International Journal of Food Microbiology 70 Ž2001.

267–281
www.elsevier.comrlocaterijfoodmicro

Nutritional factors affecting the production of two bacteriocins

from lactic acid bacteria on whey
Nelson P. Guerra, M. Luisa Rua, Lorenzo Pastrana)
´
Departamento de Bioquımica, ´ e Inmunoloxıa,
Xenetica ´ Facultade de Ciencias de Ourense, UniÕersidade de Vigo, As Lagoas,
32004 Orense, Spain
Received 27 November 2000; received in revised form 26 February 2001; accepted 21 May 2001

Abstract

The ability of Lactococcus lactis subsp. lactis CECT 539 and Pediococcus acidilactici NRRL B-5627 to produce
bacteriocins on both diluted and concentrated whey was investigated in batch fermentations. Both strains produced the
higher amounts of biomass and bacteriocin titres on diluted whey. Luedeking and Piret expression was able to model the
production of nisin, which was produced as a primary metabolite on both culture media. However, the pediocin production
could not be typified in any case due to the negligible growth of P. acidilactici. Although the whey supported the growth
and bacteriocin production by the two strains, both biomass and bacteriocin productions were lower than those obtained on
MRS broth. The effect of total sugar, nitrogen, phosphorous and buffer concentrations on the production of nisin and
pediocin was studied in diluted whey using factorial experiments and empirical modelling. The production of nisin was
greatly inhibited by the increase in nitrogen, buffer, and to a lesser extent, sugar concentration in the medium, nevertheless,
the used phosphorous source produced a light stimulatory effect on bacteriocin synthesis. In addition, the growth of Lc 1.04
was mainly affected by the nitrogen source used. On the other hand, pediocin was inhibited by the increase in buffer,
phosphorous, and to a lesser degree, by the sugar and nitrogen concentration.
The inhibitory activity of pediocin disappeared almost totally after 15 min of treatment with trypsin, papain, subtilisin
and pepsin. The activity of nisin was drastically reduced by treatment with trypsin, subtilisin and pepsin. Nevertheless, 50%
of the initial activity was retained when nisin was treated with papain. Both bacteriocins showed the highest heat stability at
acidic pH and short incubation times. q 2001 Elsevier Science B.V. All rights reserved.

Keywords: Bacteriocins; Whey; Lactococcus lactis; Pediococcus acidilactici; Nisin; Pediocin

1. Introduction bacteria associated with food spoilage and foodborne

Bacteriocins are proteinaceous antibacterial com-
pounds that may be produced by lactic acid bacteria
commonly present in foods ŽDaeschel, 1989; Ray,
1992.. They are bactericidal to many Gram-positive
)
Corresponding author. Tel.: q34-88-387-062; fax: q34-88-
387-001.
E-mail address: pastrana@uvigo.es ŽL. Pastrana..
illnesses ŽHurst, 1981; Daeschel and Klaenhammer,
1985; Bhunia et al., 1988; Klaenhammer, 1988;
Schillinger and Lucke, 1989. and retain their proper-
ties after heat treatment ŽHurst, 1981; Bhunia et al.,
1988, 1991; Pucci et al., 1988.. They are degraded
by the proteolytic enzymes of the gastrointestinal
tract and seem to be non-toxic and non-antigenic to
animals. Thus, they can be used to enhance the
safety and shelf life of many foods ŽHurst, 1981;

0168-1605r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 8 - 1 6 0 5 Ž 0 1 . 0 0 5 5 1 - 7
268 N.P. Guerra et al.r International Journal of Food Microbiology 70 (2001) 267–281
Daeschel and Klaenhammer, 1985; Bhunia et al.,
1988..
The production of bacteriocins is normally per-
formed in complex growth media: de Man Rogosa
and Sharpe ŽMRS., All Purpose with Tween ŽAPT.,
Elliker, Brain Heart Infusion ŽBHI., Tryptone Glu-
cose Extract ŽTGE., Trypticase Soy Broth ŽTSB. and
Trypticase Soy Broth Yeast Extract ŽTSBYE.. Al-
though these media promote exuberant growths and
relatively high bacteriocin levels, their high cost
make them unsuitable for a large-scale production.
Furthermore, some medium components Že.g. large
amounts of proteins, which are not totally consumed
by the producer strains at the end of fermentation.
may interfere with the subsequent bacteriocin purifi-
cation ŽBarefoot and Klaenhammer, 1984..
It seems more adequate to use raw materials like
some wastes from the food industry Žas in the pro-
duction of some antibiotics. as a basis of the culture
media. However, only few reports deal with bacteri-
ocin production from wastes, e.g. nisin production
on sugar molasses ŽEgorov et al., 1980. and mesen-
terocin 5 ŽDaba et al., 1993., pediocin PO2 ŽLiao et
al., 1993., lactocin 705 ŽVignolo et al., 1995. and a
nisinrpediocin mixture ŽGoulhen et al., 1999. on
whey.
Studies on complex media and on wastes have
demonstrated that bacteriocin production depends on
the medium composition ŽBiswas et al., 1991; Par-
ente and Hill, 1992; Daba et al., 1993; Parente and
Ricciardi, 1994; Yang and Ray, 1994.. This depen-
dence is due both to the qualitative and quantitative
nature of the nutrient sources Žmainly those of C and
N .. Additionally, the composition of the media itself
provokes different final pH values that also deter-
mine the bacteriocin titres ŽYang and Ray, 1994;
Vignolo et al., 1995; Cabo, 1998..
In the present work, we studied the kinetic be-
haviour of bacteriocin production by Lactococcus
lactis and Pediococcus acidilactici strains on whey.
Subsequently, in an effort to optimise the composi-
tion of this medium, we studied the bacteriocin
production from an empirical point of view as a
function of four factors—total sugars, nitrogen,
phosphorous and buffer concentration—which have
a known influence on bacteriocin production. We
also studied the influence of pH, temperature and
proteolytic enzymes on the stability of the bacteri-
ocin extracts produced by the two lactic acid bacte-
ria.
2. Materials and methods
2.1. Bacterial cultures and media
L. lactis subsp. lactis CECT 539 ŽLc 1.04. and
Carnobacterium piscicola CECT 4020 strain ŽCb
1.01. were obtained from the Spanish Type Culture
Collection ŽCECT, Valencia, Spain. and they were
used as nisin-producing strain and as target organ-
ism, respectively. P. acidilactici NRRL B-5627 ŽPc
1.02., the pediocin-producing strain, was obtained
from the Northern Regional Research Laboratory
ŽNRRL, Peoria, IL, USA.. The three bacteria were
grown in de Man, Rogosa and Shape ŽMRS, Merck,
Germany. broth and maintained as frozen stock held
at y40 8C in nutrient broth plus 15% Žvrv. glyc-
erol. Working cultures were maintained as slants on
MRS agar at 4 8C, and propagated twice in liquid
cultures in the same medium at 30 8C before use.
2.2. Whey preparation, inoculation and batch cul-
tures
Whey obtained from a local dairy plant was used
in two forms: as concentrated whey ŽCW: the liquid
remaining after the first cheese pressing. and as
diluted whey ŽDW: CW mixed with wash waters..
Both whey media were prepared as follows: after
adjusting the pH to 4.5 with 5N HCl, they were
heated at 121 8C for 15 min to denature the proteins,
and the precipitates were removed by centrifugation
Ž12,000 = g for 15 min.. The supernatants were
adjusted to pH 6.3, sterilised at 121 8C for 15 min
and used as culture media.
Batch cultures of Pc 1.02 and Lc 1.04 were
performed in 250 ml Erlenmeyer flasks containing
50 ml of whey on a rotary shaker ŽInnova 4330, New
Brunswick Scientific, NJ. at 30 8C for 18 h. The
inoculum consisted of 2% Žvrv. of a 12-h culture on
MRS broth. Samples were withdrawn at intervals
during incubation periods to measure growth, pH,
total sugar, nitrogen, proteins, and phosphorous and
antibacterial activity.
For comparative purposes, 18-h batch cultures of
both strains on MRS broth were also performed in
the same conditions as the cultures on whey. All
 N.P. Guerra et al.r International Journal of Food Microbiology 70 (2001) 267–281
analytical determinations of these cultures were car-
ried out at the end of the fermentation.
2.3. Analytical methods
Growth was monitored by absorbance at 700 nm
and converted to dry cell weight from a standard
curve. Cells were harvested by centrifugation
Ž12,000 = g for 15 min at 4 8C. of culture samples
and washed twice with saline Ž0.8% NaCl.. The
culture supernatants were used to determine total
sugars Žphenol–sulphuric acid method ŽDubois et al.,
1956. according to Strickland and Parsons Ž1968.
with glucose ŽPanreac, Barcelona, Spain. as stan-
dard., phosphorous Žmolybdate reaction ŽMurphy and
Riley, 1962. according to Strickland and Parsons
Ž1968. with potassium di-hydrogen phosphate ŽPan-
reac. as standard., nitrogen Žmicro-Kjeldahl, substi-
tuting distillation for the spectrophotometric method
of Havilah et al., Ž1977. with ammonium sulphate
ŽPanreac. as standard. and proteins Žmethod of Lowry
et al., Ž1951. with bovine serum albumin ŽSigma, St.
Louis, MO, USA. as standard.. Ashes were deter-
mined by calcination of aliquots of medium at 550
8C for 6 h in a muffle furnace ŽHobersal, Barcelona,
Spain.. To determine the solid residue, whey aliquots
were evaporated at 60 8C for 4 h, and subsequently,
at 100 8C until constant weight.
2.4. Extraction of antibacterial actiÕity
Aliquots from LAB cultures were adjusted to pH
3.5 with 5N HCl to avoid the adsorption of molecules
of bacteriocin onto the producer cell surfaces ŽYang
et al., 1992.. Subsequently, they were heated for 3
min to kill the cells and centrifuged at 27,200 = g
for 15 min at 4 8C. The supernatants containing
overall antibacterial activity Žbacteriocin extract.
were frozen until further use.
2.5. Bacteriocin actiÕity assays
Quantitative activities of bacteriocin extracts from
culture samples of Lc 1.04 and Pc 1.02 were esti-
mated by using a photometric assay on culture tubes
ŽCabo et al., 1999.. C. piscicola CECT 4020 ŽCb
1.01. was used as target organism. The method
consists on the determination of growth inhibition Žat
700 nm. of the target organism caused by serial
dilutions of bacteriocin extracts. The method was
carried out as follows: bacteriocin extracts were di-
269
luted as needed in distilled sterile water Žthis step
eliminated the need to correct the pH of bacteriocin
extracts.. 2.5 ml of the diluted bacteriocin extracts
were added in sterile culture tubes. Each tube was
inoculated with 2.5 ml of a culture of Cb 1.01
Ždiluted to an absorbance of 0.2 at 700 nm with
sterile buffered MRS broth ŽpH 6.3... Controls con-
sisted of three culture tubes in which the diluted
bacteriocin extract was substituted by distilled sterile
water. The tubes were incubated for 6 h at 30 8C.
Growth inhibition was measured spectrophotometri-
cally at 700 nm. Dose–response curves were ob-
tained from these data. Bacteriocin activity was cal-
culated as Bacteriocin Units ŽBU mly1 , 1 BU mly1
s amount of bacteriocin needed to obtain 50%
growth inhibition Žlethal dose 50 ŽLD50. compared
to control tubes...
2.6. Bacteriocin sensitiÕity to proteolytic enzymes
Solutions of protease in suitable buffers Žpepsin,
Sigma: potassium hydrogen phthalate-HCl 0.2 M,
pH 2.2; trypsin, Sigma, and subtilisin Carlsberg,
Sigma: TRIS 0.2 M, pH 7.6 and papain, Sigma:
phosphate 0.2 M, pH 7. containing one enzymatic
unit Ždetermined according to Murado et al., 1993.
were mixed with equal volumes of bacteriocin ex-
tracts and incubated over 30, 60 and 90 min at the
optimum temperature for the activity of each en-
zyme: 37 8C for pepsin and subtilisin, 25 8C for
trypsin and 30 8C for papain. After treatment, the
samples were adjusted to pH 6.0 to determine the
remaining antibacterial activity. Controls consisted
of samples of extracts in the same conditions without
enzymes.
2.7. Statistical designs
2.7.1. Effects of media ingredients and pH on the
production of nisin and pediocin
A complete factorial design ŽAkhnazarova and
Kafarov, 1982; Box et al., 1989. based on two levels
and four variables was used to study the effect of
four factors Žtotal sugars, nitrogen, phosphorous and
buffer concentration. on the bacteriocin production
by Lc 1.04 and Pc 1.02 on diluted whey. The design
consisted of 20 experiments with 16 Ž2 4 . factorial
points and four replicates of the central treatment.
The media were inoculated with 2% Žvrv. of a 12-h
culture of the appropriate producer strain and incu-
270 N.P. Guerra et al.r International Journal of Food Microbiology 70 (2001) 267–281

bated for 18 h at 30 8C. Bacteriocin activity ŽBU Table 1

mly1 . and cell growth Žg ly1 . were measured as Mean composition Žg ly1 . of the culture media obtained from
acidified wheys
described above.
CW DW
2.7.2. Effects of temperature and pH on the actiÕity Total sugars 48.11 20.54
of nisin and pediocin Reducing sugars 32.13 16.86
A multifactorial composite rotatable design Proteins 5.02 2.04
ŽAkhnazarova and Kafarov, 1982. based on five Total nitrogen 1.05 0.45
levels and two variables was used to study the Total phosphorus 0.43 0.25
Ashes 5.10 3.00
combined influence of pH and temperature on the Solid residue 62.70 26.60
stability of both nisin and pediocin. The design
consisted of 13 experiments with four Ž2 2 . factorial
points, four axial points to form a central composite tion. Pediocin activity was maximum after 15 h Žon
design with a s 1.267 and five centre points for DW: 58 BU mly1 . and 12 h Žon CW: 51 BU mly1 ..
replication. On these media, the profiles described by the pH
Bacteriocin extracts from 18-h cultures of Lc 1.04 seemed to influence the production of pediocin. On
and Pc 1.02 were adjusted at different pH values DW, a rapid fall of pH was observed after the first 3
with 5N HCl or NaOH, and incubated for 5 and 15 h of incubation in concomitance with a rapid in-
min at the temperatures defined in the experimental crease in pediocin production. After this time, the
matrix ŽTable 6.. After incubation, the samples were fall of pH, as well as pediocin production rate was
adjusted to pH 6.0 and monitored for remaining slower ŽFig. 1.. A similar trend in both variables was
activity Ž%RA.. Controls consisted of samples of observed on CW. In this medium, the pH fell rapidly
untreated bacteriocin extracts. after 6 h of incubation and pediocin production rate
was higher in this period. Subsequently, the pH
3. Results decline was reduced, and in parallel, the pediocin
synthesis was slower.
3.1. Batch cultures of L. lactis subsp. lactis CECT Since the best results for both strains were ob-
539 and P. acidilactici NRRL B-5627 tained on DW, this medium was chosen for further
The ability of L. lactis CECT 539 and P. acidi- production studies.
lactici NRRL B-5627 to produce bacteriocins was 3.2. Modelling of growth and bacteriocin production
first tested in both concentrated and diluted whey
Žwhose mean compositions are shown in Table 1.. To model the bacteriocin production rates, the
The results presented in Fig. 1 showed that the whey Luedeking and Piret expression ŽLuedeking and Piret,
media without supplementation are capable of pro- 1959. was used:
moting the growth and bacteriocin production by the dP dX
two producing-strains. sa qb X ;
dt dt
Maximum biomass and nisin productions du-
ring batch fermentation of whey by Lc 1.04 were or dividing by the biomass Ž X in mg mly1 . Ž 1.
obtained after 12 h Ž0.42 g ly1 on DW. or 15 h q P s am q b Ž 2.
Ž0.34 g ly1 on CW.. Then, the biomass production Where q P is the specific bacteriocin production
rates were slower. In addition, nisin production was rate, m is the specific growth rate, a is a growth-as-
sensibly lower on CW Ž8 BU mly1 . than on DW Ž23 sociated constant for bacteriocin production ŽBU
BU mly1 . in spite of the fact that the metabolic mgy1 . and b is the non-growth-associated constant
activity Žnutrients consumption. was higher in CW ŽBU mgy1 hy1 ..
ŽFig. 1 and Table 2.. Before using this model, the experimental data for
On the other hand, Pc 1.02 produced lower cell growth and bacteriocin production were
amounts of biomass than Lc 1.04 on DW Ž0.17 g smoothed with a generalised logistic equation ŽEd-
ly1 . and on CW Ž0.10 g ly1 . after 18 h of incuba- wards and Wilke, 1968.. Then, both specific growth
 N.P. Guerra et al.r International Journal of Food Microbiology 70 (2001) 267–281 271

Fig. 1. Growth kinetics of Lc 1.04 ŽI, B. and Pc 1.02 Ž`, v . on DW Žopen symbols. and CW Žclosed symbols.. X: biomass; C: total
sugars; BT: bacteriocin; Pr: protein; P: total phosphorous. Mean of three analytical replications.

Ž m . and bacteriocin production Žq P . rates were
calculated using the smoothed data.
A high deviation from linearity was observed in
plot of q P versus m for Pc 1.02. On the contrary, a
straight line with good fitting Žtypical of the primary
metabolite production. was obtained for nisin pro-
duction on diluted Ž a s 71.50, b s y0.14, r 2 s
0.982. and on concentrated whey Ž a s 27.90, b s
0.04, r 2 s 0.985..
3.3. Joint effects of the buffer, total nitrogen, phos-
phorous and sugars concentration on bacteriocins
production on whey
A complete first-order factorial design was used
to study the influence of four factors Žtotal nitrogen,
phosphorous, sugars and pH evolution. on the pro-
duction of nisin and pediocin. The input variables
used were:
Ž1. Initial concentration of total sugars in the
media Ž C .. The levels of this variable Žg ly1 . ob-
tained by supplementing the DW with lactose ŽPan-
reac. ranged from total sugars concentration of the
DW to total sugar concentration of the CW.
Ž2. Initial concentration of total nitrogen Ž N ..
The simplest amino acid, glycine ŽPanreac. was used
as a nitrogen source to reduce the additional carbon
contribution to the system and the interference with
the variable C. In addition, it has been reported that
this amino acid does not produce inhibitory action on
the production of nisin ŽDe Vuyst, 1995..
272 N.P. Guerra et al.r International Journal of Food Microbiology 70 (2001) 267–281
Table 2
Nutrient consumption, maximum biomass and bacteriocin produc-
tion obtained from cultures of Lc 1.04 and Pc 1.02 on wheys ŽCW
and DW. and on MRS broth in the maximum production time.
The initial pH of the cultures was fixed at 6.23
Lc 1.04 Pc 1.02
MRS CW DW MRS CW
Consumptions (g ly1 )
Total sugars 8.11 8.48 7.56 6.56 3.09
Total nitrogen 0.97 0.34 0.14 0.82 0.10
Total phosphorous 0.07 0.05 0.09 0.12 0.08
Maximum productions
Biomass Žg ly1 . 1.56 0.32 0.41 1.76 0.09 0.18
Bacteriocin ŽBU mly1 . 50.0 8.3 22.9 493.2 51.6 57.9
Ž3. Initial concentration of total phosphorous Ž P ..
KH 2 PO4 ŽPanreac. was used as a phosphorous
source. This salt has been recognised as the better
source for nisin production ŽBaranova and Egorov,
1969; De Vuyst and Vandamme, 1993.. This nutrient
was added as KH 2 PO4 sterile solution to the previ-
ously autoclaved media in order to avoid saline
precipitations during the sterilisation process.
Those concentrations corresponding to the DW
without supplements were taken as the inferior levels
for the last two variables. To reach the superior limit,
the media were supplemented to obtain the same
CrN and CrP relationships as the MRS medium
with variable C fixed in its maximum value Žtotal
sugars concentration of CW..
Ž4. Buffer concentration in the culture media Ž B ..
Potassium hydrogen phthalate ŽPanreac. –NaOH
ŽPanreac. was used as the buffering agent because
microorganisms do not consume it ŽGonzalez, ´ 1988;
Siso, 1989.. Since the experimental system used
makes it impossible to fix the final pH values in the
culture, the study of this variable was approached in
an indirect way. Thus, the media were buffered at
different molarities in order to generate different
final pH values in the incubation period.
The coded and uncoded factor levels are shown in
Table 3. Bacteriocin Žas BU mly1 . and biomass Žas g
ly1 . productions were used as response variables.
The results are shown in Table 4. The significance
analysis of the proposed models for growth and
bacteriocin production by Lc 1.04 and Pc 1.02 is
shown in Table 5.
3.3.1. Empirical models for nisin production
Table 4 shows the experimental matrix, as well as
the corresponding results for nisin and biomass pro-
duction and the final pH reached in the cultures. The
equations obtained for nisin and biomass production
after an 18-h incubation period were:
DW
BT Ž BU mly1 . s 9.62 y 1.96 B y 1.41C y 4.49N
2.02
q 0.71 P q 0.59BC q 2.34 BN
0.05
0.11 q 0.76CN y 0.53CNP Ž 3.
X Ž g ly1 . s 0.265 q 0.009B y 0.043C y 0.172 N
q 0.018 P q 0.014 BC y 0.013 BN
q 0.032CN y 0.01 NP q 0.019BCN
Ž 4.
The coefficients were acceptable according to the
Student’s t-test Ž a - 0.05. and the equations ob-
tained were significant in Fisher’s F-tests Ž a - 0.05.
applied to both quotients total errorrexperimental
error and lack of fittingrexperimental error.
Among the most notable characteristics of the
system studied, it should be stated that for nisin
production, all the independent variables had nega-
tive coefficients except for P ŽModel Ž3... On the
contrary, the coefficients of the binary interactions
between variables had positive effects on response.
When the corresponding response surfaces are
represented ŽFig. 2., it can be observed that the
variable C only showed a clear effect on nisin and
biomass production for N s y1 ŽFig. 2ŽA. and ŽB...
In both cases, the increase of the carbon source
inhibited the nisin synthesis, as well as the cell
Table 3
Experimental domain and codification of the variables used in the
factorial design to test the influence of the buffer Ž B ., total sugar
Ž C ., nitrogen Ž N . and phosphorous Ž P . concentrations on the
production of nisin and pediocin on whey
Codified Natural values
values B ŽM. C Žg ly1 . N Žg ly1 . P Žg ly1 .
y1 0.0 26.0 0.43 0.27
0 0.05 37.0 4.01 0.91
1 0.1 48.0 7.55 1.56
Codification: Vc s ŽVnyVo.rDVn; decodification: Vn sVoq
Ž DVnVc..
Vc: coded value; Vn: natural value; Vo: natural value in the centre
of the experimental domain; DVn: increment of Vn corresponding
to one unit of Vc.
 N.P. Guerra et al.r International Journal of Food Microbiology 70 (2001) 267–281 273

Table 4
Values of BT Žas BU mly1 ., X Žas g ly1 . and final pH in the experiments on the effect of the nutrient sources on the growth and
bacteriocin production by Lc 1.04 and Pc 1.02 on DW
Coded variables Lc 1.04 Pc 1.02
B C N P BT X Final BT X Final
pH pH
1 1 1 1 5.97 0.12 5.87 10.55 0.08 5.83
1 1 1 y1 6.13 0.11 5.87 15.99 0.09 6.09
1 1 y1 1 9.09 0.41 5.51 26.50 0.14 5.64
1 1 y1 y1 7.61 0.35 5.34 22.76 0.13 5.62
1 y1 1 1 5.69 0.08 5.95 24.42 0.08 5.84
1 y1 1 y1 5.70 0.06 5.90 25.13 0.09 5.82
1 y1 y1 1 12.50 0.56 5.42 26.64 0.14 5.64
1 y1 y1 y1 11.49 0.53 5.53 27.64 0.13 5.59
y1 1 1 1 3.89 0.06 5.10 34.38 0.11 5.09
y1 1 1 y1 3.39 0.05 5.08 53.84 0.20 4.84
y1 1 y1 1 18.68 0.40 4.70 24.89 0.19 5.01
y1 1 y1 y1 13.83 0.30 4.86 37.21 0.18 4.82
y1 y1 1 1 8.37 0.15 5.37 30.47 0.16 4.92
y1 y1 1 y1 4.79 0.13 5.41 38.72 0.19 4.90
y1 y1 y1 1 21.35 0.50 4.79 29.38 0.17 4.91
y1 y1 y1 y1 21.27 0.47 4.76 57.38 0.17 4.93
0 0 0 0 8.62 0.25 5.38 31.56 0.14 5.37
0 0 0 0 8.51 0.24 5.38 28.12 0.14 5.34
0 0 0 0 7.36 0.25 5.41 27.21 0.14 5.30
0 0 0 0 8.15 0.27 5.39 28.56 0.14 5.37

growth. In addition, the phosphorous exerted very ŽB... As there is a significant interaction between B
little influence on both nisin and biomass production and N in MODEL Ž3., the effect of both variables
Žleft and right part of Fig. 2ŽC. and ŽD... on nisin production must be analysed in a joint way.
Contrarily, it can be noted that nisin production In fact, a strong inhibitory effect of variable N was
and growth were dramatically inhibited by the in- observed for low values of B Žleft part of Fig. 2ŽB...
crease in glycine Žright and left part of Fig. 2ŽA. and In parallel, an increase of B depressed the nisin
Table 5
Significance analysis of the proposed models for growth and nisin production by Lc 1.04 and pediocin production by Pc 1.02
SS: sum of squares; fd: degrees freedom; QM: quadratic means; E: total error; Ee: experimental error; LF: lack of fitting
Lc 1.04 Pc 1.02
BT ŽBU mly1 . X Žg ly1 . BT ŽBU mly1 .
QM ErQM Ee s 5.58 2.35 3.14
F Ž a s 0.05. s F311 s 8.76 F310 s 8.79 F311 s 8.76
QM LFrQM Ee s 7.29 2.93 3.94
F Ž a s 0.05. s F38 s 8.85 F37 s 8.89 F38 s 8.85
r2s 0.964 0.994 0.945
r 2 adj s 0.929 0.989 0.904

SS fd QM SS fd QM SS fd QM
Model 531.6 8 66.5 0.535 9 0.059 2095.8 8 262.0
Error 19.9 11 1.81 0.003 10 0.003 122.8 11 11.2
Experimental error 1.0 3 0.32 0.004 3 0.001 10.7 3 3.6
Lack of fitting 18.9 8 2.37 0.003 7 0.004 112.1 8 14.4
Total 551.6 19 29.03 0.538 19 0.028 2218.6 19 116.8
274 N.P. Guerra et al.r International Journal of Food Microbiology 70 (2001) 267–281

Fig. 2. Response surfaces obtained for both bacteriocin ŽBT. and biomass Ž X . production on diluted whey for Lc 1.04, according to the
factorial plan defined in Table 3. The four input variables Ž C, N, B and P . in coded values.
 N.P. Guerra et al.r International Journal of Food Microbiology 70 (2001) 267–281 275

production only for the minimum level of N Žsee left
parts of Fig. 2ŽC. and ŽD... These partial annulments
explain the positive sign of the interaction BN,
which almost neutralises the negative effect of the
other input variables. Nevertheless, the poor influ-
ence of the buffer on biomass production Žright parts
of Fig. 2ŽC. and ŽD.. indicated a specific effect of
this variable on nisin synthesis.
To verify this fact, four series of cultures were
performed using different initial concentrations of
buffer: 0, 0.03, 0.10 and 0.25 M Žcorresponding to
codified values of B of y1.0, y0.4, 1.0 and 4.0,
respectively.. In these experiments, the media were
not supplemented with C, N and P sources. The
results showed that the increase in buffer concentra-
tion only produces a light effect on biomass produc-
tion but resulted in an increase of the final pH,
provoking an exponential fall of the bacteriocin titres
ŽFig. 3..
3.3.2. Empirical models for pediocin production
The results obtained for biomass and pediocin
production and the final pH values reached in the
cultures of Pc 1.02 are shown in Table 4. The
models developed for pediocin and biomass produc-
tion provided the following empirical equations:
BT Ž BU mly1 . s 30.1 y 7.9B y 2.1C y 4.5P
y 2.2 BN q 4.0 BP q 1.6CN
y 3.9BCN y 2.3CNP Ž 5.
X Ž g ly1 . s 0.141 y 0.031 B y 0.001C y 0.015N
y 0.006 P q 0.002 BC y 0.008 BN
q 0.008 BP y 0.005CN y 0.004CP
y 0.011 NP q 0.004 BCN q 0.004 BCP
q 0.005BNP y 0.005CNP
q 0.004 BCNP Ž 6.
The most illustrative response surfaces generated
according to MODEL Ž5. are represented in Fig. 4.
Pediocin production was mainly affected by buffer

Fig. 3. ŽA. Evolution of cultures of Lc 1.04 on unbuffered DW Žv . and on buffered DW to concentrations 0.03 Ž`., 0.10 ŽI. and 0.25 M
Ž,. with potassium hydrogen phthalate–NaOH. ŽB. Final pH, biomass concentration Ž X . and bacteriocin titres ŽBT.. Ž ) .: Amounts
produced at the end of the culture.
276 N.P. Guerra et al.r International Journal of Food Microbiology 70 (2001) 267–281

Fig. 4. Response surfaces obtained from Model Ž5. for bacteriocin production by Pc 1.02. Notations as shown in Fig. 2.

and phosphorous. In fact, the variable B ŽFig. 4ŽB., 3.4. Effects of heat and pH treatment
mainly for P s y1., as well as the variable P ŽFig.
4ŽB., mainly for B s y1. exerted a strong inhibitory Samples of nisin and pediocin Žproduced on DW
effect on pediocin synthesis. in the optimum conditions determined according to
The effect of the other two input variables did not the factorial plan used in the above section. were
have a dramatic influence on pediocin production. used to test their heat and pH stability using a
The increase in lactose concentration stimulated the second-order rotatable design. The range and codifi-
pediocin synthesis lightly only when N s 1 Žright cation of both variables are shown in Table 6. The
part of Fig. 4ŽA.., but in the other situations, C
produced a light inhibitory action Žright and left parts Table 6
of Fig. 4ŽA... This fact confirmed the low capacity Experimental domain and codification of the variables used in the
of Pc 1.02 to metabolise this sugar. On the other factorial design to test the sensitivity to the temperature and pH of
the bacteriocin extracts produced by Lc 1.04 and Pc 1.02
hand, glycine showed light positive and negative
effects ŽFig. 4ŽA.., as expected from its presence in Codified values Natural values
MODEL Ž5. forming positive and negative interac- T Ž8C. pH
tions with the rest of the input variables. y1.267 63.0 3.7
The empirical model for biomass production ŽEq. y1 70.0 4.0
Ž6.. included all independent variables and all their 0 95.5 5.2
q1 121.0 6.5
interactions. Nevertheless, this model was considered q1.267 128.0 6.8
unsatisfactory due to the reduced growth of Pc 1.02, Increments 1 25.5 1.2
and its significance analysis is not shown in Table 5.
 N.P. Guerra et al.r International Journal of Food Microbiology 70 (2001) 267–281 277

Fig. 5. Response surfaces corresponding to the %RA of bacteriocin extracts from Lc 1.04 and Pc 1.02, after both pH and temperature ŽT .
treatment for 5 and 15 min, according to the experimental plan defined in Table 6.

pH and temperature ŽT . values, as well as the time All the empirical equations obtained for remain-
of treatment were ranged at levels commonly used in ing activity allowed the reconstruction of the experi-
food treatments ŽTable 6.. mental data, being acceptable coefficients according

Fig. 6. Sensitivity of the bacteriocin extracts produced by Lc 1.04 ŽA. and Pc 1.02 ŽB. to proteolytic enzymes: pepsin Ž`., subtilisin ŽI.,
papain Ž,. and trypsin Že..
278 N.P. Guerra et al.r International Journal of Food Microbiology 70 (2001) 267–281
to the Student’s t-test with a s 0.05 and the validity
of the equations according to the Fisher F-test with
a s 0.05. Fig. 5 shows the response surfaces of
remaining activity Ž%RA. after both 5 and 15 min of
treatment. In all cases, the overall initial activity was
almost retained for temperatures below 100 8C, low
pH and short incubation times. In addition, when
extracts of nisin and pediocin adjusted to pH 3.5
were stored at temperatures between y4 and 4 8C
during 1 month, the overall initial activity was re-
tained.
3.5. Effects of proteolytic enzyme treatment
The sensitivity of nisin and pediocin to the prote-
olytic attack of subtilisin, papain, pepsin and trypsin
was determined in controlled and reproducible condi-
tions. Fig. 6 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 hydrolytic rate of protease enzymes
slowed down but in the mixtures containing nisin
with papain or pepsin, the protease activity stopped
at this time ŽFig. 6ŽA... The profiles observed for
nisin stabilities are in concordance with those re-
ported by Matsusaki et al. Ž1998. for purified sam-
ples of nisin A and Z with the exception of pepsin
treatment. The proteolytic action of subtilisin, pa-
pain, pepsin and trypsin on samples containing pe-
diocin was very similar, and after 1.5 h of incuba-
tion, almost all the antibacterial activity was lost.
4. Discussion
Several complex culture media of high cost have
been used for bacteriocin production. In the current
study, we have used an effluent from the food indus-
try Žconcentrated and diluted whey. as culture media
for the production of nisin and pediocin at low
production costs. On these media, L. lactis produced
higher amount of biomass than P. acidilactici. Lac-
tococcus species are known to have a higher ability
to ferment lactose than Pediococcus species ŽBergey,
1986..
Bacteriocin production was slightly higher Žfor Pc
1.02., and higher Žfor Lc 1.04. in DW than in CW.
Although this fact suggests the possible effect of
substrate inhibition, it could also be related to the
control that the supplied sugar substrate exerts on the
bacteriocin biosynthesis ŽDe Vuyst et al., 1989..
On the other hand, Lc 1.04 and Pc 1.02 produced
lower amounts of bacteriocins and biomass on whey
media than on MRS broth ŽTable 2.. In this way, it
was observed that the highest differences were for Pc
1.02. In fact, the productions of pediocin were 8.6
Žon DW. and 9.7 Žon CW. times lower than the
levels reached on MRS medium ŽTable 2.. The lower
bacteriocin productions observed for both strains on
whey suggest that this media lacks some essential
nutrient for growth and bacteriocin production, or
that those whey proteins have an unsuitable composi-
tion to be used as a nitrogen source. In fact, the
protein consumption observed on whey media was
lower than on MRS broth for both strains ŽTable 2..
The kinetic characterisation of bacteriocin produc-
tion was only possible for nisin. This bacteriocin was
produced as a primary metabolite on both diluted
and concentrated whey. Pediocin production could
not be typified because a lack of fitting was obtained
between the specific growth and pediocin production
rates. This fact suggests that this bacteriocin synthe-
sis depends on an additional factor such as the pH. In
this way, the evolution of pH had a higher influence
on pediocin production than on nisin production on
whey media.
In order to investigate simultaneously the influ-
ence of the total nitrogen, phosphorous and sugars,
as well as the pH evolution, and to obtain an empiri-
cal model for describing the bacteriocin production
on whey, a complete first-order factorial design was
used.
Nisin production was mainly affected by glycine
and buffer. Considering that nisin is produced on
whey as a primary metabolite, the inhibitory effect of
glycine on nisin production could be a consequence
of the inhibition that this amino acid produced on
bacterial growth. In fact, the inhibitory action of
glycine on cell growth could be related to the alter-
ations that this amino acid produces in the muropep-
tide composition of bacterial peptidoglycan, or to its
action on the peptidoglycan precursor synthesis
ŽStrominger and Birge, 1965; Hammes et al., 1973;
Schleifer et al., 1976; De Jonge et al., 1996.. These
observations disagree with those reported by De
 N.P. Guerra et al.r International Journal of Food Microbiology 70 (2001) 267–281
Vuyst Ž1995., who observed that the addition of
glycine to the culture medium had no influence on
biomass and nisin production by L. lactis subsp.
lactis NIZO 22186.
Although the buffer also produced an inhibitory
effect on nisin production, its action on biomass
production was almost void. This specific inhibitory
effect of the variable B on nisin synthesis could be
related with the final pH reached in the cultures. In
fact, the final pH values in the buffered series ŽTable
4, for B s 1. were always higher than those reached
in the unbuffered series ŽTable 4, for B s y1..
These final pHs reached in the buffered cultures
could be the improper well for bacteriocin synthesis,
well for the post-translational processing of prenisin
to produce active nisin ŽYang and Ray, 1994.. This
finding is consistent with the results of Cabo Ž1998.,
who observed higher amounts of nisin in TGE broth
than in TGE buffer broth, although the cell growth
was slightly higher in the buffered TGE medium.
The inhibitory effect that the increase in lactose
concentration produced on both nisin and biomass
production also suggests a substrate inhibition effect,
or a possible carbon source regulation involved in
the nisin biosynthesis as in the former experiment.
KH 2 PO4 is known to increase greatly the production
of nisin ŽBaranova and Egorov, 1969; De Vuyst and
Vandamme, 1993., but in the current study, the
phosphorous did not produce a relevant positive
effect on both nisin and biomass production on
whey.
Pediocin production was dramatically inhibited by
the increase in both buffer and phosphate concentra-
tion. The strong inhibitory effect of variable B on
the pediocin synthesis could be explained by a simi-
lar mechanism related with the final pH reached in
the culture as was exposed for nisin production. This
interpretation is consistent with the results of Yang
and Ray Ž1994. who observed that pediocin AcH
was produced in higher amounts in TGE broth than
in TGE buffer broth. The need for a final pH of
3.6–3.7 for high pediocin production has been re-
ported ŽBiswas et al., 1991.. This fact was found to
be due to the need of low pH for post-translational
processing of prepediocin to produce active pediocin
ŽJohnson et al., 1992..
Pediocin is not produced as a primary metabolite
on whey, as was demonstrated in the former experi-
279
ment. In this way, the inhibitory action of phosphate
could be attributed to a specific effect related to the
control that the exogenous phosphate exerts on the
regulatory mechanisms of some secondary metabo-
lites synthesis. This regulatory effect may be ex-
plained 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 bio-
synthesis, etc. ŽMartin, 1977..
Both nisin and pediocin were heat-stable particu-
larly at low pH. This fact has been observed for
other bacteriocins: lacticin ŽRyan et al., 1996.,
lactacins B and F ŽDe Vuyst and Vandamme, 1994.,
mesenterocin 5 ŽDaba et al., 1993.. Such behaviour
is attributed to the high content of glycine and to the
existence of globular structures and strong hydropho-
bic unions at the molecular level ŽDe Vuyst and
Vandamme, 1994..
5. Conclusions
Although the titres of bacteriocin were lower than
those obtained on MRS broth, this study demon-
strated the feasibility of the production of nisin and
pediocin on an effluent of low cost. The kinetic
characterisation of both nisin and pediocin produc-
tion helped to clarify the different effects produced
by the nutrient sources Žlactose, glycine and
KH 2 PO4 . used in this work. In addition, the models
developed allowed a good reconstruction of the ex-
perimental data and identified the key variables in
the production of both nisin and pediocin on both
media. The nutrient sources used in this work were
not adequate to increase the bacteriocin production
on diluted whey. Further studies based on finding
other sources Žlike yeast extract or peptones. are
needed to optimise the unbuffered whey composi-
tion.
Acknowledgements
We thank Miguel Anxo Murado Žhead of the
´ Marinas
research team of Instituto de Investigacions ˜
de Vigo, CSIC. for his aid in the elaboration of this
work. Nelson P. Guerra was the recipient of ICI and
Xunta de Galicia fellowships.
280 N.P. Guerra et al.r International Journal of Food Microbiology 70 (2001) 267–281
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