Professional Documents
Culture Documents
Influence of PH Drop On Both Nisin and Pediocin Production by Lactococcus Lactis and Pediococcus Acidilactici
Influence of PH Drop On Both Nisin and Pediocin Production by Lactococcus Lactis and Pediococcus Acidilactici
2002/362: received 25 November 2002, revised 3 February 2003 and accepted 14 March 2003
ABSTRACT
N . P . G U E R R A A N D L . P A S T R A N A . 2003.
Aims: To develop a kinetic model for describing the specific effect of pH drop on nisin and pediocin
production in whey.
Methods and Results: The effect of pH drop on both bacteriocin productions was tested in non-buffered
whey and whey buffered at initial pH 6Æ3 with 0Æ03, 0Æ10 and 0Æ25 mol l)1 of potassium hydrogen phthalate-NaOH.
An accurate description of the experimental data of nisin and pediocin obtained at different pH drops is
obtained with the proposed model.
Conclusions: The proposed model was able to typify both bacteriocins as pH-dependent primary metabolites.
Significance and Impact of the Study: The decisive role of pH drop for bacteriocin production on whey was
demonstrated and modelled. This study contributes to a better understanding of underlying metabolic regulatory
mechanisms, which could facilitate the optimization of bacteriocin production for upscaling.
In the present work, the kinetics of nisin and pediocin were harvested by centrifugation (3000 g for 15 min at 4C)
production are studied in whey buffered to initial pH 6Æ3 of culture samples and washed twice with saline (0Æ8% NaCl).
using different concentrations of the buffering agent. The Total sugars, phosphorous, nitrogen and protein content
experiments were set up to assess the quantitative effect of were determined as described by Murado et al. (1993).
pH drop on bacteriocin production. A kinetic model was set
up and fitted to the data obtained from the fermentations.
Bacteriocin activity determination
Aliquots from cultures were adjusted to pH 3Æ5 with 5 N
M A T E R I A LS A N D M E T H O D S HCl to prevent the adsorption of bacteriocin onto the
producer cell surfaces, and then heated for 10 min to kill the
Bacterial cultures and media
cells (Guerra and Pastrana 2002a). The heat-killed cells were
Lactococcus lactis subsp. lactis CECT 539 (producer of nisin) removed by centrifugation (27 200 g for 15 min at 4C) and
and Carnobacterium piscicola CECT 4020 (which was used as the supernatant was used to measure bacteriocin activity in
indicator strain) were obtained from the Spanish Type culture tubes (Cabo et al. 1999) against C. piscicola.
Culture Collection (CECT, Valencia, Spain). Pediococcus
acidilactici NRRL B-5627 (producer of pediocin), was
RESULTS
obtained from the National Center for Agricultural Utiliza-
tion Research (NCAUR, Peoria, IL, USA). The three bacteria Fig. 1a shows the time course of pH, biomass and
were grown in MRS broth and maintained as frozen stock held bacteriocin production by L. lactis subsp. lactis and
at )40C in nutrient broth plus 15% (v/v) glycerol. The P. acidilactici in a non-buffered culture and in three
working cultures were maintained as slants on de Man Rogosa buffered cultures. As expected, the increase in buffer
Sharpe (MRS) agar at 4C, and propagated twice in liquid concentration leads to an increase of the final pH, which
cultures in the same medium at 30C before use. provoked a decrease in pH drop (DpH, defined as the
Whey, which was obtained from a local dairy plant as the difference between initial and final pH). However,
liquid remaining after the first cheese pressing was proc- although biomass production by both strains was practi-
essed in the following conditions. After adjusting the pH to cally unaffected, both nisin and pediocin titres decreased
4Æ5 with 5 N HCl, whey was heated at 121C for 15 min to as pH drop decreased. As all fermentations were started
denature the proteins and the precipitates were removed by in the same initial conditions (composition and pH 6Æ3),
centrifugation (27 200 g for 15 min at 4C). This treatment the pH drop generated in each culture seems to account
only affected the protein content, which was reduced from for both nisin and pediocin synthesis. This indicates that
8Æ2 to 5 g l)1. The resulting medium contained (g l)1): total biomass produced by L. lactis and P. acidilactici was more
sugar, 48Æ51; total nitrogen, 1Æ05; total phosphorus, 0Æ43 and productive in the non-buffered cultures.
soluble proteins, 5. On the contrary, both bacteriocins were produced during
To study the influence of pH drop rate on nisin and the exponential growth phase in all cultures supporting the
pediocin production, four series of batch cultures were proposal that both bacteriocins display primary metabolite
performed on whey buffered at initial pH 6Æ3 with 0 (non- kinetics (Guerra and Pastrana 2002b). This was confirmed
buffered medium), 0Æ03, 0Æ10 and 0Æ25 mol l)1 potassium by using the Luedeking and Piret (1959) equation:
hydrogen phthalate-NaOH. This buffering agent was used to
avoid alterations in the culture media. Then the media were rBT ¼ arX þ bX ð1Þ
sterilized at 121C for 15 min and used as culture medium.
All fermentations were carried out in 250-ml Erlenmeyer where rX and rBT are respectively, the biomass (X) and
flasks containing 50 ml of whey, in a rotary shaker bacteriocin production (BT) rates, a is a growth-associated
(200 rev min)1). The media were inoculated with 2% constant (BU mg)1) and b is the non-growth-associated
(v/v) of a 12 h culture of the appropriate producing-strain constant (BU mg)1 h)1). Before using this model, the
and incubated for 18 h at 30C. experimental data for cell growth and bacteriocin production
The samples were withdrawn at intervals during incuba- were smoothed with a generalized logistic equation
tion periods to determine cell dry mass, pH, residual total (Edwards and Wilke 1968). Then, the experimental values
sugar and antibacterial activity. of l and qBT were calculated using the smoothed data and
adjusted to eqn (1). The numeric integration of eqn (1) with
respect to time provided the estimate values of BT:
Analytical methods
X
t
The growth was monitored by optical density at 700 nm and BT ¼ ðarX þ bX Þ ð2Þ
converted to dry cell mass using a standard curve. The cells t¼0
ª 2003 The Society for Applied Microbiology, Letters in Applied Microbiology, 37, 51–55
EFFECTS OF pH ON BACTERIOCIN PRODUCTION 53
6 6
pH
pH
5 5
0·3
0·10
X (g l−1)
X (g l−1)
0·2
0·05
0·1
0 0
20
3
0 0
0 5 10 15 20
Hours
(b)
60 10 50
9 40
50
Nis (BU ml−1)
8 30
40
3
7 20
30
6 10
20 5 0
0 0·3 0·6 0·9 0 1 2 3 4
∆pH
(c)
Fig. 1 (a) Time course of pH, biomass (X) 12 60
and bacteriocin production (Nis and Ped) by
L. lactis and P. acidilactici on non-buffered 9
Ped (BU ml−1)
Nis (BU ml−1)
ª 2003 The Society for Applied Microbiology, Letters in Applied Microbiology, 37, 51–55
54 N . P . G U E R R A A N D L . P A S T R A N A
ª 2003 The Society for Applied Microbiology, Letters in Applied Microbiology, 37, 51–55
EFFECTS OF pH ON BACTERIOCIN PRODUCTION 55
MRS broth (Guerra and Pastrana 2002b). This suggests that Guerra, N.P. and Pastrana, L. (2002a) Nisin and pediocin pro-
whey lack some essential nutrient for growth and bacteriocin duction on mussel-processing waste supplemented with glucose
production, or those whey proteins have an unsuitable and five nitrogen sources. Letters in Applied Microbiology 34,
composition to be used as nitrogen source. Therefore, 114–118.
Guerra, N.P. and Pastrana, L. (2002b) Modelling the influence of pH
further studies based on finding of both cheap nutrient
on the kinetics of both nisin and pediocin production and
sources to supplement whey and other culture techniques
characterization of their functional properties. Process Biochemistry
(fed batch or continue) are needed. 37, 1005–1015.
Kim, W.S., Hall, R.J. and Dunn, N.W. (1997) The effect of nisin
REFERENCES concentration and nutrient depletion on nisin production of
Lactococcus lactis. Applied Microbiology and Biotechnology 48, 449–
Biswas, S.R., Ray, P., Johnson, M.C. and Ray, B. (1991) Influence of 453.
growth conditions on the production of a bacteriocin, pediocin AcH, Luedeking, R. and Piret, E.L. (1959) A kinetic study of the lactic acid
by Pediococcus acidilactici H. Applied and Environment Microbiology fermentation. Batch process at controlled pH. Journal of Biochemical
57, 1265–1267. Microbiology Technology Engineering 1, 393–412.
Cabo, M.L., Murado, M.A., González, M.P. and Pastoriza, L. (1999) Murado, M.A., Siso, M.I.G., González, M.P., Montemayor, M.I.,
A method for bacteriocin quantification. Journal of Applied Micro- Pastrana, L. and Pintado, J. (1993) Characterization of microbial
biology 87, 907–914. biomasses and amylolytic preparations obtained from mussel pro-
Cabo, M.L., Murado, M.A., González, M.P. and Pastoriza, L. (2001) cessing waste treatment. Bioresource Technology 43, 117–125.
Effects of aeration and pH gradient on nisin production. A Parente, E. and Ricciardi, A. (1994) Influence of pH on the production
mathematical model. Enzyme and Microbial Technology 29, 264–273. of enterocin 1146 during batch fermentation. Letters in Applied
Edwards, V.H. and Wilke, C.R. (1968) Mathematical representation of Microbiology 19, 12–15.
batch culture data. Biotechnology and Bioengineering 10, 964–974. Ray, B. (1992) Bacteriocins of starter culture bacteria as food
Egorov, N.S., Baranova, I.P., Kozlova, Y.I., Volkov, A.G., Grushina, biopreservative. In Food Biopreservatives of Microbial Origin ed.
V.A., Isai, E.I., Isai, P.P. and Sidorenko, A.T. (1980) A new nutrient Ray, B. and Daeschel, M. pp. 177–205. Florida: CRC Press, Inc.
medium for Streptococcus lactis producing nisin. Antibiotiki 25, 260– van Niel, E.W.J. and Hahn-Hägerdal, B. (1999) Nutrient requirements
263. of lactococci in defined growth media. Applied Microbiology and
Goulhen, F., Meghrous, J. and Lacroix, C. (1999) Production of a nisin Biotechnology 52, 617–627.
Z/pediocin mixture by pH-controlled mixed-strain batch cultures in Yang, R. and Ray, B. (1994) Factors influencing production of
supplemented whey permeate. Journal of Applied Microbiology 86, bacteriocins by lactic acid bacteria. Food Microbiology 11, 281–291.
399–406.
ª 2003 The Society for Applied Microbiology, Letters in Applied Microbiology, 37, 51–55