Letters in Applied Microbiology 2003, 37, 51–55

Influence of pH drop on both nisin and pediocin production

by Lactococcus lactis and Pediococcus acidilactici

N.P. Guerra and L. Pastrana

Department of Biochemistry, Genetics and Immunology, Vigo University, Ourense, Spain

2002/362: received 25 November 2002, revised 3 February 2003 and accepted 14 March 2003

ABSTRACT
N . P . G U E R R A A N D L . P A S T R A N A . 2003.
Aims: To develop a kinetic model for describing the specific effect of pH drop on nisin and pediocin
production in whey.
Methods and Results: The effect of pH drop on both bacteriocin productions was tested in non-buffered
whey and whey buffered at initial pH 6Æ3 with 0Æ03, 0Æ10 and 0Æ25 mol l)1 of potassium hydrogen phthalate-NaOH.
An accurate description of the experimental data of nisin and pediocin obtained at different pH drops is
obtained with the proposed model.
Conclusions: The proposed model was able to typify both bacteriocins as pH-dependent primary metabolites.
Significance and Impact of the Study: The decisive role of pH drop for bacteriocin production on whey was
demonstrated and modelled. This study contributes to a better understanding of underlying metabolic regulatory
mechanisms, which could facilitate the optimization of bacteriocin production for upscaling.

Keywords: Lactococcus lactis, nisin, pediocin, Pediococcus acidilactici, pH drop, whey.

rate), deviations from linearity have been observed for some

INTRODUCTION
Bacteriocins are proteinaceous antibacterial compounds that
are produced by lactic acid bacteria commonly present in
foods. They are bactericidal to many Gram-positive bacteria
associated with food spoilage and food-borne illnesses and
retain their properties after heat treatment. Thus, some
bacteriocins, mainly nisin and pediocin, are used as food
preservatives (Ray 1992).
Bacteriocins are normally produced in complex media
under well-controlled conditions of temperature and pH
(Parente and Ricciardi 1994), but cheap raw materials such
as whey (Goulhen et al. 1999), sugar molasses (Egorov et al.
1980) and mussel-processing wastes (Guerra and Pastrana
2002a) have also been used as culture media.
In general, although bacteriocin synthesis has been found
to be growth-associated (a linear relationship was indeed
observed between bacteriocin production rate and growth
Correspondence to: Lorenzo Pastrana, Area de Bioquı´mica e Bioloxı´a Molecular,
Facultade de Ciencias de Ourense, Universidade de Vigo, 32004 Ourense, Spain
(e-mail: pastrana@uvigo.es).
ª 2003 The Society for Applied Microbiology
bacteriocins (Biswas et al. 1991; Kim et al. 1997). This has
been related to the complex interaction of many factors
which affect bacteriocin synthesis, such as media composi-
tion (carbohydrate, nitrogen and phosphorous sources, etc.)
and fermentation conditions (mainly pH and temperature).
In batch fermentations, when the optimal initial culture
conditions for cell growth are fixed (pH, temperature and
media composition), bacteriocin production is strongly
dependent on the pH profiles – the pH drop and the final
pH reached in the cultures. Therefore the need for a deter-
mined pH drop for high nisin (Cabo et al. 2001) and pediocin
(Yang and Ray 1994) productions have been referred. In
addition, the final pH of the cultures may influence both the
adsorption of the bacteriocins to the producer cells and the
post-translational processing of prebacteriocin to produce
active bacteriocin (Biswas et al. 1991). In fact, the influence of
pH on bacteriocin synthesis is a complex phenomenon, which
makes the metabolic typification of some bacteriocins difficult.
In these cases, Luedeking and Piret (1959) model is not always
adequate to describe bacteriocin synthesis.
52 N . P . G U E R R A A N D L . P A S T R A N A
In the present work, the kinetics of nisin and pediocin
production are studied in whey buffered to initial pH 6Æ3
using different concentrations of the buffering agent. The
experiments were set up to assess the quantitative effect of
pH drop on bacteriocin production. A kinetic model was set
up and fitted to the data obtained from the fermentations.
M A T E R I A LS A N D M E T H O D S
Bacterial cultures and media
Lactococcus lactis subsp. lactis CECT 539 (producer of nisin)
and Carnobacterium piscicola CECT 4020 (which was used as
indicator strain) were obtained from the Spanish Type
Culture Collection (CECT, Valencia, Spain). Pediococcus
acidilactici NRRL B-5627 (producer of pediocin), was
obtained from the National Center for Agricultural Utiliza-
tion Research (NCAUR, Peoria, IL, USA). The three bacteria
were grown in MRS broth and maintained as frozen stock held
at )40C in nutrient broth plus 15% (v/v) glycerol. The
working cultures were maintained as slants on de Man Rogosa
Sharpe (MRS) agar at 4C, and propagated twice in liquid
cultures in the same medium at 30C before use.
Whey, which was obtained from a local dairy plant as the
liquid remaining after the first cheese pressing was proc-
essed in the following conditions. After adjusting the pH to
4Æ5 with 5 N HCl, whey was heated at 121C for 15 min to
denature the proteins and the precipitates were removed by
centrifugation (27 200 g for 15 min at 4C). This treatment
only affected the protein content, which was reduced from
8Æ2 to 5 g l)1. The resulting medium contained (g l)1): total
sugar, 48Æ51; total nitrogen, 1Æ05; total phosphorus, 0Æ43 and
soluble proteins, 5.
To study the influence of pH drop rate on nisin and
pediocin production, four series of batch cultures were
performed on whey buffered at initial pH 6Æ3 with 0 (non-
buffered medium), 0Æ03, 0Æ10 and 0Æ25 mol l)1 potassium
hydrogen phthalate-NaOH. This buffering agent was used to
avoid alterations in the culture media. Then the media were
sterilized at 121C for 15 min and used as culture medium.
All fermentations were carried out in 250-ml Erlenmeyer
flasks containing 50 ml of whey, in a rotary shaker
(200 rev min)1). The media were inoculated with 2%
(v/v) of a 12 h culture of the appropriate producing-strain
and incubated for 18 h at 30C.
The samples were withdrawn at intervals during incuba-
tion periods to determine cell dry mass, pH, residual total
sugar and antibacterial activity.
Analytical methods
The growth was monitored by optical density at 700 nm and
converted to dry cell mass using a standard curve. The cells
ª 2003 The Society for Applied Microbiology, Letters in Applied Microbiology, 37, 51–55
were harvested by centrifugation (3000 g for 15 min at 4C)
of culture samples and washed twice with saline (0Æ8% NaCl).
Total sugars, phosphorous, nitrogen and protein content
were determined as described by Murado et al. (1993).
Bacteriocin activity determination
Aliquots from cultures were adjusted to pH 3Æ5 with 5 N
HCl to prevent the adsorption of bacteriocin onto the
producer cell surfaces, and then heated for 10 min to kill the
cells (Guerra and Pastrana 2002a). The heat-killed cells were
removed by centrifugation (27 200 g for 15 min at 4C) and
the supernatant was used to measure bacteriocin activity in
culture tubes (Cabo et al. 1999) against C. piscicola.
RESULTS
Fig. 1a shows the time course of pH, biomass and
bacteriocin production by L. lactis subsp. lactis and
P. acidilactici in a non-buffered culture and in three
buffered cultures. As expected, the increase in buffer
concentration leads to an increase of the final pH, which
provoked a decrease in pH drop (DpH, defined as the
difference between initial and final pH). However,
although biomass production by both strains was practi-
cally unaffected, both nisin and pediocin titres decreased
as pH drop decreased. As all fermentations were started
in the same initial conditions (composition and pH 6Æ3),
the pH drop generated in each culture seems to account
for both nisin and pediocin synthesis. This indicates that
biomass produced by L. lactis and P. acidilactici was more
productive in the non-buffered cultures.
On the contrary, both bacteriocins were produced during
the exponential growth phase in all cultures supporting the
proposal that both bacteriocins display primary metabolite
kinetics (Guerra and Pastrana 2002b). This was confirmed
by using the Luedeking and Piret (1959) equation:
rBT ¼ arX þ bX ð1Þ
where rX and rBT are respectively, the biomass (X) and
bacteriocin production (BT) rates, a is a growth-associated
constant (BU mg)1) and b is the non-growth-associated
constant (BU mg)1 h)1). Before using this model, the
experimental data for cell growth and bacteriocin production
were smoothed with a generalized logistic equation
(Edwards and Wilke 1968). Then, the experimental values
of l and qBT were calculated using the smoothed data and
adjusted to eqn (1). The numeric integration of eqn (1) with
respect to time provided the estimate values of BT:
X
t
BT ¼ ðarX þ bX Þ ð2Þ
t¼0
 EFFECTS OF pH ON BACTERIOCIN PRODUCTION 53

(a) L. lactis Ped. acidilactici

6 6

pH
pH
5 5

0·3
0·10

X (g l−1)
X (g l−1)
0·2

0·05
0·1

0 0

Ped (BU ml−1)

Nis (BU ml−1)
40

20
3

0 0
0 5 10 15 20
Hours

(b)
60 10 50

9 40

Ped (10 AU ml−1)

O Ped (BU ml−1)

50
Nis (BU ml−1)

8 30
40

3
7 20
30
6 10

20 5 0
0 0·3 0·6 0·9 0 1 2 3 4

∆pH

(c)
Fig. 1 (a) Time course of pH, biomass (X) 12 60
and bacteriocin production (Nis and Ped) by
L. lactis and P. acidilactici on non-buffered 9
Ped (BU ml−1)
Nis (BU ml−1)

(J), and on buffered whey at pH 6Æ3 with 0Æ03 40

(É), 0Æ10 (Ñ) and 0Æ25 mol l)1 (S) potassium
6
hydrogen phthalate-NaOH. (b) Final nisin
and pediocin titres obtained in this study and 20
in TGE (Ç) by Yang and Ray (1994). (c) 3
Experimental (symbols) and calculated (lines)
according to eqn (4) for nisin and pediocin 0 0

production. Non-buffered ( ), 0.03 (s), 0.10
0 5 10 15 0
Hours
5 10 15 20
(h), 0.25 (,) in TGE (n)

ª 2003 The Society for Applied Microbiology, Letters in Applied Microbiology, 37, 51–55
54 N . P . G U E R R A A N D L . P A S T R A N A
As expected, a null value of b was obtained in all cultures
for both bacteria, however the values of a did not remain
constant as DpH changed.
By definition, Luedeking and Piret model assumes that
the changes in the rate of product formation only depends
on changes in the biomass production rate. However, when
the time course of some variable (like pH evolution) or pH
drop produces a specific effect on product formation, it
could be impossible to correlate rBT vs rX or obtain a
constant value for coefficient a.
In this sense, Cabo et al. (2001) proposed a model that
modifies eqn (1) by including a term for the effect of pH
gradient (VpH) on bacteriocin production rate in realkalized
cultures:
rBT ¼ ðarX þ bX Þð1 þ bV pHÞ ð3Þ
where b is a constant of proportionality to be experimentally
determined.
However, in batch cultures, it has been observed that the
profiles described by the pH produce a specific effect on bac-
teriocin production kinetic (Biswas et al. 1991; Yang and Ray
1994; Guerra and Pastrana 2002b). This effect can also be
described by using eqn (3), but VpH must be substituted for
the pH drop rate (rpH ¼ DpH/Dt) (Guerra and Pastrana
2002b). So, a better correlation between experimental and fit-
ted data for nisin and pediocin production would be obtained.
In this study, the plot of the final bacteriocin (nisin and
pediocin) titres vs DpH suggests the existence of an exponen-
tial relationship between both variables (left part of Fig. 1b).
The same trend was observed for pediocin AcH production on
tryptine-glucosa-yeast extract (TGE) by Yang and Ray 1994
(right part of Fig. 1b). However, these authors also observed
the existence of a maximum DpH beyond which leuconocim
Lcm1 production declined.
As bacteriocins are usually considered as primary meta-
bolites (b ¼ 0) and a depends on DpH, all the above
observations could be satisfied by modifying of eqn (3)
including an exponential term for the effect of DpH on
bacteriocin production.
t h
X  i
BT ¼ amax ed ðDpHmax DpHÞ rX ð1 þ brpHÞ ð4Þ
t¼0
where d is a constant of proportionality, amax is the
maximum yield (BU mg)1) that corresponds to the maxi-
mum value of DpH and DpHmax is the theoretical maximum
difference between initial and final pH from which bacte-
riocin production stopped.
The fitted specific bacteriocin models for both nisin (Nis)
and pediocin (Ped) production were as follows:
t h  i
X
Nis ¼ 2356e033ð093DpHÞ rX ð1 þ 329rpHÞ ð5Þ
t¼0
ª 2003 The Society for Applied Microbiology, Letters in Applied Microbiology, 37, 51–55
t h
X  i
Ped ¼ 36226e045ð099DpHÞ rX ð1 þ 282rpHÞ
t¼0
ð6Þ
These models enable both nisin and pediocin to be typified
as pH-dependent primary metabolites.
In Fig. 1c are given the predictions of eqns (5) and (6). As
can be seen, the proposed equation yielded good fits and
higher accuracy of predictions for the experimental data
than eqn (1). For this, it would be used as a general model to
describe bacteriocin production. In the same way, from the
values of DpHmax, the minimum pH (pHmin) from which
bacteriocin production stopped could be determined. So, the
theoretical value of final pHmin for L. lactis and P. acidilactici
in whey was calculated as 5Æ30 and 5Æ24, respectively.
DISCUSSION
The ability of L. lactis and P. acidilactici to produce
bacteriocins was tested in whey buffered at initial pH of 6Æ3
with different concentrations of buffering agent. With the
kinetic model of Luedeking and Piret model, both nisin and
pediocin production were typified as primary metabolites
in each culture. However, for the two bacteria, the growth-
associated coefficients (a) increased exponentially as pH
drop increased.
In these cases, when a limiting factor affects only the
biosynthesis of metabolites (primary or secondary) without
affecting biomass production, variable a becomes strongly
dependent on this factor.
In this way, the present study has shown that bacteriocin
production can be accurately described by eqn (4), in which
there is the influence of both rpH and DpH.
Indeed, the theoretical final pHmin values obtained from
the proposed model for higher production of pediocin and
nisin on whey correspond to the final pH obtained in the
non-buffered cultures.
These results agree with those reported by Cabo et al.
(2001) and Yang and Ray (1994), who obtained higher nisin
and pediocin titres in non-buffered TGE than in buffer TGE
broth. This was related to the need for a low final pH for an
efficient post-translational processing of both prebacteriocins
to produce active bacteriocins (Yang and Ray 1994).
However, it can be pointed out that higher pH drops
enhance both nisin and pediocin production until a final pH
inappropriate for survivability and cell growth of L. lactis
and P. acidilactici (van Niel and Hahn-Hägerdal 1999) was
reached.
Finally, this study demonstrated the feasibility of the
production of both bacteriocins on a cheap effluent. Never-
theless, the nisin and pediocin titres obtained on whey were
respectively, 6Æ2 and 9Æ7 times lower than those obtained on

MRS broth (Guerra and Pastrana 2002b). This suggests that
whey lack some essential nutrient for growth and bacteriocin
production, or those whey proteins have an unsuitable
composition to be used as nitrogen source. Therefore,
further studies based on finding of both cheap nutrient
sources to supplement whey and other culture techniques
(fed batch or continue) are needed.
REFERENCES
Biswas, S.R., Ray, P., Johnson, M.C. and Ray, B. (1991) Influence of
growth conditions on the production of a bacteriocin, pediocin AcH,
by Pediococcus acidilactici H. Applied and Environment Microbiology
57, 1265–1267.
Cabo, M.L., Murado, M.A., González, M.P. and Pastoriza, L. (1999)
A method for bacteriocin quantification. Journal of Applied Micro-
biology 87, 907–914.
Cabo, M.L., Murado, M.A., González, M.P. and Pastoriza, L. (2001)
Effects of aeration and pH gradient on nisin production. A
mathematical model. Enzyme and Microbial Technology 29, 264–273.
Edwards, V.H. and Wilke, C.R. (1968) Mathematical representation of
batch culture data. Biotechnology and Bioengineering 10, 964–974.
Egorov, N.S., Baranova, I.P., Kozlova, Y.I., Volkov, A.G., Grushina,
V.A., Isai, E.I., Isai, P.P. and Sidorenko, A.T. (1980) A new nutrient
medium for Streptococcus lactis producing nisin. Antibiotiki 25, 260–
263.
Goulhen, F., Meghrous, J. and Lacroix, C. (1999) Production of a nisin
Z/pediocin mixture by pH-controlled mixed-strain batch cultures in
supplemented whey permeate. Journal of Applied Microbiology 86,
399–406.
ª 2003 The Society for Applied Microbiology, Letters in Applied Microbiology, 37, 51–55
EFFECTS OF pH ON BACTERIOCIN PRODUCTION 55
Guerra, N.P. and Pastrana, L. (2002a) Nisin and pediocin pro-
duction on mussel-processing waste supplemented with glucose
and five nitrogen sources. Letters in Applied Microbiology 34,
114–118.
Guerra, N.P. and Pastrana, L. (2002b) Modelling the influence of pH
on the kinetics of both nisin and pediocin production and
characterization of their functional properties. Process Biochemistry
37, 1005–1015.
Kim, W.S., Hall, R.J. and Dunn, N.W. (1997) The effect of nisin
concentration and nutrient depletion on nisin production of
Lactococcus lactis. Applied Microbiology and Biotechnology 48, 449–
453.
Luedeking, R. and Piret, E.L. (1959) A kinetic study of the lactic acid
fermentation. Batch process at controlled pH. Journal of Biochemical
Microbiology Technology Engineering 1, 393–412.
Murado, M.A., Siso, M.I.G., González, M.P., Montemayor, M.I.,
Pastrana, L. and Pintado, J. (1993) Characterization of microbial
biomasses and amylolytic preparations obtained from mussel pro-
cessing waste treatment. Bioresource Technology 43, 117–125.
Parente, E. and Ricciardi, A. (1994) Influence of pH on the production
of enterocin 1146 during batch fermentation. Letters in Applied
Microbiology 19, 12–15.
Ray, B. (1992) Bacteriocins of starter culture bacteria as food
biopreservative. In Food Biopreservatives of Microbial Origin ed.
Ray, B. and Daeschel, M. pp. 177–205. Florida: CRC Press, Inc.
van Niel, E.W.J. and Hahn-Hägerdal, B. (1999) Nutrient requirements
of lactococci in defined growth media. Applied Microbiology and
Biotechnology 52, 617–627.
Yang, R. and Ray, B. (1994) Factors influencing production of
bacteriocins by lactic acid bacteria. Food Microbiology 11, 281–291.