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.
Trang 1Contents lists available atScienceDirect
Carbohydrate Polymers journal homepage:www.elsevier.com/locate/carbpol
the production of short-chain fatty acids assessed by NMR spectroscopy
Bruna Higashia, Tamara Borges Marianoa, Benício Alves de Abreu Filhob,
Regina Aparecida Correia Gonçalvesa, Arildo José Braz de Oliveiraa,*
a Graduate Program in Pharmaceutical Sciences, Department of Pharmacy, State University of Maringá, Ave Colombo 5790, 87.020-900, Maringá, Brazil
b Departament of Basic Health Sciences, State University of Maringá, Ave Colombo 5790, 87.020-900, Maringá, Brazil
A R T I C L E I N F O
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)
Keywords:
Acetic acid
Co-culture
Klebsiella oxytoca
Lactic acid
Lactobacillus acidophilus
Symbiotic
A B S T R A C T 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
1 Introduction
There are several available strategies for the modulation of the
in-testinal microbiota and improve health and well-being A range of
therapeutic tools has been developed, such as the introduction of
un-ique or associated microorganisms (probiotics), the provision of
sub-strates 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 con-sidered 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 micro-organisms most commonly used as probiotics, principally due to their
https://doi.org/10.1016/j.carbpol.2020.116832
Received 22 April 2020; Received in revised form 22 July 2020; Accepted 24 July 2020
⁎Corresponding author at: Universidade Estadual de Maringá, Av Colombo 5790, Bloco K-80 CEP 87020-900, Maringá, PR, Brazil
E-mail address:ajboliveira@uem.br(A.J.B de Oliveira)
Carbohydrate Polymers 248 (2020) 116832
Available online 27 July 2020
0144-8617/ © 2020 Elsevier Ltd All rights reserved
T
Trang 2long history of safe use (Daly & Davis, 1998) They are termed lactic
acid bacteria (LAB) since the main end-product of carbohydrate
meta-bolism 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
com-pounds by probiotic microorganisms, and to ensure the rational design
of prebiotic/probiotic combinations to avoid the proliferation of
gas-trointestinal 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
chromato-graphic 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
biologicalfluids, the most widely used being gas chromatography (GC)
combining selective GC detectors, aflame 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
hy-drochloric acid (Van Eijk, Bloemen, & Dejong, 2009) In addition, their
hydrophilicity results in poor chromatographic separation and
in-sufficient 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
che-mical derivatization methods have been introduced to quantify SCFAs
while using LC–MS However, these derivatizations require longer
re-action time or specific rere-action 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
chroma-tographic 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
poten-tial 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
appropriate instrumental conditions, the NMR response is exactly pro-portional 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 pa-thogen 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 ex-tensively 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 fructooligo-saccharides) 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
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 Fundação Oswaldo Cruz (FIOCRUZ-Rio de Janeiro-Brazil) 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 withCelloto 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 followingFooks 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
Trang 3fructooligosaccharide (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
cal-culated 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
per-formed followingMogna 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
mi-croaerophilic 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
ad-justed 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 (20μL) were added to
the MacConkey agar surface Afilter 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
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 followingPamphile, 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-TEC-CPD 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
SHIMA-DZU-SS550 with an emissionfield of 12.5 kV, from a distance of 9.8
mm, provided by the Central Complex for Research Support (COMCAP,
State University of Maringá)
2.5 Short chain fatty acids by NMR
Analysis of the short organic fatty acids during fermentation was
performed by the method described byRamanjooloo et al (2009), with
modifications
The activated probiotic strain cultures were adjusted to an optical density of 0.8 (OD 600), which provided viable cell counts of ap-proximately 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 forfive 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 andfiltered 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 700μL 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 for1H NMR and 125 MHz for13C 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), Sup-plementary material 3
The SCFA concentrations after fermentation of the media containing the different carbon sources were calculated according to Eq.2
= ⎛
⎝
⎞
⎠
⎛
⎝
⎞
⎠
ID x
V
152 (3)(
1000 )]
(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
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 thefindings of studies byShah et al (2016)andYun 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
Trang 4oxytoca 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
lac-tobacilli 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
pro-biotic bacteria and ensured greater cell viability after 24 h of
incuba-tion
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
del-brueckii subsp deldel-brueckii
In the present study, changes in pH after fermentation were also
observed (Fig 2) It was found that L acidophilus lowered pH
sig-nificantly more than the other probiotic strains in co-culture with K
oxytoca A positive correlation between inhibitory activity and the
re-duction 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
3.2 Antimicrobial activity by disc diffusion test 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 glu-cose 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
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)
Trang 5et 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
pro-duction 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
lacto-bacilli strains, agreeing with studies by while Neal-McKinney et al
(2012)andShokryazdan 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
ex-hibited 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 thefirst is in coccobacilli form while the second is
in bacilli form Conglomerates of both types of bacteria were also
ob-served (Fig 3a) along with cell-cell interactions in some cases (Fig 3b)
3.4 Analysis of SCFA by1H NMR
The extracts obtained from the L acidophilus fermentation media, which most effectively inhibited K oxytoca, were analyzed by1H NMR Fig 4shows1H 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 4shows that the extract obtained from L acidophilus fermen-tation contains lactic acid, characterized by a doublet atδH1.32 (J= 1.28 Hz) and a quartet atδH4.28, acetic acid, characterized by a singlet
at δH 1.99, pyruvic acid characterized by a singlet atδH2.12, and succinic acid characterized by a singlet atδH2.57 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 Ma-terial 3) and map correlation HSQC analysis (Supplementary MaMa-terial 2)
The quantification of the SCFA shown inTable 3was 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 atδH7.36 and the methine hydrogen
atδH5.20 and there was, therefore, no overlap with the organic acid hydrogens
It can be seen inTable 3that the lactic, acetic and succinic acids are final fermentation products of L acidophilus, and the increased pro-duction 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 con-taining 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
Fig 2 pH values of supernatant fraction of the cultures of K oxytoca in monoculture and co-culture with Lactobacillus strains in different substrates (CH, FOS-P95, and Inulin)
Table 1
Inhibitory activity of untreated supernatants of probiotic strains against K
oxytoca
Probiotic strains Diameter of inhibition zone (mm) *
L acidophilus ATCC 4356 11.7 ± 0.6 a
L paracasei ATCC 335 11 ± 1 a, c
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
Trang 6produced the largest amounts of organic acids, mainly lactic acid, and
ensured greater L acidophilus cell viability afterfive 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 inTable 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
acid is derived from sugar metabolism through glycolysis and followed
by a reduction in pyruvic acid This acid is the most important inter-mediary 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 byWang 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 lacto-bacilli However, under normal physiological conditions, it does not accumulate in the colon due to the presence of species that can convert 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
Trang 7lactic acid to different organic acids, especially butyric acid (Flint,
Duncan, Scott, & Louis, 2014)
In addition, acetic acid can be absorbed and pass through the
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 inter-esting 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
4 Conclusion
The results of the present study demonstrated that some strains of Lactobacillus can inhibit K oxytoca, and that this antagonism is influ-enced 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 acid-ophilus, 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 in-testinal bacteria, probiotic viability, and SCFA production Besides, NMR spectroscopy proved to be a reliable technique for the quantitative
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
Table 2
Assignment of SCFA signals identified and subsequently utilized for
quantifi-cation by1H NMR, based in MestreNova 12.0 software prediction, HMDB, and
BMRDB (Supplementary Material 3)
Methylene Group
δ H (multiplicity,
J in Hz)
Acetic acid
Lactic acid
Pyruvic acid
Succinic acid
2 and 3 2.57 (s)
aMultiplicity: s, singlet; d, doublet
Trang 8evaluation of the production of organic acids, offering several
ad-vantages over other commonly used techniques
Credit author statement
All the authors have read, approved, and made substantial
con-tributions 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
pre-viously 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 Tecnológico (The National
Council of Scientific and Technological Development) (CNPq), the
Coordenação de Aperfeiçoamento de Pessoal de Nível 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)
Appendix A Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.carbpol.2020.116832
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