Process Biochemistry 37(2001) 1005– 1015

www.elsevier.com/locate/procbio

Modelling the influence of pH on the kinetics of both nisin and

pediocin production and characterization of their functional
properties
Nelson P. Guerra a, Lorenzo Pastrana b,*
a
Departamento de Fundamentos Quı́micos y Biológicos, Facultad de Ingenierı́a Quı́mica, Uni6ersidad de Oriente, Santiago de Cuba, Cuba
b
Departamento de Bioquı́mica, Xenética e Inmunoloxı́a, Facultade de Ciencias de Ourense, Uni6ersidade de Vigo, As Lagoas,
32004 Ourense, Spain
Received 23 October 2000; received in revised form 6 August 2001; accepted 16 October 2001

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

1. Introduction compounds are non-toxic, active in a wide pH range

Lactic acid bacteria (LAB) play a fundamental role
in food fermentation processes [1]. Raw foods, includ-
ing milk, fruits, vegetables and meats, are often pre-
served by lactic acid fermentation. The antimicrobial
effect may be due to the production of organic acids
(lactic and acetic acids), diacetyl, hydrogen peroxide
and bacteriocins or antibacterial peptides [2]. Bacteri-
ocins are proteinaceous compounds with antibacterial
activity against closely related micro-organisms [3].
However, some bacteriocins, with a broad spectrum of
activity (e.g. nisin and pediocin), appear to inhibit
several spoilage organisms and food-borne pathogens,
including Bacillus cereus, Clostridium botulinum, Ente-
rococcus faecalis, Staphylococcus aureus and Listeria
monocytogenes [4,5]. In addition, these antibacterial
* Corresponding author. Tel.: + 34-988-387-062; fax: + 34-988-
387-001.
E-mail address: pastrana@uvigo.es (L. Pastrana).
and sensitive to the proteolytic enzymes, including gas-
tric proteinases. Most retain their properties after high
heat treatment and are stable over several months,
during frozen and refrigeration storage and after dry-
ing. With such characteristics, bacteriocins have a high
potential value as food preservatives [6,7]. Nevertheless,
among the many bacteriocins of starter culture bacte-
ria, only a few have been studied as biopreservatives in
food systems. These include nisin from Lactococcus
lactis subsp. lactis and pediocins from Pediococcus
acidilactici [8].
The production, purification, genetic determinants,
culture media, operational optimization and functional
characterization of nisin and pediocin are well-docu-
mented [9–12]. Traditionally, studies of bacteriocin
production have been performed through physiological
and metabolic control of their biosynthesis on complex
media. The main variables affecting the production of
bacteriocins are temperature, pH, composition of the
nutrient media and inducers [10 –22]. Although the

0032-9592/01/$ - see front matter © 2001 Published by Elsevier Science Ltd.

PII: S 0 0 3 2 - 9 5 9 2 ( 0 1 ) 0 0 3 1 2 - 0
1006 N.P. Guerra, L. Pastrana / Process Biochemistry 37 (2001) 1005–1015

Table 1 Table 1 (Continued)

Bacterial strains used in this work
Strains Strain Source
Strains Strain Source designations
designations
Leuconostoc mesenteroides Ln 3.05 CECT 394
Carnobacterium piscicola Cb 1.01 CECT 4020 ssp. mesenteroides
Lactobacillus acidophilus Lb 1.03 NCK 56 Leuconostoc mesenteroides Ln 3.06 CECT 891
Lactobacillus bre6is Lb 2.01 CECT 216 ssp. mesenteroides
Lactobacillus casei ssp. Lb 3.01 CECT 277 Leuconostoc mesenteroides Ln 3.07 CECT 4046
casei ssp. mesenteroides
Lactobacillus casei ssp. Lb 3.02 CECT 475 Leuconostoc mesenteroides Ln 3.08 CECT 873
casei ssp. cremoris
Lactobacillus casei ssp. Lb 3.03 CECT 4040 Weissella paramesenteroides Ln 4.01 CECT 4027
casei Leuconostoc sp. Ln 5.01 WYO
Lactobacillus casei ssp. Lb 3.04 CECT 4043 Leuconostoc sp. Ln 5.03 WYO
case Pediococcus acidilactici Pc 1.01 CECT 98
Lactobacillus delbrueckii Lb 4.05 JCM 1106 Pedicoccus acidilactici b Pc 1.02 NRRL B-5627
ssp. lactis Pedicoccus acidilactici Pc 1.03 WYO LB 42-23
Lactobacillus delbrueckii Lb 4.06 JCM 1248 Pedicoccus pentosaceus Pc 2.01 CECT 923
ssp. lactis Enterococcus faecium Ec 1.01 CECT 410
Lactobacillus fermentum Lb 5.01 CECT 285 Enterococcus faecium Ec 1.02 CECT 964
Lactobacillus fermentum Lb 5.02 CECT 4007 Enterococcus hirae Ec 2.01 CECT 279
Lactobacillus hel6eticus Lb 6.02 CECT 404 Listeria monocytogenes Lm 1.01 ES
Lactobacillus hel6eticus Lb 6.03 CECT 414 Listeria monocytogenes Lm 1.02 ES
Lactobacillus hel6eticus Lb 6.04 CECT 541
a
Lactobacillus hel6eticus Lb 6.05 CECT 800 Nisin A producing strain.
b
Lactobacillus hel6eticus Lb 6.06 CECT 905 Pediocin PA-1 producing strain.
Lactobacillus hel6eticus Lb 6.07 CECT 402 CECT, Colección Española de Cultivos Tipo (Valencia, Spain);
Lactobacillus cur6atus Lb 7.01 CECT 904 NCK, Department of Food Science (North Carolina State Univer-
Lactobacillus plantarum Lb 8.01 CECT 220 sity, NC); JCM, Japan Collection of Microorganisms; MG, Instituto
Lactobacillus plantarum Lb 8.02 CECT 221 de Productos Lácteos de Asturias (Spain); NCDO, National Collec-
Lactobacillus plantarum Lb 8.03 CECT 748 tion of Dairy Organisms (UK); WYO, Department of Animal Science
Lactobacillus plantarum Lb 8.04 CECT 229 of Wyoming (Wyoming, USA); NRRL, Northern Regional Research
Lactobacillus plantarum Lb 8.05 CECT 4044 Laboratory (Peoria, IL); ES, Delegación Provincial de Sanidad (La
Lactobacillus plantarum Lb 8.06 CECT 4045 Coruña, Spain); MN, Centro Tecnológico de la Carne (Monells,
Lactobacillus sakei Lb 9.01 CECT 906 Girona, Spain).
Lactobacillus sakei Lb 9.02 MN
Lactococcus lactis ssp. Lc 1.01 CECT 697
cremoris production of nisin and pediocin are carried out under
Lactococcus lactis ssp. Lc 1.02 CECT 185 well-controlled culture conditions, there are discrepan-
lactis cies mainly concerned with the kinetic typology of their
Lactococcus lactis ssp. Lc 1.03 CECT 188 production. In this way, even though bacteriocin produc-
lactis
tion is generally associated with biomass evolution [22],
Lactococcus lactis ssp. Lc 1.04 CECT 539
lactis a their classification as primary or secondary metabolites
Lactococcus lactis ssp. Lc 1.05 CECT 540 is not clear. Nisin is considered to be a primary metabo-
lactis lite by some authors [15,18], but not by others [9,22] and,
Lactococcus lactis ssp. Lc 1.06 CECT 916 in the same way, pediocin is considered as a secondary
lactis [8,10] or a primary metabolite [23,24]. These discrepan-
Lactococcus lactis ssp. Lc 1.07 CECT 984
lactis
cies are related to pH dependent phenomena, such as the
Lactococcus lactis ssp. Lc 1.08 CECT 4041 adsorption of the bacteriocins onto cell surfaces and/or
lactis the post-translational processing of the prepeptides to
Lactococcus lactis ssp. Lc 1.09 CECT 4042 active forms [10,25]. However, kinetic analysis of both
lactis nisin and pediocin production (e.g. adjustment of exper-
Lactococcus lactis Lc 1.10 MG 1614
Lactococcus lactis spp. Lc 1.12 WYO
imental data to a kinetic production model) had not been
lactis performed until now.
Leuconostoc mesenteroides Ln 3.01 CECT 827 On the other hand, there are variations in test proce-
ssp. cremoris dures used and especially in the methodologies used to
Leuconostoc mesenteroides Ln 3.02 CECT 912 detect bacteriocin production and to determine the
ssp. dextranicum
Leuconostoc mesenteroides Ln 3.03 CECT 215
effects of temperature, pH and proteolytic enzymes on
ssp. mesenteroides bacteriocin activity. Consequently, it is difficult to make
Leuconostoc mesenteroides Ln 3.04 CECT 219 comparisons between the properties of these antibacterial
ssp. mesenteroides compounds.
 N.P. Guerra, L. Pastrana / Process Biochemistry
Modelling growth and bacteriocin production en-
ables the elucidation of underlying metabolic regulatory
mechanisms which could facilitate the optimization of
production for upscaling. On the other hand, the math-
ematical formalization in the experimental planning of
the studies of stability and activity of both nisin and
pediocin could avoid the difficulty in interpretation and
comparison of results.
In this work, a kinetic analysis of both nisin and
pediocin production, using a modification of the
Luedeking and Piret model, was developed. A multifac-
torial experimental design and response surface analysis
was used to determine the effects of pH and tempera-
ture on the stability of both bacteriocins and their
sensitivity to the action of proteolytic enzymes. In
addition, the antibacterial activity of nisin and pediocin
was compared with bacteriocin-like inhibitory sub-
stances from other lactic acid bacteria using hierarchi-
cal clustering analysis.
2. Materials and methods
2.1. Bacterial strains, media and culture conditions
The strains used in this study are listed in Table 1.
Cultures were maintained as frozen stocks at − 40 °C
in Nutrient Broth containing 15% (v/v) of glycerol and
were propagated twice in appropriate media before use.
All Lactococcus, Lactobacillus, Carnobacterium, Pedio-
coccus and Leuconostoc strains were grown on MRS
broth. Strains of Enterococcus were grown on Rothe
broth and Listeria strains were grown on Mc Bride
agar.
All bacteriocin-producing cultures of lactic acid bac-
teria (LAB) were grown in 250 ml Erlenmeyer flasks
containing 50 ml of medium, on a rotary shaker (200
rpm) at 30 °C, until the early stationary phase. All
cultivations were started with a 2% (v/v) inoculum of a
12-h culture. Samples were taken for biomass, culture
pH, antibacterial activity and analytical determinations,
as required.
2.2. Extraction of antibacterial acti6ity
Aliquots from LAB cultures were adjusted to pH 3.5
with 5 N HCl to avoid the adsorption of molecules of
bacteriocin onto the producer cell surfaces. Thereafter,
they were heated for 3 min to kill the cells and cen-
trifuged at 27,200×g for 15 min at 4 °C. The superna-
tants containing antibacterial activity (bacteriocin
extract) were adjusted to pH 6.0 and frozen until
further use.
1007
2.3. Bacteriocin acti6ity assays
Two methods were employed. For fermentation ki-
netic purposes, the quantitative bacteriocin activity of
L. lactis subsp. lactis CECT 539 (Lc 1.04) and P.
acidilactici NRRL B-5627 (Pc 1.02) was estimated by
using a photometric assay on culture tubes [26], using
C. piscicola CECT 4020 (Cb 1.01) as target organism.
Each tube contained 2.5 ml of serial dilutions of bacte-
riocin extracts (pH 6.0) and an equal volume of a
culture of Cb 1.01, adjusted with sterile buffered MRS
broth (pH 6.3) to provide an initial absorbance (at 700
nm) of 0.2. The tubes were incubated for 6 h at 30 °C.
Growth inhibition was measured spectrophotometri-
cally at 700 nm. One bacteriocin unit (BU) was defined
as the amount of bacteriocin causing 50% growth inhi-
bition (lethal dose 50: LD50 obtained from duplicate
samples), compared with a control without
bacteriocins.
For comparative purposes, an agar well-diffusion
method was used [27]. An overnight culture of indicator
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
for Lactococcus, Lactobacillus, Carnobacterium and
Leuconostoc strains indicators.) Wells (5 mm in diame-
ter) were cut into plates and 50 ml of culture superna-
tant fluid of the producing-strains placed into each well.
Plates were kept for 4 h at 4 °C to allow the diffusion
of the extracts and later they were incubated at 30 °C
for 24 h, after which the widths of the clear inhibition
zones around the well were measured. A solution of 1
g/l of nisaplin (Aplin and Barrett Ltd., Dorset, UK)
adjusted to pH 6.0, was used as bacteriocin standard.
The bacteriocin activity was quantified as a relative
inhibition index (RII) according to the expression:
RII = Ze/(Zn − 1), where Ze and Zn are the mean
diameters, respectively (obtained from triplicate experi-
ments) of the inhibition zones produced by the extract
and nisaplin solution.
2.4. Analytical methods
Samples were taken, at regular intervals, for analysis.
Growth was monitored by absorbance at 700 nm and
converted to dry cell weight from a standard curve. The
cells were harvested by centrifugation (12,000× g dur-
ing 15 min at 4 °C) of culture samples and washed
twice with saline (0.8% NaCl). The culture supernatants
were used to determine total sugars, phosphorous, ni-
trogen and proteins by methods as described previously
[28,29]. The lactic acid was determined by gas-liquid
chromatography with FI detection [30], using a SU-
PELCO SPB1701 column.
1008 N.P. Guerra, L. Pastrana / Process Biochemistry
2.5. Bacteriocin sensiti6ity to proteolytic enzymes
Solutions of protease in suitable buffers (pepsin:
potassium hydrogen phthalate– HCl 0.2 M, pH 2.2;
trypsin and subtilisin: TRIS 0.2 M, pH 7.6 and pa-
pain: phosphate 0.2 M, pH 7.0) containing 1 enzymic
unit (determined according to Murado et al. [29]) were
mixed with equal volumes of bacteriocin extracts and
incubated over 30, 60 and 90 min at each optimum of
temperature for the enzymes: 37 °C for pepsin and
subtilisin, 25 °C for trypsin and 30 °C for papain.
After the treatment, the samples were adjusted to pH
6.0 to determine the remaining antibacterial activity.
Controls consisted of samples of extracts treated un-
der the same conditions but without enzymes.
2.6. Statistical methods
A multifactorial composite orthogonal design [31]
based on five levels and two variables was used to
study the combined influence of pH and temperature
on the stability of both nisin and pediocin. The design
consisted of 13 experiments with four (22) factorial
points, four axial points to form a central composite
design with h =1.267 and five centre points for repli-
cation.
Bacteriocin extracts from 18 h cultures of Lc 1.04
and Pc 1.02, were adjusted at different pH values with
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-
ing sugars; BT: bacteriocin; Lact: lactic acid; TP: total phosphorous;
TN: total nitrogen. Means of three analytical replications.
5 N HCl or NaOH and incubated for 5 and 15 min at
the corresponding temperature, according to the ex-
perimental matrix defined by the design used. After
incubation the samples were adjusted to pH 6.0 and
monitored for remaining activity (%RA). Controls
consisted of samples of untreated bacteriocin extracts.
In order to compare the activity of nisin and pe-
diocin with other bacteriocins, a cluster analysis was
used. The classification variables used were the in-
hibitory spectrum (defined as the number of inhibited
indicator cells) and the potencies of the antibacterial
compounds (defined as RII) produced by each pro-
ducing strain on the indicator organisms. Both vari-
ables were standardized by average deviation. The
Euclidean distance was used as a measure of the simi-
larity index. The average linkage (in the variant of
unweighted pair-group average) was used as amalga-
mation (linkage) method [32].
3. Results and discussion
3.1. Nisin and pediocin production on MRS broth
Fig. 1 shows the growth of both L. lactis subsp.
lactis CECT 539 (Lc 1.04) and P. acidilactici NRRL
B-5627 (Pc 1.02) cultures. The last strain produced
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-
port, which is a function of the pH. Usually, the
optimum pH varies between 6.0 and 6.5 and decreases
rapidly at higher or lower values. For this reason, the
 N.P. Guerra, L. Pastrana / Process Biochemistry
Fig. 2. Diagram of phases for both specific rates of bacteriocin
production (qBU in BU mg/h) and growth (v in hours) and for
absolute rates of bacteriocin production (rBU in BU ml/h) and
growth (rX in g/l/h). Lc 1.04 (open symbols) and Pc 1.02 (closed
symbols).
Fig. 3. Experimental (symbols) and adjusted data (continuous lines)
according to Eq. (3). Notations as in Fig. 2.
failure to grow at acidic pH is likely to be caused by a
limitation of cytoplasmic processes (acidification of the
cytoplasm and the collapse of the motive force) [35–
37].
The role of pH in growth and bacteriocins produc-
tion is well-documented and depends on the media and
microorganisms, although there are discrepancies about
the starting and final pH values and control or not of
pH during the fermentation [9,13,18,21].
In our assays, the initial pH of the cultures (6.3) was
fixed in an adequate range (5.0–6.8) for the production
of pediocin and nisin [21]. When the pH of the culture
fell below 5, the growth of Lc 1.04 and bacteriocin
production stopped, while in the case of Pc 1.02,
growth and pediocin production only slowed down.
For nisin production, this behaviour has been observed
by Yang and Ray [21] on TGE medium. These re-
searchers also reported that nisin production was in-
creased at final pH values above 5. In contrast, the
same authors and Biswas et al. [10] reported that
pediocin production increased, in the presence of high
cell mass at final pH below 5, because of the need for
low pH for conversion of prepediocin to active
pediocin.
1009
3.2. Kinetic analysis of the bacteriocins production
The upper part of Fig. 2 shows the adjustments of
the specific rates of growth (v) and bacteriocin produc-
tion (qBU) (previously calculated by numerical deriva-
tion from smoothed experimental data with a
generalized logistic equation [38]), using the Luedeking
and Piret model [39]:
rBU =hrX +iX ; or dividing by X: qBU =hv+i (1)
A straight line, typical of a primary metabolite pro-
duction, was obtained for nisin (h=38.3, i= −0.34,
r 2 =0.9514). Nevertheless, in the case of the pediocin,
the lack of fit of specific rates (v and rBU) correspond-
ing to the first 10 h of incubation, did not allow its
production to be characterized (right upper part of Fig.
2).
In an effort to explain this behaviour, the procedure
reported by Pintado [40] was applied. Basically, the
method consists of the representation of growth rate
(rX ) versus the metabolite production rate (e.g. bacteri-
ocin: rBU) define a curve, which branches tend to open
when the ratio i/h is increased, in the general case of a
mixed metabolite (h and i"0). The maximum opening
of the curve takes place for pure secondary metabolites
and it becomes a straight line for a pure primary
metabolite (left lower part of Fig. 2). When the biosyn-
thesis of metabolites (primary or secondary) requires a
limiting level of a factor ( f ) affecting the growth and
metabolite production (generally a nutrient different
from the carbon source), the Eq. (1) must be changed
by Eq. (2) and then, even in the case of a pure primary
metabolite, a linear relationship between rX and rBU is
impossible.
rBU =(hrX +iX )F (2)
F being a linear function of factor f.
In pediocin, the opening of branches in the represen-
tation of rX versus rBU was not sufficient, according to
the profiles presented by Pintado [40], to suppose a
primary, secondary or a mixed character (lower part of
Fig. 2). This fact suggests that these rates depend on an
additional factor: the pH. According to the role of this
variable discussed in the above section, it could be
noted that the acidification of the culture (gradient of
pH: DpH) favored both bacteriocin productions, before
a pH value inhibitory for growth in the media was
reached. A simple form to model this effect consists of:
F=1+kDpH and then:
rBU =(hrX +iX)(1+kDpH) (3)
When Eq. (3) was used to adjust the experimental
production rates, a good fit was obtained for both
bacteriocins (continuous lines in Fig. 3). The calculated
parameters (Table 2) revealed the primary character for
both bacteriocins (i$0). When the adjustments were
1010 N.P. Guerra, L. Pastrana / Process Biochemistry

Table 2 duction rates) were obtained. According to the k values,

Parameter values in the expression for the pH dependence of nisin
nisin production showed only slight (and negative) de-
and pediocin production (Eq. (3))
pendence on the gradient of pH, whereas pediocin
Bacteriocin a i k production was markedly affected by the DpH. For this
reason, it can be concluded that pediocin is a pH-de-
Nisin 40.73 0.55 −0.32 pendent primary metabolite and before a final pH
Pediocin 127.28 0.22 1.11
unsuitable for growth was reached, its production rate
was favored by the acidification of the medium.
Table 3
Experimental domain and codification of the variables in the factorial 3.3. Effects of pH and temperature on the stability of
design used to study the combined influence of temperature and pH both bacteriocins
on the antibacterial activity of nisin and pediocin

Codified values Natural values

The stability of nisin, pediocin and 1 g/l of nisaplin
solution (used as reference) to temperature and pH was
T (°C) pH studied by a complete second-order orthogonal design.
The empirical models obtained with this procedure
−1.267 63.0 3.7
permitted the evaluation of the stability of the extracts
−1 70.0 4.0
0 95.5 5.2 (without the need to assay all the combinations of the
+1 121.0 6.5 variables) and to calculate the optimum conditions of
+1.267 128.0 6.8 stability. In view of the possible applications of bacteri-
Increments 1 25.5 1.2 ocins as food preservatives, the experimental domain
(Table 3) was selected regarding usual levels existing in
foods (for pH) and in their processing operations (for
Table 4 both variables).
Empirical equations obtained for remaining activity (% RA) of bacte-
The empirical equations obtained for the remaining
riocin samples after pH and heat treatment
activity (Table 4) were highly consistent, being the
Bacteriocin Empirical equations coefficients acceptable according to the Student’s t-test
samples (h=0.05) and the validity of the equations according to
the Fisher F-test (h= 0.05). Interactions between the
Nisaplin %RA (5 min) =88.68−7.93 T −7.83 pH
two variables were only observed for nisaplin and pe-
−3.53 T pH −4.25 T 2
%RA (15 min)= 78.64−10.59 T −11.68 pH diocin after 5 min of incubation and, in all cases, the
−7.24 T 2 pH had no influence on the response as a second order
Nisin %RA (5 min)= 87.74−5.89 T −7.07 pH variable.
−3.32 T 2 The response surfaces of %RA after 5 and 15 min of
%RA (15 min)= 83.48−6.10 T −7.41 pH treatment are depicted in Fig. 4. All bacteriocins
−3.66 T 2 showed similar behaviour. The stabilities were maxima
Pediocin %RA (5 min) = 88.39−8.27 T −8.02 pH at temperatures below 100 °C, low pH and short incu-
−2.47 T pH −6.95 T 2 bation times, decreasing toward high values of these
%RA (15 min)= 81.55−7.80 T −7.84 pH
variables (Table 5). While nisaplin was the most sensi-
−7.27 T 2
tive bacteriocin, especially after 15 min of incubation,
nisin showed a high mean stability in the assayed
carried out fixing the value of i as 0, little variation in experimental domain (Fig. 4). This heat-stability at acid
the values of the parameters and in the correlation pH has been described for most bacteriocins [41–44].
coefficients (between experimental and predicted pro- This behaviour is attributed to their high content of
Table 5
Optimum temperature and pH values for each bacteriocin samples

Bacteriocin samples

Nisaplin Nisin Pediocin

5 min 15 min 5 min 15 min 5 min 15 min

T (°C) 85.0 76.9 74.3 72.8 86.0 81.7

pH 3.7 3.7 3.7 3.7 3.7 3.7
Maximum RA (%) 99.3 97.3 99.3 95.4 99.5 93.6
 N.P. Guerra, L. Pastrana / Process Biochemistry 1011

was also determined. The experiments were carried out

by standardizing the enzymic units added to the reac-
tion mixtures under optimum conditions of pH and
temperature for each enzyme. Since the specific activity
of extracts was unknown, it was not possible to stan-
dardize the enzyme/substrate ratios. Extracts were
therefore selected from postincubates of the stationary
phase of the cultures containing a similar level of total
remaining proteins. For comparative purposes, a solu-
tion of 1 g/l of nisaplin was also assayed.
The results (Fig. 5) showed that, in all cases, expo-
nential decreases of activity (%RA) were produced
through the incubation time. The experimental data of
each series of experiments were adjusted to a first order
kinetic model:

%RA = 100 e − kt (4)

where t is the incubation time.

Due to the existence of an asymptotic value for
remaining activity, a lack of fit was evident, probably
due to enzyme inactivation, product inhibition or pres-
ence of compounds (mainly proteinaceous) in the sam-
ples that could act as competitive or protective agents.
In an effort to explain this behaviour, Eq. (4) was
modified, introducing the asymptote (ra) as a new
parameter, with the restriction that %RA= 100 for
t= 0:

%RA = ra + (100−ra)e − kt (5)

Fig. 4. Response surfaces corresponding to the %RA after both pH
and temperature treatment, of nisaplin (A), nisin (B) and pediocin (C)
samples for 5 and 15 min, according to the experimental plan defined Good adjustments were obtained with this new
in Table 3.
glycine and to the formation, at a molecular level, of
globular structures and strong hydrophobic interactions
in the molecule [45].
model, although the calculated values for k and ra
(Table 6) only have validity as an estimation for com-
parative purposes due to the small number of experi-
mental data.
Treatment of the samples containing nisin with pa-
3.4. Sensiti6ity of bacteriocins to proteolytic enzymes
The sensitivity of nisin and pediocin to the prote-
olytic attack of subtilisin, papain, pepsin and trypsin
pain yielded the highest values of ra and the degrada-
tion was practically completed after 1.5 h of incubation
(Fig. 5), despite that this enzyme breaks histidine–va-
line and dihydroxyalanine–lysine bonds present in the
nisin molecule [46]. This result cannot be explained by
enzyme inactivation since in the same reaction condi-
tions, papain continued the hydrolysis of pediocin (the
Table 6 value of ra for pediocin was five times lower than for
Values of k and ra for each bacteriocin sample according to Eq. (5) nisaplin or nisin). This may be explained by the produc-
tion of increasing concentrations of proteins during
Proteases Bacteriocin samples
fermentation, which competed with nisin for papain.
Nisaplin Nisin Pediocin On the other hand, the values of k and ra obtained
with subtilisin, pepsin and trypsin were slightly higher
k ra k ra k ra for nisin than for nisaplin, indicating the relevance of
the matrix in the stability of bacteriocins.
Pepsin 2.82 17.78 4.93 22.09 1.86 6.63
No significant differences were found between the
Papain 4.40 54.19 3.31 55.93 2.36 10.80
Subtilisin 3.59 16.03 3.04 19.71 1.81 6.49 action of proteases in the samples containing pediocin
Trypsin 2.99 8.07 6.37 20.34 2.36 11.32 and their parameters were lower than the samples con-
taining nisin (Table 6).
 1012
Table 7
Antibacterial activity spectrum of lactic acid cultures as determined by the agar well-diffusion assay

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 + + − − − − − − − − + − − − − + + + −

N.P. Guerra, L. Pastrana / Process Biochemistry

Lb 3.02 − + − − − − − − − − + − − − + − + + −
Lb 3.03 + + − − − + − − − − + − − − + + + + −
Lb 3.04 + + + − − + − − − − + − − − + + + + −
Lb 4.06 − + − − − − − − − − + − − − + − + + −
Lb 6.03 + + + − − − − + − − + − − − + + + + −
Lb 6.06 + + − − − − − − − − + − − − − − + + −
Lb 6.07 + + − − − − − − − − + − − − − + + + −
Lb 8.01 + + − − − − − − − − + − − − + + + + +
Lb 8.02 + + − − − + − − − − + − − − + + + + −
Lb 8.03 + + − − − − − − − − − − − − + + + + +
Lb 8.05 + + − − − − − − − − + − − − − + − + +
Lb 9.01 + + − − − − − − − − + − − − + − − + −
Lb 9.02 + − − − − − − + − + − − + − + + + + +
Lc 1.01 + + − − − − − + − + − − − − + − + − +
Lc 1.02 + + − − − − − + − − − − − − − + − + +
Lc 1.03 + + − − − − − + − − − − − − + − + + +
Lc 1.04 + + + + + + + + + + + − − − + + + + +
Lc 1.07 + + − − − − + + − − − − − − + − + − +
Lc 1.08 + + + − − − + + − − − − − − + − + − +
Lc 1.09 − + − − − − − + − − − − − − + + + + +
Lc 1.10 + + − − − − − + − − − − − − + + + + +
Lc 1.12 + + + − − − − + − − − − − − + + + − +
Ln 3.02 + + − − − − − − − − + − − − + + + + −
Ln 3.03 + + − − − − − − − − + − − − + + + + −
Ln 3.04 − + − − − − − − − − + − − − + + + + +
Ln 3.05 + + − − − − − + − − − − − − + + + + +
Ln 3.06 − + − − − − − − − − + − − − + + + + +
Ln 3.07 − + − − − − − − − − + − − − − + + − −
Ln 3.08 − + − − − + − − − − + − − − + + + + −
Ln 4.01 − + − − − − − − − + + − − − + + + + −
Ln 5.01 + + − + − + + + + + − − − − + + + + +
Ln 5.03 + + + + − + + + + + − − + − + + + + +
Pc 1.01 + − − − − − − − − − − − − − − + + + +
Pc 1.02 + + + − − + − − + + + + + + + + + + +
Pc 1.03 + − + − − − − − − − − − − − − + + − +
Pc 2.01 + − − − − − − − − − − − − − − + + + +

+, Zone of inhibition; −, no zone of inhibition.

 N.P. Guerra, L. Pastrana / Process Biochemistry 1013

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
and Pc 1.02 extracts with a set of other bacteriocin-pro-
ducing lactic acid bacteria
Usually the characterization of the bacteriocin activ-
ity is carried out by determination of the inhibition
spectrum against a set of target microorganisms (num-
ber of sensitive/number of total tested strains) by mea-
suring of the diameter of the inhibition zone in agar
plates. In these assays, no standard reference was used
and a comparison with other bacteriocins (in the same
experimental conditions) is not shown.
In this work, the inhibitory spectra of nisin and
pediocin (expressed as number of inhibited strains,
NIS) and their potencies (expressed as RII) were com-
pared with other extracts containing bacteriocin-like
inhibitory substances (BLIS) from lactic acid bacteria.
The study was divided in two steps. Firstly a set of 50
strains of lactic acid bacteria were tested as producers
against Enterococcus (Ec 1.01, 1.02, 2.01) and Listeria
monocytogenes (Lm 1.01 and 1.02) strains. Of these, 14
were found to be non-producing strains, while 36 pro-
duced bacteriocin-like inhibitory substances (BLIS)
which to various degrees inhibited the growth of the
indicator organisms. Subsequently, the antibacterial ac-
tivity of these producing strains were compared with
those produced by Lc 1.04 and Pc 1.02 using as indica-
tors the 14 non-producing strains plus the five target
organisms used in the first step.
Table 7 shows the different inhibitory activity of
these bacteriocin producers. Some strains (Lc 1.04, Pc
1.02, Ln 5.01, Ln 5.03) were inhibitory to almost all the
indicators, while others (Ln 3.07, Lb 3.02, Lb 4.06, Lb
6.06, Lb 9.01, Pc 1.01, Pc 1.03, Pc 2.01) were active
only against four or five strains. These differences in
sensitivity and resistance of a strain to the different
bacteriocins are related to the existence of different, but
specific, surface receptors for different bacteriocins in
Gram-positive bacterial cells [47].
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-
tibacterial activity of the producing bacteria. Because of
this, the data were processed by cluster analysis using
the inhibitory spectrum and potency of each extract, as
classification variables.
The pyramidal representation (Fig. 6) showed that Pc
1.02 and Lc 1.04 formed separate clusters, each one
with the highest distance indexes, revealing that nisin
and pediocin are the most potent bacteriocins assayed
and they have the broadest inhibitory spectra. In addi-
tion to this, an independence between lactic acid pro-
duction and RII was observed (even the major lactic
acid producers: Lb 3.04 and Lb 9.02, did not span the
broadest inhibitory spectrum).
Acknowledgements
We thank Miguel Anxo Murado (head of the re-
search team of Instituto de Investigacións Mariñas de
Vigo, CSIC) for their aid in the elaboration of this
work. Nelson P. Guerra was a recipient of ICI and
Xunta de Galicia fellowships.
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