production of Lactobacillus plantarum LP02 isolated
from infant feces with potential cholesterol-lowering
Hwang1* Jen-Ni Chen2 Yu-Ting Huang1
and Zhang-Yi Mao1
1Department of Food Science
and Technology, HungKuang University, No. 34, Chung-Chie
Road, Shalu District, Taichung City 43302,Taiwan, R.O.C.
2Department of Food Science
and Biotechnology, National ChungHsing University, 250,
KuoKuang Road, Taichung City 402, Taiwan, R.O.C.
*Corresponding author. E-mail:
firstname.lastname@example.org. +886-4-2631-8652 #5009; Fax:
Accepted 9 June, 2011
The potentially hypocholesterolemic strain, designated PL02, of
Lactobacillus plantarum, was isolated from infant feces.
The aim of this
was to characterize and to cultivate this isolate for biomass
production in a 5 L fermentor by batch or fed-batch
fermentation. A modified medium composition without peptone was
used to produce more viable cells with a 150 rpm agitation speed
and 0.5 vvm of aeration during incubation. A cell concentration
of 2.2 g dry cell weight (DCW) per liter in fermented broth was
reached in a 5 L fermentor after the glucose was consumed
completely during batch fermentation. In addition, biomass was
significantly improved at 28 h of fed-batch fermentation (9.45 g
DCW/l) over a constant feeding rate of 20 ml/h of feeding
solution since the glucose was consumed at batch process. A much
shorter fermentation time (15 h) and greater biomass (10.12 g
obtained by using multistep feeding rates to maintain glucose
concentration of 1 to 5 g/l during fed-batch process. The
biomass of L. plantarum PL02 produced by fed-batch
fermentation was greater than that achieved by batch
fermentation and the fed-batch method might also be suitable for
other lactic acid bacteria fermentation.
Lactobacillus plantarum, fermentation, fed-batch
Probiotics, microorganisms beneficial to human or animals, have
recently gained greater interest for use in the industry.
Numerous papers have described their physiological functions,
such as inhibiting of pathogenic microorganisms, lowering
cholesterol levels, enhancing immunity, alleviating lactose
intolerance, improving nutrition and digestion, and conferring
anticancer effects (Hirayama and Rafter, 2000; Leroy and Vuyst,
2004; Wollowski et al., 2001).
Some lactic acid bacteria (LAB) have been reported to possess a
cholesterol-lowering ability (Buck and Gilliland, 1994; Lye et
al., 2010). The possible mechanisms for this ability have been
reported by Lye et al. (2010) under
conditions that mimic
of the human gastrointestinal tract. Oral administration of
sufficient amounts of fermented Lactobacillus acidophilus
products has been claimed to decrease the concentration of blood
cholesterol in humans (Schaafsma et al., 1998). Gilliland et al.
(1985) proposed that the cholesterol might be adhered to or
absorbed into the LAB cells when these strains are cultured with
cholesterol. LAB might absorb cholesterol in the
to prevent absorption back into the body in enterohepatic
circulation (Grill et
Production of lactic acid and its derivates from batch or
fed-batch fermentation of LAB has been studied extensively (Peng
et al., 2006; Racine and Saha, 2007; Tan and Ding, 2006; Wee and
Ryu, 2009; Xu et al., 2003). Jiang et al. (2009) improved
phenyllactic acid (PLA) production from the strain
sp. SK007 by intermittent fed-batch fermentation supplemented
with a suitable precursor, phenylpyruvic acid. Production of
bacteriocins, such as nisin or pediocin, has also been studied
in fed-batch fermentation
(Castro et al., 2007;
Papagianni et al., 2007).
LAB metabolite production is generally studied in
batch or fed-batch fermentation. However, medium formulations
and fermentation conditions for LAB industrial production are
rarely reported due to the commercial advantage for the company
marketing the strains.
is one of the most common LAB found in fermented foods.
Survival, functional and probiotic properties of
in the human intestinal tract have been reviewed (de Vries et
al., 2006). Recently, Jeun et al. (2010) reported a strain of
with hypocholesterolemic effects in mice.
on growth and kinetics of
produced in different conditions during the fermentation of
edible sea weeds was studied by Gupta et al. (2010).
Many LAB related papers described the production of lactic acid
or other metabolites by batch or fed-batch fermentation;
however, there are limited papers describing LAB biomass
production for human, animal or industrial use.
The purpose of this study was to develop a suitable industrial
medium and a set of applicable conditions for producing biomass
of L. plantarum LP02 isolated from infant feces with
potential cholesterol-lowering ability by batch and fed-batch
fermentation. Biomass production from the fed-batch fermentation
of the isolated strain was carried out by controlling the
glucose concentration of the media at 1 to 5 g/l during the
Materials And Methods
Isolation and identification of potential cholesterol-lowering
Strains were isolated from healthy babies as previously reported by
Kimura et al. (1997). Fresh feces (1 g) from newborn babies at local
hospitals in Taiwan was suspended and sequentially diluted in 9 ml
sterile water. Aliquots of 100 μl of the diluted samples were spread
onto Rogosa agar plates and incubated at 37°C for 24 to 48 h. The
colonies that displayed sufficient growth were cultivated in MRS
medium at 37°C for 24 to 48 h and preserved at -70°C in a 10%
glycerol solution. The isolated strains were assayed for
characte-ristics, such as catalase activity, Gram stain type,
morphology, mobility and acid/oxgall tolerance.
The isolated strains were further screened for potential
cholesterol-lowering strains according to the procedure in Ahn et
al. (2003). The MRS agar plates containing 0.5%
(TDCA) and 0.37 g/l CaCl2 were made holes by an aseptic
glass tube with a diameter of 5 mm. Aliquots of 50 µl supernatant
from centrifuging the fresh culture of isolated strains were
inoculated into the holes in the agar plate and incubated at 37°C
for 48 h. The precipitate halo that formed around some of the holes
was considered indicative of a cholesterol-lowering strain. During
the screening process, the cholesterol-lowering strain
Lactobacillus acidophilus ATCC 43121 was used as a positive
Strain identification was performed by the Biosource Collection and
Research Center (BCRC) of the Food Industry Research and
Development Institute (FIRDI, Hsinchu, Taiwan). The design of the
carbohydrate fermentation was determined using the API 50CHL kit and
16S rRNA sequencing was performed using total DNA from the
experimental isolate and the reference strain Lactobacillus
plantarum BCRC 10069.
The compositions of culture media used in batch and fed-batch
fermentations are as follows. MRS broth supplemented with 0.05% L-cysteine
was used for strain activation and cultivation of seed culture. The
base medium used for both batch and fed-batch fermentations of L.
plantarum LP02 in 5 L fermentors consisted of (per liter)
glucose, 10 g; yeast extract, 40 g; mono sodium glutamate (MSG), 2
g; L-cysteine, 0.2 g; MgSO4·7H2O, 0.2 g; KH2PO4,
0.2 g; FeCl3, 0.05 g; and MnSO4· 4H2O,
0.05 g. All the compounds were first dissolved in appropriate
amounts of distilled water and then sterilized at 121°C for 20 min.
The feed medium composition (1 L) was composed of (per liter)
glucose, 500 g; yeast extract, 50 g; MSG, 10 g; KH2PO4,
0.2 g; MgSO4.7H2O, 0.2 g; L-cysteine,
0.2 g; MnSO4.4H2O, 0.05 g; and FeCl3,
0.05 g. The glucose
was dissolved in 700 ml deionized water and sterilized separately
before it was mixed with other compounds, which were also dissolved
and sterilized in 300 ml of water before use.
Strain activation, cultivation and
The isolated strains were incubated on a MRS agar plate for 24 to 48
h and then grown in MRS broth to produce a seed culture. For
Gram-staining, a loop of fresh culture was spread on a glass slice
and then stained with crystal violet solution for 1 min. The slide
was then washed and iodine solution was added. Then, the slide was
washed again with ethanol and stained finally with safranin for 30
s. After the stained slide dried in the air, it was examined by
microscopy. Cells with a deep blue color were identified as
Gram-positive, while a red color indicated Gram-negative cells.
Catalase activity was measured by spreading one loop of fresh
culture on a slide and then applying H2O2 (35%
concentration) to the culture; the production of bubbles indicated
positive activity, while no bubbles meant a negative activity. To
determine the strain’s motility, 50 ul of fresh culture was
inoculated onto the concave side of a slide with a glass cover slip
to observe the motility. To evaluate tolerance to acids and bile
salts, 1 ml of fresh culture was mixed with 9 ml of
phosphate-buffered saline (PBS) solution (pH 2, 2.5, 3.2 adjusted by
HCl) and incubated at 37°C, 80 rpm for 3 h. Cell viability was
determined by counting the number of colony forming units (cfu)
produced after inoculation of serially diluted culture onto MRS agar
plates. The viable colony count for the control was obtained by
serially diluting the cells with acid-free PBS buffer control at pH
7.2 PBS buffer and counting the cfu produced in agar plates. Two
milliliter aliquots of the acid-treated samples (pH 2.0 PBS for 3 h)
were centrifuged at 6,000 rpm for 10 min to test for bile salt
tolerance. The cell pellet was resuspended in pH 7.2 PBS and added
to 10 ml MRS broth containing 0.3% (W/V) of oxgall and the cultures
were grown at 37°C for 24 h. Samples were taken at time intervals
(3, 12 and 24 h) to determine viable cell counts on MRS agar plates.
All cultures were performed in triplicate in 50 ml Erlenmeyer flasks
containing 10 ml of the corresponding medium at 37°C for 24 h.
Incubation of strains in a solution of cholesterol-phosphatidylcholine
A cholesterol-phosphatidylcholine micelle solution was
prepared according to the method of
Gilliland et al. (1985).
First, 10 mg of cholesterol and 22 mg of egg phosphatidylcholine
were dissolved in chloroform and dried by nitrogen gas. Then, 10 ml
of a sucrose solution (0.4 M) was added to the dried material, and
the mixture was vortexed for 15 min total, with a pause every 5 min.
Finally, a mixture of 10 ml of freshly prepared cholesterol-phosphatidylcholine
micelles solution and 10 ml of MRS-thio broth, containing 0.2%
sodium thioglycollate and 0.3% oxgall, was inoculated with 0.2 ml of
fresh culture (incubated 16 h). After 24 h of cultivation at 37°C
and centrifugation, the cell pellet was washed twice and resuspended
in the same volume of sterile water. Both the supernatant and the
cell suspension solution were used for further analysis of the
Characteristics of L. plantarum LP02 used in this study.
Bile salt tolerance
Others: Gram-positive, catalase-negative, rod-shape,
*Counts are converted to log10 CFU/ml.
Each value represents mean ± SD from three different experiments.
Data in the same row with different letters are significantly
different at p < 0.05.
Batch and fed-batch fermentations
Batch fermentation was conducted in a 5 L fermentor (Biotop BTF-A5L,
Taiwan). Three liters of the base medium was sterilized in the
fermentor at 121°C for 30 min. Fermentation was carried out at 37°C
in media with pH 6.25 (achieved by
addition of 5 N NaOH), using the previously determined agitation and
aeration parameters. A 1% solution of seed culture, cultivated in
MRS medium for 16 h, was used as the inoculum. Samples were taken
every two hours for analysis of cell density and glucose
concentration. Fed-batch fermentation was carried out in a 5 L
fermentor with 2 L of initial working base medium. When the glucose
consumed reached approximately 1 g/l,
at a constant rate of 20 ml/h until
end of fermentation or at multi stages of feeding rates to maintain
glucose concentration of 1 to 5 g/l by measuring the glucose level
and adjusting the feeding rates immediately after each sampled time
point. The conditions of pH, temp, agitation and aeration parameters
were the same as in the batch fermentation. Volume productivity was
calculated based on the biomass production rate per liter, per hour.
During the fermentation, the cell density and glucose concentration
were measured in triplicate.
The glucose concentration was determined by the DNS method. The DNS
reagent contained the following: 3, 5-dinitrosalicyclic acid, 1 g;
potassium sodium, 30 g; and sodium hydroxide, 1.6 g in 100 ml
deionized water. A mixture of 1 ml of appropriately diluted
supernatant and 1 ml of DNS reagent was boiled for 5 min. Before
measuring the cell density at OD550 nm, 3 ml of distilled
water were added to the reaction solution. The glucose concentration
was calculated by comparing the experimental value obtained to a
glucose standard curve. The concentration of lactic acid was
measured by high-pressure liquid chromatography (HPLC) (LC-10AT,
Shimadzu, Japan) with a Sypergi 4 µ Fursion-RP80A column (250 x 4.6
mm, Phenomenex, USA) and a micro-guard column maintained at 25°C.
Cell growth was measured dry cell weight (DCW) per liter. To
determine the DCW, cells of known optical density were pelleted from
a 10 ml sample and washed once with water. The washed cell pellet
was dried at 80°C for 24 h and weighed. A conversion factor of 3.20
± 0.08 g DCW/l-OD was determined and used to estimate the biomass
from the optical density measured at 600 nm. Viable counts (cfu/ml)
were conducted by the spread plate method on MRS agar supplemented
with 0.05% L-cysteine.
Cholesterol concentration was determined according to the protocol
reported in Gilliland et al. (1985). A mixture containing 0.5 ml of
supernatant or pellet suspension, 3 ml 95% ethanol and 2 ml 50% NaOH
was heated in a 60°C water bath for 10 min. Then, 5 ml n-hexane and
3 ml distilled water were added to the solution, shaken for 20 s and
incubated for 15 min at room temperature to allow for separation.
The solution in the organic layer was dried by blowing N2
gas during incubation in a 60°C water bath. Next, 4 ml of o-phthalaldehyde
reagent was added to the dry sample, gently mixed with 2 ml
concentrated sulfuric acid and incubated for 10 min at room
temperature. The absorbance at 550 nm was measured to calculate the
cholesterol concentration by comparing the experimental value with a
range (0 to 50 ppm) of standard cholesterol solutions.
Data were expressed as mean ± standard error of the mean. Treatments
were compared using one-way analysis of variance followed by Tukey’s
tests for multiple comparisons between means. P-values less than
0.05 were considered statistically significant.
Results and Discussion
Characterization of the isolated strain
The isolated strain was rod-shaped, Gram-positive, catalase-negative,
non motile, and acid/oxgall tolerant as shown in Table 1. Based
on these results, the strain was identified as lactic acid
bacteria. The acid tolerance of this strain was similar to those
strains reported by Havenaar et al. (1992) and Marteau et al.
(1997) which were analyzed in vitro and in vivo in conditions
that mimic the environment of the digestive tract. This strain
exhibited satisfactory growth at pH values of 2.0, 2.5 and 3.2
for 3 h. An average of 0.05 to 2.8 log cfu/ml decrease also
showed that the strain was not greatly affected by the acid. To
simulate an intestinal environment, the cell pellet that was
acid tolerant to pH 2.0 was next inoculated in MRS medium
supplemented with 0.3% oxgall and incubated for 24 h. The
isolated strain is also tolerant to bile salts, as evidenced by
the similar cell viability count obtained between the experiment
and control conditions after incubation for 12 h.
Strain identification reported from FIRDI showed that partial
sequencing of the 16S rRNA from the isolate had a 99% identity
with that of L. plantarum BCRC10069. In addition, results
from a biochemical assay using the API 50CHL kit also suggested
that the isolate was a strain of L. plantarum. Therefore,
the isolate was confirmed as a strain of L. plantarum and
was designated L. plantarum LP02.
Cholesterol concentration in the cells and supernatant after
incubation of different lactic acid bacteria with a
cholesterol-containing solution for 24 h.
*Initial cholesterol concentration in culture broth was around
100 μg/ml which was estimated using the concentration of
cholesterol-phosphatidlycholine mycelles added to the media.
Each value represents the mean ± SD from three different
experiments. Data in the same column with different letters are
significantly different at p < 0.05.
Cholesterol concentration in the supernatant and the cells
The hypocholesterolemic effects of LAB in various animals and
human are strain specific. Gilliland et al. (1985) isolated
strains with the ability to reduce cholesterol concentration in
serum by screening for strains with the ability to lower the
cholesterol concentration in broth. To evaluate the
cholesterol-lowering ability of the isolated LAB, the
concentration of choles-terol in both the cells and supernatant
after incubation with cholesterol-phosphatidylcholine micelles
solution was determined and compared to that of a known
cholesterol-lowering strain, L. acidophilus BCRC17010. As shown
in Table 2, final cholesterol concentrations in the cells and
supernatant of the L. plantarum LP02 culture were 42.59 µg/ml
and 31.11 µg/ml, respectively when the strain was incubated for
24 h in a solution with an initial concentration of
approximately 100 µg/ml of cholesterol estimated using the
concentration of cholesterol-phosphatidlycholine mycelles added
to the solution. Table 2 also indicated that strains
Lactobacillus rhamnosus GG, Lactobacillus paracasei 33,
Lactobacillus bulgaricus, and Bifidobacterium longum
exhibited relatively low concentration of cholesterol in cells.
During incubation of this isolated strain, the cholesterol
concentration gradually decreased in the supernatant and
increased in the cells (Figure1); this finding supported the
hypothesis that the isolated strain had potent
cholesterol-reducing ability. The reduction of cholesterol in
the supernatant might be explained by absorption of cholesterol
onto the cell surface or into the cells. Some LAB can modulate
bile acid excretion which can cause a lowering of plasma
cholesterol levels. The cholesterol-decreasing ability may
result from LAB’s co-precipitation with the deconjugated bile
salts by strains of bacteria that produce bile salt hydrolase
(Pereira et al., 2003). Some Lactobacillus species have been
proven to reduce blood cholesterol levels and are used as
probiotics (de Vries et al., 2006). However, there are few
reports about a decrease of cholesterol concentration or
absorption of cholesterol by the strain L. plantarum. Recent
studies have demonstrated that L. plantarum may exert beneficial
cholesterol reducing effects of increasing bile acid excretion
in mice (Jeun et al., 2010). In addition, the strain L.
plantarum PH04 isolated from infant feces was also evaluated for
its potential as a cholesterol-reducing probiotic in mice
(Nguyen et al., 2007). There are five possible mechanisms for
the removal of cholesterol from media by lactobacilli (Lye et
al., 2010). In this result, cholesterol-lowering effects might
be due to the deconjugation of bile salts by the enzyme bile
salt hydrolase (Ahn et al., 2003) or the cholesterol might be
removed by either binding to the bacterial cellular surface or
by being absorbed into the cells.
To produce L. plantarum LP02 biomass for industrial use,
composition of the commercial medium, for example, the source of
carbon and nitrogen sources should be studied beforehand.
Different levels of glucose concentration (0 to 50 g/l), yeast
extract (0 to 50 g/l) and sodium glutamate (0 to10 g/l) were
tested to find the most suitable media composition for
obtaining a greater amount of biomass. Figure 2 shows the
effects of different levels of glucose, yeast extract and sodium
glutamate on the cell density of the strain L. plantarum
for 24 h incubation. Results from the use of more suitable
medium compositions are outlined in Table 3. The media
composition with 10 g/l of glucose, 40 g/l of yeast extract and
2 g/l of sodium glutamate could reach the highest cell density
of 1.6 to 2 g (DCW)/l which was similar to that of using the
commercial MRS medium. This medium contained the cheaper,
industrial quality of yeast extract and sodium glutamate,
without using the peptone, a more expensive nitrogen source.
Factors known to affect the production of LAB metabolites, such
as lactic acid or bacteriocin, during fermentation by LAB
include medium composition (carbohydrate source, sugar
concentration and growth factors), the presence of oxygen, level
of pH and concentration of the product (Burgos-Rubioetal et al.,
2000; Todorov and Dicks, 2006). However, there are few reports
describing the influences of medium composition on viable cells
production for commercial purpose. An example elucidated the
growth medium, free from animal-derived ingredients, with
potential for commercial cultivation of probiotics strains for
application in vegetarian foods (Heenan et al., 2002). However,
the optimized medium compositions for commercial production of
LAB biomass are different depending on the various
characteristics of the strains.
Optimization of the fermentation process improved the
development of higher biomass production to ensure its
economical viability. Biomass production from the strain L.
plantarum LP02, cultivated both by batch and fed-batch
fermentations in 5 L fermentors, depended on the procedural
conditions, such as agitation speed, aeration, and pH values.
Figure 3 shows that agitation speeds of 150 or 300 rpm during
batch fermentation without aeration enhanced the cell density
when compared with no agitation. This result suggested that the
isolated strain might be oxygen tolerance. However, biomass
production with an agitation speed of 150 rpm and aeration at 1
vvm exhibited a significantly lower cell density (Figure 4).
Biomass production with 0.5 vvm was similar to that
without aeration. Higher cell densities were observed in the
initial period of fermentation when the aeration was 1.0 vvm;
however, the final cell density was lower than that without
aeration or at 0.5 vvm. These data showed that the strain is
microaerophilic, because the higher oxygen concentrations
hindered cell growth. Fu and Mathews (1999) found that when L.
plantarum was cultivated for lactic acid production from
lactose, the cell yield was higher under aerobic conditions, but
the lactic acid production was higher under anaerobic
conditions. Appropriate control of aeration rate during the
process of batch or fed-batch fermentation is important for the
L. plantarum biomass production.
A time course for L. plantarum LP02 cell growth by batch
fermentation was carried out in a 5 L fermentor at 37°C, at 150
rpm, pH 6.2 and without aeration. As shown in Figure 5, cell
density and biomass productivity reached the highest levels of
2.53 g/l and 0.43 g/l-h, respectively, at 8 h of fermentation.
The glucose was almost completely consumed at 8 h, a
significantly shorter time period than that of significantly
shaking flask fermentation, which took 16 h to accomplish the
same level of glucose depletion (data not shown). In addition,
the biomass yield based on consumed glucose reached its highest
level of 0.30 g DCW/g at 4 h. All the data showed that biomass
production and glucose consumption in the 5 L fermentor was
faster than cultivating in flask. After 8 h of fermentation,
cell density, biomass yield and productivity significantly
decreased due to the limited glucose concentration in the
Medium composition for production of
biomass determined by this study.
A high concentration of medium composition (500 g/l glucose and
50 g/l yeast extract) was added at a constant rate of 20 ml/min
or at multi-stage rates to maintain an appropriate glucose
concentration. A constant rate (20 ml/min) of feeding was
started at 8 h of fermentation when the glucose concentration
was very low (1.12 g/l) and cell density was at 7 OD600nm (2.5 g
DCW/l) (Figure 6). Cell density significantly increased to 28
OD600nm (9.45 g DCW/l) at 28 h of fermentation after
maintaining the glucose concentration at an average value of
1.32 g/l. The highest productivity, 1 g/h-l, was obtained at 9 h
of fermentation, in the initial period of feeding. Biomass yield
based on the consumed glucose was found to have the highest
yield, 0.30 g/g at 4 h of fermentation. The yield dropped
closely to 0 at 8 h and sharply increased to 0.10 g/g at 9 h,
one hour after feeding and then gradually reduced during the
rest of the fermentation process. When comparing batch and
fed-batch fermentations, a significantly greater amount of
biomass and a higher productivity rate were observed during
fed-batch fermentation, although, longer incubation time and
greater amounts of materials were required.
To investigate the effect of glucose on biomass production,
different concentration of glucose were maintained in the
cultures during fermentation. Biomass production was conducted
by increasing the feeding rate to adapt the glucose
concentration between 1 and 5 g/l by manually changing the
feeding rate at every sampling time after 8 h of fermentation.
Results showed in Figure 7 indicated that cell density reached
the highest biomass of 31 OD (around 10.12 g DCW/l) at 15 h and
the highest volume productivity of 1.72 g/l-h for 10 h. The
average productivity after feeding was 1.11 g/l-h and was higher
than that achieved with a constant feeding rate (Figure 6). The
highest biomass yield (0.24 g/g) was recorded at 4 and 7.5 h of
sampling times and then gradually decreased over time. Changing
the feeding rates to maintain glucose level at 1 to 5 g/l
significantly shortened the fermentation time required to reach
the same bio-mass concentration comparing with that at a
constant feeding rate and the highest productivity was
Biomass production could be further improved by modifying
fed-batch fermentation. Hayakawa et al. (1990) obtained a large
dried biomass (40 g/l) at 34 h of continuous fed-batch
fermentation by recycling cells through cross-filters in
fermentors to remove lactic acid. Four strains of LAB were
produced by fed-batch fermentation and evaluated as feed
additives for weaned piglets (Gurrea et al., 2007). A mathematic
model was created to produce lactic acid in membrane bioreactor
through fermentation of Lactobacillus casei at a high cell
density (98.7 OD at 600 nm) (Boudrant et al., 2005); this
density was seven-times higher than the cell density achieved in
fed-batch fermentation. A cell-recycle fermentation system was
also established for the production of lactic acid (Xu et al.,
2006) and in the future, could be developed for the production
of not only metabolites but also biomass.
Characterization of L.
plantarum LP02 showed that it is acid and bile tolerant,
a characteristic important for the LAB to survive
gastrointestinal ingestion. The potential
hypocholesterolemic effects of the isolated strain L.
plantarum LP02 require the support of additional
research before application, especially in industrial or
human use. The isolated strain was definitively identified
as L. plantarum by the report of FIRDI to match the
sequence of the 16S RNA gene of the isolate, as well as
being identified by an API 50 CHL kit. Strains with
cholesterol-lowering properties as functional starter
cultures may enhance quality of end product besides
probiotic characteristics. The microaerobic characteristic
of this strain is beneficial for biomass production in
fermentors because agitation is needed during the process of
pH control or feeding. Cell density was significantly
improved from 2.53 g DCW/l, with batch fermentation to 10.12
g DCW/l with fed-batch fermentation, while controlling the
feeding rate to maintain an appropriate glucose
concentration. The lactic acid concentration should be
monitored during the process because high concentrations can
slow cell growth during the batch or fed-batch fermentation.
Modified continuous fermentation or fed-batch fermentation
with cell recycle through a membrane to remove lactic acid
might enhance the cell density. Besides, cell density and
biomass yield might be further improved through other
fed-batch strategies based on exponential feeding or with
feedback control, such as DO stat. The potential
cholesterol-reducing strain isolated in this study may be
added to fermented dairy products to achieve
hypocholesterolemic properties. The success of using a
functional starter culture in a particular food is strongly
strain dependent and is crucial for rational selection. The
applied strains will be adapted to the process conditions
and the intrinsic
factors inherent to the food.
We thank the National Science Council for the financial
support of this work through the projects of
NSC97-2622-B-241-001-CC3 and 98-EC-17-A-17-S1-128-2.
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