol 1993;43:117 – 25.
[10] Biswas SR, Ray P, Johnson MC, Ray B. Influence of growth [30] Da Silva GA. Evaluation of butanoic, pentanoic and hexanoics
conditions on the production of a bacteriocin, pediocin AcH, by acids as internal standards for analysis of acidic fermentation
Pediococcus acidilactici H. Appl Environ Microbiol end-products from Bacillus stearothermophilus by gas liquid
1991;57:1265 – 7. chromatography. Appl Microbiol Biotechnol 1995;43:626 –31.
[11] Ray B. Nisin of Lactococcus lactis ssp. lactis as a food biopreser- [31] Akhnazarova S, Kafarov V. Experiment Optimization in Chem-
vative. In: Ray B, Daeschel M, editors. Food Biopreservatives of istry and Chemical Engineering. Moscow: MIR, 1982.
Microbial Origin. Florida: CRC Press, 1992:207 – 64 Chapter 9. [32] Carrasco J, Hernan M. Estadı́stica Multivariante en las Ciencias
[12] Ray B. Pediocin(s) of Pediococcus acidilactici as a food bio- de la Vida. Madrid, España: Ciencia, 1993:3.
preservative. In: Ray B, Daeschel M, editors. Food Biopreserva- [33] Van Niel EWJ, Hahn-Hägerdal B. Nutrient requirements of
tives of Microbial Origin. Florida: CRC Press, 1992:265 – 322 lactococci in defined growth media. Appl Microbiol Biotechnol
Chapter 10. 1999;52:617 – 27.
N.P. Guerra, L. Pastrana / Process Biochemistry 1015
[34] Nes IF, Diep DB, Havarstein LS, Brurberg MB, Eijsink V, Holo riocin produced by Streptococcus lactis. Bull Acad Sci
H. Biosynthesis of bacteriocins in lactic acid bacteria. Anton 1977;45:217 – 21.
Leeuw Int JG 1996;70:113 – 28. [42] Liu W, Hansen N. Some chemical and physical properties of
[35] Poolman B, Konings WN. Relation of growth of Streptococcus nisin, a small-protein antibiotic produced by Lactococcus lactis.
lactis and Streptococcus cremoris to amino acid transport, J Appl Environ Microbiol 1990;56:2551 – 8.
Bacteriol 170:700 – 707. [43] Reddy GV, Shahani KM. Isolation of an antibiotic from Lacto-
[36] Bibal B, Goma G, Vayssier Y, Pareilleux A. Influence of pH, bacillus bulgaricus. J Dairy Sci 1971;54:748 – 52.
lactose and lactic acid on the growth of Streptococcus cremoris: [44] Ryan MP, Rea MC, Hill C, Ross P. An application in cheddar
a kinetic study. Appl Microbiol Biotechnol 1988;23:340 – 4. cheese manufactured for a strain of Lactococcus lactis producing
[37] Gonçalves LMD, Ramos A, Almeida JS, Xavier AMRB, Car- a novel broad-spectrum bacteriocin, lacticin 3147. Appl Environ
rondo MJT. Elucidation of the mechanism of lactic acid growth Microbiol 1996;62:612 – 9.
inhibition and production in batch cultures of Lactobacillus [45] De Vuyst L, Vandamme EJ. Nisin, a lanthibiotic produced by
rhamnosus. Appl Microbiol Biotechnol 1997;48:346 –50. Lactococcus lactis subsp lactis: properties, biosynthesis, fermen-
[38] Edwards VH, Wilke CR. Mathematical representation of batch tation and applications. In: De Vuyst L, Vandamme EJ, editors.
culture data. Biotechnol Bioeng 1968;10:964 –74. Bacteriocins of Lactic Acid Bacteria: Microbiology, Genetics and
[39] Luedeking R, Piret EL. Transient and steady states in continu- Applications. London: Blackie Academic and Professional,
ous fermentation. Theory and experiment. J Biochem Microbiol 1994:151 – 221.
Tech Eng 1959;1:431 –59. [46] Rodrı́guez JM. Review: antimicrobial spectrum, structure, prop-
[40] Pintado J. Producción de ácido cı́trico a partir de efluentes del erties and mode of action of nisin, a bacteriocin produced by
procesado de mejillón modalidades de cultivo y criterios de Lactococcus lactis. Food Sci Technol Int 1996;2:61 – 8.
optimización, PhD Thesis, Universidade de Santiago de Com- [47] Hanlin MB, Kalchayanad N, Ray P, Ray B. Bacteriocins of
postela, España, 1995. lactic acid bacteria in combination have greater antibacterial
[41] Kozak W, Bardowski J, Dobrzanski WT. Lactostrepcin, a bacte- activity. J Food Prot 1993;56:252 – 5.