Current Topics in

Biotechnology

Production of probiotics and bacteriocins on wastes from food

industry
Lorenzo Miguel Pastrana Castro*, Nelson Pérez Guerra, Cristina López Macías and Ana Torrado Agrasar
Departamento de Bioquímica, Xenética e Inmunoloxía, Facultade de Ciencias de Ourense, Universidade de Vigo, Campus de Ourense,
As Lagoas s/n, CP 32004, Ourense, Spain

ABSTRACT incubation times. However, the inhibitory activity of

The productions of nisin by Lactococcus lactis
subsp. lactis CECT 539 and pediocin by Pediococcus
acidilactici NRRL B-5627 were tested on cheap culture
media from two important wastes: mussel processing
wastes (MPW) and whey (W). Although both media
supported the cell growth and bacteriocin production,
the levels produced were lower than those obtained on
MRS broth. The compositions of both the MPW and
whey media were optimised using factorial experiments
and empirical modelling, as a function of four factors:
total sugars, nitrogen, phosphorous and buffer concentration.
Nisin and pediocin productions were higher than those
obtained in MRS broth when the whey and MPW
media were supplemented with yeast extract or
Casitone. Therefore, improvements in both nisin and
pediocin productions on both media were attempted by
converting the batch fermentation into a repeated fed-
batch and re-alkalized fermentation. The cultures of both
bacteriocin-producing strains were characterised with
bacteriocin titres compared with the batch process on
MRS, MPW and whey media. Mathematical models
including the effects of the main variables affecting the
cell growth and bacteriocin synthesis by both strains were
set up to describe the fed-batch production of biomass and
bacteriocin by L. lactis and Ped. acidilactici. Nisin and
pediocin obtained from the cultures on MRS broth
showed the highest heat stability at acidic pH and short
*To whom correspondence should be addressed: Departamento
de Bioquímica, Xenética e Inmunoloxía, Facultade de Ciencias de
Ourense, Universidade de Vigo, As Lagoas s/n, CP 32004,
Ourense, Spain.
e-mail address: pastrana@uvigo.es
both bacteriocins disappeared almost totally after 90
min of treatment with proteolytic enzymes.
KEYWORDS: Whey, mussel processing wastes,
Lactococcus lactis and Pediococcus acidilactici, nisin,
pediocin, probiotics, mathematical models, stability.
1: INTRODUCTION
Bacteriocins are ribosomally synthesized antimicrobial
proteins or protein complexes produced by Gram-
positive bacteria belonging to the family of so-called
beneficial bacteria, thus being considered more natural
than antibiotics. These compounds display a bactericidal
mode of action exclusively towards closely related
Gram-positive bacteria, including spoilage organisms
and food-borne pathogens such as Listeria monocytogenes,
Clostridium botulinum and Staphylococcus aureus, but
they are ineffective against Gram-negative bacteria [1].
In addition, bacteriocins are non-toxic and sensitive to
the action of digestive proteases [1,2].
Due to their antibacterial activity, there is an
increasing interest in using the bacteriocins produced
by lactic acid bacteria (LAB) as food biopreservatives
[3]. This could contribute to the reduction of the amounts
of other antimicrobial substances added to the food,
including sodium metabisulphite, butylated hydroxytoluene,
nitrite and nitrate [4] and to a decrease in the intensity
of the processing conditions (e.g. sterilization or
pasteurization) in low acidic foods [5,6]. In consequence,
the application of bacteriocins as food preservatives
could be a way for satisfying the growing consumers
demand for minimally processed, easily prepared and
ready-to-eat “fresh” food products.
26
On the other hand, many bacteriocin-producing
LAB have been used as probiotic microorganisms,
including Pediococcus, Lactobacillus, Lactococcus,
Bifidobacterium and Streptococcus species [7-9].
Probiotics are viable microorganisms and supportive
substances that once ingested by humans and animals,
produce beneficial physiology effects by assisting in
the establishment of an intestinal population which is
beneficial to both the humans and animals, and
antagonistic to harmful bacteria [10,11]. Other studies
showed that LAB produce beneficial effects on the host
throughout the modulation of the host immune response
and they can survive the intestinal motility and adhere
well to gastric mucosa [8,10].
Conditions that favored high biomass production
resulted in a comparable increase in bacteriocin production,
which is indicative of a primary metabolite [12,13]. The
main variables affecting the growth of probiotic LAB
and bacteriocin production are the temperature, aeration,
pH and the media composition.
Growth of LAB at optimal temperature (between 30
and 37ºC) usually results in optimal bacteriocin production
[12,14], however temperature stress and growth at sub-
optimal temperature may result in an increase in bacteriocin
yields [14,15].
Aeration was found to affect negatively bacteriocin
production [16-18] probably due to chemical degradation
[19]. For this reason, the most favorable conditions for
bacteriocin production have been proposed to be the
anaerobic conditions with a minimal agitation to obtain
a homogeneous cell suspension [20].
The pH is the most relevant variable in bacteriocin
production, since it may influence the bacteriocin activity
and solubility, the adsorption of bacteriocins on producer
cell surfaces [21] and the posttranslational processing of
prebacteriocin to produce active bacteriocin [22]. The
optimal pH for most bacteriocins is usually in the range
of 5.5-6.0 [12,23], which is often lower than the optimal
pH for growth [16,22,24].
Finally, with respect to the media composition,
bacteriocin is strongly affected by the type and
concentration of the carbon, nitrogen and phosphate
sources, cations, surfactants and inhibitors [19]. Glucose
and K2HPO4 have found to be the best carbon and
phosphate sources for bacteriocin production [12,18,
9,22,23]. The high quality protein hydrolizates (yeast
extract, meat extract, tryptone and peptone, casein
hydrolyzate) have proven to be good nitrogen sources
for bacteriocin production [15,25].
Since the primary goal of probiotic and bacteriocin
production is the cost-effective production of bioproductions,
Lorenzo Miguel Pastrana Castro et al.
it is important to define a suitable strategy that allows
production of biomass and the desired product to a high
concentration with productivity. Three alternatives used
usually for this purpose are: (i) the use of cheaper culture
media, (ii) the use of an efficient cultivation technique
and (iii) the design of an adequate control-system
throughout the use of accurate models describing cell
growth and product formation.
With regard to the first alternative, it has been
demonstrated the suitability of glycogen-rich mussel-
processing wastes (MPW) [26] and whey [15,27-29] to
be used as culture media for various bioproductions of
potential economic interest, including enzymes, organic
acids, hormones and bacteriocins.
With regard to the second alternative, it has been
reported that the production of some bacteriocins has
been successfully enhanced by employing fed-batch rather
than batch fermentation methods [30-32].
Finally, the third strategy consists in the development
of accurate models for describing the kinetic behaviour
of the growth and bacteriocin production by the producing
strains. These mathematical models could be used to
build algorithms for controlling the fed-batch production of
biomass and bacteriocins.
In this work, these three alternatives were used to
enhance the production of potentially probiotic biomasses
and bacteriocins by Lactococcus lactis subsp. lactis CECT
539 and Pediococcus acidilactici NRRL B-5627 on
media from mussel processing wastes and whey. Firstly,
the effect of the type and concentrations of the carbon,
nitrogen, phosphate sources and buffer concentration on the
growth and bacteriocin production by both strains was
studied. Secondly, the application of a fed-batch technique
based on successive re-alkalizations of the culture media
for was evaluated. Thirdly, mathematical models including
the effect of the main variables affecting the growth and
bacteriocin production in these cultures were developed
and validated. Finally, in view of the possible applications
of nisin and pediocin as food preservatives, the effects
of pH and temperature on the stability of both bacteriocins,
and their sensitivities to the action of proteolytic enzymes
were also studied.
2: Suitability of the mussel processing wastes (MPW)
and whey to support the growth of Lactococcus
lactis and Pediococcus acidilactici.
2.1: Preparation of the food wastes to be used as
culture media.
The wheys used in this study were obtained from
a local dairy plant in two forms: as concentrated whey
Nisin and pediocin production on wastes from food industry
27

(CW: the liquid remaining after the first cheese pressing)
and as diluted whey (DW: CW mixed with wash
waters). Two media from mussel processing wastes
(MPW) were also used: a diluted mussel processing
waste (DMPW) medium and a concentrated mussel
processing waste (CMPW) medium, which were obtained
in different dates from a local mussel processing plant.
Although both the raw MPW and whey can support
the microbial growth, usually both wastes are partially
deproteinized before using them as culture media. This
treatment is carried out to remove the protein fraction of
low assimilation that can precipitate with the decrease in
pH, thus interfering the determinations of biomass in the
cultures.
For these reasons, both wastes were previously
acidified to pH 4.5, heated at 121ºC for 15 min to denature
the proteins and the precipitates were removed by
centrifugation (12,000 x g for 15 min).
Since both the L. lactis and Ped. acidilactici strains
have not amylolytic capacity, the glycogen contained in
the MPW media was completely hydrolysed to its basic
glucose monomer unit, with a commercial preparation of
amylases. Then, the supernatants were adjusted at pH 6.3,
sterilised at 121ºC for 15 min and used as culture media. In
all cases, the pre-treatment of the wastes only affected their
protein fraction. The resulting compositions of the treated
DMPW, CMPW, DW and CW are shown in Table 1.
2.2: Batch cultures of Lactococcus lactis and Pediococcus
acidilactici on MRS broth and on media from whey
and DMPW.
To evaluate the aptitude of the DW, CW and
DMPW media for the production of probiotic biomass
and bacteriocins by L. lactis and Ped. acidilactici, a serie
of batch cultures on these media were carried out. For
comparative purposes, 18 h batch cultures of both
strains on MRS broth were also performed in the same
conditions as the cultures on DMPW and whey.
The time courses of these batch cultures are shown in
Figure 1. As it can be seen, both L. lactis and Ped.
acidilactici produced higher amounts of biomass in
DMPW than in CW and DW medium, thus showing
that glucose is a better carbon source than lactose for
both strains [15].
Although biomass production by L. lactis on
DMPW medium was slightly higher than that produced
on DW medium, the amount of nisin produced on this
last medium was remarkably higher than that obtained
in the first one (Figure 1, Table 2). Although this fact
could be related to the exhaustion of some vitamin or
cation essential for nisin synthesis on the DMPW medium,
it can also be postulated that their proteins could have a
poor concentration of some essential amino acids needed
for nisin synthesis. Additionally, the low buffer capacity of
the protein present in the DMPW medium in comparison
with the MRS, DW and CW media could have an indirect
effect on nisin production. Thus, the lowest final pH values
reached in the DMPW medium could be inadequate for
nisin synthesis. Contrarily, the biomass and pediocin
productions by Ped. acidilactici in DMPW medium
were approximately 6-fold higher than in DW medium.
Although this suggests that glucose and/or the protein
present in the DMPW medium are more suitable sources
of carbon and nitrogen for Ped. acidilactici, the increase in
pediocin production in this medium could be related to
a specific effect of pH. Thus, the highest pH drop (rpH:
difference between the initial and final pH values)
seemed to stimulate the synthesis of pediocin, until a final
pH inappropriate for survivability and cell growth of
Ped. acidilactici [33] was reached. In effect, from the
comparison between the values of rpH, final pH and
pediocin concentration obtained in the cultures of Ped.
acidilactici in the four culture media assayed, it can be
observed that the highest pediocin production was
obtained for the highest values of biomass and rpH
(right part of Figure 1).
For example, although biomass production by Ped.

Table 1. Mean composition (g/L) of the culture media

obtained from both the mussel processing waste
(MPW), and whey. DMPW: diluted mussel processing
waste, CMPW: concentrated mussel processing wastes,
CW: concentrated whey, DW: diluted whey.
DMPW CMPW CW DW
Total sugars 5.33 101.33 48.11 22.54
Reducing sugars 5.33 101.33 32.13 16.86
Proteins 1.82 3.47 5.02 2.04
Total nitrogen 0.65 0.54 1.05 0.45
Total 0.14 0.06 0.43 0.25
phosphorous
28
Lorenzo Miguel Pastrana Castro et al.

Figure 1. Growth, pH and bacteriocin production profiles by Lactococcus lactis

subsp. lactis CECT 539 and Pediococcus acidilactici NRRL B-5627 on MRS
broth (●), DW (Ο), CW (∆) and DMPW (◊) media. X: biomass concentration; BT:
bacteriocin concentration. Means of three analytical replications.

Table 2. Growth and fermentation parameters obtained from cultures of L.

lactis and Ped. acidilactici on wheys (CW and DW), DMPW medium and
MRS broth at the maximum production time. YBU/X: bacteriocin yield
coefficient expressed as the amount of bacteriocin produced (BU:
bacteriocin units) per mg of biomass produced. YBU/TSc: bacteriocin yield
coefficient expressed as the amount of bacteriocin produced per mg of total
sugars consumed. rpH: pH drop.
L. lactis Ped. acidilactici
Parameters CW DW DMPW MRS CW DW DMPW MRS
Biomass (g/L) 0.32 0.41 0.49 1.56 0.09 0.18 0.79 1.76
Bacteriocin 8.3 22.9 9.5 50.0 51.6 57.9 368.4 493.2
(BU/mL)
rpH 1.11 1.59 2.54 1.51 1.01 1.57 2.77 1.68
YBU/X (BU/mg) 25.9 55.8 19.5 32.0 573.3 321.7 466.3 280.1
YBU/TSc 1.0 3.0 6.1 6.2 16.7 28.7 179.7 75.1
(BU/mg)
Nisin and pediocin production on wastes from food industry
acidilactici was two-fold higher in MRS than in DMPW
medium, the pediocin levels were only 1.33 times lower
in the DMPW medium, probably due to the fact that the
rpH value was 1.65 times lower in the complex medium.
With regard to the cultures performed on CW and
DW, it can be noted that L. lactis produced higher amounts
of biomass than Ped. acidilactici on both media, probably
due to the Lactococcus species have a higher ability to
ferment the lactose than Pediococcus species [34].
On the other hand, from the detailed observation
of the growth kinetics of both strains on CW and DW
media (Figure 1), it can be noted that the highest biomass
and bacteriocins (nisin and pediocin) productions were
obtained on DW medium. Although this fact suggests a
possible effect of substrate inhibition, it could be also
related to the control that the supplied sugar substrate
exerts on the bacteriocin biosynthesis [35].
From the comparison between the growth kinetics
of L. lactis and Ped. acidilactici on MRS, DW, CW and
DMPW media, it can be observed that both strains
produced the highest biomass and bacteriocin levels on
MRS medium. In this way, it was observed that the
highest differences were for Ped. acidilactici, because
the productions of pediocin on DW and CW were
respectively, 8.6 and 9.7 times lower than those levels
reached on MRS medium (Table 2). Only the bacteriocin
yield coefficients expressed as the amount of bacteriocin
produced (BU: bacteriocin units) per gram of biomass
produced (YBU/X) or per gram of total sugars consumed
(YBU/TSc) obtained in the CW, DW and DMPW cultures
were comparable or higher than those obtained in MRS
broth (Table 2).
The lower bacteriocin productions observed for
both strains on the media obtained from wastes in
comparison to those obtained in the complex media
MRS could be due to the following reasons:
1. These media have unbalanced C/N or C/P
relationships in comparison with the MRS broth.
2. Their proteins have an unsuitable composition to
be used as nitrogen source for L. lactis and Ped.
acidilactici for bacteriocin production.
3. The DW, CW and DMPW media lack of some
essential nutrient for growth and bacteriocin
(nisin and pediocin) production.
2.3: Kinetic characterization of both nisin and pediocin
production on MRS broth and on media from whey
and DMPW.
Figure 2 shows the adjustments of the specific rates of
growth (µ) and bacteriocin (qBU) production (previously
calculated by numerical derivation from smoothed
29
experimental data with a generalized logistic equation
[36]), using the Luedeking and Piret model [37]:
r = α⋅r + β ⋅ X ; or dividing by X:
BT X
q = α ⋅µ + β (1)
BT
A straight line, typical of a primary metabolite
production, was obtained for nisin in MRS, DW and
CW media (left part of Figure 2). Nevertheless, a nonlinear
relationship between qBU and µ was observed in the
culture of L. lactis on DMPW medium (left lower part
of Figure 2) and in the cultures of Ped. acidilactici in
the four culture media (right part of Figure 2). Thus, in
these cases, it can be concluded that nisin and pediocin
were not produced as primary metabolites.
3: Optimization of the culture media obtained from
wastes for bacteriocin production.
Studies dealing with the effect of the type, concentration
and nature of the nutrients present in the culture media
on bacteriocins production have been reported in the
last years [19,33]. However, the effects of these variables
are traditionally studied by the one- at-a-time strategy,
i.e., varying one variable while keeping all others constant.
Although this strategy is simple and easy, without the
need of statistical analysis, it involves a relatively large
number of experiments and the interaction among
variables is often difficult to detect. Contrarily, statistically
based experimental designs are more efficient approaches
that can deal with a large number of variables
simultaneously. In addition, the interaction among
different variables can be established.
From the previous batch cultures, it was concluded
that the composition of the culture media has a strong
influence on biomass and bacteriocin production.
Additionally, the composition of the media itself
provokes different final pH values that also determine
the bacteriocin titres [15,24,32].
In an effort to optimise the composition of the
DMPW and DW media, the productions of nisin and
pediocin were studied from an empirical point of view,
as a function of four factors: total sugars, nitrogen,
phosphorous and buffer concentration. Those concentrations
corresponding to the DW without supplements were
taken as the inferior levels for the four independent (C,
N, P and B) variables. To reach the superior limit, the
media were supplemented to obtain the same C/N and
C/P relationships as the MRS medium, with the variable C
fixed in its maximum value (total sugars concentration
of CW and MRS media for DW and DMPW cultures,
respectively).
 Lorenzo Miguel Pastrana Castro et al.
30

Figure 2. Relationship between the specific rates of bacteriocin production (qBU

in BU mg-1h-1) and growth (µ in h-1) obtained from the cultures of L. lactis and
Ped. acidilactici on MRS, DW, CW and DMPW media.

The simplest amino acid glycine and KH2PO4, the
most appropriate phosphate source for nisin production
[25], were used as nitrogen and phosphorous sources,
respectively. Glucose and lactose were used as carbon
source to supplement the DMPW and the DW medium,
respectively. Since optimal pH for bacteriocin production
may also be affected by the composition of the culture
medium [38], possible interactions between pH and other
variables (C, N and P) can exist. Then, the media were
buffered with potassium hydrogen phthalate-NaOH in
order to generate different acidification rates during the
fermentation. The actual and coded factor levels for the
four variables in both media are shown in Table 3. Biomass
and bacteriocin concentration after 18 h of incubation
Nisin and pediocin production on wastes from food industry
31

Table 3. Coded and actual factor levels in the complete factorial design on
the effect of medium composition on the production of biomass and
bacteriocin by L. lactis and Ped. acidilactici on DW and DMPW media. B:
Buffer concentration (M), C: carbon source concentration (g/L), N: total
nitrogen concentration (g/L), P: total phosphorous concentration (g/L).
Natural values
DW medium DMPW medium
Codified B C N P B C N P
values
-1 0.0 26.0 0.43 0.27 0.0 5.0 0.65 0.14
0 0.05 37.0 4.01 0.91 0.05 12.5 1.89 0.39
1 0.1 48.0 7.55 1.56 0.1 20.0 3.14 0.64
Codification: Vc= (Vn - Vo)/ ∆Vn; decodification: Vn = Vo + (∆Vn * Vc).
Vc: coded value; Vn: natural value; Vo: natural value in the centre of the
experimental domain; ∆Vn: Increment of Vn corresponding to one unit of Vc.

were used as response variables. Additionally, the final
pH value reached in each culture was recorded.
3.1: Cultures of Lactococcus lactis on DW and
DMPW media.
The results of the above experimental design on
biomass (X), nisin production (BT) and final pHs of the
cultures of L. lactis on DW and DMPW media are
shown in Table 4. The corresponding response surfaces
are depicted in Figures 3 (in case of DW) and 4 (in case
of DMPW).
With respect to the cultivation on DW medium
(Figure 3), it can be observed that phosphorous and
variable C did not produce an appreciable effect on nisin
and biomass production. On the contrary, the increase
in the nitrogen concentration dramatically inhibited the
growth and nisin production. From the comparison between
biomass and nisin production, it can be observed that
the depression that glycine caused on nisin production
could be related with its inhibitor effect on biomass
production. In fact, the inhibitory action of glycine on
cell growth could be related to the alterations that this
amino acid produces in the muropeptide composition of
bacterial peptidoglycan, or to its action on the peptidoglycan
precursor synthesis [39-42].
Although the buffer also produced an inhibitory
effect on nisin production, its action on biomass production
was almost void, thus indicating a specific effect of the
variable B on nisin synthesis (Figure 2). This could be
related with the final pH reached in the cultures. In fact,
the final pH values in the buffered series (Table 4, for
B = 1) were always higher than those reached in the
unbuffered series (Table 4, for B = 0). These final pHs
reached in the buffered cultures could affect the
bacteriocin synthesis or the posttranslational processing
of prenisin to produce active nisin [24]. This finding is
consistent with the results of Cabo et al., [32], who
observed higher amounts of nisin in TGE broth than in
TGE buffer broth, although the cell growth was slightly
higher in the last medium.
With regard to the cultures performed on DMPW
medium, the results obtained (Table 4, Figure 4) showed
that also on this medium, glycine depressed biomass
production by L. lactis without affecting nisin production,
thus indicating that in this culture, nisin production did
not displayed primary metabolite kinetics.
In these cultures, the increase in the buffer
concentration stimulated nisin production in all the cases,
in contrast with the results observed in the above
cultures on DW medium. These results could be related
to the low final pH values reached in the unbufered
series in comparison with the buffered media.
In conclusion, it can be noted that the highest nisin
productions were obtained for a final pH value in the
range of 4.76-4.79 in DW (for B = -1; C= -1; N = -1;
P = -1 or P = 1) and DMPW (for B = 1; C= -1; N = -1;
P = -1) media (Table 4).
To clarify the specific effect of variable B on nisin
production, four series of cultures on DW and DMPW
media were performed using different initial concentrations
of buffer: 0, 0.03, 0.10 and 0.25 M. In these experiments,
the media were not supplemented with the carbon, nitrogen
and phosphate sources.
 Lorenzo Miguel Pastrana Castro et al.
32

Table 4. Values of bacteriocin (BT in BU/mL) and biomass (X in g/L)

concentrations and final pH in the experiments on the effect of the nutrient
sources on the growth and bacteriocin production by L. lactis on DW and
DMPW media.
Coded DW medium DMPW medium
variables
B C N P BT X Final pH BT X Final pH
1 1 1 1 5.97 0.12 5.87 10.87 0.15 4.81
1 1 1 -1 6.13 0.11 5.87 8.63 0.20 4.76
1 1 -1 1 9.09 0.41 5.51 21.29 0.38 4.68
1 1 -1 -1 7.61 0.35 5.34 17.07 0.49 4.67
1 -1 1 1 5.69 0.08 5.95 21.62 0.36 4.73
1 -1 1 -1 5.70 0.06 5.90 26.44 0.33 4.73
1 -1 -1 1 12.50 0.56 5.42 20.54 0.35 4.69
1 -1 -1 -1 11.49 0.53 5.53 30.53 0.47 4.76
-1 1 1 1 3.89 0.06 5.10 10.55 0.26 3.97
-1 1 1 -1 3.39 0.05 5.08 9.20 0.20 3.99
-1 1 -1 1 18.68 0.40 4.70 11.39 0.21 3.85
-1 1 -1 -1 13.83 0.30 4.86 6.42 0.22 3.84
-1 -1 1 1 8.37 0.15 5.37 10.99 0.24 4.03
-1 -1 1 -1 4.79 0.13 5.41 15.57 0.26 4.02
-1 -1 -1 1 21.35 0.50 4.79 7.21 0.23 3.85
-1 -1 -1 -1 21.27 0.47 4.76 9.52 0.35 3.85
0 0 0 0 8.62 0.25 5.38 15.21 0.34 4.43
0 0 0 0 8.51 0.24 5.38 13.15 0.31 4.53
0 0 0 0 7.36 0.25 5.41 13.99 0.34 4.52
0 0 0 0 8.15 0.27 5.39 12.99 0.32 4.54

The results obtained (Figure 5) showed that the
increase in buffer concentration only produced a light
effect on biomass production in both media, but resulted in
an increase in the final pH, which affected nisin synthesis
in a different way. In DW medium, the increase in the
buffer concentration provoked an exponential fall of the
nisin titres (lower parts of Figures 5A and C) in parallel
with the increase in the final pH values (upper parts of
Figures 5A and C). On the contrary, in DMPW medium
the highest nisin production (33 BU/mL) was obtained
in the culture medium buffered with 0.10 M potassium
hydrogen phthalate-NaOH (lower parts of Figure 5B
and C), in which a final pH value of 4.80 was reached.
This indicates the existence of an optimum final pH
value for nisin production on DMPW medium. Similar
observations on the existence of an optimum final pH
in bacteriocin production have been reported previously
for leuconocim Lm1 production by Leuconostoc
mesenteroides Lm1 [24].
3.2: Cultures of Pediococcus acidilactici on DW and
DMPW media.
The results obtained for biomass and pediocin
productions and the final pH values reached in the
cultures of Ped. acidilactici on DW are shown in Table
5 and the most representative response surfaces are
represented in Figure 6. In this case, the increase in
glycine and lactose concentrations have only light positive
and negative effects on pediocin production, probably
because neither this amino acid nor the lactose are good
nitrogen and carbon sources for this Pediococcus strain.
However, both the variable P and B strongly inhibited
the synthesis of pediocin. Since pediocin production on
DW did not displayed primary metabolite kinetics (Figure
2), the strong inhibitory effect of variable P on the
pediocin production could be attributed to a specific effect
related to the control that the exogenous phosphate
exerts on the regulatory mechanisms of some secondary
metabolites synthesis. This regulatory effect may be
Nisin and pediocin production on wastes from food industry
33

Figure 3. Response surfaces obtained for bacteriocin (BT) and biomass (X) production
by L. lactis on DW medium, according to the factorial plan defined in Table 3. The four
input variables (C, N, B and P) in coded values.
34 Lorenzo Miguel Pastrana Castro et al.

Figure 4. Response surfaces obtained for bacteriocin (BT) and biomass (X) production
by L. lactis on DMPW medium, according to the factorial plan defined in Table 3. The
four input variables (C, N, B and P) in coded values.
Nisin and pediocin production on wastes from food industry
35

Figure 5. Time course of biomass (X), nisin (BT) and pH by L. lactis on DW (A) and DMPW
(B) media. The cultures were performed on unbuffered media (●) and on media buffered at
concentrations of 0.03M (Ο), 0.10 M ( ) and 0.25 M (∇) with potassium hydrogen phthalate-
NaOH. C: Final pH, biomass concentration (X*) and bacteriocin titres (BT*) reached at the end
of each serie of the cultures performed on DW (open symbols) and DMPW (black symbols)
media. Means of three analytical replications.

explained by inhibition of the production or the action
of the appropriate bacteriocin synthetases, a limited
biosynthesis of necessary inducers of bacteriocin biosynthesis,
inhibition of bacteriocin precursor biosynthesis, etc. [43].
The increase in buffer concentration led to a decrease
in both the pH drop and consequently the final pHs in
the buffered cultures were higher than those reached in
the unbuffered cultures of Ped. acidilactici (Table 5).
This provoked a decrease in the final levels of pediocin.
This interpretation is consistent with the results of Yang
and Ray, [24] who observed that pediocin AcH was
produced in higher amounts in TGE broth than in TGE
buffer broth. The need of a final pH 3.6 to 3.7 for high
pediocin production has been reported [22]. This fact
was found to be due to the need of low pH for
posttranslational processing of prepediocin to produce
active pediocin [44].
In the cultivations on DMPW (Figure 7) there were
only little changes in pediocin production with increasing
nitrogen, phosphate and glucose concentrations. However,
the increase in the buffer concentration determined an
inhibition of pediocin production in all cases, as it was
observed in the cultures of Ped. acidilactici on DW
(Figure 6). On the other hand, the supplements (K2HPO4,
glycine and glucose) and buffer had a little influence on
the growth of Ped. acidilactici during the fermentation
on DMPW (right part of Figure 7). For this reason, it can
be concluded that the action of the variables studied on
pediocin production is not determined by their influence on
the growth of Ped. acidilactici.
4: Effect of the type and concentration of the nitrogen
source.
As it was showed in the previous experiments,
both the DW and DMPW media supported the growth
and bacteriocin production by L. lactis and Ped.
acidilactici strains. However, supplementing both media
with glucose (in case of DMPW) or lactose (in case of DW),
 Lorenzo Miguel Pastrana Castro et al.
36

Table 5. Values of bacteriocin (BT in BU/mL) and biomass (X in g/L)

concentrations and final pH in the experiments on the effect of the nutrient
sources on the growth and pediocin production by Ped. acidilactici on DW and
DMPW media.
Coded DW medium DMPW medium
variables
B C N P BT X Final pH BT X Final pH
1 1 1 1 10.55 0.08 5.83 66.43 0.08 5.83
1 1 1 -1 15.99 0.09 6.09 95.17 0.09 6.09
1 1 -1 1 26.50 0.14 5.64 85.68 0.14 5.64
1 1 -1 -1 22.76 0.13 5.62 92.35 0.13 5.62
1 -1 1 1 24.42 0.08 5.84 90.81 0.08 5.84
1 -1 1 -1 25.13 0.09 5.82 108.57 0.09 5.82
1 -1 -1 1 26.64 0.14 5.64 68.60 0.14 5.64
1 -1 -1 -1 27.64 0.13 5.59 111.07 0.13 5.59
-1 1 1 1 34.38 0.11 5.09 176.30 0.11 5.09
-1 1 1 -1 53.84 0.20 4.84 250.72 0.20 4.84
-1 1 -1 1 24.89 0.19 5.01 231.60 0.19 5.01
-1 1 -1 -1 37.21 0.18 4.82 242.06 0.18 4.82
-1 -1 1 1 30.47 0.16 4.92 192.98 0.16 4.92
-1 -1 1 -1 38.72 0.19 4.90 208.02 0.19 4.90
-1 -1 -1 1 29.38 0.17 4.91 275.93 0.17 4.91
-1 -1 -1 -1 57.38 0.17 4.93 363.97 0.17 4.93
0 0 0 0 31.56 0.14 5.37 168.49 0.14 5.37
0 0 0 0 28.12 0.14 5.34 165.09 0.14 5.34
0 0 0 0 27.21 0.14 5.30 174.35 0.14 5.30
0 0 0 0 28.56 0.14 5.37 175.12 0.14 5.37

Figure 6. Response surfaces

obtained for bacteriocin (BT)
production by Ped. acidilactici
on DW medium, according to
the factorial plan defined in
Table 3. The four input variables
(C, N, B and P) in coded values.
Nisin and pediocin production on wastes from food industry
37

Figure 7. Response surfaces obtained for biomass (X) bacteriocin (BT) production by
Ped. acidilactici on DMPW medium, according to the factorial plan defined in Table 3.
The four input variables (C, N, B and P) in coded values.
38 Lorenzo Miguel Pastrana Castro et al.

KH2PO4 and glycine, did not produce quantitatively
relevant effects in both biomass and bacteriocin production.
This showed that these supplements were not good sources
of nutrients for both bacteriocin production by both LAB.
This fact revealed that the productions of nisin and
pediocin have a more complex dependency on the medium
composition than the simple search of the optimum C/N
and C/P relationships.
Consequently, the use of other sources of nutrients
with a different chemical nature than those used in the
previous experiments could be a possible resource to
increase bacteriocin production on DW and DMPW. In
this way, from the above discussion, only it is reasonable to
test other nitrogen sources, which seem to have not only a
nutritional role, but also a precursor role in bacteriocin
synthesis [45].
Thus, a new series of cultivations were performed, in
which the effect of complex (yeast extract and Casitone)
and other simple nitrogen sources (glycine, ammonium
chloride and glutamic acid) on bacteriocin production
was tested. A second order rotatable factorial design [46]
based on two levels and two variables (Table 6) was
used to study the joint effect of the concentrations of the
carbon (lactose in case of DW and glucose in case of
DMPW) and nitrogen sources on the bacteriocin and
biomass productions by L. lactis and Ped. acidilactici.
In the cultures of L. lactis in DW buffered with
0.10 M potassium hydrogen phthalate-NaOH, the nitrogen
sources affected both bacteriocin and biomass production
in two different ways (Figure 8). Thus, the use of NH4Cl
and glycine inhibited biomass and nisin production, thus
showing that these two nitrogen sources were not good
nitrogen sources. On the contrary, notable high increases in
biomass and nisin productions were achieved in media
containing yeast extract or Casitone. A similar effect of
these four nitrogen sources on biomass and nisin productions
was observed in the cultures of L. lactis on DMPW
(Figure 9). Supplementing this last medium with glutamic
acid also resulted in an increase in biomass production.
However nisin production increased only slightly (Figure
9), thus indicating the role of this amino acid as a
growth stimulant of L. lactis, but not as a precursor
during nisin biosynthesis [45].
Similar effects of the nitrogen sources on biomass
and bacteriocin production were observed in the cultures of
Ped. acidilactici on DW (Figure 10) and DMPW
(Figure 11) media. However, the positive effect that the
supplements with glutamic acid produced on the growth
of Ped. acidilactici (Figure 11) was lower than that
produced by this amino acid on biomass production by
L. lactis (Figure 9).
For both strains, the high concentrations of the
carbon source in the medium counteracted the positive
effect of the complex nitrogen sources on bacteriocin
production, indicating a possible substrate inhibition as
suggested by De Vuyst and Vandamme, [47].
The poor influence of the NH4Cl would be probably
due to that lactococci and pediococci are not capable to
assimilate ammonium ions [48].
Since the magnitudes of the pH drops did not explain

Table 6. Experimental domain and codification of the variables in the

experiment on the study of the influence of the carbon (C) and
nitrogen (N) sources on the growth and bacteriocin production by
L. lactis and Ped acidilactici in DW and DMPW media.
Natural values
DW medium DMPW medium
Coded N (g/L) C (g/L) N (g/L) C (g/L)
values
-1.267 0.4 25.6 0.6 5.0
-1 1.3 28.2 0.9 6.8
0 4.4 38.1 2.0 13.4
1 7.5 48.0 3.0 20.0
1.267 8.4 50.6 3.38 21.8
Codification: Vc = (Vn-Vo)/∆Vn
Decodification: Vn+(∆Vn·Vc)
Vc: Coded value
Vn: Natural value
Vo: Natural value in the centre of the experimental domain
∆Vn: Increment of Vn corresponding to one unit of Vc
Nisin and pediocin production on wastes from food industry
Figure 8. Response surfaces corresponding to the production
of nisin (BT) and biomass (X) by L. lactis on DW
medium, as a function of the initial levels of the carbon
(lactose) and four nitrogen sources. The response surfaces
were obtained according to the experimental plan defined
in Table 6.
the high increases in nisin and pediocin productions in
the cultures supplemented with yeast extract or Casitone,
the positive effects of these nitrogen sources could be
39
Figure 9. Response surfaces corresponding to the
production of nisin (BT) and biomass (X) by L. lactis
on DMPW medium buffered with 0.10 M potassium
hydrogen phthalate-NaOH, as a function of the initial
levels of the carbon (glucose) and five nitrogen
sources. The response surfaces were obtained
according to the experimental plan defined in Table 6.

40
Figure 10. Response surfaces corresponding to the
production of pediocin (BT) and biomass (X) by Ped.
acidilactici on DW medium, as a function of the initial
levels of the carbon (lactose) and four nitrogen sources.
The response surfaces were obtained according to the
experimental plan defined in Table 6.
Lorenzo Miguel Pastrana Castro et al.
Figure 11. Response surfaces corresponding to the
production of pediocin (BT) and biomass (X) by Ped.
acidilactici on DMPW medium, as a function of the
initial levels of the carbon (glucose) and five nitrogen
sources. The response surfaces were obtained according
to the experimental plan defined in Table 6.
Nisin and pediocin production on wastes from food industry
related with their high contents in minerals, vitamins
and amino acids such as serine, cysteine and threonine.
These amino acids have a precursor role during nisin
biosynthesis without affecting the final cell yield of L.
lactis subsp. lactis NIZO 22186 [45].
In conclusion, supplementing the DW and DMPW
media with yeast extract or Casitone led to an increase
in nisin production when compared with the MRS medium
(Table 7). However, increased pediocin levels were only
obtained in the DMPW medium supplemented with the
complex nitrogen sources (Table 7).
5: Fed batch and re-alkalized cultures of L. lactis
and Ped. acidilactici on DMPW and DW.
As it was demonstrated in the previous experiments,
the option of using complex nitrogen sources was an
operative solution to the problem related with the massive
production of probiotic biomass and bacteriocins using
effluents from the food industry as culture media.
However, taking into account the cheap nature of the
DW and DMPW media, the use of these supplements
could represent an additional cost for bacteriocin
production, thus resulting uneconomical for a large-
scale process. For this reason, it is reasonable to test
other alternatives for enhancing the growth and
bacteriocins production in the media obtained from
wastes. Fed-batch cultivation technology gives the
possibility of obtaining higher amounts of biomass and
bacteriocins than batch cultures through the controlled
supply of fresh substrate [30,31,49].
In effect, this cultivation technique has been
successfully applied to the production of primary and
Table 7. Maximum biomass and bacteriocin productions by L. lactis
and Ped. acidilactici on different culture media. In the cultures
supplemented with yeast extract (YE) or Casitone, the maximum
biomass and bacteriocin values obtained in the above cultures
(Figures 9-12) are reported.
L. lactis
Culture medium BT X (g/L)
(BU/mL)
DW + YE 74.2 0.89
DW + Casitone 55.6 0.73
DW 22.9 0.41
DMPW + YE 100.3 1.01
DMPW + 95.8 0.91
Casitone
DMPW 9.5 0.49
MRS 50.0 1.56
41
secondary metabolites, such as citric acid [50] and
bacteriocins such as propionicin (Paik & Glatz, 1997),
amylovorin [31], gallidermin and epidermin [51] or
nisin [32,52]. This last author used a modified fed-
batch technique, in which the culture medium was re-
alkalized repeatedly up to the initial pH with 4N NaOH
in each nutrient feeding cycle. Using this procedure, the
active period and the productions of biomass and nisin
on TGE were higher than those observed in batch
production of nisin [32].
Then, this fed-batch fermentation technique was
used to improve nisin and pediocin production on both
DW and DMPW media. The cultures were carried out
at a controlled temperature of 30ºC and 200 rpm, in a 6
L bench top fermentor (New Brunswick Scientific, New
Jersey), which was filled with 4 L working volume of
medium. An aeration flow rate of 0.5 L/h and an agitation
speed of 200 rpm were maintained throughout the
fermentation. Foam was controlled by the addition of a
2% anti-foam A solution.
The cultivations were initiated as batch cultures
during the first 8 h (in the case of DMPW cultures) or 12 h
(in the case of DW cultures), which were the necessary
times to reach the stabilization of pH, biomass and
bacteriocins production in batch cultures. At this time, a
culture sample (100 mL) was withdrawn aseptically from
the growing culture for antibacterial activity and analytical
determinations. The total sugars determination was used to
calculate the feeding substrates volumes needed to restore
the initial total sugars concentration in the fermentation
medium. Immediately after each re-alkalization of the
fermentation media, the adequate volume of the feeding
Ped. acidilactici
BT X (g/L)
(BU/mL)
195.3 1.34
185.3 1.20
57.9 0.18
939.3 1.38
933.69 1.39
368.4 0.79
493.2 1.76
42 Lorenzo Miguel Pastrana Castro et al.

substrate was added. These periods of sampling, feeding

and re-alkalization strategies were then maintained for
as long as the producing strain was able to bring about
the decrease of pH. The volumes of the culture media
were kept constant by feeding the fermentor with 100
mL fresh substrates containing the amounts of sugars
consumed in each cycle. The result was a quasi-continue
fermentation process, so that for the volume:

dV
=0 (2)
dt

and for any other state variable:

dY
≠0 (3)
dt

5.1: Fed-batch and re-alkalized cultures of

Lactococcus lactis on DMPW and whey media.
The results corresponding to a re-alkalized cultivation
fed with a 240 g/L concentrated glucose (fermentation
I) showed that biomass and pediocin production and
nutrients consumption described wavy profiles (Figure
12). So that, there were three different fermentation
phases in the culture, whose succession was determined
by the final pH value reached at the end of each feeding
and re-alkalization cycle:

I: Phase of homolactic fermentation.

In this phase (first 48 h of incubation) the final pH
values in each feeding and re-alkalization cycle were
lower than the optimal value of 4.80 observed for nisin
production in the buffered DMPW cultures (Figure 5).
This affected negatively the biomass and nisin production
rates, which progressively decreased after 24 h of
incubation. On the other hand, as a result of the catabolic
processes, only the production of lactic acid was observed,
which increased at a constant rate of 0.21 g·L-1·h-1.
Although the glucose was consumed at a constant
rate of 0.23 g·L-1·h-1, the nitrogen and phosphorous
consumption rates in each re-alkalization cycle
paralleled with the growth rate, showing an initial step
of rapid consumption (the first 16 h of incubation)
followed by a second step of slow consumption (16-48
h of fermentation).
II: Phase of transition.
During this period (24-112 h) the final pH at the
end of each re-alkalization cycle reached values between
4.50 and 5.00, which are suitable for nisin production
Figure 12. Time course of re-alkalized cultures of L.
lactis on DMPW with feeding with a 240 g/L concentrated
glucose. X: biomass concentration; B: butane-2,3-diol;
BT: nisin titres; LA: lactic acid; AA: acetic acid; Pr, TS,
TN, TP: proteins, total sugars, phosphorous and nitrogen
concentrations I: phase of homolactic fermentation; II:
phase of transition; III: phase of mixed acid fermentation.
(Figure 5). In effect, in this phase, biomass and nisin
increased describing logistic profiles until reaching
respectively, the 56.1% and the 51.5% of the total biomass
and nisin produced during the fermentation.
This intensification in the metabolic activity of L.
lactis was accompanied by strong consumptions of the
sources of nitrogen and phosphorous. Although the glucose
consumption rate (0.15 g·L-1·h-1) was lower than that of
the previous phase, lactic acid was produced at a constant
rate of 0.21 g·L-1·h-1 until the 80 h of incubation.
Thus, although many LAB are able to degrade lactic
acid [30,53], the decrease in glucose consumption rate
Nisin and pediocin production on wastes from food industry
43

could not be related to lactic acid degradation by L.

lactis, because this implies a reduction in lactic acid
concentration. An alternative interpretation would be
that during the first 48 h of fermentation, glucose was
overconsumed to compensate the stress generated as a
consequence of both the improper final pH values for
growth and the rapid increase of pH at the beginning of
each re-alkalization period.
On the other hand, since the glucose concentration
was maintained in a level of about 5 g/L during all the
incubation, it is reasonable to suppose that a limitation
of the carbon flow was produced in the second half of
the transition phase. Thus, the carbon source could be
preferably consumed to support the intensive growth
observed between the 80 and 112 h of cultivation,
leading to a decrease in the lactic acid production rate
from the 80 h of incubation (Figure 12). This would
explain the evolution of the culture towards a behaviour
of mixed acid fermentation.

III: Phase of mixed acid fermentation.

The most noteworthy characteristics of this period
(112 h to the end of the cultivation) were the shift of
metabolism towards a mixed acid fermentation (with
the production of butane-2,3-diol and acetic acid), and
the depletion of the nitrogen and phosphorous sources.
As a consequence of this nutrient limitation, both the
growth and bacteriocin production rates decreased. In
addition, after 128 h of cultivation, the producer strain
completely lost the ability to recover the acidic pH at
the end of each re-alkalization cycle.
This shift from homolactic acid to mixed acid
fermentation has been observed before in cultures under
glucose and nitrogen limitation [54-56] and under carbon
excess conditions with lactose [57], galactose [57,58]
and maltose [59,60].
In this first cultivation, the growth stopped probably
due to the depletion of nitrogen and/or phosphorous
sources at the end of the fermentation (Figure 12) and/
or the accumulation of the mixed acid metabolites in
the medium. To test the first possibility, another fed-
batch experiment (fermentation II) was carried out using a
concentrated MPW (CMPW) medium (Table 1) as a
feeding substrate to replenish the glucose consumed and
other nutrients (nitrogen and phosphorous) in each feeding
cycle.
In this second fed-batch and re-alkalized fermentation
(Figure 13), the three fermentation phases referred in the
previous cultivation (fermentation I) were also observed.
Although, the profiles described by all the variables were
similar to those observed in the first fed-batch fermentation,
Figure 13. Time course of re-alkalized cultures of L.
lactis on DMPW with feeding with CMPW (101.33 g of
glucose/L). Et: ethanol. Other notations are as in Figure
12. I: phase of homolactic fermentation; II: phase of
transition; III: phase of mixed acid fermentation.
the productions of biomass, nisin and mixed acid
metabolites were practically linear from the 120 h of
fermentation. In addition, the bacteriocin-producing strain
was able to bring about the decrease of pH. Thus, the
final pH in each re-alkalization cycle reached values near to
the optimum final pH value for nisin production (4.80).
The consumption of phosphorous was linear once
the mixed acid fermentation phase had begun, but the
nitrogen concentration in the medium increased gradually
from 168 h to the end of the cultivation. This may be due to
an accumulation of a fraction of proteins of difficult
assimilation for L. lactis. This nitrogen accumulation
was also observed when this medium was used for
production of gibberellic acid by the fungus Gibberella
fujikuroi [61]. The glucose consumption increased from
the 104 h of cultivation, associated with the period of
greater metabolic activity (in terms of biomass and mixed
acid metabolites production).

44
Comparing the metabolites production in both
experiments, it can be observed that in the second
fermentation L. lactis produced ethanol instead butane-
2,3-diol, probably due to the production of acetic acid
and ethanol would constitute a most efficient pathway
of regeneration of NAD+ [55,56].
The fed-batch fermentation on whey (fermentation
III) was carried out using a mixture of a concentrated
whey (Table 1) and a concentrated lactose (400 g/L) to
feed the fermentor. The results of this experiment are
depicted in Figure 14. In this cultivation, the homolactic
phase (first 84 h of incubation) and the mixed acid
fermentation phase (84 h to the end of the cultivation)
were also observed.
Finally, since the three fed-batch fermentations
were carried out under aerobic conditions, formate was
not detected in the medium, indicating that pyruvate
Figure 14. Time course of re-alkalized cultures of L.
lactis on DW with feeding with concentrated whey
(48.11 g of total sugars/L) and a 400 g/L concentrated
lactose. Notations are as in Figure 12. I: phase of homolactic
fermentation; II: phase of mixed acid fermentation.
Lorenzo Miguel Pastrana Castro et al.
dehydrogenase (PDH) was the only alternative pathway
of pyruvate metabolism.
5.2: Fed-batch and re-alkalized cultures of
Pediococcus acidilactici on DMPW and DW media.
In the first fed-batch and re-alkalized culture of
Ped. acidilactici (Figure 15) fed with a concentrated
glucose (240 g/L), the behaviour of the fermentation
variables was similar to that observed in the cultures of
L. lactis. In general, the following phenomena were
observed in this culture:
1. The feeding and re-alkalization cycles led to an
increase in biomass and bacteriocin production by
Ped. acidilactici.
Figure 15. Time course of re-alkalized cultures of Ped.
acidilactici on DMPW with feeding with a 240 g/L
concentrated glucose. BT: pediocin titres. Other notations
are as in Figure 12. I: phase of homolactic fermentation; II:
phase of mixed acid fermentation.
Nisin and pediocin production on wastes from food industry
2. Both biomass and pediocin productions described
wavy profiles.
3. The bacteriocin producing strain grew displaying
two metabolic phases: a first homolactic phase
followed by a mixed acid fermentation phase.
4. The pH recovery capacity collapsed when the
growth stopped due to the exhaustion of the
nitrogen and phosphorous sources and/or the
accumulation of mixed acid metabolites.
However, there were differences with regard to
the similar cultivation of L. lactis (Figure 12). Firstly, the
sources of N and P were not exhausted simultaneously
and secondly, the synthesis of the mixed acid metabolites
was not simultaneous.
The second fed-batch culture on DMPW with
feeding with a concentrated MPW medium (Figure 16)
had the purpose of preventing the indicated nitrogen
and phosphorous exhaustion previously observed. With
respect to the preceding cultivation of Ped. acidilactici,
Figure 16. Time course of re-alkalized cultures of Ped.
acidilactici on DMPW with feeding with CMPW
(101.33 g of glucose/L). BT: pediocin titres. Other
notations are as in Figure 12.
45
the glucose consumption was lower, the phosphorous
consumption was similar, and the consumptions of
nitrogen and proteins increased. These new
consumption profiles were synchronous between them
and with those observed for biomass and pediocin
productions, which described the characteristic wavy
profiles of the previous situations.
In this second fermentation on DMPW medium,
the pediocin producing strain developed a metabolism
typically homolactic, probably as a consequence of the
joint supplement of glucose, nitrogen and phosphorous
throughout the feeding with CMPW medium.
The third fed-batch and re-alkalized culture of
Ped. acidilactici, was carried out on whey supplemented
with a 2% yeast extract (DWYE medium) using a mixture
of a concentrated glucose (400 g/L) and concentrated
Figure 17. Time course of re-alkalized cultures of Ped.
acidilactici on DWYE medium with feeding with CWYE
and a 400 g/L concentrated glucose. Notations are as in
Figures 12 and 13.

46
whey supplemented with a 2% yeast (CWYE medium)
as feeding substrates.
The results obtained (Figure 17) showed that, in
spite of the rising evolution of the final pH values, the
supplements with yeast extract and the feeding with
glucose produced an increase in both the active period
and bacteriocin levels, as compared to the previous batch
culture on DW medium (Figure 1).
In general, the following phenomena were observed
during the time course of the fed-batch and re-alkalized
cultivation of Ped. acidilactici on DWYE medium:
1. The pediocin-producing strain only developed a
mixed acid fermentation pattern, in contrast with
the two metabolic phases (homolactic and mixed
acid fermentations) observed in the previous fed-
batch and re-alkalized cultivations of L. lactis
(Figures 12, 13 and 14) and Ped. acidilactici
(Figure 15).
2. The concentrations of biomass, pediocin and
nutrients described wavy profiles, which had been
observed in the three fed-batch and re-alkalized
cultivations of Lactococcus lactis (Figures 12, 13
and 14) and in the two fed-batch and re-alkalized
cultivations of Pediococcus acidilactici NRRL B-
5627 in DMPW medium (Figure 15 and 16).
3. The feeding with glucose led to a dramatic decrease
in lactose consumption. This contributed to an
accumulation of lactose in the culture medium
(Figure 17).
4. Although at the end of the fermentation, the
phosphorous and the nitrogen sources were not
completely exhausted, the growth stopped and the
capacity to recover the acidic pH collapsed.
Table 8. Maximum biomass (X*) and nisin (BT*) productions in batch and fed-batch and
re-alkalized cultures of L. lactis.
DMPW (I): first fed-batch culture on DMPW with feeding with a 240 g/L concentrated
glucose.
DMPW (II): second fed-batch culture on DMPW with feeding with CMPW medium.
DW (III): fed-batch culture on DW medium with feeding with a mixture of CW medium and
a 400 g/L concentrated lactose.
Batch cultures
Parameter CW DW DMPW MRS
X* (g/L) 0.32 0.41 0.49
BT* (BU/mL) 8.3 22.9 9.5
Lorenzo Miguel Pastrana Castro et al.
In conclusion, the use of fed-batch techniques with
re-alkalization cycles enhanced both biomass and
bacteriocin production by L. lactis and Ped.
acidilactici in DMPW and DW media as compared to
batch fermentations in DW, CW, DMPW and MRS
broth (Tables 8 and 9). This suggests that the positive
effect that this cultivation technique produces on
nisin and pediocin production could be generalized to the
production of other bacteriocins in cheap culture media.
In addition, the biomass produced by L. lactis and
Ped. acidilactici in these cultures could be used as probiotic
additives in the formulation of feed for piglets and
chickens. These probiotic products could play an important
role in the therapy of different diseases, taking the advantage
of natural products which have no side effects.
6: Modelling the growth and bacteriocin production
by Lactococcus lactis and Pediococcus acidilactici on
DMPW and DW.
An important aspect to be considered for the
industrial production of bacteriocins is the cost-effective
production of these bioproducts. One alternative to reduce
this cost is the use of an adequate cultivation method that
enhances bacteriocin production, as it was demonstrated in
the previous fed batch and re-alkalized cultures of L.
lactis and Ped. acidilactici. However, the control-system
development for fed-batch fermentations is very difficult
due to the lack of accurate models describing cell growth
and product formation in these processes. This is one of
the reasons for which the control-system development
for fermentation is not straightforward. [62].
Fed-batch cultures
DMPW (I) DMPW (II) DW (III)
1.56 2.10 9.38 3.48
50.0 51.2 109.6 124.7
Nisin and pediocin production on wastes from food industry
47

Table 9. Maximum biomass (X*) and pediocin (BT*) productions in batch and fed-batch
and re-alkalized cultures of Ped. acidilactici.
DMPW (I): first fed-batch culture on DMPW with feeding with a 240 g/L concentrated
glucose.
DMPW (II): second fed-batch culture on DMPW with feeding with CMPW medium.
DWYE (III): fed-batch culture on DWYE medium with feeding with a mixture of CWYE
medium and a 400 g/L concentrated glucose.
Batch cultures Fed-batch cultures
Parameter CW DW DMPW MRS DMPW (I) DMPW (II) DWYE (III)

X* (g/L) 0.09 0.18 0.79 1.76 2.28 2.82 6.57

BT* (BU/mL) 51.6 57.9 368.4 493.2 1034.0 1395.1 517.6

The application of algorithms for controlling the
industrial fermentation processes could allow the design of
optimal policies to improve some bioproductions in a
cost-effective way. In this respect, the advances in
instrumentation and the use of on-line computers for
measuring key variables in fermentation technologies
could offer new opportunities for the application of
stoichiometric and kinetic models for monitoring and
controlling the processes [49].
Usually, logistic-type models and Luedeking and
Piret model [37] have been respectively used to model
bacterial growth and bacteriocin production during batch
fermentations [14,63]. However, these models have
found to be unsatisfactory to describe the biomass and
bacteriocin productions in fed-batch cultures [31] and
in fed-batch cultures with re-alkalization cycles [32].
This indicates that these models did not include all the
variables affecting both biomass and bacteriocin production.
For these reasons, some modifications for both the
logistic-type and Luedeking and Piret models have
been suggested recently [31,32].
6.1: Modelling the growth of Lactococcus lactis and
Pediococcus acidilactici.
As it was discussed above, the growth rate of L.
fermentation. In addition, nisin and pediocin
production seemed to be affected by the final pH
reached in each re-alkalization cycle. Therefore, in a first
step, a modified logistic equation was set up to describe
the effects of the main factors affecting the growth of
both LAB. In addition, a modification of the Luedeking
and Piret model including a term for the specific effect
of pH on nisin and pediocin production was developed.
Effect of pH
Although the optimal pH for growth and
product formation by lactic acid bacteria has found to
be around 6.0, a decline in the growth is produced as
pH decrease [38,64-67]. An inadequate pH can cause the
precipitation of necessary nutrients, ions, etc, the
denaturation of enzymes and structural proteins or
cause membrane disruption or inactivation of transport
or other protein functions [64,68-70].
Since the growth rate is a function of pH (Passos,
& al., 1994), the model proposed by Presser et al., [66]
was set up to describe the relationship between both
variables, so that:
⎛ 10 pH min ⎞
R = b ⋅ ⎜⎜ 1 − ⎟ ; being: b = r ⋅ 10 − pH min
X 0 ⎜ pH t ⎟⎟ 0 X

lactis and Ped. acidilactici growing in DMPW medium ⎝ 10 ⎠

seems to be a function of pH change, nutrient limitation (4)
(nitrogen and phosphorous) and product inhibition
(lactic acid, acetic acid, ethanol and butane-2,3-diol), where RX is the growth rate (g·L-1·h-1) affected by the
variables that change in concentration in the course of the pH, pHmin is the minimum pH value beyond which no
 Lorenzo Miguel Pastrana Castro et al.
48

growth is possible, pHt is the pH over time, rX is the The inhibitory effect of organic acids lies in the
growth rate (in g·L-1·h-1) in each re-alkalization cycle. reduction of pH, as well as in the presence of the
The numeric integration of equation (4) with respect undissociate form of the molecules, which acts by
to time provided the estimates values of biomass chelating elements essential for growth, by collapsing
concentration over time (XR): the electrochemical proton gradient. In addition, they
can produce alterations in the cell membrane permeability,
thus producing the disruption of the substrate transport
t =t t =t ⎛ pH min ⎞ system [67].
⎜ 10 ⎟
X = ∑ R = ∑ b ⋅ ⎜1 − ⎟⎟ (5) The ability of ethanol to produce perturbation in
R X 0 ⎜ pH t
t =0 t =0 ⎝ 10 ⎠ cell membranes or the alteration in fatty acid composition
contributes to an increase in the uptake of the undissociated
Effect of nutrient (nitrogen and phosphorous) form of both acetic and lactic acid [71,72]. On the other
limitation hand, although the antimicrobial effect of butane-2,3-
diol is unknown, it has been reported that this compound is
From the observation that biomass production by able to deactivate enzymes from several microorganisms
L. lactis and Ped. acidilactici and both nitrogen and [73].
phosphorous consumption are directly correlated Then, the negative influence of the amounts of lactic
(Figures 12 to 17), the simplest forms to describe these acid, acetic acid, ethanol and butane-2,3-diol produced
relationships are: in the cultures can be described as follows [69]:

γ[rNc ] = (1 + b1 ⋅ rNc ) (6)

⎛
γ[LA] = ⎜⎜ 1 −
[LA] ⎞⎟ (9)
⎟
⎝ [LAmax ] ⎠
γ[rPc ] = (1 + b2 ⋅ rPc ) (7)

⎛
γ[AA] = ⎜⎜ 1 −
[AA] ⎞⎟ (10)
where γ[rNc] and γ[rPc] are functions that account for ⎝ [AAmax ] ⎟⎠
the effect of the nitrogen and phosphorous consumption
rates on the growth of L. lactis and Ped. acidilactici, b1
and b2 are constants of proportionality, rNc and rPc are ⎛
γ[Et ] = ⎜⎜ 1 −
[Et ] ⎞⎟ (11)
the nitrogen and phosphorous comsumption rates
(g·L-1·h-1). ⎝ [Et max ] ⎟⎠
Combining the effects of pH and the rates of
nitrogen and phosphorous consumption, the overall form
of the model is:
⎛
γ[2,3B ] = ⎜⎜ 1 −
[2,3B] ⎞⎟ (12)
⎝ [2,3Bmax ] ⎟⎠
⎛ pH min ⎞
t =t t =t ⎜
X = ∑ R = ∑ b ⋅ ⎜1 −
R X 0 ⎜
10
pH t ⎟⎟( 1
)(
⎟ ⋅ 1 + b ⋅ rNc ⋅ 1 + b ⋅ rPc
2
)
t =0 t =0 ⎝ 10 ⎠ where: γ[LA], γ[AA], γ[Et] and γ[2,3B] are the inhibitory
(8) functions for the inhibition of the lactic acid, acetic
acid, ethanol and butane-2,3-diol, respectively. [LA],
Effect of product formation [AA], [Et] and [2,3B] are the total concentration (mM)
Bacterial growth inhibition by organic acids (lactic of lactic acid, acetic acid, ethanol and butane-2,3-diol.
and acetic acid) and other products (ethanol or butane- [LAmax], [AAmax], [Etmax] and [2,3Bmax] are respectively,
2,3-diol) is important in complex fermentation process the maximum concentrations (mM) of lactic acid, acetic
such as fed-batch cultivation [67]. Thus, the effects of acid, ethanol and butane-2,3-diol, which completely
these variables may be also considered in the growth prevent the growth.
model. Thus, including equations (9), (10), (11) and (12)
Nisin and pediocin production on wastes from food industry
49

into equation (8) gives:

⎛ 10 pHmin ⎞
R
t=t
X = ∑ b ⋅ ⎜1 −
0 ⎜ pHt ⎟ 1 2
( )( )
⎟ ⋅ 1 + b ⋅ rNc ⋅ 1 + b ⋅ rPc ⋅ γ LA ⋅ γ AA ⋅ γ 2,3B ⋅ γ Et[ ] [ ] [ ] [ ]
t =0 ⎝ 10 ⎠
(13)
Then, if µ was constant, equation (13) could be
expressed in the integral form:
K
X =
R ⎛ ⎛ ⎛ pHmin ⎞ ⎞ ⎞
⎜ c − µ ⋅ ⎜ b ⋅ ⎜ 1 − 10 ⎟ ⎛
⋅ ⎜ 1 + b ⋅ rNc ⎞⎟ ⋅ ⎛⎜ 1 + b ⋅ rPc ⎞⎟ ⋅ γ[LA] ⋅ γ[AA] ⋅ γ[2,3B ] ⋅ γ[Et ]⎟ ⋅ t ⎟
⎜ ⎜ 0 ⎜⎜ pHt ⎟⎟ ⎝ 1 ⎠ ⎝ 2 ⎠ ⎟ ⎟
1+ e ⎝ ⎝ ⎝ 10 ⎠ ⎠ ⎠
(14)

where K and X0 are respectively, the maximum and the From these observations, the effect of final pH on
initial biomass concentration (g/L), µ is the specific nisin synthesis may be modelled as follows:
growth rate (h-1), t is the time (h).

6.2: Modelling bacteriocin production by L. lactis

t =t
(
BTNis = ∑ α ⋅ R + β ⋅ X
X R
)⋅ ⎛⎜⎜ 1 − b ⋅ pH pH− pH
3
opt t
⎞
⎟
⎟
and Ped. acidilactici. t =0 ⎝ opt ⎠
The introduction of the values of RX and XR into (17)
the Luedeking and Piret expression [37] results in:
where BTNis is the nisin concentration (in BU/mL), b3 is
a constant of proportionality, pHopt is the optimum final
r = α⋅R + β⋅ X (15)
BT X R pH, from which bacteriocin production stopped and pHt
is the final pH value in each re-alkalization cycle. Other
where rBT is the bacteriocin production rate (in BU·mL- terms are as previously defined.
·h ), α is a growth-associated constant (BU/mg) and β
1 -1
As it was discussed above, in batch (Figures 1, 6
is the non-growth-associated constant (BU·mg-1·h-1). and 7) and fed-batch cultures of Ped. acidilactici
Other terms are as previously defined. (Figures 15, 16 and 17), pediocin production increased
Therefore, numeric integration of equation (15) with the increase in pH drop. Thus, pediocin synthesis
results in equation (16), which provides the bacteriocin
was modelled considering that the increase in pediocin
estimates (BT):
concentration is proportional to the increase in the pH
drop rate (∂pH), which is less than the maximum ∂pH
t =t
(
BT = ∑ α ⋅ R + β ⋅ X
X R
) (16) from which bacteriocin synthesis stopped. Then, the
pediocin estimates (BTPed) were obtained from the
t =0
following model:
Specific effect of the optimum final pH value on nisin
and pediocin production
t =t
(
BTPed = ∑ α ⋅ R + β ⋅ X
X R
)⋅ ⎛⎜⎜ 1 + e − b4 ⋅ ( ∂pH max − ∂pH) ⎞
⎟
⎟
In batch cultures of L. lactis on DMPW media t =0 ⎝ ⎠
buffered at initial pH 6.30 with different concentrations (18)
of the buffering agent (Figure 5), nisin synthesis was
maximal at a final pH of 4.80 (optimum value),
decreasing for higher and lower final pH values. pH i − pH min
being: ∂pH max = (19)
Contrarily, in batch cultures of this strain on DW ∆t
media, the higher nisin titres were obtained on the
unbuffered DW, which provided the lowest final pH (or where, b4 is a constant of proportionality, ∂pHmax is the
the highest pH drop). maximum decrease in pH drop rate, from which bacteriocin
 Lorenzo Miguel Pastrana Castro et al.
50

production stopped, pHi is the initial pH in each re-

alkalization cycle, pHmin is the theoretical minimum
value of pH below which bacteriocin production stopped,
∆t is the time between each re-alkalization (h). Other terms
are as previously defined.
Then, the effects of all factors affecting biomass
production by Lactococcus lactis and Pediococcus
acidilactici were tested by fitting model (14) to the
experimental cell growth data. From the biomass
formation estimates obtained with this model, nisin and
pediocin production were then modelled using models
(17) and (18), respectively.
For optimal estimation of the model parameters, a
non-linear regression technique (method of Newton by
using a minimal non-linear squares method) was used
to minimise the deviations between model predictions
and experimental fed-batch data.
In Figures 18 and 19 are represented the predictions
of the equations used for describing the bacterial growth
(left part) and bacteriocin production (right part) by L.
lactis and Ped. acidilactici. Models (14), (17) and (18)
were found to be suitable for the simulation of re-alkalized
cultures of the two LAB on both DMPW and DW media
(Tables 10 and 11). Although these models have little
biological meaning because their empirical nature, they
could be used for developing an appropriate control
strategy for optimizing fed-batch production of both
nisin and pediocin.
The inhibitory effects of lactic acid, acetic acid,
butane-2,3-diol and ethanol on the growth of L. lactis Figure 18. Experimental data (symbols) of biomass (left
and Ped. acidilactici seem to be very low, since the part) and nisin (right part) production by L. lactis obtained
inhibitory levels obtained from model (14) for both in the fed-batch and re-alkalized cultures on DMPW
strains were higher than those levels reached at the end medium with feeding with a 240 g/L concentrated glucose
of the three fed-batch cultivations (Tables 10 and 11, (A), with feeding with CMPW (B), and on DW medium
Figures 12-17). with feeding with CW and a 400 g/L concentrated lactose.
With regard to bacteriocin production, model (17) The curves drawn through the biomass (X) data were
showed that the optimal final pH for nisin synthesis was obtained according to model (14). The curves drawn
3.86 for the fermentations I and II on DMPW (Table through the nisin (BT) data were obtained according to
10). In whey culture, the optimum final pH for nisin model (17).
production obtained from model (17) was 4.18. These
values are in perfect agreement with the actual final pH of ∂pHmax calculated with model (18), the minimum pH
values (3.86, 3.88 and 4.18) obtained in the first re- (pHmin) from which pediocin synthesis stopped was
alkalization cycle of each fed-batch fermentations on determined as 4.03 in the two fermentations on DMPW.
DMPW (Figures 12 and 13) and DW (Figure 14), in However, for fed-batch and re-alkalized culture on whey,
which the highest nisin production rates were obtained. the pHmin value obtained from model (18) was calculated as
However, these values were different to those observed 4.84. These values are very similar to the actual final
in batch cultures of L. lactis on DMPW (pHopt = 4.80; pH values (4.00, 4.06 and 4.82) obtained in the first re-
Figure 5) and DW (pHopt = 4.60; Figure 5), thus alkalization cycle for the three fed-batch fermentations, in
showing that the optimum final pH for nisin production which the highest pediocin production rates were obtained
depends on the culture conditions. (Figures 15, 16 and 17). These results showed that pediocin
In the case of pediocin production, from the values production was stimulated by the highest final pHs in
Nisin and pediocin production on wastes from food industry
51

Figure 19. Experimental data (symbols) of

biomass (left part) and pediocin (right part)
production by Ped. acidilactici obtained in the
fed-batch and re-alkalized cultures on DMPW
medium with feeding with a 240 g/L
concentrated glucose (A), with feeding with
CMPW (B), and on DWYE medium with
feeding with CWYE and a 400 g/L
concentrated glucose. The curves drawn
through the biomass (X) data were obtained
according to model (14). The curves drawn
through the pediocin (BT) data were obtained
according to model (18).

Table 10. Parameters in the models for fed-batch production of biomass

(model 14) and nisin (model 17) by L. lactis on DMPW and DW media.
r2X and r2Nis are the correlation coefficients between expected and experimental
biomass and nisin values.
Parameter DMPW (I) DMPW (II) DW (III)
K (g/L) 2.64 8.36 5.57
-1
µ (h ) 0.014 0.009 0.014
pHmin 3.57 3.34 3.52
b1 902.28 8.99 4.75
b2 0.01 0.01 0.01
LAmax (mM) 207.26 636.53 169.56
AAmax (mM) 190.01 277.56 27.50
2,3Bmax (mM) 120.94 - 213.83
Etmax (mM) - 591.05 -
α (BU/mg) 21.28 20.35 39.64
β (BU·mg ·h )
-1 -1
0.28 0.12 0.11
b3 1.79 2.45 1.30
pHopt 3.86 3.86 4.18
2
rX 0.9935 0.9978 0.9892
2
r Nis 0.9946 0.9917 0.9980
 Lorenzo Miguel Pastrana Castro et al.
52

Table 11. Parameters in the models for fed-batch production of biomass

(model 14) and pediocin (model 18) by Ped.acidilactici on DMPW and DWYE
media.
r2X and r2Ped are the correlation coefficients between expected and experimental
biomass and pediocin values.
Parameter DMPW (I) DMPW (II) DWYE (III)
K (g/L) 1.94 2.94 7.02
-1
µ (h ) 0.058 0.031 0.023
pHmin 3.88 3.88 3.48
b1 0.36 2.67 625.67
b2 3.70 0.45 157.12
LAmax (mM) 277.93 519.85 40.26
AAmax (mM) - - 37.98
2,3Bmax (mM) 341.26 - -
Etmax (mM) 150.45 - 12.10
α (BU/mg) 2253.55 2620.54 637.84
β (BU·mg-1·h-1) 0 0 0.92
b4 11.54 3.33 44.56
∂pHmax 0.27 0.27 0.18
2
rX 0.9851 0.9861 0.9881
r2Ped 0.9836 0.9747 0.9808

Table 12. Experimental domain and codification of the variables in

the factorial design used to study the combined influence of
temperature and pH on the stabilities of nisin and pediocin.
Natural values
Codified values
T (º C) pH
- 1.267 63.0 3.7
-1 70.0 4.0
0 95.5 5.2
+1 121.0 6.5
+ 1.267 128.0 6.8

each culture, as it was observed in all the previous
batch cultures of Ped. acidilactici. These observations
are in perfect agreement with those reported by other
researchers [22,24].
All these results support the proposal about the
existence of an optimum final pH for nisin [24] and
pediocin [22,24] productions.
Finally, since in the fed-batch cultures of L. lactis
and Ped. acidilactici, the value of parameter α was
found to be much higher than β, and both nisin and
pediocin productions were dependent on the final pH
reached in each re-alkalization cycle, these bacteriocins
can be classified as pH-dependent primary metabolites.
7: Effects of pH and temperature on the stability of
both nisin and pediocin
The stability of nisin and pediocin (previously
produced in MRS broth) and 1 g/L of Nisaplin solution
(used as reference) with respect to temperature and pH
was studied by a complete second-order rotatable design.
The remaining activity (%RA) was used as output variable.
The range and codification of both variables are
shown in Table 12. The pH and temperature (T), as well
as the time of treatment were ranged at levels commonly
used in food treatments.
The response surfaces of %RA after 5 and 15 min of
treatment are depicted in Figure 20. All bacteriocins
Nisin and pediocin production on wastes from food industry
showed a similar behaviour. The stabilities were
maxima at temperatures below 100ºC, low pH and short
incubation times, decreasing towards high values of
these variables. While Nisaplin was the most sensitive
bacteriocin, especially after 15 min of incubation, nisin
showed a high mean stability in the assayed
experimental domain (Figure 20). This heat-stability at
acid pH has been described for most bacteriocins
[74,75]. This behaviour is attributed to their high
content of glycine and to the formation, at a molecular
level, of globular structures and strong hydrophobic
interactions in the molecule [76].
8: Effects of proteolytic enzymes treatment on the
activity of nisin and pediocin
The sensitivity of nisin and pediocin to the proteolytic
53
Figure 20. Response surfaces
corresponding to the %RA after both pH
and temperature treatment of 1 g/L
Nisaplin solution and samples of nisin
and pediocin produced on MRS broth by
L. lactis and Ped. acidilactici for 5 and
15 min. The response surfaces were
obtained according to the experimental
plan defined in Table 12.
attack of subtilisin, papain, pepsin and trypsin was
determined in controlled and reproducible conditions.
Figure 21 shows the results obtained in this assay. In all
cases the remaining activity of bacteriocin samples fell
rapidly after 0.5 h of protease treatment, producing
losses of bacteriocin activity of 30% or higher. Then,
the hydrolysis rate of the proteases slowed down, but in
the mixtures containing nisin with papain the protease
activity stopped after 0.5 h of treatment (Figure 21 A
and B). The profiles observed for nisin stabilities are in
concordance with those reported by Matsusaki et al.,
[77] for purified samples of nisin A and Z, with the
exception of pepsin treatment. The proteolytic action of
subtilisin, papain, pepsin and trypsin on samples
containing pediocin was very similar, and after 1.5 h of
incubation almost all the antibacterial activity was lost.

54
Figure 21: Sensitivity of 1 g/L Nisaplin solution (A), and the nisin (B) and
pediocin (C) produced by L. lactis and Ped. acidilactici on MRS broth to
proteases enzymes: pepsin ( Ο ), subtilisin ( ), papain (∇) and trypsin (◊).
ACKNOWLEDGEMENTS
The research presented in this paper was financially
supported by the Instituto Nacional de Investigación y
Tecnología Agraria y Alimentaria (INIA), Spain (project
CAL01-045-C2-2) and The Xunta de Galicia, Spain
(project PGIDT00BIO1E). We thank COREN, S.C.L.
for their collaboration in the elaboration of this work.
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