Short Communication

Direct lactic acid fermentation of Jerusalem artichoke tuber extract using

Lactobacillus paracasei without acidic or enzymatic inulin hydrolysis
Hwa-Young Choi
a
, Hee-Kyoung Ryu
a
, Kyung-Min Park
a
, Eun Gyo Lee
b
, Hongweon Lee
b
,
Seon-Won Kim
c
, Eui-Sung Choi
a,
a
Industrial Biotechnology Research Center, KRIBB, Daejeon, Republic of Korea
b
Biotechnology Process Engineering Center, KRIBB, Daejeon, Republic of Korea
c
Division of Applied Life Science (BK21 Program), EB-NCRC and PMBBRC, Gyeongsang National University, Jinju, Republic of Korea
a r t i c l e i n f o
Article history:
Received 28 December 2011
Received in revised form 23 March 2012
Accepted 24 March 2012
Available online 2 April 2012
Keywords:
Lactic acid
Lactobacillus paracasei
Jerusalem artichoke
Inulin
a b s t r a c t
Lactic acid fermentation of Jerusalem artichoke tuber was performed with strains of Lactobacillus
paracasei without acidic or enzymatic inulin hydrolysis prior to fermentation. Some strains of L. paracasei,
notably KCTC13090 and KCTC13169, could ferment hot-water extract of Jerusalem artichoke tuber more
efciently compared with other Lactobacillus spp. such as L. casei type strain KCTC3109. The L. paracasei
strains could utilize almost completely the fructo-oligosaccharides present in Jerusalem artichoke. Inulin-
fermenting L. paracasei strains produced c.a. six times more lactic acid compared with L. casei KCTC3109.
Direct lactic fermentation of Jerusalem artichoke tuber extract at 111.6 g/L of sugar content with a
supplement of 5 g/L of yeast extract by L. paracasei KCTC13169 in a 5 L jar fermentor produced 92.5
ce:hsp sp="0.25"/>g/L of lactic acid with 16.8 g/L fructose equivalent remained unutilized in 72 h. The
conversion efciency of inulin-type sugars to lactic acid was 98% of the theoretical yield.
2012 Elsevier Ltd. All rights reserved.
1. Introduction
Lacticacidis widelyusedinthefood, pharmaceutical andchemical
industries and is a platform chemical used for manufacturing of a
variety of chemicals including the bio-degradable polymer, poly-lac-
tic acid (Gao et al., 2011). The recent demand for the large-scale fer-
mentative production of lactic acid for poly-lactic acid requires the
supply of lowcost rawmaterials. Rawmaterials such as lignocellulo-
sics are cheap renewable substrate for lactic acid fermentation, but
use of lignocellulosic biomass requires extensive pretreatment and
saccharication(Abdel-Rahmanet al., 2011). Therefore, anadditional
carbon resource like fructans that can be produced from non-grain
crop plants such as Jerusalem artichoke should be considered for
cost-effective production of commodity chemicals suchas lactic acid.
In addition to sucrose and starch, fructans contribute to the pool of
storage carbohydrates in plants. Unlike the grain crops, Jerusalem
artichoke can grow well in non-fertile land and is resistant to plant
diseases, not competing with grain crops for arable land (Bajpai and
Bajpai, 1991). However, there have been few studies on lactic acid
production from Jerusalem artichoke. It has been considered that
inulin-type fructans in Jerusalem artichoke cannot be converted to
lactic acid by Lactobacillus sp. (Ge et al., 2009a). Therefore, acid
pretreatment of Jerusalem artichoke (Ge et al., 2009b) or mixed-cul-
tivation with an inulinase-secreting microorganismsuch as Aspergil-
lus niger (Ge et al., 2009a) was employed for efcient lactic acid
fermentation with Jerusalem artichoke.
On the other hand, there have been reports that certain species
of lactobacilli can ferment fructo-oligosaccharides (Kaplan and
Hutkins, 2000) and that a strain of L. paracasei can degrade even
long chain inulin-type fructans (Makras et al., 2005). The fructo-
oligosaccharide utilization operon of L. paracasei has been reported
(Goh et al., 2007) and the beta-fructosidase gene in this operon was
expressed for optimal production of bioethanol from grass fructan
(Martel et al., 2010). However, there has been no report that
L. paracasei can actually ferment Jerusalem artichoke without pre-
treatment for inulin hydrolysis.
We report in this study that strains of L. paracasei can efciently
ferment fructo-oligosaccharides with degree of polymerization
(DP) of up to 13 in Jerusalem artichoke without pretreatment for
inulin hydrolysis. The selected strains could also utilize inulin with
higher DP such as the one from dahlia.
2. Methods
2.1. Jerusalem artichoke
The Jerusalem artichoke dry powder was purchased from local
market. The hot-water extract of Jerusalem artichoke tuber was
0960-8524/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biortech.2012.03.075

Corresponding author. Address: Industrial Biotechnology Research Center,

KRIBB, 125 Gwahangno, Yuseong-gu, Daejeon 305-806, Republic of Korea. Tel.:
+82 42 860 4453; fax: +82 42 860 4489.
E-mail address: choi4162@kribb.re.kr (E.-S. Choi).
Bioresource Technology 114 (2012) 745747
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prepared by 30 min autoclave of the tuber powder in distilled
water at 150 or 180 g/L, respectively, for ask and fermentor culti-
vation. The extract was centrifuged at 10,000g for 15 min to re-
move debris. The monomeric sugar content of the extract was
determined by HPLC analysis after enzymatic hydrolysis of the ex-
tract with 1/50 volume of inulinase (Sigma Cat. No. I2017) at 37 C
for 15 h. The monomeric sugar content (glucose plus fructose) of
the hot-water extract for ask and fermentor cultivation was
90.6 and 111.6 g/L, respectively. The pH value of the suspension
was around 6.0. For ask cultures, pH of the suspension was ad-
justed by addition of 50 g/L of CaCO
3
. For fermentor study, the
pH was adjusted to 6.0 by addition of 10 N NaOH.
2.2. Strains and cultivations
Strains of Lactobacillus were purchased from the Korean Collec-
tion for Type Culture (Daejeon, Korea). The strains were main-
tained in Difco Lactobacilli MRS medium (Difco Laboratories).
For ask cultures, 50 mL of medium in 250 mL ask was inocu-
lated with overnight culture to give 0.1 OD
600
and incubated stat-
ically at 37 C for 72 h. Ten milliliter of distilled water or MRS
medium was added to 40 mL of hot-water extract of Jerusalem
artichoke tuber with 50 g/L of CaCO
3
for pH control. For assessment
of the capability of fermentation of high molecular weight inulin,
inulin from dahlia tubers (Sigma Cat. No. I3754) was added at
40 g/L to MRS medium in place of glucose. The ask experiment
was done in duplicate. For fermentor cultures, a seed culture
grown in 200 mL MRS medium was inoculated into a 5 L jar fer-
mentor (KoBiotech, Incheon, Korea) with 1.8 L of hot-water extract
of Jerusalem artichoke tuber with a supplementation of 5 g/L of
yeast extract (Difco Laboratories). The cultures were carried-out
at 37
o
C with agitation at 150 rpm without aeration for 72 h. The
content of sugars in the broth remained unutilized after fermenta-
tion was determined by inulinase treatment and shown as fructose
equivalent.
2.3. Analysis
The growth of cells was monitored by measuring the optical
density at 600 nm (OD
600
) using a UVICON930 spectrophotometer
(Kontron Analytical, Lucerne, Switzerland). Quantitative analysis of
lactic acid and sugars (glucose, fructose and sucrose) was per-
formed by HPLC (Agilent Technologies 1200 series) using an HPX
87H column (Bio-Rad, Hercules, CA, USA) and an RI detector. The
change in fructo-oligosaccharides prole during Jerusalem arti-
choke fermentation was analyzed qualitatively by HPLC (Agilent
Technologies 1200 series) on a Shodex Asahipak NH2P-50 column
with a mobile phase containing water and acetonitrile (35:65) at a
ow rate of 1 ml/min. Column temperature was set to 40 C and
the elution was monitored by RI detector. Fructo-oligosaccharides,
1-kestose (GF2), nystose (GF3) and 1-fructofuranosylnystose (GF4),
were from Wako Pure Chemical Industries (Osaka, Japan). The opti-
cal purity of the lactic acid produced was determined in triplicate
using Megazyme D-Lactate and L-Lactate Assay Kit (Megazyme
International Ireland Ltd., Wicklow, Ireland).
3. Results and discussion
3.1. Lactobacillus strains fermenting fructo-oligosaccharides in
Jerusalem artichoke
Six strains of Lactobacillus paracasei (KCTC3165, 3166, 3169,
3510, 13090 and 13169) and the Lactobacillus casei type strain
(KCTC3109) were tested for lactic acid fermentation of fructo-oli-
gosaccharides in Jerusalem artichoke. First, the strains were grown
in MRS medium. All the tested strains grew very well in MRS med-
ium without any signicant differences in growth or lactic acid
productivity (>17 g/L lactic acid from 20 g/L of glucose) (Table 1).
These strains were then cultivated in hot-water extract of Jerusa-
lem artichoke tuber without any supplement except for CaCO
3
for pH adjustment for 72 h. The lactic acid production was, how-
ever, rather inefcient (ranging from 6.0 to 16.5 g/L) (data not
shown), considering the high concentration of sugar (72.5 g/L) in
the tuber extract. This low productivity have been caused by lim-
ited growth of the strains in tuber extract (data not shown) possi-
bly due to a shortage of nutrients required for proper growth of
lactic acid bacteria in the tuber extract. When the Jerusalem arti-
choke tuber extract was supplemented with 1/5 strength of MRS,
most L. paracasei strains except for KCTC3166 efciently produced
lactic acid (Table 1). Notably, the strains KCTC13090 and
KCTC13169 showed more efcient production of lactic acid com-
pared with other L. paracasei strains. In contrast, the lactic acid pro-
duction was considerably low in the L. casei strain, conforming to
the previous study where acid pretreatment of the Jerusalem arti-
choke tuber for inulin hydrolysis was required for efcient lactic
fermentation with L. casei (Ge et al., 2009b). L. paracasei
KCTC13169, selected as the best inulin-fermenting strain, pro-
duced c.a. six times more lactic acid compared with L. casei strain
from the Jerusalem artichoke under the condition without any
pretreatments.
To assess the enhanced lactic acid production observed for L.
paracasei KCTC13169 in Jerusalem artichoke, the change in fructan
prole of the 72 h culture broth was analyzed by HPLC using ami-
no-column. L. paracasei KCTC13169 strain could almost completely
utilize the fructo-oligosaccharides with DP of up to 13, in contrast
to L. casei KCTC3109 strain that fermented mostly glucose and fruc-
tose, but not the fructo-oligosaccharides with higher DP (data not
shown).
3.2. Pronounced difference in inulin-fermenting capability with high
molecular weight inulin from dahlia as substrate
For assessment of the capability of fermentation of high molec-
ular weight inulin, MRS medium with dahlia inulin in place of glu-
cose was used. As shown in Table 1, the strains L. paracasei
KCTC3165, KCTC13090 and KCTC13169 produced 1317 g/L of lac-
tic acid from dahlia inulin in 48 h, whereas L. paracasei strains
KCTC3166, 3169, 3510 and L. casei KCTC3109 produced less than
2 g/L of lactic acid. It is not surprising that L. casei KCTC3109 could
not grow well in inulin-containing medium. It seemed that L. para-
casei strains KCTC3166, 3169 and 3510 could utilize, as carbon
source, the low molecular weight fructo-oligosaccharides in Jeru-
salem artichoke tuber extract, but are incapable of utilizing higher
molecular weight fructo-oligosaccharides in dahlia inulin. The
strain L. paracasei KCTC13169 was again the best among the tested
strains in the inulin-containing medium as well as in Jerusalem
artichoke tuber extract.
3.3. Fermentation from Jerusalem artichoke extract using L. paracasei
KCTC13169
Lactic fermentation of Jerusalem artichoke tuber extract by L.
paracasei KCTC13169 strain was studied in a 5 L jar fermentor
(Fig. 1). The fermentation medium at 111.6 g/L sugar content was
supplemented with yeast extract (5 g/L). In a preliminary experi-
ment, yeast extract supplementation at 5 g/L was found to ade-
quately, though not sufcient, support the growth of the strain in
Jerusalem artichoke tuber extract. The fermentation proceeded
very rapidly at the early stage of fermentation but slowed down
at the later stage producing 92.5 g/L of lactic acid in 72 h. During
fermentation, the concentration of fructose released from inulin
746 H.-Y. Choi et al. / Bioresource Technology 114 (2012) 745747
increased to ca 20 g/L followed by a slow decrease to 11 g/L at the
nal stage, leaving 16.8 g/L fructose equivalent unutilized, possibly
due to the shortage of certain nutrients. This may indicate that the
rate of inulin hydrolysis by this strain is higher than the rate of
conversion of sugars into lactic acid. The conversion efciency of
inulin-type sugars (94.8 g/L) to lactic acid (92.5 g/L) was 98% of
the theoretical yield. The optical purity was found 93.2% of L(+)-lac-
tic acid.
Selected strains of L. paracasei, notably the KCTC13169 strain
that does not require the addition of inulinase or heterologous
expression of inulinase for inulin-type sugar utilization can be a
more useful option in lactic acid fermentation with low cost fruc-
tan-containing biomass especially for food applications. Economic
evaluation of this process in comparison with the processes with
acidic or enzymatic pretreatment remains to be studied.
4. Conclusions
The carbohydrates derived from Jerusalem artichoke could be
converted efciently to lactic acid by L. paracasei strains such as
KCTC13169 and KCTC13090 without acidic or enzymatic hydroly-
sis prior to fermentation. L. paracasei KCTC13169 produced approx-
imately six times more lactic acid compared with the type strain L.
casei. It was shown that the selected L. paracasei strains could uti-
lize fructo-oligosaccharides almost completely (up to DP of 13
detectable in Jerusalem artichoke), whereas the L. casei KCTC3109
was found incapable of utilizing the fructo-oligosaccharide. Direct
lactic fermentation of Jerusalem artichoke tuber extract yielded
92.5 g/L of lactic acid with 98% conversion efciency.
Acknowledgements
This study was supported by the National Research Foundation
of Korea Grant funded by the Korean Government (MEST) (NRF-
2010-C1AAA001-0029084) and by the Bio-industry Technology
Development Program funded by the Ministry for Food, Agricul-
ture, Forestry and Fisheries, Republic of Korea.
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Table 1
Lactic acid fermentation with different Lactobacillus strains in inulin-containing media.
Strain MRS
a
Tuber extract + MRS
b
Inulin + MRS
c
OD
600
Lactate (g/L) Lactate (g/L) OD
600
Lactate (g/L)
L. casei KCTC3109 10.0 17.2 7.7 0.9 1.6
L. paracasei KCTC3165 10.6 17.2 40.4 9.3 13.9
L. paracasei KCTC3166 11.4 19.2 19.9 2.2 2.1
L. paracasei KCTC3169 11.1 17.4 42.6 0.6 1.8
L. paracasei KCTC3510 10.1 17.3 41.6 1.0 1.6
L. paracasei KCTC13090 9.1 17.4 45.5 8.0 13.0
L. paracasei KCTC13169 9.4 17.3 50.6 7.8 16.7
a
Samples from 72 h cultures were analyzed.
b
Hot-water extract of Jerusalem artichoke tubers with supplements of 1/5 strength MRS and 50 g/L CaCO
3
. Optical density was not measured because of CaCO
3
. Samples
from 72 h cultures were shown.
c
MRS medium containing 40 g/L inulin from dahlia tubers in place of glucose without a supplement of CaCO
3
. Samples from 48 h cultures were analyzed.
Fig. 1. Fermentation of Jerusalem artichoke tuber extract with L. paracasei
KCTC13169 in a 5 L jar fermentor. The medium (2 L) containing 111.6 g/L of sugar
was used with the pH adjustment to 6.0 with 10 N NaOH. The culture was carried-
out at 37 C with agitation at 150 rpm without aeration.
H.-Y. Choi et al. / Bioresource Technology 114 (2012) 745747 747