Lactate removal by anionic-exchange resin on nisin production by Lactococcus lactis
Pak-Lam Yu1*, Noel W. Dunn2 & Woojin S. Kim2
1Biotechnology
Group, Institute of Technology and Engineering, College of Sciences, Massey University,
Palmerston North, New Zealand
2Department of
Biotechnology, University of New South Wales, Sydney, NSW 2052, Australia
*Author for
correspondence (Fax: +64-6-350 5604; E-mail: P.Yu@massey.ac.nz)
Key words: anionic
resin, bacteriocin, lactate, Lactococcus
lactis, sucrose utilisation, nisin production
Abstract
When lactate was removed from sucrose fermentation in situ by using the anionic-exchange
resin Amberlite IRA-67 on Lactococcus
lactis growing in batch culture, nisin production increased by two-fold
when compared to the alkali pH-controlled fermentation. In comparison to
sucrose, lactate removal increased nisin production 1.5-fold and 0.3-fold when
galactose and glucose were used as carbon sources respectively.
Nisin is a 3354 Da antimicrobial peptide or bacteriocin produced by
certain strains of Lactococcus lactis.
It belongs to a class of bacteriocins that are referred to as lantibiotics
because of their characteristic thioester bridges consisting of meso-lanthionine and
3-methyl-lanthionine. Nisin is used as a food preservative in more than 50
countries (Delves-Broughton et al.
1996). Its antimicrobial activity is effective against numerous Gram-positive
bacteria. Some of its applications include preventing outgrowth of clostridia
and bacilli spores in processed cheese spreads and canned foods, extending the
shelf-life of pasteurized milk and controlling the lactic acid bacteria in beer
production (Helander et al. 1997).
Nisin production has been studied in terms of fermentation parameters,
such as media composition, optimal pH and host specificity. Nisin is regarded
as a primary metabolite and nisin production is affected by the type and level
of carbon, nitrogen and phosphate sources and other nutritional factors (De
Vuyst et al.1990, De Vuyst &
Vandamme 1992, De Vuyst & Vandamme 1993, De Vuyst 1995, Kim et al. 1997a). Maximum nisin production
is directly related to biomass formation and production is closely associated
with growth. The final concentration of nisin produced is host specific (Kim et al. 1977b). Continuous production of
nisin in a bioreactor system coupled to a microfiltration module increases
nisin productivity because it serves to maintain a low concentration of lactic
acid (Taniguchi et al.1994). Over a
four-fold increase in nisin production was reported in such a continuous system
when compared to that obtained in batch culture. Another novel method using a
mixed-culture of L. lactis and the
yeast Kluyveromyces marxianus has
also been used to keep the pH at constant by the assimilation of lactate by the
yeast (Shimizu et al. 1999). By
keeping the lactate concentrations low, nisin in the medium was 1.7 times
higher than that in the anaerobic culture with pH control via addition of NaOH.
In this study, L. lactis
LM0230(TnNip) was used as a nisin
producer, and lactate was kept to a low concentration by adsorption on to an
anionic resin. Glucose, galactose and sucrose were used as carbon sources in a
medium that supports high growth rate and high biomass formation. The effect of
lactate removal on biomass and nisin production in batch fermentations was
compared to the pH-controlled cultures which were controlled by the addition of
NaOH.
Materials and methods
Bacterial
strains, medium, growth and resin
Lactococcus lactis subsp. lactis LM0230(TnNip) (Kim & Dunn 1997) was used. Nisin was assayed using Micrococcus luteus ATCC 9341 as
indicator organism. Both strains were grown on a modified M17 medium (Terzaghi
& Sandine 1975) at 30 °C. It is composed of (g/l):
polypeptone peptone, 14; lab lemco, 7; yeast extract, 3.5; MgSO4.7H2O,
0.35; and ascorbic acid, 0.7. Glucose, sucrose or galactose was added to give
40 g/l. The anionic exchange resin used in this study was Amberlite IRA-67 (Supelco,
USA).
Batch
fermentation
Batch fermentations were conducted in 1-L
Quickfit-type fermenters at 30 °C with the pH
maintained at 6.5 by automatically-feeding 5 M NaOH. Alternatively, the
anionic resin was manually added to the fermenters to maintain the pH at
6.5. The fermentations were continually
mixed with a magnetic stirrer. Overnight cultures were used as inoculum at 10 %
v/v. Samples (1 ml) were collected hourly for turbidity and lactate measurements
and for nisin assay. The samples for turbidity measurement were appropriately
diluted in saline and measured at 620 nm. The samples for nisin assay were
adjusted to give a pH of 2.0 + 0.5 with a few
drops of conc HCl and were stored at 4°C.
Analytical
methods
Nisin assays were performed by an agar
diffusion method (Tramer & Fowler 1964). All assays were performed in
duplicate and averaged results are shown. Nisin concentrations are stated as
Relative Nisin Concentration (RNC). Biomass concentration was determined
turbidometrically (at 620 nm). A correlation factor of 0.3 was used to convert
the absorbance values into biomass concentrations of dry cell weight in mg/ml.
Lactate determination
In addition to determining lactate concentration by NaOH consumption,
the lactate concentration was determined using a YSI 2300 (USA). The samples
were diluted with saline to keep concentrations below 10 mM lactate.
Results and Discussion
Comparison of growth of the
nisin-producing strain with the original host
The
nisin-producing L. lactis strain LM0230(TnNip) was constructed from the
plasmid-free host strain LM0230. Growth on glucose of the parent and the
nisin-producing derivative were studied (Figure 1). At 40 g/l glucose (222 mM),
complete conversion of glucose to lactic acid was achieved after 15 h of growth
at 30 oC by LM0230. A
biomass of 2.76 mg/ml was reached at the stationary phase by LMO230. In
contrast, the nisin-producing strain LM0230(TnNip) reached a biomass of only 0.81 mg/ml after 15 h growth. The
cell density continued to increase before it levelled off at a biomass of 1.2
mg/ml at 24 hours (data not shown). After 15 h of growth, LM0230(TnNip) used only half of the glucose
available and the lactate concentration was one quarter of that produced by
LM0230. The large differences between the two strains
show that the introduction of transposon TnNip
into LM0230 had a significant effect on growth.
Fig.
1. Growth of LM0230 and LM0230(TnNip) on glucose. Biomass, ■ ; glucose conc.,
▲
; lactate conc.,=. Solid lines are
LM0230 and dash lines are LM0230(TnNip).
Growth of the
nisin-producing strain on different sugars
LM0230(TnNip) was
cultivated using different sugars, and growth and nisin production were
measured. This was conducted in the presence and absence of the anionic resin
IRA-67. As shown in Figure 2, the type of carbon source used in the
fermentation had a significant effect on the maximum biomass achieved. At 40
g/l, sucrose provided the highest growth when compared to galactose and glucose.
Sucrose, a disaccharide, was
rapidly utilized by the strain, probably because it possesses a very efficient
phosphoenolpyruvate-dependent phosphotransferase system for sucrose uptake,
transport and metabolism (Hengstenberg 1977). A genetic linkage between sucrose
utilization and nisin production has been demonstrated (Gasson 1984). At biomass of 1.8 mg/ml and above, the addition of resin
showed a beneficial effect to the cell growth, as shown by a further increase
of biomass compared to the pH-controlled culture. A maximum biomass of 4 mg/ml
was reached in the sucrose fermentation with the addition of resin. This was
approximately seven-fold higher in cell density than that of glucose
fermentation. While the galactose and sucrose fermentations
required approximately 14 and 8 hours, respectively, to reach their maximum
biomass concentrations, the glucose fermentation showed a steady increase in
biomass for approximately 24 hours before it levelled off. At 40 g/l sugar
concentration, incomplete utilization of the glucose and galactose were
observed while sucrose was completely metabolised in the period tested. Based
on the amount of lactate produced, about 3/4 and 1/2 of the glucose and
galactose were not used respectively.
Fig.
2. Growth of L.lactis LM0230 (TnNip) on 40 g/l glucose, galactose or sucrose, with lactate removal
by anionic resin IRA-67 or pH-controlled at 6.5 with NaOH. Biomass for glucose,
♦ ; galactose, ▲
;
sucrose,=. Solid lines are
pH-controlled with NaOH. Dash lines indicate the use of the anionic resin.
Effect
of lactate removal on nisin production
Lactate progressively inhibits cell growth (Bibal et al. 1988). Growth is inversely
proportional to lactate concentration. There is a direct relationship between growth
and nisin production, however, it is not known whether nisin production is
directly affected by lactate or indirectly via growth.
Lactate at 250 and 450 mM were produced in the
pH-controlled fermentations with galactose and sucrose, respectively (Figure
3). By the addition of resin, much of the lactate was removed from the
fermentation broth, resulting in a low level of residue lactate (50-80 mM) in
all the fermentation runs. Complete removal of lactate was not possible with
the resin.
Fig.
3. Lactate concentrations with or without
the removal lactate by anionic resin IRA-67 in the L. lactis LM0230 (TnNip)
fermentation of 40 g/l glucose, galactose or sucrose in pH 6.5 at 30 oC.
Lactate conc. on glucose, ■ ; galactose, ▲
;
sucrose, ♦. Solid lines indicate no
removal of lactate. Dash lines indicate the removal of lactate.
The nisin concentrations were shown to increase when
lactate was removed in all three sugar fermentations (Figure 4). The sucrose fermentation produced the highest
biomass and gave the highest nisin concentration when compared to those of
galactose and glucose. In the sucrose fermentation, the removal of lactate by
the resin produced a RNC of 10 at the early
stationary phase, and this was nearly double that of the alkali pH-controlled
fermentation. The nisin concentration decreased rapidly in the sucrose
fermentation after the highest cell density was reached at the eighth
hour. Nisin, being an oligopeptide, was perhaps being utilized as a nitrogen
source for growth. Even in a proteinase-negative L. lactis LM0230, peptidases are present which can digest the nisin
peptide. A drop in the nisin concentration, although at a lower magnitude, was
also observed with the galactose fermentation. This was not the case in the
glucose fermentation, which showed low cell growth. The overall nutrient level
remaining high and there was no observable decrease in the nisin level in
samples with or without lactate removal.
Fig.
4. Nisin production of L.lactis
LM0230 (TnNip) as expressed as
Relative Nisin Concentration in the fermentations of 40 g/l of glucose,
galactose or sucrose with or without the removal of lactate by anionic resin
IRA-67. Relative nisin conc. with glucose, ▲
;
galactose, ♦ ; sucrose =. Solid lines indicate no
removal of lactate. Dash lines indicate the removal of lactate.
The lactate concentration in the medium has a direct effect on nisin
production. At low lactate concentrations, without
limiting growth, samples with similar biomass concentrations taken from the
lactate removal fermentation, consistently showed higher nisin concentrations
than those samples taken from the alkali pH-controlled fermentation. For
example, after five hours of the sucrose fermentation, the biomass
concentration was 1.9 mg/ml for the samples from the lactate removal
fermentation and the alkali pH-controlled fermentation, yet the nisin
concentrations were 5 and 3 RNC, respectively (Figures 2 & 4).
Although lactate concentration can alter growth, a direct link between lactate
removal and nisin production is suggested.
Acknowledgement
The scientific discussions with Mallika Boonmee and Wallace Bridge were
gratefully acknowledged.
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