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Food Microbiology 27 (2010) 741e748
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Food Microbiology
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Isolation and characterization of acid-sensitive Lactobacillus plantarum
with application as starter culture for Nham production
Pornpan Jaichumjai a, b, Ruud Valyasevi c, Apinya Assavanig a, Peter Kurdi c, *
a
Department of Biotechnology, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand
Center of Excellence for Agricultural Biotechnology: AG-BIO/PERDO-CHE, Commission on Higher Education, Ministry of Education, Thailand
c
Food Biotechnology Laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Paholyothin Road, Klong 1, Klong Luang, Pathumthani 12120, Thailand
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 November 2009
Received in revised form
22 March 2010
Accepted 23 March 2010
Available online 30 March 2010
The aim of this study was to derive new starter culture variants that are unable to grow below pH 4.6, the
desirable pH of the Thai fermented pork sausage, Nham, specified by Thailand Food Standard, and apply
them in Nham fermentation. Several acid-sensitive mutants of one of the commercial Nham starter
cultures, Lactobacillus plantarum BCC 9546, were isolated as spontaneous neomycin-resistant mutants.
The growth of three representative mutants was characterized in MRS broth, which revealed that their cell
numbers and acid production were lower than that of the wild-type. The Hþ-ATPase activities of the
three mutants were found significantly lower than that of the wild-type under either neutral or acidic
conditions. Consequently, internal pH values of the mutants appeared to be lower, especially in acidic
environment (pH 5). The most acid-sensitive mutant was applied in experimental Nham production and
the pH of Nham fermented with the mutant had significantly higher pH at the end of fermentation (3 days)
and after an additional 4 days of storage at 30 C. These results indicate that the use of acid-sensitive
L. plantarum as starter culture can reduce the severity of post-acidification and increase the shelf life of
Nham at ambient temperature.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Acid-sensitive
Hþ-ATPase activity
Internal pH
Lactobacillus plantarum
1. Introduction
Nham is a Thai-style fermented pork sausage that is made
of minced pork, boiled pork rinds, cooked rice, garlic, salt, sugar,
pepper, chili and sodium nitrite. It is packed in banana leaves or
plastic sheets and allowed to ferment for approximately 3e4 days
at ambient temperature (about 30e35 C in Thailand), after which
period the pH of the product turns to 4.5e4.6 (Valyasevi and Rolle,
2002). Nham is either fermented spontaneously, or starter cultures
are used to initiate fermentation for better quality control in
terms of consistency and microbiological safety of the product. A
hazard analysis found that the Nham fermentation’s critical limit
for product safety is pH 4.6 or lower (Paukatong and Kunawasen,
2001). Lactobacillus plantarum BCC 9546 is one of the commercial
Nham starter cultures available in the Thai market.
Nham producers generally do not store the product at their
factories, storage usually takes place at the retailer’s facilities in
refrigerated conditions. Many Nham manufacturers tend to keep
the product at room temperature during product handling, transportation or storage which can result in prolonged fermentation
* Corresponding author. Tel.: þ66 2 564 6700x3752; fax: þ66 2 564 6590.
E-mail address: [email protected] (P. Kurdi).
0740-0020/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fm.2010.03.014
(over-fermentation). This process is mainly caused by lactic
acid bacteria that produce organic acids, mostly lactic acid, which
contribute to a variety of undesired flavor and texture changes in
Nham such as water dripping, discoloration and off-flavor development. Since Nham producers prefer reducing the product loss
caused by over-fermentation there is a considerable interest in an
economic solution to prevent over-fermentation. A mutated starter
culture whose growth is sensitive to high acidity, i.e. pH 4.6,
may offer a strategy to the manufacturer to overcome the above
problem.. The potential new acid-sensitive starter strain would
possibly have reduced energy metabolism and would be unable to
continue the fermentation below pH 4.6, thereby over-fermentation could be prevented.
There are several reports about acid-sensitive mutants of lactic
acid bacteria such as an acid-sensitive Lactobacillus delbrueckii subsp.
bulgaricus in yoghurt fermentation has reduced post-acidification
that prolongs the viability of Bifidobacterium breve in yoghurt during
refrigerated storage (Ongol et al., 2007). An Oenococcus oeni mutant
that lacks malolactic activity (Galland et al., 2003), as well as an acidsensitive variant strain of Lactobacillus helveticus (Yamamoto et al.,
1996) and Streptococcus bovis mutants (Miwa et al., 2000) had
reduced Hþ-ATPase activity. These mutants were obtained with the
use of neomycin as a selective tool for isolating Hþ-ATPase deficient
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P. Jaichumjai et al. / Food Microbiology 27 (2010) 741e748
strains. In Escherichia coli, a correlation between resistance to
neomycin (Kanner and Gutnick, 1972) and defects in ATPase activity
has been reported. Some of the independently obtained aminoglycoside antibiotic-resistant ATPase mutants of Escherichia
coli appeared to have membranes leaky to protons, and a decrease
in proton gradient across such membranes was demonstrated
(Tsuchiya and Rosen, 1975; Humbert and Altendorf, 1989). It was
postulated that defective Hþ-ATPase would lead to cellular energy
shortage that would result in a reduced uptake of neomycin into the
cell, thereby leading to resistance to the antibiotic (Humbert and
Altendorf, 1989). Hþ-ATPase plays an important role in cytoplamic
pH regulation in lactic acid bacteria by expelling protons out of the
cell utilizing the energy released by ATP hydrolysis. Mutants defective in Hþ-ATPase are impaired for survival at low extracellular
pH (Nannen and Hutkins, 1991), likely because many cytoplasmic
enzymes have their pH optima in a neutral range (Hutkins and
Nannen, 1993).
Besides the Hþ-ATPase there are also other enzymes that may
contribute to pH homeostasis of lactic acid bacteria such as glutamate decarboxylase and arginine deiminase. It was demonstrated
that in Lactobacillus ssp. glutamate decarboxylase catalyze the
decarboxylation of glutamate, resulting in the stoichiometric release
of the end products g-aminobutyrate (GABA) and CO2 (Hanaoka,
1967). The reaction product, GABA is exported from the cell via
the Glu2/GABA antiporter, leading to the decrease of cytoplasmic
Hþ ion concentration and a slight increase of the internal pH.
The arginine deiminase (ADI) pathway may also contribute to pH
homeostasis converting arginine to ammonia, ornithine and carbon
dioxide and generating 1 mol of ATP per mol of arginine. Ammonia
production (contingent upon arginine availability) may contribute
to survival at acidic environment by neutralizing the pH. This
process has been reported in oral streptococci (Marquis et al., 1987;
Casiano-Colòn and Marquis, 1988; Curran et al., 1995) as well as
in Streptococcus lactis and Streptococcus cremoris (Poolman and
Konings, 1988) and in a L. plantarum strain isolated from orange
peel (Arena et al., 1999). All the above three mechanisms reduce
acidification of the internal compartment and are important in
maintaining acid resistance for the survival of lactic acid bacteria.
L. plantarum is one of the most intensively studied Lactobacillus
strains, however the application of its mutant as an acid-sensitive
starter have not been reported to date. In this study we attempted
to isolate an acid-sensitive L. plantarum strain with mutation in
either Hþ-ATPase, glutamate decarboxylase or arginine deiminase
and employ it in Nham production to hinder the fermentation
below pH 4.6 and subsequently prolong the shelf life during storage
at ambient temperature (30 C).
2. Materials and methods
2.1. Bacterial strains: acid isolation of acid-sensitive mutants
L. plantarum BCC 9546 and Pediococcus acidilactici BCC
9545 were obtained from BIOTEC Culture Collection, Pathumthani,
Thailand. Lactobacillus brevis LSF 8-13 was a gift from Chulalongkorn University, Thailand.
Bacteria were grown in MRS medium until mid-exponential
phase was reached (A660 about 0.8e0.9). Then 0.1 ml aliquots of
10-and 100-fold dilution of the culture was spread onto half strength
MRS plates containing 600 or 750 mg/ml neomycin sulfate for acidsensitive mutant generation and incubated at 30 C for 2 days. After
that neomycin plates were used as master plates for replica-plating
on half strength MRS pH 4.5 and on half strength MRS plates. After
incubation at 30 C for 2 days, the acid-sensitive colonies, identified
as the ones unable to grow on half strength MRS pH 4.5 plates, were
picked up from the half strength MRS replica plates. They were
cultured in MRS broth for 15 h and adjusted to A660 about 0.8 then
were used as inoculum (2%) for the growth experiments in MRS
broth. The pH of the culture broth was monitored for 24e72 h and
the isolates with the highest culture broth pH were stored in 20%
glycerol at 80 C. To obtain isolates with higher acid-sensitivity,
mildly acid-sensitive mutants (pH 4.2) were re-streaked onto half
strength MRS containing 1500 mg/ml neomycin plates and incubated
at 30 C for 24e48 h then each single colony was inoculated to MRS
broth, incubated at 30 C for 15 h. The cultures were adjusted to A660
around 0.8 then inoculated (2%) to MRS broth. The pH of the culture
broth was monitored for 24e72 h and the isolates with the highest
culture broth pH were kept and stored in 20% glycerol at 80 C.
2.2. Measurement of growth characteristics
Growth parameters such as viable count, culture pH, and titratable acidity were measured during 72 h culture in MRS broth at
30 C. Samples were taken from the culture broth at various time
points and were serially diluted with sterile saline and spread onto
half strength MRS plates. Viable counts were determined after 2 days
of incubation at 30 C. Another portion of the sample was diluted
50-fold with CO2-free distilled water and titratable acidity was
measured by the method of AOAC (2000). The pH of the culture
broth was measured using a pH meter (Mettler Toledo, Switzerland).
Growth was monitored by measurement of absorbance at 660 nm
(Helios-a spectrophotometer, Thermo Electron Corp., UK).
2.3. Measurement of Hþ-ATPase activity
Activity of Hþ-ATPase was assayed using the method of
Matsumoto et al. (2004) with minor modifications. Cells cultivated
in MRS broth overnight at 30 C were harvested by centrifugation
(8000 g for 5 min) and washed three times in sterile saline, then
harvested cells were suspended in sterile saline to an A660 around
27. Each bacterial suspension (1.0 ml) was added to 10 ml of MRS
broth with different pH (4.0, 5.0, 6.0 and 7.0 adjusted with L-lactic
acid) and then was incubated for 1 h at 30 C. Cells were
centrifuged at 8000 g for 5 min, resuspended in 1 ml of 75 mM
TriseHCl buffer (pH 7) containing 10 mM MgSO4 and permeabilized by the addition of 30 ml of toluene:acetone mixture
(1:9, v:v; Lauret et al., 1996) and vigorous mixing for 5 min. Then
permeabilized cells were kept at 80 C before use. Hþ-ATPase
activity assay was conducted as described by Belli and Marquis
(1991) with minor modifications. Permeabilized cells (100 ml)
were mixed with 1 ml of 50 mM Trisemaleate buffer (pH 6.0)
containing 10 mM MgSO4 and 10 ml of 0.4 M N,N0 -dicyclohexyl
carbodiimide (DCCD; Wako Pure Chemicals Industries Ltd., Osaka,
Japan) in ethanol or 10 ml of ethanol (as control). Mixtures were
incubated at 37 C for 1 h, then a 10 ml aliquot of 0.5 M ATP (Fluka)
was added to start the assay. Mixtures were incubated at 37 C for
20 min and subsequently put on ice to stop the reaction. Liberated
inorganic phosphate (Pi) in the reaction mixtures was measured
using the Phosphor C test kit (Wako). Results of the assay are
expressed as the amount of Pi produced per minute per mg protein
of permeabilized cells. Protein content of the cells was determined
by the Bradford method (Bradford, 1976).
2.4. Internal pH measurement
Bacterial intracellular pH was measured as outlined by Kurdi et al.,
(2000). Strains were cultured in MRS broth to mid-exponential phase
(6e7 h) at 30 C, harvested and washed two times in ice cold 150 mM
KPO4, 1 mM MgSO4, pH 7.0 buffer then resuspended in 150 mM KPO4,
1 mM MgSO4, pH 7.0 buffer to A660 w 10. The cell suspension was
diluted to a final volume of 9 ml with prewarmed buffer (A660 w 0.5),
P. Jaichumjai et al. / Food Microbiology 27 (2010) 741e748
and then preloaded with the membrane permeable precursor probe
5 (and 6-)-carboxyfluorescein diacetate succinimidyl ester (Molecular Probes Inc., Eugene, OR, USA) at 30 C for 1 h. After that the cell
suspension was centrifuged (8,000 g for 5 min) and the cells were
resuspended in 150 mM KPO4, 1 mM MgSO4 buffer, pH 7.0, then 1 M
glucose was added to a final concentration of 10 mM and the mixture
was incubated at 30 C for 1 h, divided into two parts and centrifuged
(8000 g for 5 min). One part was resuspended in the same buffer
(pH 7.0), while the other part was resuspended in the same buffer
with pH 5.0. The two cell suspensions were incubated at 30 C
for another 30 min. After washing once, the cells were resuspended
in the same buffer followed by internal pH measurements using
a spectrofluorometer (FP 6500, Jasco Co., Tokyo, Japan) with excitation and emission wavelength of 490 and 520 nm, respectively.
Calibration of the fluorescent signal was carried out using de-energized cells in buffers pH 5.0 and pH 7.0. De-energization was achieved
by adding valinomycin (Fluka) and nigericin (Fluka), both from
a 2 mM stock to a final concentration of 2 mM, while DCCD (0.01 mM
final concentration) was added to the cells in the cuvette (30 C)
placed in the spectrofluorometer when its effect on the internal pH of
the cells was investigated.
2.5. Measurement of arginine deiminase activity
Every strain was grown to stationary phase (15e24 h) in
GYPeArg broth, while L. plantarum BCC 9546 was also inoculated to
GYP broth and used as Arg-free control. The GYP medium contains
(g/l): 0.2, glucose; 3, yeast extract; 5, peptone; 1 ml, Tween 80; 5 ml,
salt solution. The salt solution comprised of (g/l): 40, MgSO4$7H2O;
2, MnSO4$4H2O; 2, FeSO4$7H2O; 2, NaCl. Such GYP medium was
supplemented with 3.484 g/l, arginine (20 mM) (GYPeArg) and both
media were adjusted to pH 6.5 with 6 N HCl before sterilization at
121 C for 15 min. All L. plantarum strains and P. acidilactici BCC 9545
as positive control were harvested by centrifugation at 8000 g for
5 min at 25 C suspended in the same media to A660 around 1, then
these cell suspensions were used to inoculate (1%) GYPeArg broth
and GYP broth. Fifty ml samples were taken at 0, 9, 24, 48 and 72 h.
The arginine deiminase activity was detected in terms of ammonia
production from arginine by using NHþ
4 ion-selective electrode
(Mettler Toledo, Switzerland), and was defined as the amount of
ammonium ion produced per mg of protein. The protein concentrations of the cell suspensions were determined by Bradford
method using bovine serum albumin as standard (Bradford, 1976).
2.6. Determination of glutamate decarboxylase activity
Bacterial strains were cultured in GYPeGlu medium (per liter:
glucose, 5 g; proteose peptone, 5 g; yeast extract, 5 g; Tween 80,
1 ml; salt solution (as described above), 5 ml; monosodium glutamate, 9.36 g (50 mM)) for 24 h. Cells were harvested by centrifugation (8000 g for 5 min), then incubated at A660 w 10 in 50 mM
sodium acetate buffer pH 4.5 containing 50 mM monosodium
glutamate for 24 h. After centrifugation (8000 g for 5 min), 10 ml
supernatants were subjected to TLC analysis on Silica gel 60 F254
plates (Merck) using 1-butanol:glacial acetic acid:H2O ¼ 3:2:1 as
solvent. The TLC plate was developed with 0.5% ninhydrin in
acetone. A known g-aminobutyric acid (GABA) producer L. brevis
LSF 8-13 was used as positive control. The amount of glutamate that
Lactobacillus strains consumed was measured by a biochemical
analyzer (YSI 7100MBS, YSI Inc., Yellow Springs, Ohio, USA) using
glutamate specific membrane. Assuming that consumed glutamate
was fully converted to GABA by the intracellular glutamate
decarboxylase enzyme the GABA production of our strains was
calculated. The protein concentrations of the cell suspensions were
determined as mentioned above.
743
2.7. Nham fermentation
One loopful of stock cultures of L. plantarum BCC 9546 and N7501 were cross-streaked on half strength MRS agar and half strength
MRS agar plates containing 1500 mg/ml neomycin, respectively and
were incubated at 30 C for 24e48 h. A single colony of bacteria was
transferred into 5 ml of MRS broth and incubated at 30 C for 15 h.
Cells were harvested by centrifugation at 25 C at 8000 g for 5 min
and washed with 5 ml of 0.1% sterile peptone water. Finally, the cell
concentration was adjusted to 107 cfu/ml with 0.1% sterile peptone
water. Nham sausages were mainly produced at Product Development Division, Department of Livestock, Pathumthani using the
standard recipe (Visessanguan et al., 2004), and transported to
the laboratory for further analysis. Nham was prepared by mixing
ground pork (52%), cooked pork rind (35%), sucrose (0.4%), garlic
(4.3%), salt (1.9%), cooked rice (4.3%), sodium erythorbate (0.2%),
sodium tripolyphosphate (0.2%), monosodium glutamate (0.2%),
whole bird chili (2%), and potassium nitrite (0.01%). Half of the
sausages (two batches) were prepared with a modified formula
which contained the same ingredients but only 3% of garlic. The
ingredients were thoroughly mixed and stuffed into plastic casings
(3.0 cm diameter, approximately 200 g each). A total of four batches
of Nham were prepared by combining two kinds of starter cultures
(the wild-type and N750-1) with two kinds of Nham formulations
(normal formulation and reduced garlic (3%) formulation). Nham
sausages were incubated at 30 C for 7 days. Samples were taken at
0 h, 12 h and every 24 h up to 7 days.
Nham sample (25 g) was aseptically transferred to a sterile
plastic bag and pummeled at 230 rpm for 30 s in a stomacher Model
400 (Seward, England), with 225 ml of sterile peptone water.
Appropriate decimal dilutions of the samples were prepared using
the same diluent and 0.1 ml of each dilution was plated in triplicates
on half strength MRS agar containing 0.5% CaCO3 incubated at 30 C
for 1 day for total LAB count. After that, all colonies were replicaplated on half strength MRS-neomycin plates (1500 mg/ml of
neomycin sulfate) containing 0.5% CaCO3 and incubated at 30 C for
2 days for neomycin-resistant mutant count. Direct pH measurements were carried out using a pH meter.
2.8. Statistical analysis
Results are expressed as means standard deviations of triplicate analyses for each sample unless otherwise stated. A one-way
analysis of variance and Duncan’s multiple range tests were used to
establish the significance of differences among the mean values
at the P < 0.05 significance level. The statistical analyses were
performed using SPSS version 15.0 for Windows (2006).
3. Results and discussion
3.1. Isolation of acid-sensitive mutants of L. plantarum BCC 9546
Approximately 6,700 spontaneous neomycin-resistant mutants
(600 mg/ml neomycin sulfate) were obtained with a frequency of
105e106. Only one mutant was found to have its culture broth pH
near the desired pH 4.6 after 72 h of growth (strain R5 with a culture
broth pH of 4.4). Whereas, among the mutants resistant to 750 mg/ml
neomycin sulfate, strain N750 had a culture broth pH of 4.5 at 24 h
which decreased to pH 4.19 at 72 h. To generate mutants with
resistance to higher neomycin concentration, R5 and N750 were restreaked on half strength MRS plates containing 1500 mg/ml neomycin
sulfate. Twenty-six colonies of R5 mutants were found not to lower
the pH of the MRS broth below 4.4 during 72 h of incubation at 30 C.
In addition, four of N750 mutants resistant to 1500 mg/ml neomycin
sulfate had their culture pH above 4.4 after 72 h of incubation at 30 C.
P. Jaichumjai et al. / Food Microbiology 27 (2010) 741e748
From the above mutants with culture broth pH not lower than 4.4
after 72 h, three representative strains were selected for further
studies, namely: R5 (resistant to 600 mg/ml neomycin), R5-18 (an
R5-derivative resistant to 1500 mg/ml neomycin) and N750-1
(an N750-derivative resistant to 1500 mg/ml neomycin).
different pHs (pH 4e7, this pH range resembles that of in Nham
between the beginning and the end of fermentation). Table 1 shows
the ATPase activity of L. plantarum BCC 9546 and its acid-sensitive
mutants at different acidic pHs in the presence (A) and absence (B)
of DCCD, a relatively specific inhibitor of the F0 part of the F0F1ATPase. The difference in ATPase activity with or without DCCD was
also calculated and since it is the activity inhibited by DCCD this
value can be considered as the activity of Hþ-ATPase (Table 1C). The
ATPase activity of all mutant strains were significantly lower than
that of wild-type strain at all conditions and at pH 4 the ATPase
activity of all mutants decreased below the values at other pHs
(5e7). Since the Hþ-ATPase activity (DCCD inhibited) of the wildtype strain was significantly higher than those of all mutant strains
at the above pH interval it indicates that the mutants had reduced
membrane-bound Hþ-ATPase activity. It is postulated that mutants
with reduced Hþ-ATPase activity do not generate sufficient energy
to concentrate neomycin, therefore they are resistant to neomycin
because neomycin uptake by bacterial cells is energy dependent
process (Kanner and Gutnick, 1972). Such defect in the energy
metabolism in our acid-sensitive mutants might explain their
growth characteristics and reduced acid production in MRS broth.
3.2. Growth characteristics of the representative mutants
Growth and acid production of the three acid-sensitive mutants
compared to parent strain BCC 9546 are shown in Fig. 1 A and B,
respectively. After 8 h of culturing, the cell number of R5-18 and
N750-1 were similar to that of the wild-type strain, while the viable
count of R5 was slightly lower. Afterwards, until 48 h of cultural
time, the viable count of the three mutants declined by 3 to 10 fold
and were lower than that of the wild-type strain. However, after 48 h
the cell number of R5 and R5-18 increased again and surpassed that
of the wild-type strain while the viable count of N750-1 remained
stagnant (Fig. 1A). The acid production of all strains increased until
24 h culture time followed by virtually no or substantially less acid
production in the next 24 h. While no more acids were produced by
the parent strain and N750-1 the acidity of the culture broths of
R5 and R5-18 were increased considerably between 48 and 72 h
(Fig. 1B). Overall, N750-1 produced about half as much acid as the
wild-type after 72 h, while the acid production of the other two
mutants did not differ much from that of N750-1 until 48 h. The
tendencies observed in acid production were also reflected in the pH
values of the culture broths (Fig. 1B). The pH of the culture broth of all
mutants differed from that of the wild-type strain by one pH unit at
8 h and the pH of their culture broths did not decrease between 24
and 48 h. Yet after 48 h the culture broth pH of R5 and R5-18
decreased nearly to the pH value of the wild-type strain’s culture.
Only the culture broth pH of N750-1 (w4.5) was substantially higher
than that of the wild-type at the end of the 72 h culturing period.
Taken together these observations the cell number increase of R5
and R5-18 accompanied by increased acid production and culture pH
reduction between 48 and 72 h suggest that the mutations that
made R5 and R5-18 acid-sensitive are unstable and can be reversed
after 48 h of continuous culture. This makes R5 and R5-18 not suitable for the application as Nham starter cultures. On the other hand,
the acid-sensitivity of N750-1 is probably caused by a stable
mutation and seem to be at the desired level, since this strain appear
to reduce the pH of the culture broth no lower than about 4.5, what
makes it a good candidate for a new Nham starter culture.
3.4. Measurement of internal pH
In order to characterize the pH homeostasis of acid-sensitive
mutants, their internal pH upon energization with glucose
was investigated. Internal pH (pHi) measurements revealed that
the mutants had significantly lower pHi (by about 0.2 pH unit) at
external pH 7.0 than that of wild-type (Table 2). At acidic condition
(pH 5.0) the internal pH of the mutants were also lower than the
pHi of the wild-type however only that of N750-1 did by a significant margin. At pH 5.0 the difference between external and internal
pH (DpH) of the parent strain was one unit, whereas N750-1 could
maintain a DpH of only 0.43, and it is likely that acidification of
the cytoplasm under acidic condition affects the physiology of the
latter strain considerably. Lactic acid is a weak organic acid with
increased proportion of its protonated (neutral) form at low pH that
can freely pass the cell membrane, then dissociating at the more
alkaline cytoplasmic pH (Kashket, 1987). This possibly results in the
disturbance of the internal pH regulation in N750-1, at least, due to
its reduced Hþ-ATPase activity, therefore it cannot maintain its
cytoplasmic pH near neutral at external pH 5.0 as the wild-type
strain.
In order to reveal the overall effectiveness of other internal pH
maintenance processes DCCD was applied during the internal pH
measurements to inhibit Hþ-ATPase. A representative experiment
shown in Fig. 2 demonstrates that after the cells were first energized by addition of glucose (10 mM), addition of DCCD resulted in
3.3. ATPase activity under acidic conditions
To investigate the cause of acid-sensitivity, we measured the
ATPase activity of the wild-type and the three mutant strains in
9.5
6.0
pH (solid lines)
B 6.5
log cfu/ml
A 10.0
9.0
8.5
8.0
7.5
7.0
2.5
2.0
5.5
1.5
5.0
4.5
1.0
4.0
.5
3.5
0
24
48
Time (h)
72
0.0
0
24
48
Total Acidity (%, dashed lines)
744
72
Time (h)
Fig. 1. Viable cells (A), pH and acidity (B) in the MRS culture broth of L. plantarum strains. C, B: L. plantarum BCC 9546; ;, 7: R5; A, >: R5-18; -, ,: N750-1. The black and
white symbols in Fig. 1 B represent pH and acidity, respectively. The symbols represent means standard deviation (error bars) obtained from three independent experiments.
P. Jaichumjai et al. / Food Microbiology 27 (2010) 741e748
Table 1
ATPase activity of L. plantarum BCC 9546 and its acid-sensitive mutants at different
pHs (pH 4e7).
pH 4.0
pH 5.0
pH 6.0
pH 7.0
(A) ATPase activity (nmol Pi min1 mg protein1) assayed in the absence of DCCD
69.28 4.56bcC 73.99 7.83cC
Wild-type 50.15 6.75aB 63.85 4.36bC
R5
3.53 0.48aA
6.93 1.04bA
9.13 0.54cA
8.62 0.54cA
R5-18
6.50 2.23aA 15.35 1.67bB
13.59 0.57bB 14.03 1.61bB
N750-1
3.37 0.69aA
4.65 2.06abA
8.55 1.17cA
6.32 2.16bcA
Strain
pH 5.0
pH 6.0
(B) ATPase activity assayed in the presence of DCCD
41.61 3.56bcC
Wild-type 19.70 4.21aA 37.60 3.11bC
R5
0.57 0.46aB
2.41 1.42bA
3.86 0.42cA
R5-18
2.37 0.60aB
9.93 2.21cB
7.15 0.72bB
N750-1
1.90 1.20aB
4.21 1.86abA
6.12 1.45bAB
Strain
pH 4.0
pH 5.0
pH 6.0
pH 7.0
27.67 6.62aB
5.27 0.52bA
6.44 1.03aA
2.43 0.57bA
28.54 5.37aB
5.67 0.71bA
6.44 2.63aA
1.50 1.15abA
a decline in internal pH. After energization the cytoplasm became
more alkaline than the extracellular buffer, and assuming
that DCCD inhibited proton translocating ATPase, decrease in the
internal pH upon DCCD addition was likely the result of a net
proton leak through the cell membrane. When the rate of internal
pH decrease was measured following the DCCD addition, the
results revealed that (Table 2) the wild-type had the smallest
proton leak among the strains at both tested pHs, while the internal
pH decrease rate was highest in N750-1 at acidic condition (pH 5.0).
This also implies that the mechanisms involved in pH homeostasis
excluding the Hþ-ATPase are probably the weakest in N750-1
among the strains at least at acidic conditions. Moreover, that is
coupled with the lowest Hþ-ATPase activity in N750-1 which is
probably the main reason behind that this strain has the smallest
internal pH at external pH 5.0. Since these parameters indicate
disturbed pH homeostasis and smaller cellular energy level
(smallest DpH), it is reasonable to assume that all these factors
combined have also a profound impact on the survival of this strain
in acidic environment.
Table 2
Internal pH and proton leak in L. plantarum BCC 9546 and its acid-sensitive mutants
at extracellular pH (pHex) 7.0 and 5.0.
Internal pHa
Proton leakb
pHex
Wild-type
R5
R5-18
N750-1
DCCD
7.20
Val
7.15
7.10
0
45.45 4.78cC
2.94 0.32bcA
7.59 1.91bcB
4.82 1.89bAB
Hþ-ATPase ¼ (Total ATPase in the absence of DCCD) - (Total ATPase in the presence
of DCCD).
Mean values and standard deviations obtained from three independent experiments. Different capital letters (AeC) in the same column as well as different letters
(aec) in the same row indicate significant differences (P < 0.05).
Strain
Glu
7.25
7.00
pH 7.0
DCCD inhibited
(C) ATPase activity inhibited by DCCD
26.25 4.35aC
Wild-type 30.45 8.09aB
R5
2.96 0.45aA
4.52 1.70bAB
R5-18
4.13 2.07aA
5.43 2.91aB
N750-1
1.46 1.06abA
0.44 0.43aA
7.30
pH 7.0
pH 5.0
pH 7.0
pH 5.0
7.46 0.02
7.22 0.04*
7.22 0.03*
7.25 0.01*
6.02 0.16
5.83 0.38
5.58 0.23
5.43 0.03*
0.007 0.009
0.023 0.016
0.009 0.001
0.022 0.001
0.014 0.006
0.024 0.007
0.027 0.015
0.052 0.015
Results are means standard deviation obtained from three (a) or two (b)
independent experiments.
*Statistically different (P < 0.05) from that of the wild-type strain at the same pH.
a
Measured as described in Section 2.
b
Unit of internal pH decrease (per minute) in energized cells after a few minutes
of DCCD addition, see Fig. 2.
Nig
7.05
þDCCD
pH 4.0
+
H -leak
7.35
I nternal pH
DCCD
7.40
5
10
15
Time (min)
Fig. 2. Proton leakage in the mutant N750-1 at pH 7.0 upon DCCD addition. Glucose
(10 mM, Glu) energized the cells preloaded with cFSE in 150 mM KPO4 buffer containing
1 mM MgSO4 followed by the addition of Hþ-ATPase inhibitor DCCD (final concentration
10 mM) that triggered an internal pH decline attributed to Hþ-leak (arrow). The DJ and
DpH component of the proton motive force was dissipated by the addition of 2 mM
valinomycin (Val) and 2 mM Nigericin (Nig), respectively. This figure is a representative of
two independent experiments that gave similar results.
3.5. Measurement of arginine deiminase activity
The arginine deiminase activity may also contribute to the pH
homeostasis in some lactic acid bacteria (Marquis et al., 1987)
including a L. plantarum strain from orange (Arena et al., 1999)
therefore the ammonium ion production from arginine by our
L. plantarum strains was investigated. Fig. 3 shows that wild-type
and mutant L. plantarum strains produced very small amounts of
ammonium ion. Their ammonium ion production profiles were not
significantly different from each other at every sampling point. The
ammonium ion concentrations in the culture broths of L. plantarum
strains were slightly higher after 24 h of culturing (by 30e50%)
than at the beginning of the cultivation, while the culture broth
pH of these strains were dropped to 4.7 at 9 h without a change
onwards (data not shown). However, this small ammonium ion
concentration increase might not be the result of arginine deiminase activity, since similar ammonium ion concentration pattern
was observed cultivating the wild-type strain, L. plantarum BCC
9546, in arginine-free GYP medium (43% increase). The virtually no
ammonia production by any of our L. plantarum strains and their
7
NH4+ (µmol/mg protein)
Strain
745
6
5
4
3
2
1
0
0
24
48
72
Time (h)
Fig. 3. Ammonium ion production by L. plantarum and P. acidilactici strains in GYPeArg
medium at 30 C for 72 h. C: L. plantarum BCC 9546; ;: R5; A: R5-18; -: N750-1;
:: P. acidilactici BCC 9545; B: L. plantarum BCC 9546 in GYP medium. The symbols
represent means standard deviation (error bars) obtained from two independent
experiments.
746
P. Jaichumjai et al. / Food Microbiology 27 (2010) 741e748
slightly decreasing culture broth pH is a sharp contrast to the
ammonium ion production by the positive control (P. acidilactici)
which also coupled with culture broth pH increase to pH 8.3 (data
not shown). Therefore, considering all these findings, we conclude
that arginine cannot be metabolized by our L. plantarum strains.
3.6. Measurement of glutamate decarboxylase activity
The glutamate decarboxylase catalyzes the a-decarboxylation of
glutamic acid to yield g-aminobutyric acid and carbon dioxide. This
intracellular reaction results in the decrease of cytoplasmic Hþ-ion
concentration and a slight increase of the cytoplasmic pH. Fig. 4
demonstrates that all of our L. plantarum strains were able to
produce GABA from glutamate, albeit at a seemingly lower extent
than the positive control L. brevis LSF 8-13. To quantify the GABA
production the amount of glutamate consumed by L. plantarum
strains were measured by biochemical analyzer. It was found that
N750-1 showed the lowest GABA production (0.07 0.12 mmol/mg
protein/24 h), while the wild-type produced nearly twice as much
GABA (0.12 0.04 mmol/mg protein/24 h). The other two mutants,
R5 and R5-18, produced about twofold more GABA than the wildtype strain (0.26 0.02 and 0.24 0.05 mmol/mg protein/24 h,
respectively). According to the Hþ-ATPase activity measurements
(Table 1C) N750-1 showed less activity than R5 and R5-18
which probably result in less available cellular energy for solute, e.
g. glutamate, transport in this strain. That would provide a possible
explanation for this strain’s lowest glutamate decarboxylase
activity although the likelihood of down-regulation or mutation in
the glutamate decarboxylase enzyme could not be ruled out. On
the other hand, R5 and R5-18 had higher glutamate decarboxylase
activity than that of the wild-type, which might be due to a slight
up- regulation as a compensatory response to their acid-sensitivity.
Nevertheless, these results indicate that the moderate/weak
glutamate decarboxylase activity of these strains might play only
a marginal role in their pH homeostasis and this effect might be
realized over a longer period of time (24 h), therefore it might not
provide effective protection against the much faster cytoplasmic
acidification. This latter notion is also supported by the growth
experiment data (Fig. 1), which showed almost no difference in
cell number, total acidity and culture broth pH among the tree acidsensitive mutants in MRS culture up to 48 h. This suggest that in
spite of glutamate most likely available in MRS broth the higher
glutamate decarboxylase activity of R5 and R5-18 is not able to
make these mutants more acid-resistant than N750-1 with the
least glutamate decarboxylase activity (until 48 h).
3.7. Nham fermentation
From among the mutants only N750-1 was selected for Nham
fermentation trials because of its lowest Hþ-ATPase activity,
mutation stability and because its MRS broth pH was the highest
after 72 h (around pH 4.48, Fig. 1B). This mutant and the wild-type
strain (WT, L. plantarum BCC 9546) were used in two formulas, (1):
normal formula (using 4.3% garlic) and (2): limiting carbon source
in Nham by decreasing concentration of garlic to 3%.
Table 3 shows the number of total lactic acid bacteria (LAB) and
of the mutant during Nham fermentation. The mutant was counted
by replica-plating on half strength MRS-neomycin plates (1500 mg/
ml of neomycin sulfate) to confirm that most of the colonies that
grew on these plates were N750-1. The initial inoculation level
was approximately 104 cfu/g Nham in all four treatments. The
viable count of total LAB and N750-1 increased until 12 h then the
bacterial counts of all Nham formulations were slightly decreased
until the end of incubation time (7 days). However, the number of
Fig. 4. Glutamate conversion to g-aminobutyric acid by Lactobacillus strains as demonstrated with TLC. Glu: glutamate (negative control), GABA: g-amino butyric acid, L. b. 8-13:
Lactobacillus brevis LSF 8-13 (positive control), WT: wild-type (L. plantarum BCC 9546). This TLC profile is a representative of three independent experiments that gave similar
results.
P. Jaichumjai et al. / Food Microbiology 27 (2010) 741e748
747
Table 3
Number of LAB and acid-sensitive mutant starter culture cells (log cfu/g) in Nham during 7 days at 30 C.
Time (day)
0
0.5
1
2
3
4
5
6
7
N750-1-normal
N750-1-3% G
WT-normal
WT-3% G
Total LAB
N750-1
Total LAB
N750-1
Total LAB
Total LAB
4.26 0.02a
8.41 0.34a
8.41 0.33abc
8.42 0.23abc
8.20 0.32ab
8.03 0.21b
8.08 0.12bc
8.00 0.18a
8.09 0.12a
4.00 0.06bc
8.10 0.35a
8.04 0.33c
8.08 0.05bc
7.97 0.19b
7.73 0.07c
7.85 0.11cd
7.72 0.10b
7.77 0.22b
4.25 0.03a
8.50 0.19a
8.53 0.16ab
8.42 0.29abc
8.42 0.26a
8.06 0.18ab
8.05 0.21bc
8.07 0.08a
8.06 0.11a
3.98 0.03c
8.10 0.19a
8.23 0.12bc
8.05 0.17c
8.02 0.15b
7.72 0.09c
7.74 0.10d
7.74 0.17b
7.73 0.18b
4.16 0.14ab
8.45 0.28a
8.81 0.07a
8.48 0.16a
8.43 0.04a
8.31 0.10a
8.44 0.17a
8.23 0.04a
8.06 0.11a
4.13 0.14abc
8.41 0.30a
8.55 0.28ab
8.45 0.21ab
8.32 0.17ab
8.30 0.10a
8.32 0.14ab
8.02 0.20a
7.95 0.11ab
Mean values and standard deviations obtained from three independent experiments. Different letters (aed) in the same row indicate significant differences (P < 0.05). N7501-normal: N750-1 acid-sensitive starter culture was used in normal Nham formulation. N750-1-3% G: N750-1 acid-sensitive starter culture was used in 3% garlic Nham
formulation. WT-normal: wild-type starter culture was used in normal Nham formulation. WT-3% G: wild-type starter culture was used in 3% garlic Nham formulation.
total LAB in Nham fermentation using the wild-type or N750-1
starter were about three times higher than the viable count of the
N750-1 starter culture strain in Nham. This means that the number
of N750-1 starter cells represents about one third of the total LAB
cell number in Nham. This affirms that in Nham inoculated by the
mutant starter acid-sensitive L. plantarum cells play a major (or
dominant) fermentative role. Comparing the pH of Nham (Table 4)
in all trials shows that the pH values at 0 and 12 h in both wild-type
and mutant fermented sausages were not significantly different but
after 24 h there is a significant difference in pH of Nham fermented
by different starters until 7 days. In Nham fermented by the wildtype the pH decreased lower than 4.6 and faster than Nham
fermented with mutant in normal formulation. N750-1 as a new
starter culture gave Nham a consistent final pH about 4.5 until 7
days (in normal formulation) while wild-type fermented Nham had
a pH about 4.6 between the 1st and the 2nd days. Using 3% of garlic
did not result in a product with significantly higher pH than that of
produced with the traditional 4.3% garlic content neither when the
acid-sensitive mutant nor when the wild-type strain was used as
starter. Although, when the mutant was used as a starter at the end
of 7 days of fermentation the pH of Nham formulated with 3% garlic
was higher than that of Nham formulated with 4.3% garlic by about
0.2 units. This implies that garlic amount reduction in the formulation of Nham may contribute to less acid production in Nham
fermented with the mutant. Concerning the safety of Nham fermented by the mutant, the sausages formulated with 3% garlic were
above the critical pH limit of 4.6 (Paukatong and Kunawasen, 2001),
therefore these are not considered to be safe. However the mutant
fermented Nham with 4.3% garlic content had safe pH (4.6 or
Table 4
Change in pH of Nham during 7 days at 30 C.
Time (day)
N750-1-normal
N750-1-3% G
WT-normal
WT-3% G
0
0.5
1
2
3
4
5
6
7
6.10 0.03a
5.78 0.12a
5.21 0.09a
4.84 0.11a
4.69 0.13a
4.60 0.15a
4.55 0.18a
4.53 0.16a
4.48 0.15a
6.07 0.10a
5.81 0.17a
5.22 0.07a
4.88 0.05a
4.77 0.11a
4.68 0.15a
4.66 0.13a
4.67 0.16a
4.67 0.19a
6.07 0.06a
5.76 0.07a
4.97 0.05b
4.45 0.04b
4.32 0.05b
4.28 0.04b
4.22 0.02b
4.19 0.03b
4.19 0.02b
6.07 0.04a
5.76 0.15a
4.97 0.05b
4.47 0.08b
4.33 0.07b
4.30 0.07b
4.28 0.04b
4.25 0.09b
4.24 0.06b
Mean values and standard deviations obtained from three independent experiments.
Different letters (aeb) in the same row indicate significant differences (P < 0.05).
N750-1-normal: N750-1 acid-sensitive starter culture was used in normal Nham
formulation. N750-1-3% G: N750-1 acid-sensitive starter culture was used in 3% garlic
Nham formulation. WT-normal: wild-type starter culture was used in normal Nham
formulation. WT-3% G: wild-type starter culture was used in 3% garlic Nham
formulation.
below) from the 4th day onwards. Therefore, the above results
indicate that acid-sensitive L. plantarum has a potential for use as
starter culture for Nham production to prevent over-fermentation.
4. Conclusions
Spontaneous acid-sensitive L. plantarum BCC 9546 mutants
were isolated which appeared to have reduced Hþ-ATPase activity.
Experimental Nham fermented by one of the acid-sensitive mutant
starters (N750-1) had significantly higher pH than Nham fermented
by the wild-type strain and could be kept at ambient temperature
for four days without over-fermentation.
Acknowledgements
Authors would like to thank the National Center for Genetic
Engineering and Biotechnology (BIOTEC) for the financial support
for the project BT-B-02-NG-BC-5003 and this research is partially
supported by the Center of Excellence on Agricultural Biotechnology, Postgraduate Education and Research Development Office,
Commission on Higher Education, Ministry of Education, Thailand.
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