International Journal of Food Science and Technology 2014, 49, 565–570 565

Original article
Acid adaptation to improve viability and X-prolyl dipeptidyl
aminopeptidase activity of the probiotic bacterium Lactobacillus
fermentum HA6 exposed to simulated gastrointestinal tract
conditions

Son Chu-Ky,* Thi-Khanh Bui, Tien-Long Nguyen & Phu-Ha Ho*

Department of Food Technology, School of Biotechnology and Food Technology, Hanoi University of Science and Technology, 1 Dai Co
Viet, Hai Ba Trung, Hanoi, 10000, Vietnam
(Received 25 May 2013; Accepted in revised form 12 August 2013)

Summary In this work, the viability of the probiotic bacterium Lactobacillus fermentum HA6 isolated from naturally
fermented vegetables in Vietnam was improved by growing the bacterium into a mild acid condition (pH
4.0). Viability and probiotic functionality [X-prolyl dipeptidyl aminopeptidase (PepX) activity] of the
acid-adapted bacterium exposed to simulated gastrointestinal conditions were investigated. After 180 min
in the simulated gastric juice (0.3 g/L pepsin, pH 2.0), the viability of acid-adapted L. fermentum HA6
(11.5%) was higher than that of control L. fermentum HA6 (2.2%). Specific PepX activity of acid-adapted
cells (24.5 U/mg) was higher than that of control cells (17.8 U/mg). After 180-min exposure to the simu-
lated small intestinal medium (0.3 g/L bile salts, 0.1 g/L pancreatin, pH 8.0), the viability of acid-adapted
L. fermentum HA6 (13.5%) was twofold as high as that of control L fermentum HA6 (8.0%). Our results
suggested that acid adaptation has a key role in acquiring cross-protection mechanism, which in this study
resulted in higher survival of L. fermentum HA6 after simulated gastrointestinal stresses. The strategy of
acid adaptation could be valuable for the production of robust probiotics.
Keywords Acid adaptation, cross-protection, Lactobacillus fermentum HA6, probiotic, simulated gastrointestinal condition, viability,
X-prolyl dipeptidyl aminopeptidase probiotic.

may have more favourable technological and sensorial

Introduction
Probiotics are live microorganisms that when adminis-
tered in adequate amounts confer a health benefit on
the host. A probiotic strain is required to meet criteria
such as appropriateness, technically suitable competi-
tiveness and functionality (FAO/WHO, 2002). In other
words, it need to be safe, properly identified, stable,
technically amenable to mass production and storage,
as well as to give desirable organoleptic quality when
applied to food. Essential criteria are to survive in the
human gut condition, adhere onto the colon and
promote the host’s health. Probiotic strains can be
isolated from human compartments or foods (Ho &
Adams, 2006; Kaushik et al., 2009). Naturally fer-
mented foods are abundant sources for selection of
health benefit bacteria. Probiotics originated from foods
*Correspondent: Fax: +84 4 3868 2470;
emails: ha.hophu@hust.edu.vn; son.chuky@hust.edu.vn
characteristics than that from human and therefore
have greater suitability in probiotic food processing.
Dairy products have been intensively reported as food
vehicles for probiotic supplementation, whereas non-
dairy probiotic foods are in a less number (Granato
et al., 2010). Probiotic bacteria isolated from nondairy
sources such as fermented vegetables could have
potential in incorporation back into nondairy food
matrix with high survival rate and suitable technologi-
cal properties.
In Vietnam, bacteria isolated from fermented foods
in recent years have been studied mostly in the view of
bacteriocin source (Cintas et al., 2001; Cao-Hoang
et al., 2013). These data on human probiotic applica-
tion in Vietnam are still limited. Recently, a potential
probiotic Lactobacillus fermentum HA6 strain, isolated
from fermented vegetables, was identified and was
found to have antifungal activity against eight
among ten tested food-originated fungi. This strain

doi:10.1111/ijfs.12338
© 2013 Institute of Food Science and Technology
566 Improved viability and PepX activity S. Chu-Ky et al.
demonstrated inhibitory ability towards proliferation
of four types of human cancer cell in vitro (Ho &
Adams, 2006). This strain was also reported to have
X-prolyl dipeptidyl aminopeptidase (PepX, EC
3.4.14.5) activity to digest exorphin products of incom-
plete digestion of food proteins, which may lead to the
accumulation of opioid peptides in brain and resulting
neurological disorders for example autism spectrum
disorders (Ho et al., 2011; Muro Urista et al., 2011).
Reichelt et al. (1997) described abnormal peptide con-
tent in the urine of most autistic patients. Exorphin
opioids (e.g. casomorphins, gluteomorphins and gliad-
omorphins) derived from casein and gluten were found
to cross the blood–brain barrier and cause social indif-
ference symptoms. It was suggested that autism was
based on genetic errors of peptide digestion, possibly
of enzyme dipeptidyl peptidase IV (DPP IV or PepX,
EC 3.4.14.5), and autistic symptoms could be
explained by stimulant activity of exorphin towards
the brain (Reichelt et al., 1997; Shattock & Whiteley,
2002; Reichelt & Knivsberg, 2003; Shattock et al.,
2004). Although conflicting reports exist (Hunter et al.,
2003), addition of ingestible genomeceutical com-
pounds, which increase the user’s expression of DPP IV
or like substances, or supplementing with probiotics to
improve autism spectrum disorders has been proposed
(Brudnak, 2002, 2004).
The selection of potentially probiotic strains requires
the demonstration of their resistance to the stress con-
ditions present in the transit through the upper gastro-
intestinal tract in order to ensure their optimal
functionality (Kaushik et al., 2009). The acidic envi-
ronment of the stomach and the bile salts secreted in
the duodenum are major impediments to the survival
of ingested bacteria. The acid resistance of probiotics
is also critical in food applications, as acidity is
believed to be one of the main detrimental factors
affecting the viability of bifidobacteria in fermented
foods. It has been recognised that exposure to a mild
stress can result in improved resistance to subsequent
exposures, either to more extreme forms of the same
stress or to other stresses. These phenomena are
referred to as acquired stress resistance and cross-pro-
tection (Bourdineaud et al., 2003; van Bokhorst-van
de Veen et al., 2011; Mills et al., 2011; den Besten
et al., 2013).
Collado & Sanz (2007) have isolated acid-resistant
derivative Bifidobacterium species by an overnight cul-
ture in DeMan–Rogosa–Sharpe (MRS) medium at pH
2.0 and characterised these acid-resistant strains to
determine the changes associated with the acquisition
of an acid-tolerant phenotype. The acid-resistant cells
showed better ability to grow in the presence of bile
salt (1–3%) and NaCl (6–10%) and higher resistance
at elevated temperatures (60–70 °C, 10 min) than the
parental strains. Moreover, the acid-resistant strains
International Journal of Food Science and Technology 2014
displayed higher fermentative ability and enzymatic
activities. The successful use of prolonged exposures to
acid stress to improve the stability of human bifido-
bacteria indicates that this strategy could be useful for
the production of robust probiotic strains.
In this work, acid adaptation techniques have been
used to improve the viability of the probiotic bacte-
rium strain Lactobacillus fermentum HA6, isolated
from Vietnamese traditionally fermented vegetables,
through the exposure to simulated gastrointestinal
tract conditions. In addition, the effect of acid adapta-
tion on the probiotic functionality, PepX activity of
control and acid-adapted L. fermentum HA6 was also
examined after they were exposed to simulated gastro-
intestinal tract conditions.
Materials and methods
Bacterial strains, media and adaptation protocols
Lactobacillus fermentum HA6 isolated from naturally
fermented vegetables in Vietnam was cultured at 37 °C
in MRS medium. Stock cultures (kept frozen
at 80 °C) were grown until the stationary phase, then
diluted into MRS medium and incubated for 24 h at
37 °C. To obtain control cells, second subculture was
used to inoculate MRS medium at pH 6.2 at an initial
optical density of 0.1 (OD600), and L. fermentum HA6
were harvested at OD600 = 6. To obtain acid-adapted
cells, second subculture was used to inoculate MRS
medium at pH 4.0 at an initial OD600 = 0.1, and acid-
adapted cells were harvested at OD600 = 4. Both
OD600 = 6 and 4 for control and acid-adapted cells
were correspondent to the beginning of stationary
phase (Fig. 1).
Figure 1 Growth of Lactobacillus fermentum HA6 under control
(pH 6.2) conditions (control cells) and at pH 5.0, 4.0 and 3.0 (acid-
adapted cells).
© 2013 Institute of Food Science and Technology

Exposure of Lactobacillus fermentum HA6 strain to
simulated gastrointestinal tract conditions
This essay was to evaluate the ability of control (non-
adapted) and acid-adapted L. fermentum HA6 strain
to survive the passage through the gastrointestinal
tract as well as to express PepX activity. Cell suspen-
sions (109–108 cells/mL) were exposed to simulated
gastric juice (0.3 g/L pepsin), adjusted at pH 2.0 with
HCl, in sterile saline solution (0.5% w/v NaCl) and,
subsequently, to simulated small intestinal juice (pan-
creatin (0.1 g/L) and bile salts (0.3 g/L) in sterile saline
solution (0.5% w/v NaCl) adjusted at pH 8.0). Cell
suspensions were incubated at 37 °C in simulated gas-
tric medium, and samples were taken at initial time
(0 min) and 180 min after gastric exposition to deter-
mine the viability by plate counting on MRS agar and
PepX activity. Gastric-exposed cells were harvested by
centrifugation and washed twice with sterile saline
solution (0.5% w/v NaCl). Cell pellets were then sus-
pended in simulated small intestinal medium and were
incubated at 37 °C. Samples were taken at 180 and
360 min during small intestinal exposition to deter-
mine the bacterial viability and PepX activity.
Determination of viability
Cell viability was determined using the plate count
method on MRS agar medium. Cell suspension was
taken at appropriate times and diluted in appropriate
dilutions, which were spread on Petri dishes containing
MRS agar medium. These Petri dishes were then incu-
bated at 37 °C for 24 h. The viability was determined
by comparing the numbers of shocked and control
L. fermentum HA6 colonies. Each experiment was per-
formed with three independent cultures.
Preparation of cell-free extract
Lactobacillus fermentum HA6 were cultured in MRS
medium at volume of 20 mL at 37 °C, then centri-
fuged at 2000 g at 4 °C and washed three times with
cold PBS buffer. The pellet was resuspended in 2 mL
of Tris–HCl 50 mM at pH 7.5 and then subjected to
sonication on ice for three intervals of 3 min using
ultrasonic homogeniser. Suspensions of sonicated cells
were centrifuged at 15 000 g at 4 °C for 30 min, and
the resulting supernatants were transferred to new
1.5-mL Eppendorf tubes and stored at 20 °C.
Determination of PepX activity
PepX activity was determined using spectrophotometric
method as previously described (Gallo et al., 2005) with
some modifications as follows: PepX activity was mea-
sured in terms of p-nitroaniline released from glycyl-
prolyl-4-nitroanilide (Gly-PropNA) substrate (Sigma).
© 2013 Institute of Food Science and Technology
Improved viability and PepX activity S. Chu-Ky et al. 567
The assay mixture contained 100 lL of 1 mM Gly-Pro-
pNA in 50 mM Tris–HCl buffer at pH 7.5 and 100 lL
of enzyme preparation, cell-free extract or cell suspen-
sion. The mixture was incubated at 37 °C for 30 min.
The absorbance was measured at 415 nm with a
microplate reader (Bio-Rad). One unit (U) of PepX
activity was defined as the amount of enzyme required
to liberate 1 nmol of p-nitroaniline per min under the
assay conditions. These data obtained were compared
with a standard curve setup using p-nitroaniline (con-
centration from 0.01 to 1 mM). Protein concentration
was determined using Bradford assay (Bradford,
1976). Specific enzyme activity was expressed in units
of PepX activity per mg of protein.
Statistical analysis
The mean values (cell viability and PepX activity) and
standard deviation were calculated from three indepen-
dent experiments. The significance of the difference
between the mean values was determined using the
analysis of variance (ANOVA). The confidence interval
for a difference in the means was set at 95% (P ≤ 0.05)
for all comparisons.
Results and discussion
Effects of acid stress condition on the growth of
Lactobacillus fermentum HA6
To test different sublethal acid conditions, Lactobacillus
fermentum HA6 cells were cultivated in the MRS media
at different pH, 6.2 (control), 5.0, 4.0 and 3.0. The
growth of L. fermentum HA6 at different pH is pre-
sented in Fig. 1. The specific growth rate (l) of cells
cultured under control conditions (pH 6.2) was 0.19 1/
h, whereas those of cells cultured at pH 5.0, 4.0 and 3.0
decreased to the values of 0.17, 0.11 and 0.06 1/h,
respectively. Three acid conditions tested have an
impact on the growth of L. fermentum HA6, especially
on the specific growth rate and the final biomass: the
lower the pH of adaptation, the smaller the specific
growth rate and the final biomass. The pH 4.0 was cho-
sen to harvest acid-adapted cells for further experi-
ments as a clear effect of acid stress condition was
observed at pH 4.0, and the final biomass obtained by
growth in pH 4.0 represented approximately 70% of
the final biomass obtained in control conditions
(Fig. 1). Acid-adapted cells were harvested for further
analyses at the beginning of the stationary phase.
Effects of acid adaptation on the survival of Lactobacillus
fermentum HA6 during the exposure to the simulated
gastrointestinal tract conditions
To assess the effects of acid adaptation on the viability
of L. fermentum HA6 during the passage through
International Journal of Food Science and Technology 2014
568 Improved viability and PepX activity S. Chu-Ky et al.
the gastrointestinal tract, cell suspensions (108–109
cells/mL) were exposed to simulated gastrointestinal
conditions, which composed of two successive stresses:
a gastric stress (0.3 g/L pepsin and pH 2.5) and an
intestinal stress (0.1 g/L pancreatin and 0.3 g/L bile
salts at pH 8.0). Cell suspensions were withdrawn at
different times during gastrointestinal expositions to
determine the viability by plate count on MRS agar.
The results are shown in Fig. 2.
Effects of acid adaptation on the viability of Lactobacillus
fermentum HA6 during the exposure to the simulated
gastric condition
Cell viability was determined at the beginning (0 min)
and 180 min during the exposure to simulated gastric
condition to assess the impact of an acid adaptation
on the survival of L. fermentum (Fig. 2).
We found that an acid adaptation induced a
decreased growth of L. fermentum HA6. In control
condition (pH 6.2), the biomass of L. fermentum HA6
reached 1.9 109 CFU/mL, but the biomass of acid-
adapted L. fermentum HA6 only reached 2.0
109 CFU/mL. When control and acid-adapted cells
exposed to simulated gastric condition, we observed
significant decreases in survival of both types of cells and
their survivals were 3.8 107 and 2.3 107 CFU/mL,
representing a reduced viability of 1.7 log and 0.9 log,
respectively. In other words, only 2.2% of control cells
survived during 180 min of simulated gastric stress.
On the other hand, a total of 11.5% of acid-adapted
cells survived during 180 min of the same shock. It is
worth noting that acid adaptation significantly
increased the survival rate of L. fermentum HA6.
These results are in accordance with those obtained
by Collado & Sanz (2007) and confirmed the impor-
10
Control cells
Bacterial count (log CFU/ml)
Acid-adapted cells
8
6 this acid adaptation allowed L. fermentum HA6 to
4 acid stress but also to a stress of another nature (high
2
0 Effects of acid adaptation on the PepX activity of
0 min 180 min 360 min
Figure 2 Survival rates expressed by log CFU/mL of control and
acid-adapted Lactobacillus fermentum HA6 when exposed to simu-
lated gastric tract (during 0–180 min) and subsequently small intesti-
nal juice (during 180–360 min) conditions.
International Journal of Food Science and Technology 2014
tance of an adaptation of a microorganism to a mild
stress condition increase its survival to a subsequent
stress of the same nature (Bourdineaud et al., 2003;
Collado & Sanz, 2007; To et al., 2011). Cruz et al.
(2010, 2012a,b) have investigated the effect of increased
glucose oxidase concentration as a technological option
to decrease oxidative stress on the probiotic bacteria
during the processing of probiotic yogurts. In another
work recently conducted by the same group (Cruz
et al., 2013), these authors suggested that the use of
packaging systems with different oxygen permeability
rates coupled with the addition of glucose oxidase pre-
sented an interesting technological option to minimise
the oxidative stress in probiotic yogurts.
Effects of acid adaptation on the viability of Lactobacillus
fermentum HA6 during the exposure to the simulated
small intestinal condition
Control and acid-adapted cells survived to simulated
gastric condition were harvested by centrifugation and
subjected to simulated small intestinal condition. Cells
viability was determined at 180 and 360 min during
the exposure to simulated small intestinal condition in
order to evaluate the impact of an acid adaptation on
the survival of L. fermentum HA6 (Fig. 2). When con-
trol and acid-adapted cells exposed to simulated small
intestinal condition, we did not observe significant
changes in survival of both types of cells, and after
180 min of exposure, their survivals reached the same
value of 2.9 106 CFU/mL, representing a reduced via-
bility of 1.1 log and 0.9 log for control and acid-
adapted cells, respectively. In other words, only 8.0%
of control cells survived during 180 min of simulated
small intestinal stress. On the other hand, a total of
13.5% of acid-adapted cells survived during 180 min
of the same stress. In term of viability expressed in
CFU/mL, no significant difference was observed. How-
ever, the survival rate of acid-adapted cells was signifi-
cantly higher than that of control cells (13.5% vs.
8.0%). These results are in accordance with those
obtained by Chu-Ky et al. (2013) and could suggest a
cross-protection effect of an acid adaptation. Indeed,
improve its survival when exposed to a subsequent
pH, bile salts in this work) (van Bokhorst-van de Veen
et al., 2011; Singh & Jiang, 2012).
L. fermentum HA6 during the exposure to the simulated
gastrointestinal conditions
Results of experiments in the previous part demon-
strated positive effects of acid adaptation on the
viability of the probiotic bacterium L. fermentum HA6
© 2013 Institute of Food Science and Technology

Table 1 PepX activities of control and acid-adapted Lactobacillus
fermentum HA6 exposed to simulated gastrointestinal tract
conditions
PepX specific activity (U/mg)
Small intestinal
Parameters Initial Gastric condition condition
Control cells 39.5  0.2 a
17.8  0.2b
30.8  3.2c
Acid-adapted cells 37.4  0.1a 24.5  0.7b 23.0  1.1c
Means with different letters in the same row are significantly different
(P < 0.05) by ANOVA, and PepX activity was measured before exposure
(Initial), at 180 min of exposure to gastric condition and subsequently
at 180 min of exposure to small intestinal condition.
exposed to simulated gastrointestinal tract conditions.
The fact whether it would convey the same impact on
functionality of the test bacterium should be taken in
consideration. In this study, PepX activity, one of a
number of probiotic properties of L. fermentum HA6
previously characterised by Ho el al. (Ho & Adams,
2006), was investigated during transition through sim-
ulated gastrointestinal tract conditions with control
and acid-adapted cells. Results on PepX activities are
shown in Table 1. Both control and acid-adapted bac-
terial cells showed similar patterns of enzyme activity
when cells were exposed to gastric intestinal juices. A
decrease in specific PepX activity was observed after
180-min exposure to simulated gastric condition; how-
ever, the specific activity of acid-adapted bacterium
after gastric exposure remained at a higher level (24.5
U/mg) in comparison with that of control cells (17.8
U/mg). This result also suggested a positive effect of
acid adaptation on PepX activity of the probiotic bac-
terium when exposed to a low pH medium such as
simulated gastric condition.
Interestingly, when cells subsequently exposed to
simulated small intestinal condition for another
180 min, specific PepX activity of control cells was
found to increase significantly from 17.8 to 30.8 U/mg,
whereas specific PepX activity of control cells was
found to decrease slightly from 24.5 to 23.0 U/mg.
This finding indicated the complication of cross-pro-
tection mechanism. Although higher viability of acid-
adapted bacterial cells exposed to simulated small
intestinal condition was observed, it was not necessary
to observe the same impact on cell metabolism.
In conclusion, acid adaptation of L. fermentum HA6
by growing the probiotic bacterium at pH 4.0 was
found to have a significantly positive impact on the
viability and the PepX activity of L. fermentum HA6
during exposure to simulated gastrointestinal tract
conditions. The results suggested that acid adaptation
could be a useful strategy for the production of robust
probiotic strains.
© 2013 Institute of Food Science and Technology
Improved viability and PepX activity S. Chu-Ky et al. 569
Acknowledgments
We thank Vietnam National Foundation for Science
and Technology Development (NAFOSTED) for
funding this work.
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