Effects of fructans and probiotics on the inhibition of Klebsiella oxytoca and the production of short-chain fatty acids assessed by NMR spectroscopy

i An update to this article is included at the end

Carbohydrate Polymers 248 (2020) 116832

Contents lists available at ScienceDirect

Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Effects of fructans and probiotics on the inhibition of Klebsiella oxytoca and
the production of short-chain fatty acids assessed by NMR spectroscopy

T

Bruna Higashia, Tamara Borges Marianoa, Benício Alves de Abreu Filhob,
Regina Aparecida Correia Gonỗalvesa, Arildo Josộ Braz de Oliveiraa,*
a
b

Graduate Program in Pharmaceutical Sciences, Department of Pharmacy, State University of Maringá, Ave. Colombo 5790, 87.020-900, Maringá, Brazil
Departament of Basic Health Sciences, State University of Maringá, Ave. Colombo 5790, 87.020-900, Maringá, Brazil

A R T I C LE I N FO

A B S T R A C T

Chemical compounds studied in this article:
Agar (PubChem CID: 76645041)
Ammonium citrate (PubChem CID: 6435836)
Calcium carbonate (PubChem CID: 10112)
Deuterium oxide (PubChem CID: 24602)

Dipotassium hydrogen phosphate (PubChem
CID: 24450)
DL-Mandelic acid (PubChem CID: 1292)
Ethanol (PubChem CID: 702)
Glucose (PubChem CID: 5793)
Glycerol (PubChem CID: 753)
Inulin from chicory (PubChem CID:16219508)
L-Cysteine hydrochloride (PubChem
CID:60960)
Magnesium sulfate (PubChem CID: 24083)
Manganese sulfate (PubChem CID: 24580)
Sodium acetate (PubChem CID: 517045)
Sodium chloride (PubChem CI: 5234)
Sodium hydroxyl (PubChem CID: 14798)
Sucrose (PubChem CID: 5988)
Sulfuric acid (PubChem CID: 1118)
Tween 80 (PubChem CID: 86289060)

Generally, the selection of fructans prebiotics and probiotics for the formulation of a symbiotic has been based
on arbitrary considerations and in vitro tests that fail to take into account competitiveness and other interactions
with autochthonous members of the intestinal microbiota. However, such analyzes may be a valuable step in the
development of the symbiotic. The present study, therefore, aims to investigate the effect of lactobacilli strains
and fructans (prebiotic compounds) on the growth of the intestinal competitor Klebsiella oxytoca, and to assess
the correlation with short-chain fatty acids production. The short-chain fatty acids formed in the fermentation of
the probiotic/prebiotic combination were investigated using NMR spectroscopy, and the inhibitory activities
were assessed by agar diffusion and co-culture methods. The results showed that Lactobacillus strains can inhibit
K. oxytoca, and that this antagonism is influenced by the fructans source and probably associated with organic
acid production.

Keywords:

Acetic acid
Co-culture
Klebsiella oxytoca
Lactic acid
Lactobacillus acidophilus
Symbiotic

1. Introduction
There are several available strategies for the modulation of the intestinal microbiota and improve health and well-being. A range of
therapeutic tools has been developed, such as the introduction of unique or associated microorganisms (probiotics), the provision of substrates that promote the growth of resident microorganisms beneficial

⁎

to host health (prebiotics), or the combination of both (symbiotic)
(Krumbeck Maldonado-Gomez, Ramer-Tait & Hutkins, 2016).
Fructans such as inulin and fructooligosaccharides (FOS) are considered important prebiotics and the best-documented oligosaccharides
for the effect on intestinal microbiota (Gibson, 2004; Rastall, 2010).
Lactobacilli such as Lactobacillus acidophilus are among the microorganisms most commonly used as probiotics, principally due to their

Corresponding author at: Universidade Estadual de Maringá, Av. Colombo 5790, Bloco K-80 CEP 87020-900, Maringá, PR, Brazil.
E-mail address: (A.J.B. de Oliveira).

/>Received 22 April 2020; Received in revised form 22 July 2020; Accepted 24 July 2020
Available online 27 July 2020
0144-8617/ © 2020 Elsevier Ltd. All rights reserved.


Carbohydrate Polymers 248 (2020) 116832

B. Higashi, et al.

appropriate instrumental conditions, the NMR response is exactly proportional to the number of nuclei present in the molecules, and can be
considered the same for all chemical components, including the internal
standard (Caligiani, Acquotti, Palla, & Bocchi, 2007).
Klebsiella oxytoca is present in both the environment and in humans
and has been found in faeces samples of 17 % of healthy infants (Savino
et al., 2009). Although individuals infected with K. oxytoca remain
asymptomatic, the microorganism is emerging as an opportunistic pathogen of the intestine which can colonize healthy individuals. It has
been implicated in antibiotic-associated diarrhea and is the main cause
of antibiotic-associated hemorrhagic colitis (Alikhani et al., 2016;
Högenauer et al., 2006; Beaugerie et al., 2003; Hoffmann et al., 2010;
Zollner‐Schwetz et al., 2008).
While the activities of probiotics and prebiotics have been extensively studied, little is known about their effects on K. oxytoca.
Therefore, the present study the two aims were to investigate the effects
of lactobacilli strains combined with fructans (inulin and fructooligosaccharides) on the growth of K. oxytoca and SCFA production by
quantitative determination using 1H NMR spectroscopy as a green
analytical chemistry alternative to existing methods.

long history of safe use (Daly & Davis, 1998). They are termed lactic
acid bacteria (LAB) since the main end-product of carbohydrate metabolism is lactic acid (Holzapfel & Wood, 2014).
Research into the effects of certain probiotic microorganisms in
combination with prebiotics in human intestinal pathogens is limited
(Ambalam et al., 2015; Fooks & Gibson, 2002; Tzortzis, Baillon, Gibson,
& Rastall, 2004; Valdés-Varela, Hernández-Barranco, Ruas-Madiedo, &
Gueimonde, 2016). These studies are important for understanding the
effects of the carbon source on the production of antimicrobial compounds by probiotic microorganisms, and to ensure the rational design
of prebiotic/probiotic combinations to avoid the proliferation of gastrointestinal pathogens (Tzortzis et al., 2004).
The evaluation of a prebiotic should not be limited to its impact on
bacterial growth, but should also consider activities associated with
these bacteria, such as metabolic products that result from its use, such

as organic acids (Gibson et al., 2017). The main methods for the
quantification of short-chain fatty acids (SCFA) include chromatographic techniques such as high-performance liquid chromatography
(Duarte et al., 2017), gas-liquid chromatography (Madhukumar &
Muralikrishna, 2010) and gas chromatography (Kalidas et al., 2017).
Various separation techniques have been used to determine SCFA in
biological fluids, the most widely used being gas chromatography (GC)
combining selective GC detectors, a flame ionization detection (FID) or
mass spectrometry (MS) (Ahn et al., 2018; McGrath, Weir, Maynard, &
Rowlands, 1992). Regarding the unique physicochemical properties of
SCFAs, low vapor pressure and relatively high solubility in the aqueous
phase cause difficulties in the sample preparation (Park, Lee, Lee &
Hong, 2017), and after this, its needs to be transformed in volatile
derivatives. However, their procedures include the steps of chemical
reaction or concentration, and it can lead to serious analyte loss due to
the high volatility of SCFAs (Kim, Kwon, Choi, & Ahn, 2019). Other
alternatives involving the use of solid-phase extraction (SPE) followed
by chromatographic separation with acid-resistant columns (i.e. poly
(ethylene glycol) or acidic Carbowax 20 M) (Kim et al., 2019).
Liquid chromatography-mass spectrometry (LC–MS) has often been
used in metabolomics studies with minimal sample preparation as
compared with GC or GC–MS. However, the quantitation of SCFAs
without chemical derivatization requires harsh experimental conditions
in LC–MS, such as an aqueous mobile phase containing 1.5 mM hydrochloric acid (Van Eijk, Bloemen, & Dejong, 2009). In addition, their
hydrophilicity results in poor chromatographic separation and insufficient ionization in electrospray ionization (ESI) (Van Eijk et al.,
2009). Thus, it was difficult to detect SCFAs by LC–MS, because their
masses were in the lower mass range in mass spectra, where numerous
interfering peaks from solvents and additives were present (Song, Lee,
Kim, Back, & Yoo, 2019). To overcome these problems, several chemical derivatization methods have been introduced to quantify SCFAs
while using LC–MS. However, these derivatizations require longer reaction time or specific reaction conditions (Song et al., 2019).
In addition to chromatography, the NMR technique does not require

derivatization of the sample (GC, GC/MS), use of a very specific LC
detector (the pulse amperometric, electrochemical, refractive index and
mass spectrometry), GC chromatographic columns acid-resistant and,
therefore, considerably reduces handling time and is not destructive. It
is also considered environmentally friendly, as it allows the use of less
solvents and generation of waste compared, for example, to chromatographic methods. (Ramanjooloo, Bhaw-Luximon, Jhurry, & Cadet,
2009; Rodrigues et al., 2011).
There are few studies in literature, however, that use Nuclear
Magnetic Resonance Spectroscopy (NMR) to evaluate prebiotic potential. This technique can be quick and easy and provide quantitative
monitoring of the fermentation of sugars into lactic and other organic
acids (Ramanjooloo et al., 2009; Rodrigues et al., 2011).
One of the major advantages of quantitative analysis with NMR is
that unlike chromatography, it is possible to employ a single internal
standard for all the chemical substances. This is because, under

2. Materials and methods
2.1. Microorganisms and culture conditions
The composition of the De Man Rogosa and Sharpe (MRS) basal
media used in the present study was as follows: 10 g/L of protease
peptone, 10 g/L of meat extract, 5 g/L of yeast extract, 1 g/L of Tween
80, 2 g/L of ammonium citrate, 5 g/L of sodium acetate, 0.2 g/L of
magnesium sulfate, 0.05 g/L of manganese sulfate, 2 g/L of dipotassium
hydrogen phosphate and 0.05 g/L of cysteine hydrochloride. The
medium was adjusted to pH 5.7 with HCl (0.1 M).
The probiotic strains used were Lactobacillus acidophilus ATCC 4356,
L. fermentum ATCC 23271, L. paracasei ATCC 335 and L. brevis ATCC
367, supplied by the Fundaỗóo Oswaldo Cruz (FIOCRUZ-Rio de JaneiroBrazil). These strains were stored at −20 °C in MRS basal broth with 20
% glycerol and 2 % of glucose.
The probiotic strains were examined for their antagonistic activities
against Klebsiella oxytoca isolated from the rhizosphere of Aspidosperma

polyneuron in accordance with Celloto et al. (2012). Culture stocks of
the indicator strains were maintained in Muller Hinton (MH) (Difco,
Detroit, MI) with 20 % glycerol at −80 °C.
Before the assays, the probiotic strains were sub-cultured twice in
the MRS basal broth with 2 % (w/v) of glucose. The inoculum was
prepared by centrifuging the active cultures at 3000 rpm for 20 min to
collect the cell pellet. The cells were washed twice with sterile saline
solution (NaCl 8.5 g/L). The cell pellet was then resuspended in sterile
saline and adjusted at 600 nm (OD 600) with a Varian Cary Model 1E
UV–vis spectrometer to obtain the suspension of cells with the required
optical density for each assay. All the cell suspensions were freshly
prepared before each experiment.
2.2. Antimicrobial study by co-culture
The assay was performed following Fooks and Gibson (2002) with
modifications. To establish a rational symbiotic design, the effect of
different prebiotic and probiotic combinations in co-culture with K.
oxytoca was evaluated.
For the assay, each bacterial suspension, adjusted to an optical
density of 0.8 (OD 600), was inoculated at 2 % in both monoculture and
co-culture (inoculated with pure cultures of one of the probiotic strains
and K. oxytoca) in tubes containing 7 mL of MRS basal media. The MRS
broth contained 1 % of the following prebiotics: inulin (Orafti® GR ∼92
%, granulated inulin powder, Mw = 2500 and Mn = 1500, average DP
≥ 10, Beneo-Orafti, Belgium); fructooligosaccharides obtained from
Cichorium endivia (76 %, DP – 2-8, FOS-CH) and Orafti® P95
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B. Higashi, et al.

modifications.
The activated probiotic strain cultures were adjusted to an optical
density of 0.8 (OD 600), which provided viable cell counts of approximately 9 log CFU/mL. The suspensions were then inoculated at 1
% in 100 mL of a medium containing 50 g/L calcium carbonate, 10 g/L
of yeast extract, and 50 g/L of the carbon source. The assays included a
negative control without a carbon source (basal medium), positive
control with glucose as an optimal carbon source, and the prebiotics
fructans FOS-CH, FOS-P95 and inulin.
The culture was incubated at 37 °C for five days in an orbital shaker
(Marconi MA-420) with a shaking speed of 150 rpm. After incubation, a
1 mL aliquot was taken to count the CFU using the pour plate method on
MRS agar.
For NMR analysis, the solutions were centrifuged and filtered to
separate the excess calcium carbonate. They were then acidified with
sulfuric acid (1 M) to pH 1.6 and filtered again. Purification of the
fermented solution was performed by extraction with ethyl ether at an
extract/solvent ratio of 1:2 (v/v). This extraction was performed twice.
The organic phase was solubilized in 700 μL of D2O and DL-mandelic
acid (about 0.005 g) was added as an internal standard. The analyses
were performed with a Bruker Avance III HD Spectrometer operating at
500 MHz for 1H NMR and 125 MHz for 13C NMR. The chemical shifts
(δ) were expressed in parts per million (ppm). SCFA assignments were
confirmed by HSQC and HMBC correlations, published data (Caligiani
et al., 2007; Fan, 1996; Jacobs et al., 2008), prediction by MestreNova
12.0 software data bank and the reference spectra from the Human
Metabolome Database (HMDB) and Biological Magnetica Resonance
Data Bank (BMRDB) (Wishart, Jewison, Guo, & Wilson, 2012), Supplementary material 3.
The SCFA concentrations after fermentation of the media containing

the different carbon sources were calculated according to Eq. 2.

fructooligosaccharide (95 %, DP = 2-8, Beneo-Orafti, Belgium). The
tests were performed in triplicate. Incubation was performed at 37 °C
under microaerophilic conditions.
Samples were removed after 24 h of incubation to determine viable
cell count and measure pH. A 0.1 mL aliquot of each system was used to
prepare serial dilutions and then poured onto the appropriate agar
plates, i.e. MRS agar was used for counting the strains of Lactobacillus
while MacConkey agar (Difco) was used for K. oxytoca. Plates were
incubated at 37 °C for 24 h and colonies were counted. Each experiment
was conducted in triplicate. The percentage of inhibition of K. oxytoca
in co-culture with Lactobacillus strains in different substrates was calculated by Eq. 1.
Log CFU /mL in control - Log CFU /mL in co-incubation culture
Inhibition (%) = × 100 CFU /mL in control

2.3. Agar diffusion test
The production of inhibitory substances by probiotics was performed following Mogna et al. (2016) with modifications.
Bacterial suspensions of Lactobacillus strains adjusted to an optical
density of 0.3 (OD 600) were inoculated at 2 % in 7 mL of MRS broth
containing 1 % of glucose and incubated at 37 °C for 24 h under microaerophilic conditions.
After incubation, the probiotic cultures were centrifuged at 3000
rpm for 20 min and the supernatants resulting from the centrifugation
were divided into two parts. Half of the resultant supernatant was adjusted to pH 7.0 with 1 M NaOH, while the other was kept at its original
pH and sterilized by filtration using 0.22 μm filter tops (TPP,
Switzerland).
Bacterial suspensions of K. oxytoca adjusted to an optical density of
0.3 (OD 600) were used to seed MacConkey agar plates evenly using a
sterile swab, to obtain homogeneous growth throughout the Petri dish.
To determine antimicrobial activity, filter paper discs (6 mm in

diameter) soaked in the probiotic supernatants (20 μL) were added to
the MacConkey agar surface. A filter paper disc soaked in sterile culture
medium (MRS) was used as a control and placed on the MacConkey
agar plates.
The plates were placed in the refrigerator for 2 h to diffuse the
compounds in the medium and incubated at 37 °C. After incubation,
inhibitory activity was evaluated based on the formation of a clear zone
around the paper disc.

IA
m
n 1000
mOA = [ ⎛
x1,67⎞ ⎛ MA ⎞ ( )(
)]
V
⎝ ID
⎠ ⎝ 152 ⎠ 3

(2)

mOA = molar mass of SCFA,
IA = intensity of protons of organic acids,
ID = intensity of phenyl protons of mandelic acid,
mMA = mass of mandelic acid,
n = number of hydrogens
V = volume of solution analyzed (mL)
2.6. Statistical analysis
The results were evaluated using the Microsoft Excel 2007 software
program (Microsoft, Redmond, WA, USA) and Statistica 10 (StatSoft).

Values of P < 0.05 were considered statistically significant.

2.4. Scanning electron microscopy (SEM)
The combination of prebiotics and probiotics that achieved the best
result in the previous test was incubated in co-culture with the target
microorganism following the methodology described above. Scanning
electron microscopy was performed following Pamphile, Gai, Pileggi,
Rocha, and Pileggi (2008), with slight modifications, using ethanol
gradient instead of acetone (30, 50, 70, 90 and 100 %).
The co-culture sample was dried in a critical point dryer (BAL-TECCPD 030), undergoing seven cycles, and assembled in stubs with SEM
adhesive tape conductors. The sample was then covered with a thin
layer of gold (50 mA, at 27 °C) for three cycles, in a metallic coating
apparatus (BAL-TEC SCD 050 - Sputter Coater). The gold-covered
sample was observed using a scanning electron microscope SHIMADZU-SS550 with an emission field of 12.5 kV, from a distance of 9.8
mm, provided by the Central Complex for Research Support (COMCAP,
State University of Maringá).

3. Results and discussion
3.1. Antimicrobial study by co-culture
The effect of lactobacilli strains on the growth of K. oxytoca was
investigated in co-culture when growing in different substrates
(Fig. 1A). K. oxytoca counts were significantly reduced (P < 0.05) after
incubation for 24 h in co-culture with probiotics strains. The cell counts
of K. oxytoca were reduced by up to 3.12 log CFU/mL. In contrast, there
was no change (P < 0.05) in the population numbers of the probiotic
strains in co-culture with K. oxytoca, in comparison with monoculture
growth (data not shown). Therefore, the growth of the probiotic strains
was not influenced by the presence of K. oxytoca, a result which agrees
with the findings of studies by Shah et al. (2016) and Yun et al. (2009).
There were significant differences (P < 0.05) in the K. oxytoca

counts depending on the lactobacilli strain used (Fig. 1A and B). L.
acidophilus was the most effective inhibitor of K. oxytoca growth, with a
significant reduction (P < 0.05) of 3.12 log (CFU/mL) when FOS-CH
was used as a substrate. This means that L. acidophilus reduced the K.

2.5. Short chain fatty acids by NMR
Analysis of the short organic fatty acids during fermentation was
performed by the method described by Ramanjooloo et al. (2009), with
3


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B. Higashi, et al.

Fig. 1. A) Viable cell counts of K. oxytoca in monoculture and co-culture with Lactobacillus strains in different substrates (FOS-CH, FOS-P95, and Inulin). Total viable
cells were expressed as log CFU/mL. Values with a different lower case letter in each column differ significantly at the 5 % level. B) Inhibition percentage of K.
oxytoca in co-culture with Lactobacillus strains in different substrates (FOS-CH, FOS-P95, and Inulin).

3.2. Antimicrobial activity by disc diffusion test

oxytoca population by 26 %, Fig. 1B.
It is also noticeable that the antimicrobial potential exhibited by the
probiotics used in this study seemed to depend on the substrate used.
When inulin was used as a substrate the inhibition caused by the lactobacilli strains was reduced. As previously reported by Lopes et al.
(2016), the rate of consumption of FOS and inulin increases when the
degree of polymerization (DP) decreases. Thus, FOS, which has a lower
DP than Inulin, was metabolized more rapidly by almost all the probiotic bacteria and ensured greater cell viability after 24 h of incubation.
There are few studies regarding the antagonistic activity of K.
oxytoca, with the most available research using Klebsiella pneumoniae.

Mogna et al. (2016) reported that the cell count of K. pneumoniae was
reduced by more than 5 log CFU/mL when co-cultured with L. delbrueckii subsp. delbrueckii.
In the present study, changes in pH after fermentation were also
observed (Fig. 2). It was found that L. acidophilus lowered pH significantly more than the other probiotic strains in co-culture with K.
oxytoca. A positive correlation between inhibitory activity and the reduction of the pH of the medium was therefore observed. These results
indicate that the inhibitory effect could be a consequence of the ability
of the probiotics to ferment FOS/inulin, producing organic acids.

Supernatants of pure cultures of lactobacilli strains adjusted to pH 7,
and those kept at the original pH, were tested for their ability to inhibit
K. oxytoca by the disc diffusion test. In the present study, inhibition was
related to the supernatant pH and was particularly marked when the
supernatants were not adjusted. No lactobacilli supernatants adjusted
to pH 7.0 caused inhibition zones around the discs (Fig. S1).
In contrast, the untreated supernatants inhibited the target strain,
except for L. fermentum. The supernatant of L. acidophilus exhibited the
highest rates of inhibition against K. oxytoca growth, with an inhibition
halo of 11.7 ± 0.6 mm (Table 1). Interestingly, L. fermentum and L.
brevis, which exhibited lower inhibition in both the disc diffusion and
co-culture tests, are considered heterofermentative. Heterofermentative
bacteria metabolize glucose through the 6-phosphogluconate pathway,
with 1 mol of glucose resulting in an equimolar amount of lactic acid
(Tejero-Sariñena, Barlow, Costabile, Gibson, & Rowland, 2012).
Homofermentative bacteria such as L. acidophilus metabolizes the glucose through the Embden-Meyerhof pathway, where 1 mol of hexose
results in 2 mol of lactic acid (Champagne, Gardner, & Roy, 2005;
Holzapfel & Schillinger, 2002).
Very few studies have assessed the antagonistic activity of K.oxytoca
by the agar diffusion test, with most using Klebsiella pneumoniae. Mogna
4

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B. Higashi, et al.

Fig. 2. pH values of supernatant fraction of the cultures of K. oxytoca in monoculture and co-culture with Lactobacillus strains in different substrates (FOS-CH, FOSP95, and Inulin).

3.4. Analysis of SCFA by 1H NMR

Table 1
Inhibitory activity of untreated supernatants of probiotic strains against K.
oxytoca.
Probiotic strains

Diameter of inhibition zone (mm) *

L.
L.
L.
L.

–
11.7 ± 0.6 a
11 ± 1 a, c
9.3 ± 0.6 c, b

fermentum ATCC 23271
acidophilus ATCC 4356
paracasei ATCC 335
brevis ATCC 367

The extracts obtained from the L. acidophilus fermentation media,
which most effectively inhibited K. oxytoca, were analyzed by 1H NMR.
Fig. 4 shows 1H NMR spectroscopy for the different substrates. The acid
concentrations reveal that there are variations in the relative intensities
between the substrates, but in general, the same signals appeared in all
spectra.
Fig. 4 shows that the extract obtained from L. acidophilus fermentation contains lactic acid, characterized by a doublet at δH 1.32 (J=
1.28 Hz) and a quartet at δH 4.28, acetic acid, characterized by a singlet
at δH 1.99, pyruvic acid characterized by a singlet at δH 2.12, and
succinic acid characterized by a singlet at δH 2.57. The assignment of
the signals identified and subsequently utilized for quantification is
shown in Table 2 was confirmed by comparison with Standards of
short-chain fatty acids (SCFAs) 1H NMR spectra (Supplementary Material 3) and map correlation HSQC analysis (Supplementary Material
2).
The quantification of the SCFA shown in Table 3 was determined by
comparing the intensity of the aromatic hydrogens of mandelic acid
with the methyl hydrogens of lactic acid, pyruvic acid, and acetic acid
or the methylene hydrogens of succinic acid. The aromatic hydrogens of
the mandelic acid were observed at δH 7.36 and the methine hydrogen
at δH 5.20 and there was, therefore, no overlap with the organic acid
hydrogens.
It can be seen in Table 3 that the lactic, acetic and succinic acids are
final fermentation products of L. acidophilus, and the increased production of these acids during growth is directly associated with intense
metabolic activities (Ríos-Covián et al., 2016). As expected, there was
greater cell viability and concentration of organic acids in media containing a carbon source than in the negative control. Differences in the
amounts of organic acids produced for each substrate tested were also
observed. This can be explained by the fact that the fermentation rate
depends mainly on the enzymatic system of the bacteria and the
structure of the fructan, and its degree of polymerization (Lopes et al.,

2016).
Among the prebiotics evaluated, FOS-CH followed by FOS-P95

Measurements expressed in mm are the mean of three replicates ± deviation.
Different letters indicate statistically significant differences (P < 0.05) between
the lines. * The diameter measurement includes 6 mm of the paper disc.

et al. (2016) reported that L. delbrueckii subsp. delbrueckii exhibited the
greatest antagonistic effect against K. pneumoniae, with an inhibition
halo close to 10.0 mm, while Shokryazdan et al. (2014) reported an
inhibition halo of up to 12.7 mm by lactobacilli strains.
The antagonistic activity has mostly been attributed to the production of SCFA (mainly lactic and acetic acids), hydrogen peroxide,
and bacteriocins. The results of the present study indicate that acidity
and organic acids can correlate with the inhibitory activity of lactobacilli strains, agreeing with studies by while Neal-McKinney et al.
(2012) and Shokryazdan et al. (2014). Also, lactic acid appears to play a
key role in this inhibition, as homofermentative bacteria exhibited
greater inhibitory activity.

3.3. Scanning electron microscopy (SEM)
The interaction between K. oxytoca and L. acidophilus which exhibited the best results in the previous tests using FOS-CH as a substrate
was investigated by SEM. After 12 h of incubation, it was observed that
the amount of L. acidophilus present was relatively higher than K.
oxytoca (Fig. 3), since the first is in coccobacilli form while the second is
in bacilli form. Conglomerates of both types of bacteria were also observed (Fig. 3a) along with cell-cell interactions in some cases (Fig. 3b).

5


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Fig. 3. Scanning electron microscopy of L. acidophilus and K. oxytoca in co-culture after 12 h of incubation at a) 5000 and b) 20,000 times magnification at 12.50 kV
acceleration voltage.

acid is derived from sugar metabolism through glycolysis and followed
by a reduction in pyruvic acid. This acid is the most important intermediary in several metabolic pathways during fermentation, which can
lead to the formation of organic acids (Sauer, Russmayer, Grabherr,
Peterbauer, & Marx, 2017). The generation of pyruvic acid derived from
glucose or the reversal of acetic or lactic acid to pyruvic acid may be a
mechanism of the bacterium to protect itself from the acidity of the
culture (Wu, Li, Cai, & Jin, 2014).
Our results from co-culture studies agree with other studies that
have been reported that lactic acid has antibacterial activities against
certain Gram-positive and Gram-negative bacteria (Chotigarpa et al.,
2018; Wang, Chang, Yang, & Cui, 2015) and a possible mechanism for
lactic acid bacteriostatic effect as suggested by Wang et al. (2015) could
result in great leakage of proteins of these bacteria probably caused by
physiological and morphological changes in bacterial cells.
Lactic acid is the main metabolite of lactic bacteria such as lactobacilli. However, under normal physiological conditions, it does not
accumulate in the colon due to the presence of species that can convert

produced the largest amounts of organic acids, mainly lactic acid, and
ensured greater L. acidophilus cell viability after five days of incubation.
Inulin was the least favorable substrate for the growth of L. acidophilus
and the formation of a higher concentration of organic acids, as can be
seen in Table 3.
The FOS-CH samples isolated from C. endivia roots showed a similar
chemical profile and degree of polymerization (DP) to commercial FOS
(Orafti® P95) and total sugar contents were similar (p > 0.05), 100 %

for Orafti® P95 and 85.4 % for the FOS samples (Lopes et al., 2016;
Mariano, 2017). According to available literature, short degree of
polymerization fructans as FOS-CH and Orafti® P95, with an average
DP < 10 and DP > 10, are more easy metabolized by prebiotic
bacteria, however, these FOS could be induced L. acidophilus to produce
diverse levels of SCFA (Al-Sheraji et al., 2013).
Lactic acid was the main SCFA produced by L. acidophilus, which
agrees with the study by Gullón, Romaní, Vila, Garrote, and Parajó
(2012). Probably it occurred because some Lactobacillus species, such as
L. acidophilus, are homofermentative organisms. The increase of lactic
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B. Higashi, et al.

Fig. 4. B. 1H NMR spectra (500 MHz, D2O) of the extracts containing as sole carbon source (A) Glucose, (B) FOS-P95, and (C) FOS-CH, fermented by L. acidophilus
ATCC 4356.

peripheral circulation to be metabolized and act as a substrate for lipid
biosynthesis in peripheral tissues such as the brain, heart, muscle, and
adipose tissue (Belenguer, Duncan, Holtrop, Flint, & Lobley, 2008).
Studies have also shown that acetic acid has a direct role in the central
regulation of the appetite, and thus may aid in body weight control
(Frost et al., 2014). The production of large amounts of acetic acid can
also feed other colon bacteria involved in the production of butyric acid
(De Vuyst et al., 2014).
Although a few studies in the literature use NMR spectroscopy to
evaluate prebiotic potential, this technique proved to be a very interesting choice, providing quick results with a high level of accuracy.

Also, the NMR technique does not require derivatization of the sample,
considerably reducing handling time, and is non-destructive. It is also
considered to be environmentally friendly, as it allows lower solvent
use and waste generation (Emwas, 2015; Ramanjooloo et al., 2009).
Furthermore, the small volumes of solutions used for analysis do not
disturb the general concentration of the fermentation medium.

Table 2
Assignment of SCFA signals identified and subsequently utilized for quantification by 1H NMR, based in MestreNova 12.0 software prediction, HMDB, and
BMRDB (Supplementary Material 3).
Compound

Methyl/
Methylene
Group

δH (multiplicity,
J in Hz)

1

1.99 (s)

1

1.32 (d, 1.28)

1

2.12 (s)

Acetic acid

Lactic acid

4. Conclusion
The results of the present study demonstrated that some strains of
Lactobacillus can inhibit K. oxytoca, and that this antagonism is influenced by the carbohydrate source and are probably associated with
organic acid production. The combination of L. acidophilus and FOS-CH
most effectively inhibited K. oxytoca, and the fructooligosaccharide
obtained from Cichorium endivia was an effective substrate for L. acidophilus, resulting in greater viability and increased the production of
SCFA, mainly lactic acid.
This study may serve as a tool for the rational selection of symbiotic
constituents, considering as it does competitiveness with other intestinal bacteria, probiotic viability, and SCFA production. Besides,
NMR spectroscopy proved to be a reliable technique for the quantitative

Pyruvic acid
2 and 3

2.57 (s)

Succinic acid

a

Multiplicity: s, singlet; d, doublet.

lactic acid to different organic acids, especially butyric acid (Flint,
Duncan, Scott, & Louis, 2014).
In addition, acetic acid can be absorbed and pass through the

7


Carbohydrate Polymers 248 (2020) 116832

B. Higashi, et al.

Table 3
Cell viability and production of SCFA by L. acidophilus ATCC 4356 on different substrates after 5 days of fermentation.
Substrate

Cell viability (log CFU/mL)

Lactic acid (mM)

Acetic acid (mM)

Succinic acid (mM)

Pyruvic acid (mM)

Negative Control
Glucose
FOS-P95
FOS-CH
Inulin

6.34
8.53
8.48

8.50
7.69

1566
127.44
70.82
105.94
54.81

0
1.09
5.41
4.56
5.58

2.65
1.63
3.58
3.52
5.48

6.51
7.77
1.62
7.63
2.73

evaluation of the production of organic acids, offering several advantages over other commonly used techniques.

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Credit author statement
All the authors have read, approved, and made substantial contributions to the manuscript. None of the original material contained in
this manuscript has been previously published nor is currently under
review for publication elsewhere. The manuscript has not been previously published, is not currently submitted for review to any other
journal.
Declaration of Competing Interest
The authors have no conflicts of interest.
Acknowledgments
The present study was supported by grants from the Conselho
Nacional de Desenvolvimento Científico e Tecnolúgico (The National
Council of Scientic and Technological Development) (CNPq), the
Coordenaỗóo de Aperfeiỗoamento de Pessoal de Nớvel Superior (The

Coordination for the Improvement of Higher Education Personnel)
(CAPES), the Pharmaceutical Sciences Graduate Program of the State
University of Maringá (UEM) and the Complexo de Centrais de Apoio a
Pesquisa (the Central Research Support Complex) (COMCAP).
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9


Update
Carbohydrate Polymers
Volume 260, Issue , 15 May 2021, Page
DOI: />

Carbohydrate Polymers 260 (2021) 117568

Contents lists available at ScienceDirect

Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Corrigendum to: “Effects of fructans and probiotics on the inhibition of
Klebsiella oxytoca and the production of short-chain fatty acids assessed by
NMR spectroscopy” [Carbohyd. Polym. 248 (2020) 116832, doi: 10.1016/j.
carbpol.2020.116832]
Bruna Higashi a, Tamara Borges Mariano a, Benício Alves de Abreu Filho b,
Regina Aparecida Correia Gonỗalves a, Arildo Jose Braz de Oliveira a, *
a
b

Graduate Program in Pharmaceutical Sciences, Department of Pharmacy, State University of Maring´
a, Ave. Colombo 5790, 87.020-900, Maring´
a, Brazil
Departament of Basic Health Sciences, State University of Maring´
a, Ave. Colombo 5790, 87.020-900, Maring´
a, Brazil

With regard to their article “Effects of fructans and probiotics on the
inhibition of Klebsiella oxytoca and the production of short-chain fatty
acids assessed by NMR spectroscopy”, the authors regret about the
inappropriate use of the word “symbiotic”.
The term “symbiotic” has to be changed into “synbiotic” through all
the text, being this the correct term to denote a form of synergism arising
from the combined use of probiotics and prebiotics, as disclosed by the
finding of the study.

This change would not affect any of the conclusions of the whole

DOI of original article: />* Corresponding author.
E-mail address: (A.J. Braz de Oliveira).
/>Available online 25 February 2021
0144-8617/© 2020 Elsevier Ltd. All rights reserved.

manuscript.
The authors would like to apologize for any inconvenience caused
and to acknowledge prof. Amin Abbasi (Department of Food Science and
Technology, Faculty of Nutrition & Food Sciences, Nutrition Research
Center, Tabriz University of Medical Sciences, Tabriz, Iran) and prof.
Nayyer Shahbazi (Department of Food Science, Faculty of Agriculture
Engineering, Shahrood University of Technology, Shahrood, Iran) for
their advices on this matter.