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

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.

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 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: ajboliveira@uem.br (A.J.B de Oliveira) https://doi.org/10.1016/j.carbpol.2020.116832 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 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, g/L of yeast extract, g/L of Tween 80, g/L of ammonium citrate, g/L of sodium acetate, 0.2 g/L of magnesium sulfate, 0.05 g/L of manganese sulfate, 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 % 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 % (w/v) of glucose The inoculum was prepared by centrifuging the active cultures at 3000 rpm for 20 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 % in both monoculture and co-culture (inoculated with pure cultures of one of the probiotic strains and K oxytoca) in tubes containing mL of MRS basal media The MRS broth contained % 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 Carbohydrate Polymers 248 (2020) 116832 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 log CFU/mL The suspensions were then inoculated at % 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 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 The SCFA concentrations after fermentation of the media containing the different carbon sources were calculated according to Eq 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 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 % in mL of MRS broth containing % 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 and the supernatants resulting from the centrifugation were divided into two parts Half of the resultant supernatant was adjusted to pH 7.0 with 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 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 ⎠ (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) 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 Carbohydrate Polymers 248 (2020) 116832 B Higashi, et al Fig 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 % 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 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 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 mol of hexose results in 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 Carbohydrate Polymers 248 (2020) 116832 B Higashi, et al Fig 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 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 ± 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 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 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 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 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 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 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) Carbohydrate Polymers 248 (2020) 116832 B Higashi, et al Fig 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 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 Carbohydrate Polymers 248 (2020) 116832 B Higashi, et al Fig 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 not disturb the general concentration of the fermentation medium Table 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) 1.99 (s) 1.32 (d, 1.28) 2.12 (s) Acetic acid Lactic acid 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 and 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 Carbohydrate Polymers 248 (2020) 116832 B Higashi, et al Table Cell viability and production of SCFA by L acidophilus ATCC 4356 on different substrates after 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 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 Gonỗalves, R A C (2012) Biosynthesis of indole-3-acetic acid by new Klebsiella oxytoca free and immobilized cells on inorganic matrices The Scientific World Journal, 2012, 1–7 https://doi.org/10.1100/2012/495970 Champagne, C P., Gardner, N J., & Roy, D (2005) Challenges in the addition of probiotic cultures to foods Critical Reviews in Food Science and Nutrition, 45, 61–84 https://doi.org/10.1080/10408690590900144 Chotigarpa, R., Lampang, K N., Pikulkaew, S., Okonogi, S., Ajariyakhajorn, K., & Mektrirat, R (2018) Inhibitory effects and killing kinetics of lactic acid rice gel against pathogenic bacteria causing bovine mastitis Scientia Pharmaceutica https:// doi.org/10.3390/scipharm86030029 Daly, C., & Davis, R (1998) The biotechnology of lactic acid bacteria with emphasis on applications in food safety and human health Agricultural and Food Science, 7, 251–265 De Vuyst, L., Van Kerrebroeck, S., Harth, H., Huys, G., Daniel, H M., & Weckx, S (2014) Microbial ecology of sourdough fermentations: Diverse or uniform? Food Microbiology, 37, 11–29 https://doi.org/10.1016/j.fm.2013.06.002 Duarte, F N D., Rodrigues, J B., da Costa Lima, M., Lima, M S., Pacheco, M T B., Pintado, M M E., de Souza, E L (2017) Potential prebiotic properties of cashew apple (Anacardium occidentale L.) agro-industrial byproduct on Lactobacillus species Journal of the Science of Food and Agriculture, 7, 3712–3719 https://doi.org/10.1002/ jsfa.8232 Emwas, A H M (2015) The strengths and weaknesses of NMR spectroscopy and mass spectrometry with particular focus on metabolomics research Methods in molecular biology New York, NY: Humana Presshttps://doi.org/10.1007/978-1-4939-23779_13 Fan, T W M (1996) Metabolite profiling by one- and two-dimensional NMR analysis of complex mixtures Progress in Nuclear Magnetic Resonance Spectroscopy, 28, 161–219 https://doi.org/10.1016/0079-6565(95)01017-3 Flint, H J., Duncan, S H., Scott, K P., & Louis, P (2014) Links between diet, gut microbiota composition and gut metabolism The Proceedings of the Nutrition Society, 74, 13–22 https://doi.org/10.1017/S0029665114001463 Fooks, L J., & Gibson, G R (2002) In vitro investigations of the effect of probiotics and prebiotics on selected human intestinal pathogens FEMS Microbiology Ecology, 39, 67–75 https://doi.org/10.1016/S0168-6496(01)00197-0 Frost, G., Sleeth, M L., Sahuri-Arisoylu, M., Lizarbe, B., Cerdan, S., Brody, L., Bell, J D (2014) The short-chain fatty acid acetate reduces appetite via a central homeostatic mechanism Nature Communications, https://doi.org/10.1038/ncomms4611 Gibson, G R (2004) Fibre and effects on probiotics (the prebiotic concept) Clinical Nutrition, Supplement, 1, 25–31 https://doi.org/10.1016/j.clnu.2004.09.005 Gibson, G R., Hutkins, R., Sanders, M E., Prescott, S L., Reimer, R A., Salminen, S J., Reid, G (2017) Expert consensus document: The International Scientific Association for Probiotics and Prebiotics (ISAPP) consensus statement on the definition and scope of prebiotics Nature Reviews Gastroenterology & Hepatology, 14, 491–502 https://doi org/10.1038/nrgastro.2017.75 Gullón, P., Romaní, A., Vila, C., Garrote, G., & Parajó, J C (2012) Potential of hydrothermal treatments in lignocellulose biorefineries Biofuels Bioproducts and Biorefining, 6, 219–232 https://doi.org/10.1002/bbb.339 Hoffmann, K M., Deutschmann, A., Weitzer, C., Joamig, M., Zechner, E., Högenauer, C., & Hauer, A C (2010) Antibiotic-associated hemorrhagic colitis caused by cytotoxinproducing Klebsiella oxytoca Pediatrics, 125, 960–963 https://doi.org/10.1542/peds 2009-1751 Högenauer, C., Langner, C., Beubler, E., Lippe, I T., Schicho, R., Gorkiewicz, G., Hinterleitner, T A (2006) Klebsiella oxytoca as a causative organism of antibioticassociated hemorrhagic colitis The New England Journal of Medicine, 1, 370–376 https://doi.org/10.1056/NEJMoa054765 Holzapfel, W H., & Schillinger, U (2002) Introduction to pre- and probiotics Food Research International, 35, 109–116 https://doi.org/10.1016/S0963-9969(01) 00171-5 Holzapfel, W H., & Wood, B J B (2014) Lactic acid bacteria: Biodiversity and taxonomy Lactic acid Bacteria: Biodiversity and taxonomy John Wiley & Sonshttps://doi.org/10 1002/9781118655252 Jacobs, D M., Deltimple, N., van Velzen, E., van Dorsten, F A., Bingham, M., Vaughan, E E., & van Duynhoven, J (2008) 1H NMR metabolite profiling of feces as a tool to assess the impact of nutrition on the human microbiome NMR in Biomedicine, 21, 615–626 https://doi.org/10.1002/nbm.1233 Kalidas, N R., Saminathan, M., Ismail, I S., Abas, F., Maity, P., Islam, S S., Shaari, K (2017) Structural characterization and evaluation of prebiotic activity of oil palm kernel cake mannanoligosaccharides Food Chemistry, 234, 348–355 https://doi.org/ 10.1016/j.foodchem.2017.04.159 Kim, H., Kwon, J., Choi, S Y., & Ahn, Y G (2019) Method development for the quantitative determination of short chain fatty acids in microbial samples by solid phase extraction and gas chromatography with flame ionization detection Journal of Analytical Science and Technology, 10, 2–6 https://doi.org/10.1186/s40543-019- 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 Tecnológico (The National Council of Scientific 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) 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 References Ahn, Y G., Jeon, S H., Lim, H B., Choi, N R., Hwang, G S., Kim, Y P., & Lee, J Y (2018) Analysis of polycyclic aromatic hydrocarbons in ambient aerosols by using one-dimensional and comprehensive two-dimensional gas chromatography combined with mass spectrometric method: A comparative study Journal of Analytical Methods in Chemistry, https://doi.org/10.1155/2018/8341630 Alikhani, M Y., Shahcheraghi, F., Khodaparast, S., Mozaffari Nejad, A S., Moghadam, M K., & Mousavi, S F (2016) Molecular characterization of Klebsiella oxytoca strains isolated from patients with antibiotic-associated diarrhoea Arab Journal of Gastroenterology, 17, 95–101 https://doi.org/10.1016/j.ajg.2016.03.005 Al-Sheraji, S H., Ismail, A., Manap, M Y., Mustafa, S., Yusof, R M., & Hassan, F A (2013) Prebiotics as functional foods: A review Journal of Functional Foods, 5, 1542–1553 https://doi.org/10.1016/j.jff.2013.08.009 Ambalam, P., Kondepudi, K K., Balusupati, P., Nilsson, I., Wadström, T., & Ljungh (2015) Prebiotic preferences of human lactobacilli strains in co-culture with bifidobacteria and antimicrobial activity against Clostridium difficile Journal of Applied Microbiology, 119, 1672–1682 https://doi.org/10.1111/jam.12953 Beaugerie, L., Metz, M., Barbut, F., Bellaiche, G., Bouhnik, Y., Raskine, L., Petit, J C (2003) Klebsiella oxytoca as an agent of antibiotic-associated hemorrhagic colitis Clinical Gastroenterology and Hepatology, 1, 370–376 https://doi.org/10.1053/ S1542-3565(03)00183-6 Belenguer, A., Duncan, S., Holtrop, G., Flint, H., & Lobley, G (2008) Quantitative analysis of microbial metabolism in the human large intestine Current Nutrition and Food Science, 4, 109–126 https://doi.org/10.2174/157340108784245957 Caligiani, A., Acquotti, D., Palla, G., & Bocchi, V (2007) Identification and quantification of the main organic components of vinegars by high resolution 1H NMR spectroscopy Analytica Chimica Acta, 585, 110–119 https://doi.org/10.1016/j.aca.2006.12 016 Celloto, V R., Oliveira, A J B., Gonỗalves, J E., Watanabe, C S F., Matioli, G., & Carbohydrate Polymers 248 (2020) 116832 B Higashi, et al by nuclear magnetic resonance (NMR) Spectroscopy Journal of Agricultural and Food Chemistry, 59, 4955–4961 https://doi.org/10.1021/jf104605r Sauer, M., Russmayer, H., Grabherr, R., Peterbauer, C K., & Marx, H (2017) The efficient clade: Lactic acid Bacteria for industrial chemical production Trends in Biotechnology, 35, 756–769 https://doi.org/10.1016/j.tibtech.2017.05.002 Savino, F., Cordisco, L., Tarasco, V., Calabrese, R., Palumeri, E., & Matteuzzi, D (2009) Molecular identification of coliform bacteria from colicky breastfed infants Acta Paediatrica, International Journal of Paediatrics, 98, 1582–1588 https://doi.org/10 1111/j.1651-2227.2009.01419.x Shah, N., Patel, A., Ambalam, P., Holst, O., Ljungh, A., & Prajapati, J (2016) Determination of an antimicrobial activity of Weissella confusa, Lactobacillus fermentum, and Lactobacillus plantarum against clinical pathogenic strains of Escherichia coli and Staphylococcus aureus in co-culture Annals of Microbiology, 66, 1137–1143 https://doi.org/10.1007/s13213-016-1201-y Shokryazdan, P., Sieo, C C., Kalavathy, R., Liang, J B., Alitheen, N B., Faseleh Jahromi, M., & Ho, Y W (2014) Probiotic potential of Lactobacillus strains with antimicrobial activity against some human pathogenic strains BioMed Research International, 2014, 1–16 https://doi.org/10.1155/2014/927268 Song, H E., Lee, H Y., Kim, S J., Back, S H., & Yoo, H J (2019) A facile profiling method of short chain fatty acids using liquid chromatography-mass spectrometry Metabolites, 9, 2–11 https://doi.org/10.3390/metabo9090173 Tejero-Sariñena, S., Barlow, J., Costabile, A., Gibson, G R., & Rowland, I (2012) In vitro evaluation of the antimicrobial activity of a range of probiotics against pathogens: Evidence for the effects of organic acids Anaerobe, 18, 530–538 https://doi.org/10 1016/j.anaerobe.2012.08.004 Tzortzis, G., Baillon, M L A., Gibson, G R., & Rastall, R A (2004) Modulation of antipathogenic activity in canine-derived Lactobacillus species by carbohydrate growth substrate Journal of Applied Microbiology, 96, 552–559 https://doi.org/10.1111/j 1365-2672.2004.02172.x Valdés-Varela, L., Hernández-Barranco, A M., Ruas-Madiedo, P., & Gueimonde, M (2016) Effect of Bifidobacterium upon Clostridium difficile growth and toxicity when co-cultured in different prebiotic substrates Frontiers in Microbiology, 7, 38 https:// doi.org/10.3389/fmicb.2016.00738 Van Eijk, H M H., Bloemen, J G., & Dejong, C H C (2009) Application of liquid chromatography-mass spectrometry to measure short chain fatty acids in blood Journal of Chromatography B, Analytical Technologies in the Biomedical and Life Sciences, 138, 43–53 https://doi.org/10.1016/j.jchromb.2009.01.039 Wang, C., Chang, T., Yang, H., & Cui, M (2015) Antibacterial mechanism of lactic acid on physiological and morphological properties of Salmonella enteritidis, Escherichia coli and Listeria monocytogenes Food Control https://doi.org/10.1016/j.foodcont 2014.06.034 Wishart, D S., Jewison, T., Guo, A C., Wilson, M., et al (2012) HMDB 3.0—The human metabolome database in 2013 Nucleic Acids Research, 41(D1), D801–D807 Wu, J., Li, Y., Cai, Z., & Jin, Y (2014) Pyruvate-associated acid resistance in bacteria Applied and Environmental Microbiology, 80, 4108–4113 https://doi.org/10.1128/ AEM.01001-14 Yun, J H., Lee, K B., Sung, Y K., Kim, E B., Lee, H G., & Choi, Y J (2009) Isolation and characterization of potential probiotic lactobacilli from pig feces Journal of Basic Microbiology, 49, 220–226 https://doi.org/10.1002/jobm.200800119 Zollner‐Schwetz, I., Högenauer, C., Joainig, M., Weberhofer, P., Gorkiewicz, G., Valentin, T., Krause, R (2008) Role of Klebsiella oxytoca in antibiotic‐associated diarrhea Clinical Infectious Diseases, 47, e74–e78 https://doi.org/10.1086/592074 0184-2 Lopes, S M S., Francisco, M G., Higashi, B., de Almeida, R T R., Krausová, G., Pilau, E J., Braz de Oliveira, A J (2016) Chemical characterization and prebiotic activity of fructo-oligosaccharides from Stevia rebaudiana (Bertoni) roots and in vitro adventitious root cultures Carbohydrate Polymers, 152, 718–725 https://doi.org/10 1016/j.carbpol.2016.07.043 Krumbeck, J A., Maldonado-Gomez, M X., Ramer-Tait, A E., & Hutkins, R W (2016) Prebiotics and symbiotics: Dietary strategies for improving gut health Current Opinion in Gastroenterology, 32, 110–119 https://doi.org/10.1097/MOG 0000000000000249 Madhukumar, M S., & Muralikrishna, G (2010) Structural characterisation and determination of prebiotic activity of purified xylo-oligosaccharides obtained from Bengal gram husk (Cicer arietinum L.) and wheat bran (Triticum aestivum) Food Chemistry, 118, 215–223 https://doi.org/10.1016/j.foodchem.2009.04.108 Mariano, T B (2017) Extraction, chemical characterization and evaluation of the prebiotic activity of fructo-oligosacarides obtained from roots of “escarola” (Cichorium endivia) Brazil: State University of Maringá, Maringá-PR (Language Portuguese) [Master dissertation] McGrath, L T., Weir, C D., Maynard, S., & Rowlands, B J (1992) Gas-liquid chromatographic analysis of volatile short chain fatty acids in fecal samples as pentafluorobenzyl esters Analytical Biochemistry, 207, 227–230 https://doi.org/10.1016/ 0003-2697(92)90004-q Mogna, L., Deidda, F., Nicola, S., Amoruso, A., Del Piano, M., & Mogna, G (2016) Vitro inhibition of Klebsiella pneumoniae by Lactobacillus delbrueckii subsp Delbrueckii LDD01 (DSM 22106) an innovative strategy to possibly counteract such infections in humans? Journal of Clinical Gastroenterology, 50, S136–S139 https://doi.org/10 1097/MCG.0000000000000680 Neal-McKinney, J M., Lu, X., Duong, T., Larson, C L., Call, D R., Shah, D H., & Konkel, M E (2012) Production of organic acids by probiotic lactobacilli can be used to reduce pathogen load in poultry PloS One, https://doi.org/10.1371/journal.pone 0043928 e43928 Pamphile, J A., Gai, C S., Pileggi, M., Rocha, C L M S C., & Pileggi, S A V (2008) Theory chapters plant-microbe interactions between host and endophytes observed by scanning electron microscopy (SEM) In S Sorvari, & A M Pirttilä (Eds.) Prospects and applications for plant-associated microbes A laboratory manual, part a: Bacteria1 (pp 84–189) Finland: BBI (BioBien Innovations) Park, N H., Kim, M S., Lee, W., Lee, M E., & Hong, J (2017) An in situ extraction and derivatization method for rapid analysis of short-chain fatty acids in rat fecal samples by gas chromatography tandem mass spectrometry Analytical Methods, 9, 2351–2356 https://doi.org/10.1039/c7ay00168a Ramanjooloo, A., Bhaw-Luximon, A., Jhurry, D., & Cadet, F (2009) 1H NMR quantitative assessment of lactic acid produced by biofermentation of cane sugar juice Spectroscopy Letters, 42, 296–304 https://doi.org/10.1080/00387010903178632 Rastall, R A (2010) Functional oligosaccharides: Application and manufacture Annual Review of Food Science and Technology, 1, 305–339 https://doi.org/10.1146/annurev food.080708.100746 Ríos-Covián, D., Ruas-Madiedo, P., Margolles, A., Gueimonde, M., De los Reyes-Gavilán, C G., & Salazar, N (2016) Intestinal short chain fatty acids and their link with diet and human health Frontiers in Microbiology, 7, 185 https://doi.org/10.3389/fmicb 2016.00185 Rodrigues, D., Santos, C H., Rocha-Santos, T A P., Gomes, A M., Goodfellow, B J., & Freitas, A C (2011) Metabolic profiling of potential probiotic or symbiotic cheeses Update Carbohydrate Polymers Volume 260, Issue , 15 May 2021, Page DOI: https://doi.org/10.1016/j.carbpol.2020.117568 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 Jos´e 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: https://doi.org/10.1016/j.carbpol.2020.116832 * Corresponding author E-mail address: ajboliveira@uem.br (A.J Braz de Oliveira) https://doi.org/10.1016/j.carbpol.2020.117568 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  ... 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”... 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. .. 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