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Detailed kinetic and physiological characterisation of eight mannitol-producing lactic acid bacteria, Leuconostoc citreum ATCC 49370, L. mesenteroides subsp. cremoris ATCC19254, L. mesenteroides subsp. dextranicum ATCC 19255, L. ficulneum NRRL B-23447, L. fructosum NRRL B-2041, L. lactis ATCC 19256, Lactobacillus intermedius NRRL 3692 and Lb. reuteri DSM 20016, was performed using a carob-based culture medium, to evaluate their different metabolic capabilities. Cultures were thoroughly followed for 30 h to evaluate consumption of sugars, as well as production of biomass and metabolites. All strains produced mannitol at high yields (>0.70 g mannitol/g fructose) and volumetric productivities (>1.31 g/l h), and consumed fructose and glucose simultaneously, but fructose assimilation rate was always higher. The results obtained enable the studied strains to be divided mainly into two groups: one for which glucose assimilation rates were below 0.78 g/l h (strains ATCC 49370, ATCC 19256 and ATCC 19254) and the other for which they ranged between 1.41 and 1.89 g/l h (strains NRRL B-3692, NRRL B-2041, NRRL B-23447 and DSM 20016). These groups also exhibited different mannitol production rates and yields, being higher for the strains with faster glucose assimilation. Besides mannitol, all strains also produced lactic acid and acetic acid. The best performance was obtained for L. fructosum NRRL B-2041, with maximum volumetric productivity of 2.36 g/l h and the highest yield, stoichiometric conversion of fructose to mannitol.
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Mannitol production by lactic acid bacteria grown
in supplemented carob syrup
Florbela Carvalheiro Patrı
´cia Moniz
´s C. Duarte M. Paula Esteves
Francisco M. Gı
Received: 5 April 2010 / Accepted: 26 July 2010
ÓSociety for Industrial Microbiology 2010
Abstract Detailed kinetic and physiological character-
isation of eight mannitol-producing lactic acid bacteria,
Leuconostoc citreum ATCC 49370, L. mesenteroides
subsp. cremoris ATCC19254, L. mesenteroides subsp.
dextranicum ATCC 19255, L. ficulneum NRRL B-23447,
L. fructosum NRRL B-2041, L. lactis ATCC 19256, Lac-
tobacillus intermedius NRRL 3692 and Lb. reuteri DSM
20016, was performed using a carob-based culture med-
ium, to evaluate their different metabolic capabilities.
Cultures were thoroughly followed for 30 h to evaluate
consumption of sugars, as well as production of biomass
and metabolites. All strains produced mannitol at high
yields ([0.70 g mannitol/g fructose) and volumetric pro-
ductivities ([1.31 g/l h), and consumed fructose and glu-
cose simultaneously, but fructose assimilation rate was
always higher. The results obtained enable the studied
strains to be divided mainly into two groups: one for which
glucose assimilation rates were below 0.78 g/l h (strains
ATCC 49370, ATCC 19256 and ATCC 19254) and the
other for which they ranged between 1.41 and 1.89 g/l h
(strains NRRL B-3692, NRRL B-2041, NRRL B-23447
and DSM 20016). These groups also exhibited different
mannitol production rates and yields, being higher for the
strains with faster glucose assimilation. Besides mannitol,
all strains also produced lactic acid and acetic acid. The
best performance was obtained for L. fructosum NRRL
B-2041, with maximum volumetric productivity of 2.36
g/l h and the highest yield, stoichiometric conversion of
fructose to mannitol.
Keywords Carob Lactic acid bacteria Lactobacillus
Leuconostoc Mannitol production
Mannitol, a naturally occurring polyol, is widely used in
medicine, and in pharmaceutical, food and chemical
industries. It can be produced by chemical, microbiological
or enzymatic processes [1,2]. Industrial chemical pro-
duction is based on hydrogenation of fructose/glucose
mixtures at high temperature and pressure using Raney-
nickel catalyst. This process yields a mixture of mannitol
and sorbitol, an isomer, which has less interesting proper-
ties and hence significantly lower market price. Moreover,
separation of mannitol and sorbitol is rather difficult. The
enzymatic process requires the use of redox co-factors
[1,3], which, in general, renders it unattractive. Because of
these problems, mannitol production by microbial route
has become attractive, even at the industrial scale.
Heterofermentative lactic acid bacteria (LAB) belonging
to the genera Lactobacillus,Leuconostoc and Oenococcus
produce mannitol from fructose [4,5] and are among the
most efficient microorganisms for mannitol production. In
general, they can specifically produce mannitol from
glucose/fructose mixtures, without making sorbitol as
by-product [2], making it unnecessary to use purified
substrates or complex processes for product purification.
Metabolically, under adequate oxygen availability condi-
tions, glucose can be used as an energy and carbon source,
and fructose as an electron acceptor, as it can be reduced to
This article is part of the BioMicroWorld 2009 Special Issue.
F. Carvalheiro (&)P. Moniz L. C. Duarte
M. P. Esteves F. M. Gı
Unidade de Bioenergia, LNEG-Laborato
Nacional de Energia e Geologia,
Estrada do Pac¸o do Lumiar, 22,
1649-038 Lisbon, Portugal
J Ind Microbiol Biotechnol
DOI 10.1007/s10295-010-0823-5
mannitol by means of a specific mannitol dehydrogenase
(EC Depending on the microorganism, up to
2 mol mannitol can be produced from 1 mol glucose (and
2 mol fructose), if sugar assimilation is simultaneous. This
will also lead to formation of lactic acid and acetic acid
and/or ethanol and carbon dioxide as other metabolic
Carob (Ceratonia siliqua L.) is a Mediterranean
perennial tree producing pods that contain seeds (10%)
and sugar-rich pulp (90%), which exhibits higher sugar
content than sugar cane [6]. The pulp is currently an
under-utilised by-product of carob locust bean gum pro-
duction, and its main application is as animal feed, and to
a lesser extent in traditional bakery products and bever-
ages. Therefore higher-added-value applications are nee-
ded. Carob pulp mainly contains sucrose (the major
sugar), glucose and fructose that can be water extracted
and easily used to obtain sugar-rich syrups [7,8]. These
syrups are a cheap carbon source that has already been
used for formulation of culture media, e.g. for xanthan,
ethanol and citric acid production [911]. Given its sugar
content, a carob-based medium may be advantageous for
mannitol production.
In a previous work [12], 30 bacterial strains from genera
Lactobacillus,Leuconostoc and Weissella were screened
for mannitol production in carob-based syrups. From these,
a group of eight strains were identified as potential man-
nitol producers that must be studied deeply to unveil their
potential advantages and limitations concerning use as
industrial mannitol producers using carob syrup medium.
In this work, a detailed kinetic and physiological char-
acterisation of these eight strains using a carob-based cul-
ture medium was carried out, to evaluate their performance
and metabolic capabilities, and to select the most promis-
ing strain.
Materials and methods
Raw material
Carob pulp was obtained from a local de-seeding factory
(Chorondo e Filhos L.da, Loule
´, Portugal). Carob pods
were purposely collected to contain the two main varieties
grown in Portugal, Mulata and Galhosa (80% and 20%,
respectively) to produce a defined and representative lot.
The pods were industrially de-seeded into kibbles and then
stored at room temperature in burlap sacks. On average, the
kibbles present the following composition (g/100 g dry
weight): sucrose, 42.68; glucose, 8.54; fructose, 5.97;
pinitol, 5.22; lignin, 18.86; cellulose, 7.64; hemicellulose,
0.26; protein, 4.70; fat, 0.61; ash, 3.11; and others (by
difference), 2.41 [12].
Medium preparation
Carob syrup was obtained by aqueous extraction using an
orbital incubator (Infors Unitron HT, Switzerland), at 50°C
for 5 h as described elsewhere [12] and stored at -20°C.
Before use, precipitates were removed by centrifugation at
7,500 gfor 25 min in a Beckman Coulter centrifuge
(Fullerton, USA) followed by filtration using Whatman no.
41 filter paper.
Sucrose hydrolysis and sterilisation of carob syrup were
performed by correcting syrup pH to 3 using 5 M HCl,
followed by 15 min at 121°C in an autoclave. Carob syrups
were chemically characterised as described below.
The pH of hydrolysed carob syrups was corrected to 6.5
by addition of sterile NaOH. These hydrolysates were sup-
plemented with MRS nutrients, except ammonium citrate,
which was replaced by 50 mM sodium citrate buffer (pH
6.2) to keep pH values in a range acceptable for bacterial
growth. Besides sodium citrate, the final medium contained
(per litre): 10 g peptone, 8 g beef extract, 4 g yeast extract,
1 ml Tween 80, 5 g NaCH
COO, 0.2 g MgSO
, 0.05 g
and 2 g K
. The supplements and citrate
buffer were sterilised separately (121°C, 15 min) and added
aseptically to the pH-corrected carob hydrolysates, yielding
a dilution of the original carob syrup hydrolysate of 1:2.
Microorganisms and culture conditions
Eight heterofermentative lactic acid bacteria (LAB) were
used in this study. The strains and the corresponding
growth temperatures are summarised in Table 1. Stock
cultures were maintained in liquid nitrogen, and inocula
were prepared using MRS growth medium. Pre-inoculum
was obtained by re-suspending the cells in MRS growth
medium and incubation at the required temperature for
48 h. For inocula preparation, 100-ml Erlenmeyer flasks
containing 50 ml MRS medium were inoculated with 1 ml
pre-inoculum. Both pre-inoculum and inoculum were
incubated without agitation.
One millilitre of 16 h inoculum culture was used to seed
100-ml Erlenmeyer flasks containing 50 ml culture medium.
All cultures were incubated in an orbital shaker (100 rpm)
for 30 h at the same temperature as the inoculum. Samples
were withdrawn at a specific time to determine cell growth,
consumption of sugars and formation of products.
All experiments were performed, at least, in duplicate.
Replicates always differed by less than 10%, and typically
by less than 5%.
Analytical methods
Glucose, fructose, mannitol and pinitol were analysed by
high-performance liquid chromatography (HPLC; Waters,
J Ind Microbiol Biotechnol
Milford, USA) using a Sugar-SP0810 column (Shodex,
Japan). The column was maintained at 80°C, and the sugars
were eluted with ultra-pure water at flow rate of 1 ml/min.
The HPLC system was a Waters LC1 module l plus
(Millfort, MA, USA) equipped with both a refractive index
(RI) and an ultraviolet (UV) detector set at 280 nm. For
lactic acid, acetic acid, iso-butyric acid, citric acid, ethanol
and hydroxymethylfurfural (HMF) analyses, an Aminex
HPX-87H column (Bio-Rad, Hercules, CA, USA) with a
de-ashing Micro-guard pre-column (Bio-Rad) was used.
The column was maintained at 50°C, and the mobile phase
was 5 mM H
at flow rate of 0.6 ml/min. All com-
pounds were analysed with a RI detector, and HMF was
also analysed by the UV detector. All samples were filtered
through 0.22-lm Gelman membrane filters prior to
Total phenolic compounds were determined by using the
Folin–Ciocalteu colorimetric method according to Single-
ton and Rossi [13]. Briefly, 100 ll sample was mixed with
5 ml 1/10 (v/v) diluted Folin–Ciocalteu reagent and 4 ml
7.5% Na
. Absorbance was measured at 765 nm after
15 min incubation at 45°C. Total phenolic compounds are
expressed as g gallic acid equivalents (GAE)/l. Assays
were carried out in triplicate. Cell growth was evaluated by
measuring absorbance at 600 nm. At the beginning and at
the end of fermentations, biomass dry weight was deter-
mined gravimetrically. Two millilitres of each sample was
centrifuged in dried Eppendorf tubes, washed twice with
2 ml filtered water and dried overnight at 100°C.
Volumetric consumption rate of sugars (Q
, g/l h) was
calculated as total monosaccharides (glucose and fructose)
consumed in a defined time interval. The specific growth
rate (l,h
) was calculated by linear regression of the
curve of ln(OD/ODi) versus time for the exponential
growth phase. The biomass (Q
) and mannitol (Q
volumetric production rates (g/l h), hereinafter referred to
as the biomass and mannitol productivities, were calculated
as the increase in cell mass and mannitol concentrations,
respectively, for a designated time interval. The mannitol
yield (Y
g/g) was calculated as the ratio between
mannitol increase and fructose consumption, for a desig-
nated time interval.
Results and discussion
Composition of hydrolysed carob pulp syrups
and carob pulp base medium
Carob syrups used in this work were obtained by water
extraction of carob pods under operational conditions
previously optimised, and typically contain sucrose
(accounting for more than 50% of total sugars) together
with free fructose, glucose and pinitol in a total close to
200 g/l [12]. As a high content of free fructose and glucose
are required for efficient mannitol production, carob syrups
have to be hydrolysed. Table 2summarises the composi-
tion of the hydrolysed carob pulp syrups used in this work
for culture medium preparation after the sterilising hydro-
lysis. More than 90% of the original sucrose was converted
to fructose and glucose, leading to an increase of concen-
tration of both sugars, at least twofold. Under these mild
conditions, some degradation of hexoses also occurred, and
HMF was found as the main degradation product, although
at low concentration. Phenolic compounds were also found,
but in relative low amounts.
After supplementation, the concentration of components
in the hydrolysates was reduced to a half, and carob-pulp-
based medium had average composition (per litre) as fol-
lows: 44.4 g fructose, 47.6 g glucose, 7.7 g pinitol, 2.4 g
acetic acid, 0.85 g iso-butyric acid and 0.34 g HMF.
Organic acids, such as iso-butyric acid, and furan deriva-
tives such as HMF, may act as fermentation-inhibiting
Table 1 Lactic acid bacteria
strains used in this study Microorganism Strain Growth
temperature (°C)
Lactobacillus intermedius NRRL B-3692 37
Lactobacillus reuteri DSM 20016 37
Leuconostoc fructosum NRRL B-2041 30
Leuconostoc ficulneum NRRL B-23447 30
Leuconostoc citreum ATCC 49370 30
Leuconostoc lactis ATCC 19256 30
Leuconostoc mesenteroides subsp. cremoris ATCC 19254 30
Leuconostoc mesenteroides subsp. dextranicum ATCC 19255 30
J Ind Microbiol Biotechnol
compounds. However, at these low levels and initial pH
they may not exert a strong inhibition effect, suggesting the
suitability of hydrolysates for fermentation purposes;
hence, no detoxification procedure was applied.
Kinetic profiles
To evaluate mannitol production by selected strains using a
carob-based medium, the kinetic profiles for growth, sug-
ars, mannitol and other co-products were studied. Figures 1
and 2show the results obtained for Lactobacillus and
Leuconostoc strains, respectively. Amongst Lactobacillus
strains, fructose and glucose were simultaneously con-
sumed, but fructose assimilation rate was always higher.
Lb. intermedius displayed the fastest growth and maximum
specific growth rate, two times higher than those for
Lb. reuteri, which was also confirmed by the higher final
cell density achieved. The maximum mannitol concentra-
tions obtained were similar for both microorganisms,
although mannitol accumulation by Lb. intermedius
occurred at a higher rate, which is consistent with fructose
depletion. As expected, lactic acid and acetic acid were
also produced, mainly when glucose consumption was
higher. Ethanol was also found, but only in minor amounts,
typically a maximum concentration around 1 g/l.
Lb. intermedius was an exception, as it presented more
significant production (5 g/l) for the final fermentation
stages, mainly after fructose depletion and onset of single
glucose metabolism. Ethanol production from glucose has
been already described for L. intermedius strain [4]
All Leuconostoc strains preferably consumed fructose
over glucose, in similar trend to lactobacilli. The remaining
glucose concentration varied depending on the strain.
Higher fructose consumption rates were found for L. ficul-
neum and L. fructosum, which together with Lactobacillus
strains were the best mannitol producers. Maximum specific
growth rate were similar for all Leuconostoc strains
(*0.2 h
), but the final biomass concentration achieved
also depended on the strain.
As for Lactobacillus, these strains also produced lactic
acid and acetic acid. For all LAB tested, at the maximum
mannitol concentrations, aliphatic acids ranged between
9.9 and 18.5 g/l and 14.9 and 27.0 g/l for acetic acid and
lactic acid, respectively.
Kinetic and stoichiometric parameters
Table 3shows the results for kinetic and stoichiometric
parameters obtained for Lactobacillus and Leuconostoc
strains grown in carob-based medium. All strains produced
mannitol at high yields ([0.70 g mannitol/g fructose) and
relatively high volumetric productivities ([1.31 g/l h), and
displayed high fructose consumption ([91%). Independent
of the genus, strains could be divided into two groups: one
for which glucose assimilation rates were always below
1.05 g/l h (glucose consumption lower than 55%), and a
Table 2 Composition of the carob syrup hydrolysates used for cul-
ture medium preparation
Compound Concentration (g/l)
Sucrose 11.4 ±2.1
Fructose 89.2 ±1.9
Glucose 96.0 ±1.8
Pinitol 15.6 ±0.3
Acetic acid 0.70 ±0.10
Iso-butyric acid 2.05 ±0.08
Hydroxymethylfurfural 0.67 ±0.02
Phenolic compounds 1.62 ±0.04
The concentrations of these compounds in the culture medium are
half of those reported in this table, due to the addition of the required
Time (h)
Glc, Fru, ManOH, HAc, HLac (g/l)
EtOH (g/l), Ln (Abs/Abs0)
Time (h)
0 5 10 15 20 25 30 0 5 10 15 20 25 30
Glc, Fru, ManOH, HAc, HLac (g/l)
EtOH (g/l), Ln (Abs/Abs0)
Fig. 1 Fermentation profiles for mannitol production by Lactobacil-
lus strains in carob-syrup-based medium. Lb. intermedius (a);
Lb. reuteri (b). Glucose (filled squares), fructose (filled triangles),
mannitol (open triangles), lactic acid (open squares), acetic acid
(open diamonds), ethanol (open circles), cell density (plus symbols).
Data points are the average of at least two independent replicates.
Lines are guides to the eye only and do not have any statistical
J Ind Microbiol Biotechnol
second for which they ranged between 1.41 and 1.89 g/l h
(glucose consumption ranging from 63% to 96%). The
group exhibiting the faster glucose assimilation
(Lb. intermedius,Lb. reuteri,L. fructosum and L. ficulne-
um) also exhibited higher mannitol production rates and
yields as well as ratio of mannitol to aliphatic acids (lactic
acid and acetic acid) above 1. In fact, the rates for glucose
consumption and mannitol production were positively
correlated (r=0.86, data not shown).
These results clearly indicate that these two groups may
have different metabolic capabilities, with strains that
exhibited slower glucose consumption producing, in
general, higher acetic acid concentrations. The highest ratio
of mannitol to other products was obtained for Lb. reuteri
DSM 20016, but this is not due to higher mannitol pro-
duction but rather to higher biomass production yield and
lower lactic acid production.
The ratios of mannitol to other co-products (lactic and
acetic acids) obtained in this work were, however, lower
than the values (higher than 2), reported by Saha [14]. It is
worth noting that those reported values were obtained for
higher fructose concentrations and different ratio of glu-
cose to fructose, which is also a key parameter for efficient
mannitol production. Von Weymarn et al. [15] described
Time (h)
0 5 10 15 20 25 30
Glc, Fru, ManOH, HAc, HLac (g/l)
EtOH (g/l), Ln (Abs/Abs
2.0 B
Time (h)
Glc, Fru, ManOH, HAc, HLac (g/l)
Time (h)
Glc, Fru, ManOH, HAc, HLac (g/l)
2.0 D
Time (h)
Glc, Fru, ManOH, HAc, HLac (g/l)
Time (h)
Glc, Fru, ManOH, HAc, HLac (g/l)
2.0 F
Time (h)
Glc, Fru, ManOH, HAc, HLac (g/l)
0 5 10 15 20 25 30
0 5 10 15 20 25 30 0 5 10 15 20 25 30
0 5 10 15 20 25 30
0 5 10 15 20 25 30
EtOH (g/l), Ln (Abs/Abs
)EtOH (g/l), Ln (Abs/Abs
EtOH (g/l), Ln (Abs/Abs
) EtOH (g/l), Ln (Abs/Abs
) EtOH (g/l), Ln (Abs/Abs
Fig. 2 Fermentation profiles for mannitol production by Leuconostoc
strains in carob-syrup-based medium. L. fructosum (a), L. ficulneum
(b), L. citreum (c), L. lactis (d), L. mesenteroides subsp. cremoris (e),
L. mesenteroides subsp. dextranicum (f). Glucose (filled squares),
fructose (filled triangles), mannitol (open triangles), lactic acid (open
squares), acetic acid (open diamonds), ethanol (open circles), cell
density (plus symbols). Data points are the average of at least two
independent replicates. Lines are guides to the eye only and do not
have any statistical significance
J Ind Microbiol Biotechnol
for L. mesenteroides an optimum ratio of glucose to fruc-
tose of 0.55:1. Also, Saha [16] reported that lactic acid
production by Lb. intermedius surpassed mannitol con-
centration when sugar cane molasses (ratio of glucose to
fructose of 1:1) were used for culture medium preparation.
The maximum mannitol concentration (43.7 g/l) was
obtained with L. fructosum (Fig. 2a). This strain also
exhibited the highest volumetric productivity (2.36 g/l h, at
15 h) and conversion efficiency of fructose to mannitol of
100%. This productivity is higher than the obtained for
L. pseudomesenteroides ATCC 12291 grown in media
containing glucose and fructose (100:50) under similar
culture conditions [17], and the yield was higher than
values previously reported for Lb. intermedius B-3693
grown in fructose-based medium [4] and the same
L. pseudomesenteroides [17]. Although stoichiometric
conversion of fructose to mannitol has been also previously
reported for Lb. sanfranciscensis [5,18], that strain
exhibited lower productivity. Given that the initial perfor-
mances with those strains were further optimised, namely
using pH-controlled bioreactors (preventing use of citrate
buffer that was typically partially consumed, data not
shown), or using different operation modes, such as fed-
batch, cell recycling and optimised culture media, it is
expected that L. fructosum may also present a significant
performance improvement [14,15,19,20].
The mannitol volumetric productivities obtained for the
group exhibiting lower mannitol production (L. citreum,
L. lactis, L. mesenteroides subsp. cremoris and L. mesen-
teroides subsp. dextranicum) were similar to values reported
for L. mesenteroides ATCC 9135, L. pseudomesenteroides
ATCC 12291, Lb. brevis ATCC 8287 and Lb. fermentum
NRRL 1932 [15]. According to the present results, these
syrups, although containing acetic and iso-butyric acids,
HMF and some phenolics that were also solubilised during
extraction process of sugars, did not have an apparent
inhibitory effect on growth of these microorganisms. For
this reason, increasing the syrup concentration seems to be a
possibility to achieve higher performance. Furthermore, the
levels attained by the produced lactic and acetic acids did not
hinder fructose consumption. As these acids can be recov-
ered from the fermentation broth (e.g. by electrodialysis [4]),
their co-production may be an advantageous trait, as it will
be possible to design a multi-product process.
The eight mannitol-producing LAB strains tested were able
to grow and produce mannitol with relatively high effi-
ciency in carob-syrup-based medium. Kinetic and physio-
logical characterisation of these strains was performed, and
the results obtained suggest that the studied strains can be
divided into two groups based on their glucose consump-
tion and mannitol production rates, as well as ratio of
mannitol to aliphatic acids. The highest volumetric pro-
ductivity was obtained for L. fructosum, which was also
one of the most robust strains tested. This strain is currently
being used for mannitol process optimisation in carob-
based media.
Given that the levels attained by the produced acids did
not hinder fructose consumption, their co-production may
be an advantageous trait. The design of a multi-product
process is now possible, as these acids are also marketable
compounds, in particular lactic acid, which has a large and
expanding market for industrial production of bioplastics.
Table 3 Kinetic and stoichiometric parameters for growth and formation of products for Lactobacillus and Leuconostoc strains in carob-syrup-
based medium
Microorganism Consumption (%) Q
(g/l h)
(g/l h)
(g/l h)
(HLac ?HAc)
Glc Fru
Lb. intermedius 96 100 3.74 0.27 1.68 0.91 1.6 1.1
Lb. reuteri 69 98 3.27 0.16 1.79 0.96 2.3 1.4
L. fructosum 76 91 3.25 0.14 1.82 1.06 1.6 1.0
L. ficulneum 63 98 3.12 0.09 1.65 0.89 1.7 1.2
L. citreum 38 97 2.65 0.07 1.31 0.70 1.4 0.8
L. lactis 33 93 2.34 0.08 1.39 0.82 1.4 0.8
L. mesenteroides subsp. cremoris 32 95 2.27 0.003 1.37 0.83 1.4 0.8
L. mesenteroides subsp. dextranicum 55 94 2.73 0.003 1.50 0.89 1.4 0.8
Glc glucose, Fru fructose, Q
consumption rate of sugars, Q
biomass productivity, Q
mannitol productivity, Y
mannitol yield on
fructose, ManOH/HLac ratio of mannitol to lactic acid, ManOH/(HLAc ?HAc) ratio of mannitol to lactic acid ?acetic acid. All values
calculated after 24 h
J Ind Microbiol Biotechnol
Acknowledgments This work was supported by AdI, Project Val-
orAlfa (70/00326). ARS Culture Collection (National Centre for
Agricultural Utilization Research, Peoria, IL, USA) is gratefully
acknowledged for supplying bacterial strains. The authors thank
´lia Marques, Carlos Barata and Ce
´u Penedo for technical support.
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... Heterofermentative LAB of Leuconostoc, Oenoccocus, and Lactobacillus have been reported as efficient mannitol producers (Ortiz et al., 2013). Among the first genus, the Grampositive bacterium Leuconostoc mesenteroides has been used for mannitol production in different studies (Carvalheiro et al., 2011;Fontes et al., 2009Fontes et al., , 2013Von Weymarn et al., 2003). Besides, Lactobacillus fermentum has been reported as a mannitol producer (Rodriguez et al., 2012). ...
... On the other hand, the yields obtained in media A and B were identical (0.98 ± 0.04 and 0.92 ± 0.11 mol mannitol mol initial fructose −1 , respectively). In the study carried out by Carvalheiro et al. (2011), it was reported that the strains ATCC 19254 and ATCC 19255 of Leuconostoc mesenteroides produced mannitol and reached volumetric productivities of 2.27 and 2.73 g L −1 h −1 , respectively. The yields obtained were 0.83 and 0.89 g mannitol g fructose −1 , respectively (equivalent to 0.82 and 0.88 mol mannitol −1 mol fructose −1 , respectively). ...
This study developed a non-structured mathematical model for describing the effect of carbon sources and culture pH on the mannitol production by three heterofermentative lactic acid bacteria (LAB), including Leuconostoc mesenteroides NRRL B-512F and NRRL B-523 as well as Lactobacillus fermentum NRRL B-1840. Mannitol production was studied in batch culture using four culture media containing the same carbon sources (glucose and fructose, but different glucose concentration) and different nitrogen sources (yeast extract, peptone, and meat extract) with diverse concentrations. The results obtained in the early stages of the estimation algorithm suggested that cell growth on fructose could be neglected. Therefore, the initially proposed mathematical model was redefined and subsequently confirmed. The strains NRRL B-512F, NRRL B-523, and NRRL B-1840 reached the highest volumetric productivities (2.26 ± 0.05, 2.30 ± 0.05, and 1.95 ± 0.05 g L⁻¹h⁻¹; respectively) and mannitol yields (0.95 ± 0.01, 0.98 ± 0.04, and 1.00 ± 0.01 mol mannitol mol initial fructose⁻¹; respectively) in medium A. The mannitol and biomass yields suggested a higher carbon concentration favored mannitol production and growth, but a lower nitrogen concentration directly impacted both yields. Mannitol production was associated with cell growth according to the estimated parameters.
... Fructophilic nature is advantageous for the survival of bacterial strains in a fructose-rich environment and in the intestine of high fructose consuming insects (Neveling et al. 2012;Pachla et al. 2018). On the other hand, the survivability of Limosilactobacillus reuteri was significantly decreased upon increased fructose in the media, which might be attributed to its non-fructose origin, i.e. human faeces (Sriramulu et al. 2008;Carvalheiro et al. 2011). ...
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Fructophilic Lactic Acid Bacteria (FLAB), Fructobacillus fructosus DPC7238 and pseudofructophilic Leuconostoc mesenteroides DPC7261 and non-FLAB Limosilactobacillus reuteri DSM20016 strains were studied for their growth and morphological evolution as a function of increased fructose concentrations (0, 25, and 50% w/v) in the media. A comparison of the genomics of these strains was carried out to relate observed changes and understand fructose-rich adaptations. The viability of FLAB strains were reduced by approx. 50% at a 50% fructose concentration, while the Limosilactobacillus reuteri strain was reduced to approx. 98%. Electron microscopy demonstrated that FLAB strain, Fructobacillus. fructosus and pseudofructophilic Leuc. mesenteroides, were intact but expanded in the presence of high fructose in the medium. Limosilactobacillus reuteri, on the other hand, ruptured as a result of excessive elongation, resulting in the formation of cell debris when the medium contained more than 25% (w/v) fructose. This was entirely and quantitatively corroborated by three-dimensional data obtained by scanning several single cells using an atomic force microscope. The damage caused the bacterial envelope to elongate lengthwise, thus increasing width size and lower height. The cell surface became comparatively smoother at 25% fructose while rougher at 50% fructose, irrespective of the strains. Although Fructobacillus fructosus was highly fructose tolerant and maintained topological integrity, it had a comparatively smaller genome than pseudofructophilic Leuc. mesenteroides. Further, COG analysis identified lower but effective numbers of genes in fructose metabolism and transport of Fructobacillus fructosus, essentially needed for adaptability in fructose-rich niches.
... and Lactobacillus spp. produce small amounts of mannitol (Carvalheiro et al., 2011;Lee et al., 2020a;Otgonbayar et al., 2011). Thus, mannitol content is high in kimchi with a high ratio of Weissella spp. ...
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The taste of kimchi is greatly affected by the salt type used during fermentation. Here, we investigated the effects of salts with different mineral contents on the microbial community and metabolite profiles of fermented kimchi using multivariate statistical analysis. We evaluated different types of salt used to prepare kimchi, namely, solar salt aged for 1 year, solar salt aged for 3 years, dehydrated solar salt, and purified salt. The main microorganisms detected in kimchi were Weissella koreensis, Leuconostoc mesenteroides, and Latilactobacillus sakei. Leuconostoc and Weissella were mainly present in kimchi supplemented with solar salt. However, a high proportion of L. sakei was present in kimchi supplemented with purified salt and dehydrated salt. Additionally, using GC-MS-based metabolite analysis, we revealed that the content of free sugars, organic acids, and amino acids differed in kimchi fermented with different salt types. Therefore, we demonstrated that salt type had a pronounced effect on the resultant microbial community and the type and concentration of metabolites present in fermented kimchi.
... In recent years, carob has attracted considerable attention because of its high carbohydrate and mineral content 66 . Using carob via fermentation process produced many high value-added products such as lactic acid 67 , mannitol 68 , citric acid 69 , and pullulan 70 . Ethanol has also been produced using carob pod extract. ...
... Carvalheiro et al. [85] obtained a sugar-rich syrup from an agro-industrial residue, the carob pulp, which contained sucrose as the primary sugar. It was hydrolyzed under acidic conditions to obtain free fructose and glucose. ...
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Obesity, diabetes, and other cardiovascular diseases are directly related to the high consumption of processed sugars with high caloric content. The current food industry has novel trends related to replacing highly caloric sugars with non-caloric or low-calorie sweeteners. Mannitol, a polyol, represents a suitable substitute because it has a low caloric content and does not induce a glycemic response, which is crucial for diabetic people. Consequently, this polyol has multiple applications in the food, pharmaceutical, and medicine industries. Mannitol can be produced by plant extraction, chemical or enzymatic synthesis, or microbial fermentation. Different in vitro processes have been developed regarding enzymatic synthesis to obtain mannitol from fructose, glucose, or starch-derived substrates. Various microorganisms such as yeast, fungi, and bacteria are applied for microbial fermentation. Among them, heterofermentative lactic acid bacteria (LAB) represent a reliable and feasible alternative due to their metabolic characteristics. In this regard, the yield and productivity of mannitol depend on the culture system, the growing conditions, and the culture medium composition. In situ mannitol production represents a novel approach to decrease the sugar content in food and beverages. Also, genetic engineering offers an interesting option to obtain mannitol-producing strains. This review presents and discusses the most significant advances that have been made in the mannitol production through fermentation by heterofermentative LAB, including the pertinent and critical analysis of culture conditions considering broth composition, reaction systems, and their effects on productivities and yields.
... The calculations for the specific growth rate (µ, h − 1 ), volumetric productivity of D-lactic acid (Q D-LA : g/Lh), and D-lactic acid yield (g D-LA / g Sugars ) were accomplished as described in Carvalheiro et al. (2011). The specific growth rate of lactogenic E. coli JU15 in a minimal medium with glucose or xylose (µ, h − 1 ), was calculated by linear regression of the curve of optical density versus time for the exponential growth phase. ...
In this study, glucan-rich solids, and xylose-rich hydrolysates obtained from Cistus ladanifer distillery residues (CLR) were used for d-lactic acid (d-LA) production by the d-lactogenic Escherichia coli strain JU15. Firstly, hemicellulosic hydrolysates obtained by the autohydrolysis process were submitted to dilute sulfuric acid-catalysed post-hydrolysis. The influence of operational conditions on oligosaccharides hydrolysis was assessed by the combined severity parameter (CS) in the range of 1.1–2.3. The optimum post-hydrolysis conditions were found for CS of 1.6 (300 mM H2SO4, 15 min, 121 °C). Subsequent detoxification procedures on post hydrolysed liquors were carried out, where 9.1% (w/v) powdered activated charcoal enabled a full removal of furfural, 5-hydroxymethylfurfural (HMF), and phenolic compounds together with a reduction of acetic acid (37%), and formic acid (27%). Diverse fermentation modes using detoxified and non-detoxified hydrolysates, as well as using previously NaOH delignified glucan-rich solids alone (SHF or SSF) or together with pentoses liquors (SSCF) (5% loading) were performed. For all the tested conditions, both hemicellulose- and cellulose-derived sugars can be efficiently used as the carbon source to produce d-lactic acid by E. coli JU15 with a d-LA yield always surpassing 92 gd-LA/100 g sugars.
... Using cluster and dendrogram analysis, mannitol producer (9.46 0.27 g/l) strain F. tropaeoli was discovered from fruits by Ruiz Rodriguez et al. (2017). With a maximum volumetric productivity of 2.36 g/l h and the highest yield, of mannitol was obtained for L. fructosum NRRL B-2041 according to Carvalheiro et al. (2011). Also, different Leuconostoc pseudomesenteroides strains were studied for its higher mannitol production potential at different growth conditions (Bhatt et al. 2013). ...
Lactic acid bacteria (LAB) is considered as food-grade microorganism with Generally Recognized as Safe (GRAS) status. Both hetero-fermentative and homo-fermentative LAB have the ability to produce mannitol as metabolic end product in normal fermentation. Ten LAB isolates selected were detected for its mannitol production potential using colorimetric assay in preliminary study. Later selected four isolates of LAB with better mannitol production were further optimized at different culture conditions. A total of 540 mannitol combinations were obtained after optimization. All the observations were statistically analyzed using response surface methodology. Biochemical and molecular assays were carried out to identify the isolates. The isolate L8 produced mean mannitol content of 1.635 g/L, 0.345967 cell densities, at pH 7.0 and temperature 42 °C with agitation of 100 rpm was selected with optimum response surface optimization because of its higher mannitol production. Biochemical and molecular assays identified higher mannitol producers, L4 as Enterococcus faecium strain Gr17, L6 as Lactobacillus rhamnosus strain 6870, L8 as Leuconostoc pseudomesenteroides culture IMAU: 11666 and L9 as Lactobacillus plantarum subsp. plantarum strain NMB8.
Mannitol is a natural polyol used in the food, medical and pharmaceutical industries. In this work, a simplified low-cost culture medium using fructose syrup as a carbon source was formulated for mannitol production by Fructobacillus sp. CRL 2054 and F. tropaeoli CRL 2034. To this end, the effect of different medium components in bacterial growth and mannitol synthesis was assessed by applying the Plackett-Burman statistical design. The formulated FSYP-min was used for fermentations at constant pH of 5.0 in a 2 L bioreactor, where two concentrations of fructose syrup (10 and 20 %, w/v, of total sugars) were evaluated. High mannitol concentrations (ca. 90 g/L) and volumetric productivities (3.7 g/L∗h) were achieved by both strains using 20 % (w/v) of sugars; these mannitol values are one of the highest reported for LAB to date. Finally, high-purity mannitol crystals were successfully extracted after fermentation with the formulated culture medium.
Sugar alcohols are polyols that are widely employed in the production of chemicals, pharmaceuticals, and food products. Chemical synthesis of polyols, however, is complex and necessitates the use of hazardous compounds. Therefore, the use of microbes to produce polyols has been proposed as an alternative to traditional synthesis strategies. Many biotechnological approaches have been described to enhancing sugar alcohols production and microbe-mediated sugar alcohol production has the potential to benefit from the availability of inexpensive substrate inputs. Among of them, microbe-mediated erythritol production has been implemented in an industrial scale, but microbial growth and substrate conversion rates are often limited by harsh environmental conditions. In this review, we focused on xylitol, mannitol, sorbitol, and erythritol, the four representative sugar alcohols. The main metabolic engineering strategies, such as regulation of key genes and cofactor balancing, for improving the production of these sugar alcohols were reviewed. The feasible strategies to enhance the stress tolerance of chassis cells, especially thermotolerance, were also summarized. Different low-cost substrates like glycerol, molasses, cellulose hydrolysate, and CO2 employed for producing these sugar alcohols were presented. Given the value of polyols as precursor platform chemicals that can be leveraged to produce a diverse array of chemical products, we not only discuss the challenges encountered in the above parts, but also envisioned the development of their derivatives for broadening the application of sugar alcohols.
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Batch cultures of Xanthomonas campestris after 50 h growth produced the same amount of xanthan (6.8 g l-1) when grown both in defined medium and in carob extracts. However, a greater amount of cells were formed in the syrup (3 g l-1 than in the salt medium (1.4 g l-1). This fact suggest that carbo extracts are a suitable cell culture medium. However, the concentration of isobutyric acid in the broth increased from an initial value of 0.075 g l-1 up to 0.15 g l-1 containing a concentration of 9.6 × 10-5 g l-1 undissociated molecules during the fed-batch stage of carbo extract fermentation of X. campestris at pH 7. This accumulation of acid results in the decrease in cell production rate from 0.076 to 0.014 g h-1, whereas in cultures using defined medium the fed-batch operation cell production rate increses from 0.026 to 0.036 g h-1. Also, the xanthan formation is affected and the shift from batch to fed-batch in carob medium produces only a small 0.3 times increase in the xanthan production rate, compared with the 1.7 times rise in xanthan production when using the same procedure with defined medium. However, the specific xanthan production rate in carob extracts, initially of 1.3 g gt-1 of cells, is improved to 9 g g -1 of cells, which is very similar to that obtained in the defined medium feb-batch 9.6 g g-1 of cells, originated from 4.8 g g-1 of cells in batch culture. Fed-batch process of carob extract makes this raw material more suitable for xanthan production than the batch mode which is itself more adequate for cell growth.
The production of ethanol from carob pods extract by Saccharomyces cerevisiae in static and shake flask fermentation was investigated. Shake flask fermentation proved to be a better fermentation system for the production of ethanol than static fermentation. The external addition of nutrients into the carob pods extract did not improve the production of ethanol. The maximum concentration of ethanol (75 g/l) was obtained at an inoculum amount of 0.3%, a pH of 4.5, 30°C and an initial sugar concentration of 200 g/1. Under the same fermentation conditions both sterilized and non‐sterilized carob pods extract gave the same final ethanol concentration.
The production of citric acid from carob pod extract by cell recycle of Aspergillus niger at different pHs was investigated. Best results in terms of citric acid concentration, productivity, yield and sugar utilization were obtained with a substrate pH of 5.0. The citric acid concentration (85.5 g/l) and the productivity (4 g/ld) remained constant up to the second and third cycles, respectively.
Two multistage methods of sugar extraction from carob pods were compared with a single stage extraction/pressing process. A multistep extraction/pressing process produced a yield improvement of 6·6%. A second process based on the recycling of press liquor through a multistage series of vessels was not only considerably simpler and more economical in use of labour but also showed a further improvement of 1·4% to give a final sugar yield of 60%.A comparison of the calculated diffusion coefficients showed that the enhanced yield in the recycle flow process resulted from a decrease of a diffusion limitation observed in static operation. A diffusion coefficient of 9·9 × 103 m2/h was obtained at the highest yield.
The mannitol dehydrogenase of Leuconostoc pseudomesenteroides ATCC 12291 was studied for its characteristics and capacity for enzymatic mannitol synthesis, coupled to a coenzyme regeneration system. The producing strain was cultivated for 6.5 hours on a medium containing both D-glucose and D-fructose as carbon sources. Efficient release of the intracellular enzyme was obtained by cell treatment with toluene, Triton permeabilisation, or sonication. The crude enzyme extract showed optimal D-fructose reduction at pH 5.5 and 50°C, whereas D-mannitol oxidation was best at pH 8.0 and 40°C. The polyol dehydrogenase was stable up to 40°C and within the pH-range of 6.5–8.5. The crude enzyme extract displayed high substrate specificity. With NADPH, enzyme activity was only 30% of that observed with NADH. The molecular weight of the native enzyme was found to be 136 kDa. The mannitol dehydrogenase preparation was tested for its capacity to produce D-mannitol from D-fructose. Since the mannitol dehydrogenase is NADH-dependent, efficient coenzyme regeneration was needed. Regeneration of NADH from NAD+ was accomplished by formate dehydrogenase-mediated oxidation of formate into CO2. The best initial reaction rates were obtained with 0.5 mM NAD+ and 100 mM substrate.
D-Mannitol is a sugar alcohol with many applications in food, pharmaceuticals, medicine, and chemistry. Mannitol crystallizes in small white needles with a melting point of 165–170°C. Mannitol has a sweet cool taste owing to its high negative heat of solution (−121 kJ/kg). It is about half as sweet as sucrose. Mannitol has a low solubility in water of only 18% (w/v) at 25°C. Especially in alkaline solutions, it is a powerful sequestrant of metallic ions.