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Soluble arabinoxylans extracted from soft and hard wheat show a differential prebiotic effect in vitro and in vivo
Journal of Cereal Science 93 (2020) 102956
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Soluble arabinoxylans extracted from soft and hard wheat show a differential prebiotic effect in vitro and in vivo Candela Paesani a, Alicia Laura Degano c, Emiliano Salvucci a, Maria Ines Zalosnik c, ~o Paulo Fabi d, Lorena Susana Sciarini a, Gabriela Teresa Perez a, b, * Joa a
ICYTAC (Instituto de Ciencia y Tecnología C� ordoba), CONICET-UNC, C� ordoba, Argentina C� atedra de Química Biol� ogica, Facultad de Ciencias Agropecuarias, UNC, C� ordoba, Argentina CIQUIBIC (Centro de Investigaciones en Química Biol� ogica C� ordoba), Departamento de Química Biol� ogica Ranwel Caputto, CONICET-UNC, C� ordoba, Argentina d Laborat�orio de Química, Bioquímica e Biologia Molecular de Alimentos, Faculdade de Ci^encias Farmac^euticas, USP, S~ ao Paulo, Brazil b c
A R T I C L E I N F O
A B S T R A C T
Keywords: Arabinoxylans Whole wheat flour Prebiotic effect
Arabinoxylans (AX) are part of dietary fiber. They are currently under study due to their potential prebiotic effect. Wheat whole grain flours contain all the grain layers and, therefore, present a higher arabinoxylan content than white flour. It is known that the chemical structure of these compounds varies with the type of wheat cultivar and the tissue from which they are extracted. In this work, water soluble extractable arabinoxylans (WEAX) from two types of wheat whole flours (hard and soft) were extracted. We characterized the molecular size distribution and the potential prebiotic effect of those extracts. The prebiotic effect was evaluated in vitro and confirmed in vivo. Bacterial group abundance (Lactobacillus, Bifidobacterium, Clostridium, Enterococcus, Bacteriodes and total bacteria) was determined by quantitative RT-PCR. The molecular size of AX from hard wheats was significantly higher than AX from soft wheats. Both extracts showed potential prebiotic activity by increasing the growth of beneficial bacteria in vitro and in vivo, decreasing the pathogens in the profile of intestinal microor ganisms and increasing the amount of short chain fatty acids in the intestine. WE-AX from hard wheat showed a higher prebiotic activity. Prebiotic effect assessed in vitro and in vivo assays showed a significant correlation between both types of analysis. This finding suggests that the in vitro indices performed allow predicting the potential prebiotic effect in vivo.
1. Introduction Arabinoxylan (AX) structures are part of dietary fiber and one of the most important non-starch polysaccharides in the cell walls of cereals. The AX are classified into water extractable (WE-AX) and water unex tractable (WU-AX) according to their solubility. AX are formed by xylose (X) chains linked by a β1-4 bond, substituted by arabinose units (A) linked by 1α-2 and/or 1α-3 along the xylose chain. The xyloses can be disubstituted (1α-2 and 1α-3), mono-substituted (1α-2 or 1α-3) or unsub stituted (Saulnier et al., 2007). It has been also shown that ferulic acid can be esterified to the hydroxyl groups at the C-5 position of the arabinose (Mendis and Simsek, 2014). However, it is not yet clear how these substitutions influence the heterogeneous nature of the AX
(Saulnier et al., 2007). Different AX fractions have been isolated from wheat and structur ally characterized (Zach et al., 2014) and they show a high degree of structural and functional diversity. However, no reports have been made regarding AX extracted from whole wheats grains, as shown in the present work. The hardness of the grain, defined as the fracture properties or the resistance to deformation during grinding, is one of the characteristics for classifying wheat and determining the flour end use (Campbell et al., 2001). In hard wheat, the peel separation during the grinding is cleaner and the endosperm remains intact. Conversely, in soft wheat the pe ripheral cells of the endosperm tend to fragment and some cells remain �mez et al., 2008). The grain texture is attached to the bran (Go
Abbreviations: A, arabinose; AX, arabinoxylans; AXOS, hydrolysates of arabinoxylans; MS, molecular size; PA, prebiotic activity; PI, prebiotic index; RG, relative growth; SCFA, short chain fatty acids; WE-AX, water extractable arabinoxylans; WU-AX, water unextractable arabinoxylans; X, xylose. * Corresponding author. Av Valparaíso s/n, Ciudad Universitaria, 5000, C� ordoba, Argentina. E-mail addresses: [email protected] (C. Paesani), [email protected] (A.L. Degano), [email protected] (E. Salvucci), mi.zalosnik@ gmail.com (M.I. Zalosnik), [email protected] (J.P. Fabi), [email protected] (L.S. Sciarini), [email protected] (G.T. Perez). https://doi.org/10.1016/j.jcs.2020.102956 Received 5 November 2019; Received in revised form 6 March 2020; Accepted 10 March 2020 Available online 14 March 2020 0733-5210/© 2020 Elsevier Ltd. All rights reserved.
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genetically determined and controls the degree of adhesion between the protein matrix and the starch granules of the endosperm (Campbell et al., 2001). In previous studies, AX was isolated from the wheat bran and from hydrolysates of arabinoxylans (AXOS) (Maes and Delcour, 2002; Saulnier et al., 2007). However, to our knowledge, a comparison between AX extraction from wheat varieties with different grain hard ness to compare their possible biological functions has not been done. Therefore, we hypothesize that WE-AX extracted from either hard or soft whole wheat grain would present differential structures that may affect their biological functions, such as the prebiotic activity. Some studies reported the prebiotic effect of WE-AX by in vitro (Hughes et al., 2007; Lagaert et al., 2010; Vardakou et al., 2008) and in vivo assays (Damen et al., 2011; Neyrinck et al., 2011). However, there is no enough data about the predictive prebiotic capacity of the WE-AX analyzed with both methodologies. Prebiotics are compounds that stimulate the growth of bacteria, which are associated with health effects and reduce the risks of diseases mediated by microbiota alterations (Gibson et al., 2017). The prebiotic effect in the intestine can be assessed by Lactobacilli and Bifidobacteria proliferation, the decrease of intestinal pathogens (Gibson and Fuller, 2000), the increased production of metabolites derived from the activity of beneficial bacteria such as short chain fatty acids (SCFA), and the decrease of toxic metabolites such as polyamines and ammonium (Al-sheraji et al., 2013). Fractions of WE-AX have the potential to pro duce this type of beneficial effects in humans (Crittenden et al., 2002). However, the AX molecules have a high degree of structural diversity that makes it difficult to establish structure-effect relationships. Few studies have been made in this respect, and the association between the structure and the biologic effect, in particular, the production of SCFA as well as the modulatory effect on the microbiota in human or animal models, remain to be established. The aim of this work was to analyze the structural differences of the WE-AX extracted from whole wheat flour (hard and soft Argentinian varieties) and to correlate those findings with their ability to exert a prebiotic effect in vitro and in vivo.
filtered and the WE-AX precipitated with acetone: alcohol (1:1). The final extract was dried in an oven for 2 h at 50 � C. 2.2. Quantification and characterization of WE-AX Twenty-five mg of the extracts were hydrolyzed with 2 mL of tri fluoroacetic acid for 3 h at 100 � C. The hydrolysate was centrifuged and neutralized with sodium carbonate, filtered through a 22 μm pore membrane and injected into HPLC-RID as reported elsewhere (Paesani et al., 2019). The quantification of WE-AX and sugar composition determination was carried out. A calibration curve was made for each sugar with SUPELCO standards (Monosaccharide Kit 47267) treated in the same way as the samples. The samples were processed in duplicate. The arabinose/xylose ratio (A/X) was calculated in order to assess the level of substitution of the WE-AX. The protein content of the extracts was determined by Kjeldahl method. The analysis of the molecular size profile was performed by HPSECRID using the Infinity 1250 System (Agilent, Santa Clara, CA) according to (Prado et al., 2017). Samples were diluted at 10 mg/mL in the mobile phase (0.2 M NaNO3) and 25 μL was injected. The separation was carried out through four columns PL-aquagel-OH MIXED-M (300 � 7.5 mm, 8 μm) in tandem (Agilent, Santa Clara, CA), at 35 � C with a 0.6 mL/min flow. The molecular size was estimated using the T dextran series as an external standard. Each sample was evaluated in triplicate and the curves were made with the average values obtained. 2.3. In vitro prebiotic effect evaluation The bacterial strains used, the culture medium and the values calculated for this analysis were performed using the protocol reported in Paesani et al. (2019). The weight of the extract used for functionality analyzes was corrected with the purity. Lactobacillus reuteri ATCC23272, Bifidobacterium breve 286 (ICYTAC), Bacteroides fragilis 6292 and Clos trodium perfringens 4168 (Microbiology Section, Hospital Aleman, Argentina) were grown in MRS media. Escherichia coli ATCC25922 were grown in a Brain Heart Infusion (BHI) broth. For prebiotic effect assays, a semi-defined medium was used and the test was carried out following the method of Huebner et al. (2007). Relative growth (RG¼ (log CFU/mL bac AX24 – log CFU/mL bac AX0)/(log CFU/mL bac G24 – log CFU/mL bac G0)), prebiotic activity (PA ¼(RG bacteria/RG E. coli)) and prebiotic index (PI¼(CFU/mL Lb/total CFU)-(CFU/mL Bac/total CFU)þ (CFU/mL Bif/total CFU)-(CFU/mL Cl/total CFU)) were calculated ac cording to Huebner et al. (2007) and Palframan et al. (2003) respec tively, as described in Paesani et al. (2019).
2. Materials and methods 2.1. Extraction of water-soluble arabinoxylans (WE-AX) For WE-AX extraction we used two pools of whole grain flours. One of hard wheat (AX h) (commercial Argentinian cultivars: Klein Yarara, ACA 315 and Guerrero) and another pool of soft Argentinian wheat (AX s) (Experimental lines: PM 647, PM 675, PM 690 and PM 692). All soft experimental lines carry Pina-D1a and Pinb-D1a alleles which were determined using allele specific primers as reported previously by Moiraghi et al. (2013). The grain hardness of experimental lines (soft wheat) was measured by Particle Size Index (Method 55–30.01, AACC 2010) and the range of PSI values varied between 21% and 35% PSI. These values allowed us to classify them into categories raging from medium soft to very soft. On the other hand, the grain hardness of commercial cultivars (hard wheats) was determined by near infrared spectroscopy (Vignola et al., 2016) and all genotypes were classified into categories raging from hard to very hard (the range of hardness varied between 62% and 78%). The tempering moisture contents, 16 and 15% (w. b.) respectively, were ground in a roller mill (AG AQC 109, Agro matic, Switzerland) without sieves to obtain whole flour. The proximate chemical composition of whole flours and water soluble, insoluble and total AX of wheat samples is shown in Table 1 of the supplementary material. Equal weights of each cultivar flour were mixed in the different pools. The water-soluble AX extraction was set up according to Buksa et al. (2010). Briefly, the extraction with water was carried out at 50 � C, the slurry was centrifuged (1250 �g, 10 min) and the supernatant treated with α-amilase thermo resistant (Sigma, 350 kU) and pronase E (from Streptomyces griseus, Sigma Type XIV 3.5 U/mg). Then the solution was
2.4. In vivo prebiotic effect analysis 2.4.1. Animals Twenty C57BL/6 male mice (21 days old) were obtained from INIMEC-CONICET (Argentina) and then housed at the CIQUIBICCONICET animal facilities. At that moment, mice were separated into four cages (5 mice/cage) and they were left to acclimatize for 2 weeks before starting the feeding protocol. The animals were kept in a room with filtered air and controlled conditions of light/dark (12 h/12 h) and temperature (22 � 2 � C). During the entire treatment, the mice had free access to food (Standard Diet Cooperation, Buenos Aires, Argentina) and water and their consumption was recorded per group. Animal proced ures were done with the approval of our Institutional Animal Care and Use Committee (IACUC from the Faculty of Chemistry, National Uni �rdoba, EXP-UNC: 47764-2019, resolution nº 2335), which versity of Co follows guidelines from the National Institute of Health (NIH, USA). 2.5. Supplement feedings Two grams of the standard feed pellet were placed into a sterile plastic Petri dish and then pulverized and hydrated with either sterile 2
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water or suspensions of the different supplements (inulin or WE-AX). The weight of extract used for functionality analyzes was corrected with the purity (1 mg of inulin, (1/0.46) mg of AX h and (1/0.33) mg of AX s were used to avoid that the differences between the effects corre sponded to the amount thereof). Starting at 35 days of age, freshly prepared Petri dishes containing enough supplement for five mice, were added to each cage every 3 days for a period of 3 weeks, as follows: Group 1: Mice fed with feed pellets minced in sterile water (control); Group 2: Mice fed with minced feed pellets supplemented with inulin (1 mg/g of mice weight); Group 3 and Group 4: Mice fed with minced feed pellets supplemented with 1 mg/g of mice weight of AX s and AX h, respectively.
Table 1 Composition of WE-AX extracts from hard and soft wheat. The mean of the value found with its respective standard deviation is indicated. Different letters in the same line indicate significance differences with a p < 0,05. WE-AX (mg/g) Proteins (mg/g) Arabinose (mg/g) Xylose (mg/g) Glucose (mg/g) A/X
AX soft
AX hard
331 � 8 a 148 � 7 a 115 � 9 a 228 � 9 a 41 � 5 a 0.50 � 0.01 a
456 � 6 a 168 � 1 a 135 � 4 a 285 � 1 a 34 � 3 a 0.47 � 0.01 a
the results were compared by the Fisher Method, at a significance level of 0.05. The values were reported as the arithmetic mean and each letter indicates significant differences. The correlations between the measured parameters were evaluated by the Pearson Method and the reported correlation coefficient values were considered significant with p < 0.05 and/or p < 0.01. These analyses were carried out using the INFOSTAT �rdoba, Argentina). software (School of Agricultural Sciences, UNC, Co
2.5.1. Analysis of the intestinal microbiota profile On day 0 and 21 of the treatment, fecal samples were taken and kept at 80 � C until the moment they were processed. The DNA extraction was performed using the commercial Kit Stool DNA Extraction Kit AccuPrep (Bioneer K-3036) and total DNA was quantified through UVspectrophotometry using the equipment and software Gen5, Take 3 (BioTec SRL, Argentina). Real-time PCR was carried out on an iCycler (Bio-Rad) using a reaction mixture with SYBR Green as the fluorescent dye (Applied Biosystems). A 1/10 vol of the DNA preparation was the template, and 250 nM of each primer (sequences available in Table 2, € et al., 2011; Shukla et al., supplementary material) were used (Rinttila 2015). The bacterial groups evaluated were: total Bacteria (16S rDNA), Bifidobacterium, Lactobacillus, Enterococcus, Bacteroidetes (Bacteroide s-Prevotella-Porphyromonas) and Clostridium Cluster I. The PCR protocol was: 95 � C for 180 s (1 time); 95 � C for 30 s; 52–58 � C for 30 s (as appropriate for each primer); 72 � C for 30 s (40 times), and a final step of 95 � C for 60 s (once). Samples were subjected to a melting-curve analysis to confirm the amplification specificity. The change in fluorescence of SYBR Green dye was monitored in every cycle and the threshold cycle (Ct) was calculated above the background for each reaction. The cali bration curves were carried out by amplifying cDNA from pure bacterial cultures of the different genera using regular PCR (Castillo et al., 2006). Serial dilutions of cDNA were made and amplified under the same conditions as the samples. The same procedure was done with the samples of caecal content extracted at the time of mice sacrifice. The concentration found was normalized with the amplification of 16S rDNA gen. Bacterial groups were expressed as number of copies per gram of fecal matter or caecal content. Differences for bacterial groups at the beginning and at the end of the treatment were calculated.
3. Results and discussion 3.1. Characterization of WE-AX extracts from hard and soft whole wheat flour WE-AX fractions were extracted from pools of three varieties of hard (AX h) and four of soft (AX s) whole wheat flour. For each pool, proteins, sugars and WE-AX were quantified (Table 1). The final content of WE-AX in the extracts from hard wheat was significantly higher (46%) than that from soft wheat (33%), as reported by Buksa et al. (2010). This result could be due to the higher AX content and the lower average particle size of hard wheat with respect to the soft ones (Table 1, supplementary material). Considering the evaluated pa rameters, no significant difference was observed in the general compo sition of WE-AX fractions obtained from hard and soft wheats. Arabinose and xylose values, as well as the A/X ratio were similar to those reported by Saulnier et al. (2007). Maes and Delcour, (2002), observed that WE-AX extracted from wheat bran had an A/X ratio of 0.45, but the gradual precipitation of AX with ethanol changed the ratio significantly from 0.31 to 0.85, depending on the percentage of ethanol used. Therefore, the type of extraction can influence A/X ratio. Lempereur et al. (1997) reported that arabinoxylans from hard wheats were char acterized by higher levels of arabinose (indicative of higher substitution) and therefore higher molecular size. In the present work, no significant differences were found in the A/X ratio between the two types of wheat. Saulnier et al. (2007) have shown structural and functional differ ences in the WE-AX extracted from the different layers of the wheat grain. The variations in the structure of the AX are related to the tissue of origin and it is different in each variety. In the present work, however, WE-AX was obtained from all the layers of the grain, including the endosperm, so the heterogeneity of the analyzed AX is greater. Regarding the molecular size (MS) profile, AX h showed a peak of higher MS (>50 KDa) that corresponds to 25% of the total molecules that is not observed in AX s. Between 50 and 60% of the molecules correspond to a MS ranging from 29 to 32 KDa in hard wheat extracts. This is lower than the MS of soft wheat, whereas a third peak of 6 - 4 KDa was observed in both extracts, which comprises 18% of the total (Fig. 1). Dervilly et al. (2000) reported smaller molecular sizes for wheat AX extracted with another method. Maes and Delcour (2002) found a 50 KDa molecular weight AX population that precipitated with different ethanol concentrations less than 40% and another with a lower molec ular size which precipitated with higher ethanol concentrations (be tween 50 and 80%). They also reported that WE-AX precipitated with 40% ethanol showed a molecular weight of approximately 20 KDa, similar to that found in this work. Rumpagaporn et al. (2015) observed a wide-range of molecular size distribution when analyzing extracts of AX
2.5.2. Analysis of short chain fatty acids (SCFA) in caecal content At the end of the treatment (day 22) the animals were anesthetized by intraperitoneal administration of ketamine (100 mg/kg of animal) and xylazine (10 mg/kg) followed by euthanasia. Caeca from individual mice were extracted and preserved at 80 � C until use. Aliquots of 200 mg from caecal content were resuspended in 1.5 mL of 0.05% 2-methylvaleric acid in acetonitrile and homogenized in a vortex for 90 s. Sam ples were centrifuged for 20 min at 10,000 x g at 4 � C and the super natant was filtered using a 0.22 μm filter. The quantification was performed by gas chromatography (CG Agilent Technologies 7890B GC System, USA) with flame ionizer detector (FID) using a fused silica column CP 7747 (WCTO, Varian, Palo Alto, CA, USA). The temperature of the injector was 250 � C at a constant pressure of 2.8 KPa. The initial temperature was 110 � C for 2 min followed by an increase of 2 � C/min up to 140 � C and then 40 � C/min up to the final temperature of 200 � C. A calibration curve was made with the standard mix of SCFA (Volatile Free Acid Mix, COD: CRM46975, Supelco, Bellefonte, PA, USA) in concen trations of 2–8 mM (adapted from Menezes et al., 2010). 2.6. Statistical analysis All results were obtained by duplicate or triplicate. The data ob tained were treated statistically by analysis of variance (ANOVA) and 3
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Fig. 1. Molecular size distribution of WE- AXs extracted from two types of wheat Vo is the dead volume time, and Ve the elution volume. The points on the curve indicate the molecular size of the standards used for the calibration curve (y ¼ 0.1876 x þ 9.5449, R2 ¼ 0.998). 1, 2 and 3 indicate the main peaks observed for each extract.
L. reuteri and B. breve (probiotic strains) cultured in the presence of inulin generated RG values > 1 and PA values > 0 (Table 2), as expected for this compound which has been shown to have prebiotic effect (Huebner et al., 2007). Both extract AX h and AX s showed RG values greater than one, that is, higher than inulin for both probiotic strains. The AX s induced significant differences in the RG and PA of L. reuteri, but no difference was observed for B. breve. It should be noted that both PA and RG of C. perfringes were significantly lower, referring to a lower growth of possible opportunistic pathogen strains and, therefore, beneficial effects on health (Table 2). In the case of B. fragilis, both RG and PA were high (Table 2), possibly due to a great capacity of this strain to metabolize the AX (De Filippo et al., 2010). The increase in Bacter oides is associated with the production of SCFA (Huebner et al., 2007) at the gut level. In addition, other authors have shown that commensal non-toxigenic Bacteroides fragilis confers powerful health benefits to the host and is a promising probiotic candidate (Xu et al., 2018). The growth of cell wall-degrading Bacteroides strains increases the presence of oli gosaccharides available for fermentation by Bifidobacterium strains. Although the PI found for AX h was the highest (Table 2), it was not statistically significant with respect to inulin and AX s. PI for AX s and AX h were 2.66 and 2.96, respectively. These are higher than results reported for AX by Palframan et al. (2003). The values obtained in our study were similar to those reported by Gong et al. (2019), who calcu lated the PI with the same formula used in our work. They showed that the PI values using AX extracts obtained from the whole grain flours were significantly higher than the PI obtained with refined flours extract from the same wheat cultivar. Vardakou et al. (2008) obtained PI of 1.15 and 2.42 for xylanase treated and untreated wheat AX, respectively. WE-AX that showed different molecular size and crosslinking levels may affect the growth selectivity of different bacterial groups, as observed in this work. In this regard, Shin et al. (2003) have charac terized a β-xylosidase and an arabinofuranosidase in Bifidobacterium breve; thus, it would be interesting to confirm the presence of these or related enzymes in the strain used in our work. Our results confirmed the efficacy of WE-AX as potential prebiotics because they promoted the growth of probiotic strains (L. reuteri and B. breve) but did not affect the growth of possible pathogens such as C. perfringes.
Table 2 Relative Growth¼(log CFU/mL x AX24 – log CFU/mL x AX0)/(log CFU/mL x G24 – log CFU/mL x G0); Prebiotic Activity¼(RG x bacteria)- (RG E. coli); Pre biotic Index¼(CFU/mL Lb/total CFU)-(CFU/mL Bac/total CFU)þ(CFU/mL Bif/ total CFU)-(CFU/mL Cl/total CFU). The mean of the value found with its respective standard deviation is indicated. Different letters in the same line indicate significant differences with p < 0.05 (Fisher). Relative Growth
L. reuteri B. breve B. fragilis Cl. perfringes
Prebiotic Activity
L. reuteri B. breve B. fragilis Cl. perfringes
Prebiotic Index
Inulin
AX soft
AX hard
0.99 a 1.47 a 1.28 a 0.99 c
� 0.03
1.27 � 0.03 c 1.70 � 0.03 b 1.68 � 0.03 b 0.81 � 0.02 b
1,04 � 0,01 b
0.08 a 0.65 a 0.46 a 0.17 c
� 0.01
0.51 � 0.01 c 0.94 � 0.01 b 0.91 � 0.07 b 0.05 � 0.01 b
0,34 � 0,03 b
2.66 � 0.09 a
2.96 � 0.29 a
� 0.01 � 0.03 � 0.07
� 0.06 � 0.08 � 0.02
2,58 � 0,06 a
1,68 � 0,05 b 1,70 � 0,01 b 0,32 � 0,01 a
0,98 � 0,09 b 1,00 � 0,05 b a
0,38 � 0,04
from different grains. The different substitution patterns of the side chain present within the set of molecules (Adams et al., 2004) could account for these differences. Thus, the structural differences found in our work between AX are possibly due to intrinsic differences in the AX structures between hard and soft wheats, although other factors, such as flour particle size, could be involved in differential extractability. 3.2. In vitro prebiotic effect In order to evaluate whether the WE-AX extracted from hard and soft wheats have the ability to induce a prebiotic effect, we performed cul tures from gut-representative bacterial strains in the presence or absence of AX h, AX s and inulin as a positive control. Using the rate of growth under different conditions, we calculated indices of Relative Growth (RG) and Prebiotic Activity (PA).
3.3. In vivo prebiotic effect Considering that AX h and AX s showed prebiotic effect in vitro, we analyzed the potential in vivo prebiotic effect, using C57BL6 mice. For 4
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Fig. 2. Profile of intestinal microorganisms Differences between the final and initial amount (Tf-Ti) of each bacterial group after treatment. Bifidobacterium (A), Lactobacillus (B), Clostridium (C), Enterococcus (D) and Bacteroidetes-Porphyromonas (E). Different letters indicate significant differences between groups of treatment with p < 0.05.
5
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of arabinose or ferulic acid and fermentability (Wang et al., 2019). In the present work, the extraction of the AX was made from whole grain flours; therefore, the WE-AX used may present a structure and molecular size distribution resulting from all the grain layers. The intestinal microbial profile was also analyzed in the caecal content of the same mice. The results obtained are shown in Table 3. It could be observed that animals fed with WE-AX extracts showed sig nificant differences in relation to the negative control. In both cases we observed a higher proportion of Bifidobacterium and Lactobacillus, similar to the profile shown by inulin fed mice. Although the increase measured during the experiment in fecal matter was higher in AX h fed mice, the caecal samples from AX s fed mice showed a final amount of both beneficial bacterial groups significantly higher than inulin (Fig. 2A and B). Hughes et al. (2007) reported a bifidogenic effect of AX in vitro, while Neyrinck et al. (2011) showed the supplementation with AX in a quantitative analysis of caecal content induced an increase in the number of bifidobacteria. However, the number of lactobacilli decreased with this diet, while the number of Bacteroides-Prevotella spp. was not significantly modified. In our work, Bacteroidetes did not show a significant variation between treatments. Different parts of AX appear to be a relatively selective substrate because they can be used by some Bifidobacterium and Lactobacillus species, but not by E. coli or Clostridium species (Moura et al., 2007; van Laere et al., 2000).
Table 3 Number of copies per gram of caecal content of the different group of bacteria. Bifidobacterium (A), Lactobacillus (B), Clostridium (C), Enterococcus (D) and Bac teroidetes-Porphyromonas (E). The mean of the value found with its respective standard deviation is indicated. Different letters indicate significant differences between groups with p < 0.05. Bacteria
Group
Copy numbers/g
Bifidobacterium
control inulin AX s AX h
3.2 6.3 2.4 5.0
Lactobacillus
control inulin AX s AX h
3.7 � 0.9 E 03 a 0.10 � 0.03 b 0.29 � 0.07 bc 0.14 � 0.03 b
Clostridium
control inulin AX s AX h
0.020 � 0.010 � 0.010 � 0.010 �
Enterococcus
control inulin AX s AX h
8.1 4.7 1.8 2.3
Bacteroidetes
control inulin AX s AX h
0.15 � 0.17 � 0.43 � 1.35 �
� 0.9 � 1.1 � 0.8 � 1.0
E E E E
04 04 03 04
a ab c ab
0.005 b 0.004 a 0.003 a 0.002 a
� 0.8 � 0.6 � 0.5 � 0.4
E E E E
05 04 03 03
a ab ab ab
0.03 a 0.05 a 0.09 ab 0.14 ab
3.3.2. Short chain fatty acid (SCFA) quantification in the caecal content of mice Gut microbiota-derived metabolites, such as SCFAs, are important mediators of beneficial effects in the host (Wong et al., 2006). Therefore, the production of acetic, propionic, and butyric acids in the caecal content of the mice was determined. The concentration of acetic acid was significantly higher in mice fed with inulin or WE-AX. Acetic acid was the SCFA found in the highest concentration (Fig. 3A). Rumpaga porn et al. (2015) reported that acetate was the most abundant SCFA produced during fermentation of AX extracted from different cereals (corn, wheat, rice, and sorghum). Similarly, the concentration of butyric acid was higher in the caecal content of mice fed with WE-AX compared to the control group (Fig. 3C). Mice fed with AX s showed the highest levels of this SCFA. This effect can be related to the higher final level of bifidobacteria in the caecal content of mice fed with AX s (Table 3). It has been shown that cross-feeding interactions between bifidobacteria and butyrate-producing colon bac teria, such as Faecalibacterium prausnitzii (clostridial cluster IV), Anae rostipes, Eubacterium, and Roseburia species (clostridial cluster XIVa), result in an enhancement of butyrate production (Rivi� ere et al., 2016). Symbiotic combinations of probiotic strains of Lactobacillus acidophilus and Bifidobacterium animalis with prebiotics has shown also significant increase of SCFA levels, especially acetic and butyric acid (van Zanten et al., 2012). The concentration of propionic acid (7.05 mM) produced by the control group was higher than all the other treated groups (Fig. 3B). A decrease in propionic concentration with long-chain AX intake was also reported by Abbeele et al. (2011); this is a very interesting result since the accumulation of propionic is related to a negative effect at gut-brain axis levels. In previous reports, in vivo assays of structurally different AXOS showed that polysaccharide structures have a strong influence in the prebiotic potential and in the SCFA profile. In general, smaller AXOS resulted in a greater increase of butyrate concentrations and a signifi cant bifidogenic effect (Van Craeyveld et al., 2008). The larger com pounds led mainly to the formation of SCFA at low concentrations and the influence of the A/X ratio seemed to be limited. In the present work, we described how the extracts obtained from the two wheat genotypes showed differences in molecular sizes and in biological functionality. A positive correlation was observed between the final concentration of the metabolites of the probiotic strains (SCFA) and the amount of
this, we established 4 experimental groups; we used a feeding protocol consisting on adding minced standard feed pellet hydrated with either sterile water (control) or with suspensions of the different supplements (inulin, AX s or AX h). During the treatment, we found no significant differences in body weight nor in food or water consumption among the different groups (Table 3, supplementary material). 3.3.1. Intestinal microbiota profile The probiotic strains grew efficiently in the presence of the WE-AX as carbon source in the mice intestines. Mice feeding with diet supple mented with WE-AX increase the number of probiotic strains in gut microbiota (Fig. 2A and B). The growth promotion was significantly higher in mice that received AX h supplemented diet. Interestingly, the molecular size distribution of this extract, showed a higher molecule size peak, but also the highest percentage of smaller size molecules, in comparison with AX s (Figs. 1 and Fig. 2A and B). In addition, while the percentage of Clostridium decreased in the intestinal microbiota from mice with AX s, AX h and inulin supple mented diet (Fig. 2C), Bacteroidetes and Enterococcus did not show sig nificant variations during the treatment (Fig. 2D and E). The increase in bifidobacteria and lactobacilli as well as the decrease in clostridia when animals were fed with WE-AX confirm a prebiotic effect of WE-AX in the mice gut microbiota. Yang (2012) also observed an increase in Bifidobacterium and a reduction of Clostridium in fermentation studies using different dietary fiber as substrates (pectin, inulin and β-glucan). The differences in the structure and molecular size of the WE-AX extracted from different wheat genotypes would also explain the variations induced in the profile of the intestinal microbiota from each mouse group. It has been shown that the structural charac teristics of AX lead to different patterns of degradation by colonic bac teria. Rumpagaporn et al. (2015) observed that wheat and corn WE-AX showing higher amount of terminal xylose were fermented more slowly. It was also reported that the fermentability of highly branched-AX from wheat bran was lower than that of a slightly branched-AX from aleurone cells (Arrigoni et al., 2003). However, the distribution of the arabinose substitution or the esterification thereof with feluric acid could result in a larger molecular size and could explain the AX h result. Thus, branching pattern and ferulic acid substitution play crucial roles in AX fermentability. There is a negative correlation between the substitution 6
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Fig. 3. Concentration of SCFA in caecal content of mice Concentrations of acetic (A), propionic (B) and butyric acid (C) in the caecal content of mice at the end of treatments. Different letters indicate significant differences with p < 0.05 between the columns.
these bacteria (the three SCFA showed correlations with r > 0.95; p < 0.01 with lactobacillus and bifidobacterial amount). In turn, a negative correlation was observed between SCFA levels and the bacterial groups that are not considered probiotic, such as Enterococcus and Bacteroides (r < 0.94; p < 0.05). This result suggests that the increase of these compounds is in part due to the metabolism of the increasing bacterial groups considered traditional probiotics in the intestine of mice. Gong et al. (2019), also found positive correlations between both Lactobacillus and Bifidobacterium and the SCFA levels, as well as a negative correlation with Clostridium counts, as van Zanten et al. (2012) did. The fact that the groups of mice fed with WE-AX from hard and soft Argentine wheat presented higher amounts of Lactobacillus and Bifido bacterium, and a lower percentage of Clostridium confirm the potential prebiotic effect of these polysaccharide extracts. Treated groups also presented an increased concentration of acetic and butyric acids in caecal content. These results demonstrate that AX have the capacity to modulate the intestinal microbiota in a beneficial way. The hypothesis of this work was correct, since the arabinoxylans of both wheats had prebiotic effects and they were even greater than the effect observed for
the WE-AX extracted from the hard wheats. 3.3.3. Correlations between the in vitro and in vivo prebiotic effect After having analyzed the in vitro and in vivo prebiotic effect with the same WE-AX extracts, we evaluated whether a correlation between them exists. A positive correlation was found between the RG and the PA of Lactobacillus in relation to the final amount of this strain measured in caeca at the end of the in vivo treatment (r 0.99 and 0.98 with a p < 0.01, respectively). Also, we found a positive correlation between RG of Bifidobacteria and its concentration in vivo (r 0.95 with a p < 0.01). This result reflects a high predictive capacity of the in vitro prebiotic assay performed here, since the result had a similar effect as the one obtained from the in vivo study. In turn, negative correlations were observed between RG and PA of Lactobacillus, and the amount of the Bacteroides and Enterococcus in vivo (r - 0.98 and - 0.97 with a p < 0.01, respec tively). These analyses showed that the increase in the metabolism of the Lactobacillus strain was related to the lower development of strains that are expected not to increase their metabolism with a prebiotic com pound such as WE-AX. 7
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To our knowledge, this is the first study that reports a strong corre lation between the prebiotic capacity in vitro and in vivo induced by the same WE-AX samples. Considering that both in vivo and in vitro tests were performed without any sample modification, we can establish the efficiency of the in vitro analysis to estimate and predict the potential in vivo prebiotic effect for a given compound.
References Abbeele, P. Van Den, G� erard, P., Rabot, S., Bruneau, A., Aidy, S. El, Derrien, M., Kleerebezem, M., Zoetendal, E.G., Smidt, H., Verstraete, W., Wiele, T. Van De, Possemiers, S., 2011. Arabinoxylans and inulin differentially modulate the mucosal and luminal gut microbiota and mucin-degradation in humanized rats. Environ. Microbiol. 13, 2667–2680. https://doi.org/10.1111/j.1462-2920.2011.02533.x. Adams, E.L., Kroon, P.A., Williamson, G., Gilbert, H.J., Morris, V.J., 2004. Inactivated enzymes as probes of the structure of arabinoxylans as observed by atomic force microscopy. Carbohydr. Res. 339, 579–590. https://doi.org/10.1016/j. carres.2003.11.023. Al-sheraji, S.H., Ismail, A., Yazid, M., Mustafa, S., 2013. Prebiotics as functional foods : a review. J. Funct. Foods 5, 1542–1553. https://doi.org/10.1016/j.jff.2013.08.009. Arrigoni, E., Amad, R., Amrein, T.M., Gr, P., 2003. In Vitro Digestibility and Colonic Fermentability of Aleurone Isolated from Wheat Bran, vol. 36, pp. 451–460. https:// doi.org/10.1016/S0023-6438(03)00036-7. Buksa, K., Nowotna, A., Praznik, W., Gambu�s, H., Ziobro, R., Krawontka, J., 2010. The role of pentosans and starch in baking of wholemeal rye bread. Food Res. Int. 43, 2045–2051. https://doi.org/10.1016/j.foodres.2010.06.005. Campbell, G.M., Bunn, P.J., Webb, C., 2001. On predicting roller milling performance Part II. The breakage function. Power Technol. 115, 243–255. https://doi.org/ 10.1016/S0032-5910(00)00349-1. Castillo, M., Martín-Orúe, S.M., Manzanilla, E.G., Badiola, I., Martín, M., Gasa, J., 2006. Quantification of total bacteria, enterobacteria and lactobacilli populations in pig digesta by real-time PCR. Vet. Microbiol. 114, 165–170. https://doi.org/10.1016/j. vetmic.2005.11.055. Crittenden, R., Karppinen, S., Ojanen, S., Tenkanen, M., Ma, J., Poutanen, K., 2002. In Vitro Fermentation of Cereal Dietary Fibre Carbohydrates by Probiotic and Intestinal Bacteria, vol. 789, pp. 781–789. https://doi.org/10.1002/jsfa.1095. Damen, B., Verspreet, J., Pollet, A., Broekaert, W.F., Delcour, J.A., Courtin, C.M., 2011. Prebiotic effects and intestinal fermentation of cereal arabinoxylans and arabinoxylan oligosaccharides in rats depend strongly on their structural properties and joint presence. Mol. Nutr. Food Res. 55, 1862–1874. https://doi.org/10.1002/ mnfr.201100377. De Filippo, C., Cavalieri, D., Di Paola, M., Ramazzotti, M., Poullet, J.B., Massart, S., Collini, S., Pieraccini, G., Lionetti, P., 2010. Impact of diet in shaping gut microbiota revealed by a comparative study in children from Europe and rural Africa. Proc. Natl. Acad. Sci. U.S.A. 107, 14691–14696. https://doi.org/10.1073/ pnas.1005963107. Dervilly, G., Saulnier, L., Roger, P., Thibault, J.F., 2000. Isolation of homogeneous fractions from wheat water-soluble arabinoxylans. Influence of the structure on their macromolecular characteristics. J. Agric. Food Chem. 48, 270–278. https://doi.org/ 10.1021/jf990222k. Gibson, G.R., Fuller, R., 2000. Aspects of in vitro and in vivo research approaches directed toward identifying probiotics and prebiotics for human use. J. Nutr. 130, 391S–395S. https://doi.org/10.1093/jn/130.2.391s. Gibson, G.R., Hutkins, R., Sanders, M.E., Prescott, S.L., Reimer, R.A., Salminen, S.J., Scott, K., Stanton, C., Swanson, K.S., Cani, P.D., Verbeke, K., Reid, G., 2017. Expert consensus document: the International Scientific Association for Probiotics and Prebiotics (ISAPP) consensus statement on the definition and scope of prebiotics. Nat. Rev. Gastroenterol. Hepatol. 14, 491–502. https://doi.org/10.1038/ nrgastro.2017.75. G� omez, M., Oliete, B., Rosell, C.M., Pando, V., Fern� andez, E., 2008. Studies on cake quality made of wheat-chickpea flour blends. LWT - Food Sci. Technol. (Lebensmittel-Wissenschaft -Technol.) 41, 1701–1709. https://doi.org/10.1016/j. lwt.2007.11.024. Gong, L., Chi, H., Wang, J., Zhang, H., Sun, B., 2019. In vitro fermentabilities of whole wheat as compared with refined wheat in different cultivars. J. Funct. Foods 52, 505–515. https://doi.org/10.1016/j.jff.2018.11.027. ~ 2007. Functional activity of commercial Huebner, J., Wehling, R.L., Hutkins, R.W.A., prebiotics. Int. Dairy J. 17, 770–775. https://doi.org/10.1016/j. idairyj.2006.10.006. Hughes, S.A., Shewry, P.R., Li, L., Gibson, G.R., Sanz, M.L., Rastall, R.A., 2007. In vitro fermentation by human fecal microflora of wheat arabinoxylans. J. Agric. Food Chem. 55, 4589–4595. https://doi.org/10.1021/jf070293g. Lagaert, S., Pollet, A., Delcour, J.A., Lavigne, R., Courtin, C.M., Volckaert, G., 2010. Substrate specificity of three recombinant α-l-arabinofuranosidases from Bifidobacterium adolescentis and their divergent action on arabinoxylan and arabinoxylan oligosaccharides. Biochem. Biophys. Res. Commun. 402, 644–650. https://doi.org/10.1016/j.bbrc.2010.10.075. Lempereur, I., Rouau, X., Abecassis, J., 1997. Genetic and agronomic variation in arabinoxylan and ferulic acid contents of durum wheat (Triticum durum l.) grain and its milling fractions. J. Cereal. Sci. 25, 103–110. https://doi.org/10.1006/ jcrs.1996.0090. Maes, C., Delcour, J.A., 2002. Structural characterisation of water-extractable and waterunextractable arabinoxylans in wheat bran. J. Cereal. Sci. 35, 315–326. https://doi. org/10.1006/jcrs.2001.0439. Mendis, M., Simsek, S., 2014. Arabinoxylans and human health. Food Hydrocolloids 42, 239–243. https://doi.org/10.1016/j.foodhyd.2013.07.022. Menezes, E.W., Dan, M.C.T., Cardenette, G.H.L., Go~ ni, I., Bello-p�erez, L.A., Lajolo, F.M., 2010. Vitro Colonic Fermentation and Glycemic Response of Different Kinds of Unripe Banana Flour 379–385. https://doi.org/10.1007/s11130-010-0190-4. Moiraghi, M., Vanzetti, L., Pflüger, L., Helguera, M., P� erez, G.T., 2013. Effect of high molecular weight glutenins and rye translocations on soft wheat flour cookie quality. J. Cereal. Sci. 58, 424–430. https://doi.org/10.1016/j.jcs.2013.08.007.
4. Conclusions Although information on soluble AX fermentation extracted from different layers of wheat grain and AXOS is found in the literature, no studies have been found on WE-AX extracted from the hard and soft wheat whole grain flours, the composition and prebiotic activity of which have been studied in this work. The WE-AX extracted from whole wheat flour of two pools of ge notypes (soft and hard) showed similar A/X ratio but an important heterogeneity in molecular size profile. The WE-AX of hard wheat pre sented a fraction of large molecular size molecules not observed in soft wheat. The extracts of WE-AX showed, in both in vitro and in vivo tests, the ability to increase the number of beneficial bacteria (Lactobacillus and Bifidobacterium) in the profile of intestinal microorganisms and to decrease the number of possible pathogens (Clostridium). They also proved to be able to increase the amount of short chain fatty acids (SCFA) in the intestine of mice. No other studies have evaluated the prebiotic effect in vitro and in vivo using the same extracts of WE-AX. The correlations found in the present work suggest that in vitro indices allow predicting the potential prebiotic effect in vivo. The consumption of WE-AX could maintain the balance and/or modulate more favorable profiles in the intestinal microbiota. The WEAX extracted from Argentine hard wheats, which is the type grown and most consumed in Argentina, showed more important beneficial effects than the AX derived from soft wheats. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. CRediT authorship contribution statement Candela Paesani: Methodology, Validation, Formal analysis, Investigation, Writing - original draft, Visualization. Alicia Laura Degano: Conceptualization, Methodology, Resources, Writing - review & editing, Supervision. Emiliano Salvucci: Conceptualization, Meth odology, Writing - review & editing, Funding acquisition. Maria Ines ~o Paulo Fabi: Resources, Su Zalosnik: Resources, Methodology. Joa pervision, Project administration. Lorena Susana Sciarini: Resources, Writing - review & editing, Funding acquisition. Gabriela Teresa Perez: Conceptualization, Methodology, Writing - review & editing, Supervi sion, Project administration, Funding acquisition. Acknowledgements The authors acknowledge Gabriela Díaz Cort� ez for providing useful suggestions to improve the English language manuscript. This work was supported by CONICET (Consejo Nacional de Investigaciones Científicas �n Científica y Tec y T�ecnicas) and the Agencia Nacional de Promocio nol� ogica (Grant PICT 2015-0606) of Argentina. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.jcs.2020.102956. 8
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Journal of Cereal Science 93 (2020) 102956 polymerase chain reaction: an evidence of dysbiosis. Dig. Dis. Sci. 60, 2953–2962. https://doi.org/10.1007/s10620-015-3607-y. Van Craeyveld, V., Swennen, K., Dornez, E., Van de Wiele, T., Marzorati, M., Verstraete, W., Delaedt, Y., Onagbesan, O., Decuypere, E., Buyse, J., De Ketelaere, B., Broekaert, W.F., Delcour, J.A., Courtin, C.M., 2008. Structurally different wheatderived arabinoxylooligosaccharides have different prebiotic and fermentation properties in rats. J. Nutr. 138, 2348–2355. https://doi.org/10.3945/ jn.108.094367. van Laere, K.M.J., Hartemink, R., Bosveld, M., Schols, H.A., Voragen, A.G.J., 2000. Fermentation of plant cell wall derived polysaccharides and their corresponding oligosaccharides by intestinal bacteria. J. Agric. Food Chem. 48, 1644–1652. https://doi.org/10.1021/jf990519i. van Zanten, G.c., Knudsen, A., Roytio, H., Forssten, S., Lawther, M., Zanten, G.C. Van, Knudsen, A., Ro, H., Blennow, A., Lahtinen, S.J., Jakobsen, M., Svensson, B., Jespersen, L., 2012. The effect of selected synbiotics on microbial composition and short-chain fatty acid production in a model System of the human colon. PloS One 7. https://doi.org/10.1371/journal.pone.0047212. Vardakou, M., Palop, C.N., Christakopoulos, P., Faulds, C.B., Gasson, M.A., Narbad, A., 2008. Evaluation of the prebiotic properties of wheat arabinoxylan fractions and induction of hydrolase activity in gut microflora. Int. J. Food Microbiol. 123, 166–170. https://doi.org/10.1016/j.ijfoodmicro.2007.11.007. Vignola, M.B., Moiraghi, M., Salvucci, E., Baroni, V., P�erez, G.T., 2016. Whole meal and white flour from Argentine wheat genotypes: mineral and arabinoxylan differences. J. Cereal. Sci. 71, 217–223. https://doi.org/10.1016/j.jcs.2016.09.002. Wang, M., Wichienchot, S., He, X., Fu, X., Huang, Q., Zhang, B., 2019. In vitro colonic fermentation of dietary fibers: fermentation rate, short-chain fatty acid production and changes in microbiota. Trends Food Sci. Technol. 88, 1–9. https://doi.org/ 10.1016/j.tifs.2019.03.005. Wong, J.M.W., Souza, R. De, Kendall, C.W.C., Emam, A., Jenkins, D.J.A., 2006. Colonic Health : fermentation and short chain Fatty Acids. J. Clin. Gastroenterol. 40, 235–243. Xu, W., Su, P., Zheng, L., Fan, H., Wang, Y., Liu, Y., 2018. In Vivo Imaging of a Novel Strain of Bacteroides Fragilis via Metabolic Labeling, vol. 9, pp. 1–7. https://doi.org/ 10.3389/fmicb.2018.02298. Yang, J., 2012. Influence of Dietary Fibers and Whole Grains on Fecal Microbiota during in Vitro Fermentation. Zach, J., Hroudov� a, J., Sedlmajer, M., Korjenic, A., 2014. Study of heat transfer process in structure of thermal insulating materials based on natural fibers. Adv. Mater. Res. 1000, 227–230. https://doi.org/10.4028/www.scientific.net/AMR.1000.227.
Moura, P., Barata, R., Carvalheiro, F., Gírio, F., Loureiro-Dias, M.C., Esteves, M.P., 2007. In vitro fermentation of xylo-oligosaccharides from corn cobs autohydrolysis by Bifidobacterium and Lactobacillus strains. LWT - Food Sci. Technol. (LebensmittelWissenschaft -Technol.) 40, 963–972. https://doi.org/10.1016/j.lwt.2006.07.013. Neyrinck, A.M., Possemiers, S., Druart, C., van de Wiele, T., de Backer, F., Cani, P.D., Larondelle, Y., Delzenne, N.M., 2011. Prebiotic effects of wheat Arabinoxylan related to the increase in bifidobacteria, roseburia and bacteroides/prevotella in diet-induced obese mice. PloS One 6. https://doi.org/10.1371/journal. pone.0020944. Paesani, C., Salvucci, E., Moiraghi, M., Fernandez Canigia, L., P� erez, G.T., 2019. Arabinoxylan from Argentinian whole wheat flour promote the growth of Lactobacillus reuteri and Bifidobacterium breve. Lett. Appl. Microbiol. 68, 142–148. https://doi.org/10.1111/lam.13097. Palframan, R., Gibson, G.R., Rastall, R.A., 2003. Development of a quantitative tool for the comparison of the prebiotic effect of dietary oligosaccharides. Lett. Appl. Microbiol. 37, 281–284. https://doi.org/10.1046/j.1472-765X.2003.01398.x. Prado, S.B.R. Do, Ferreira, G.F., Harazono, Y., Shiga, T.M., Raz, A., Carpita, N.C., Fabi, J. P., 2017. Ripening-induced chemical modifications of papaya pectin inhibit cancer cell proliferation. Sci. Rep. 7, 1–17. https://doi.org/10.1038/s41598-017-16709-3. Rinttil€ a, T., Lyra, A., Krogius-Kurikka, L., Palva, A., 2011. Real-time PCR analysis of enteric pathogens from fecal samples of irritable bowel syndrome subjects. Gut Pathog. 3, 1–9. https://doi.org/10.1186/1757-4749-3-6. Rivi� ere, A., Selak, M., Lantin, D., Leroy, F., De Vuyst, L., 2016. Bifidobacteria and butyrate-producing colon bacteria: importance and strategies for their stimulation in the human gut. Front. Microbiol. 7 https://doi.org/10.3389/fmicb.2016.00979. Rumpagaporn, P., Reuhs, B.L., Kaur, A., Patterson, J.A., Keshavarzian, A., Hamaker, B.R., 2015. Structural features of soluble cereal arabinoxylan fibers associated with a slow rate of in vitro fermentation by human fecal microbiota. Carbohydr. Polym. 130, 191–197. https://doi.org/10.1016/j.carbpol.2015.04.041. Saulnier, L., Sado, P.E., Branlard, G., Charmet, G., Guillon, F., 2007. Wheat arabinoxylans: exploiting variation in amount and composition to develop enhanced varieties. J. Cereal. Sci. 46, 261–281. https://doi.org/10.1016/j.jcs.2007.06.014. Shin, H., Park, S., Sung, J.H., Kim, D., 2003. Purification and characterization of ␣ - L -arabinopyranosidase and ␣ - L -arabinofuranosidase from Bifidobacterium breve K110 , a human intestinal anaerobic bacterium Metabolizing Ginsenoside Rb2 and Rc, 69, 7116-7123. https://doi.org/10.1128/AEM.69.12.7116. Shukla, R., Ghoshal, U., Dhole, T.N., Ghoshal, U.C., 2015. Fecal microbiota in patients with irritable bowel syndrome compared with healthy controls using real-time
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Soluble arabinoxylans extracted from soft and hard wheat show a differential prebiotic effect in vitro and in vivo Candela Paesani a, Alicia Laura Degano c, Emiliano Salvucci a, Maria Ines Zalosnik c, ~o Paulo Fabi d, Lorena Susana Sciarini a, Gabriela Teresa Perez a, b, * Joa a
ICYTAC (Instituto de Ciencia y Tecnología C� ordoba), CONICET-UNC, C� ordoba, Argentina C� atedra de Química Biol� ogica, Facultad de Ciencias Agropecuarias, UNC, C� ordoba, Argentina CIQUIBIC (Centro de Investigaciones en Química Biol� ogica C� ordoba), Departamento de Química Biol� ogica Ranwel Caputto, CONICET-UNC, C� ordoba, Argentina d Laborat�orio de Química, Bioquímica e Biologia Molecular de Alimentos, Faculdade de Ci^encias Farmac^euticas, USP, S~ ao Paulo, Brazil b c
A R T I C L E I N F O
A B S T R A C T
Keywords: Arabinoxylans Whole wheat flour Prebiotic effect
Arabinoxylans (AX) are part of dietary fiber. They are currently under study due to their potential prebiotic effect. Wheat whole grain flours contain all the grain layers and, therefore, present a higher arabinoxylan content than white flour. It is known that the chemical structure of these compounds varies with the type of wheat cultivar and the tissue from which they are extracted. In this work, water soluble extractable arabinoxylans (WEAX) from two types of wheat whole flours (hard and soft) were extracted. We characterized the molecular size distribution and the potential prebiotic effect of those extracts. The prebiotic effect was evaluated in vitro and confirmed in vivo. Bacterial group abundance (Lactobacillus, Bifidobacterium, Clostridium, Enterococcus, Bacteriodes and total bacteria) was determined by quantitative RT-PCR. The molecular size of AX from hard wheats was significantly higher than AX from soft wheats. Both extracts showed potential prebiotic activity by increasing the growth of beneficial bacteria in vitro and in vivo, decreasing the pathogens in the profile of intestinal microor ganisms and increasing the amount of short chain fatty acids in the intestine. WE-AX from hard wheat showed a higher prebiotic activity. Prebiotic effect assessed in vitro and in vivo assays showed a significant correlation between both types of analysis. This finding suggests that the in vitro indices performed allow predicting the potential prebiotic effect in vivo.
1. Introduction Arabinoxylan (AX) structures are part of dietary fiber and one of the most important non-starch polysaccharides in the cell walls of cereals. The AX are classified into water extractable (WE-AX) and water unex tractable (WU-AX) according to their solubility. AX are formed by xylose (X) chains linked by a β1-4 bond, substituted by arabinose units (A) linked by 1α-2 and/or 1α-3 along the xylose chain. The xyloses can be disubstituted (1α-2 and 1α-3), mono-substituted (1α-2 or 1α-3) or unsub stituted (Saulnier et al., 2007). It has been also shown that ferulic acid can be esterified to the hydroxyl groups at the C-5 position of the arabinose (Mendis and Simsek, 2014). However, it is not yet clear how these substitutions influence the heterogeneous nature of the AX
(Saulnier et al., 2007). Different AX fractions have been isolated from wheat and structur ally characterized (Zach et al., 2014) and they show a high degree of structural and functional diversity. However, no reports have been made regarding AX extracted from whole wheats grains, as shown in the present work. The hardness of the grain, defined as the fracture properties or the resistance to deformation during grinding, is one of the characteristics for classifying wheat and determining the flour end use (Campbell et al., 2001). In hard wheat, the peel separation during the grinding is cleaner and the endosperm remains intact. Conversely, in soft wheat the pe ripheral cells of the endosperm tend to fragment and some cells remain �mez et al., 2008). The grain texture is attached to the bran (Go
Abbreviations: A, arabinose; AX, arabinoxylans; AXOS, hydrolysates of arabinoxylans; MS, molecular size; PA, prebiotic activity; PI, prebiotic index; RG, relative growth; SCFA, short chain fatty acids; WE-AX, water extractable arabinoxylans; WU-AX, water unextractable arabinoxylans; X, xylose. * Corresponding author. Av Valparaíso s/n, Ciudad Universitaria, 5000, C� ordoba, Argentina. E-mail addresses: [email protected] (C. Paesani), [email protected] (A.L. Degano), [email protected] (E. Salvucci), mi.zalosnik@ gmail.com (M.I. Zalosnik), [email protected] (J.P. Fabi), [email protected] (L.S. Sciarini), [email protected] (G.T. Perez). https://doi.org/10.1016/j.jcs.2020.102956 Received 5 November 2019; Received in revised form 6 March 2020; Accepted 10 March 2020 Available online 14 March 2020 0733-5210/© 2020 Elsevier Ltd. All rights reserved.
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genetically determined and controls the degree of adhesion between the protein matrix and the starch granules of the endosperm (Campbell et al., 2001). In previous studies, AX was isolated from the wheat bran and from hydrolysates of arabinoxylans (AXOS) (Maes and Delcour, 2002; Saulnier et al., 2007). However, to our knowledge, a comparison between AX extraction from wheat varieties with different grain hard ness to compare their possible biological functions has not been done. Therefore, we hypothesize that WE-AX extracted from either hard or soft whole wheat grain would present differential structures that may affect their biological functions, such as the prebiotic activity. Some studies reported the prebiotic effect of WE-AX by in vitro (Hughes et al., 2007; Lagaert et al., 2010; Vardakou et al., 2008) and in vivo assays (Damen et al., 2011; Neyrinck et al., 2011). However, there is no enough data about the predictive prebiotic capacity of the WE-AX analyzed with both methodologies. Prebiotics are compounds that stimulate the growth of bacteria, which are associated with health effects and reduce the risks of diseases mediated by microbiota alterations (Gibson et al., 2017). The prebiotic effect in the intestine can be assessed by Lactobacilli and Bifidobacteria proliferation, the decrease of intestinal pathogens (Gibson and Fuller, 2000), the increased production of metabolites derived from the activity of beneficial bacteria such as short chain fatty acids (SCFA), and the decrease of toxic metabolites such as polyamines and ammonium (Al-sheraji et al., 2013). Fractions of WE-AX have the potential to pro duce this type of beneficial effects in humans (Crittenden et al., 2002). However, the AX molecules have a high degree of structural diversity that makes it difficult to establish structure-effect relationships. Few studies have been made in this respect, and the association between the structure and the biologic effect, in particular, the production of SCFA as well as the modulatory effect on the microbiota in human or animal models, remain to be established. The aim of this work was to analyze the structural differences of the WE-AX extracted from whole wheat flour (hard and soft Argentinian varieties) and to correlate those findings with their ability to exert a prebiotic effect in vitro and in vivo.
filtered and the WE-AX precipitated with acetone: alcohol (1:1). The final extract was dried in an oven for 2 h at 50 � C. 2.2. Quantification and characterization of WE-AX Twenty-five mg of the extracts were hydrolyzed with 2 mL of tri fluoroacetic acid for 3 h at 100 � C. The hydrolysate was centrifuged and neutralized with sodium carbonate, filtered through a 22 μm pore membrane and injected into HPLC-RID as reported elsewhere (Paesani et al., 2019). The quantification of WE-AX and sugar composition determination was carried out. A calibration curve was made for each sugar with SUPELCO standards (Monosaccharide Kit 47267) treated in the same way as the samples. The samples were processed in duplicate. The arabinose/xylose ratio (A/X) was calculated in order to assess the level of substitution of the WE-AX. The protein content of the extracts was determined by Kjeldahl method. The analysis of the molecular size profile was performed by HPSECRID using the Infinity 1250 System (Agilent, Santa Clara, CA) according to (Prado et al., 2017). Samples were diluted at 10 mg/mL in the mobile phase (0.2 M NaNO3) and 25 μL was injected. The separation was carried out through four columns PL-aquagel-OH MIXED-M (300 � 7.5 mm, 8 μm) in tandem (Agilent, Santa Clara, CA), at 35 � C with a 0.6 mL/min flow. The molecular size was estimated using the T dextran series as an external standard. Each sample was evaluated in triplicate and the curves were made with the average values obtained. 2.3. In vitro prebiotic effect evaluation The bacterial strains used, the culture medium and the values calculated for this analysis were performed using the protocol reported in Paesani et al. (2019). The weight of the extract used for functionality analyzes was corrected with the purity. Lactobacillus reuteri ATCC23272, Bifidobacterium breve 286 (ICYTAC), Bacteroides fragilis 6292 and Clos trodium perfringens 4168 (Microbiology Section, Hospital Aleman, Argentina) were grown in MRS media. Escherichia coli ATCC25922 were grown in a Brain Heart Infusion (BHI) broth. For prebiotic effect assays, a semi-defined medium was used and the test was carried out following the method of Huebner et al. (2007). Relative growth (RG¼ (log CFU/mL bac AX24 – log CFU/mL bac AX0)/(log CFU/mL bac G24 – log CFU/mL bac G0)), prebiotic activity (PA ¼(RG bacteria/RG E. coli)) and prebiotic index (PI¼(CFU/mL Lb/total CFU)-(CFU/mL Bac/total CFU)þ (CFU/mL Bif/total CFU)-(CFU/mL Cl/total CFU)) were calculated ac cording to Huebner et al. (2007) and Palframan et al. (2003) respec tively, as described in Paesani et al. (2019).
2. Materials and methods 2.1. Extraction of water-soluble arabinoxylans (WE-AX) For WE-AX extraction we used two pools of whole grain flours. One of hard wheat (AX h) (commercial Argentinian cultivars: Klein Yarara, ACA 315 and Guerrero) and another pool of soft Argentinian wheat (AX s) (Experimental lines: PM 647, PM 675, PM 690 and PM 692). All soft experimental lines carry Pina-D1a and Pinb-D1a alleles which were determined using allele specific primers as reported previously by Moiraghi et al. (2013). The grain hardness of experimental lines (soft wheat) was measured by Particle Size Index (Method 55–30.01, AACC 2010) and the range of PSI values varied between 21% and 35% PSI. These values allowed us to classify them into categories raging from medium soft to very soft. On the other hand, the grain hardness of commercial cultivars (hard wheats) was determined by near infrared spectroscopy (Vignola et al., 2016) and all genotypes were classified into categories raging from hard to very hard (the range of hardness varied between 62% and 78%). The tempering moisture contents, 16 and 15% (w. b.) respectively, were ground in a roller mill (AG AQC 109, Agro matic, Switzerland) without sieves to obtain whole flour. The proximate chemical composition of whole flours and water soluble, insoluble and total AX of wheat samples is shown in Table 1 of the supplementary material. Equal weights of each cultivar flour were mixed in the different pools. The water-soluble AX extraction was set up according to Buksa et al. (2010). Briefly, the extraction with water was carried out at 50 � C, the slurry was centrifuged (1250 �g, 10 min) and the supernatant treated with α-amilase thermo resistant (Sigma, 350 kU) and pronase E (from Streptomyces griseus, Sigma Type XIV 3.5 U/mg). Then the solution was
2.4. In vivo prebiotic effect analysis 2.4.1. Animals Twenty C57BL/6 male mice (21 days old) were obtained from INIMEC-CONICET (Argentina) and then housed at the CIQUIBICCONICET animal facilities. At that moment, mice were separated into four cages (5 mice/cage) and they were left to acclimatize for 2 weeks before starting the feeding protocol. The animals were kept in a room with filtered air and controlled conditions of light/dark (12 h/12 h) and temperature (22 � 2 � C). During the entire treatment, the mice had free access to food (Standard Diet Cooperation, Buenos Aires, Argentina) and water and their consumption was recorded per group. Animal proced ures were done with the approval of our Institutional Animal Care and Use Committee (IACUC from the Faculty of Chemistry, National Uni �rdoba, EXP-UNC: 47764-2019, resolution nº 2335), which versity of Co follows guidelines from the National Institute of Health (NIH, USA). 2.5. Supplement feedings Two grams of the standard feed pellet were placed into a sterile plastic Petri dish and then pulverized and hydrated with either sterile 2
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water or suspensions of the different supplements (inulin or WE-AX). The weight of extract used for functionality analyzes was corrected with the purity (1 mg of inulin, (1/0.46) mg of AX h and (1/0.33) mg of AX s were used to avoid that the differences between the effects corre sponded to the amount thereof). Starting at 35 days of age, freshly prepared Petri dishes containing enough supplement for five mice, were added to each cage every 3 days for a period of 3 weeks, as follows: Group 1: Mice fed with feed pellets minced in sterile water (control); Group 2: Mice fed with minced feed pellets supplemented with inulin (1 mg/g of mice weight); Group 3 and Group 4: Mice fed with minced feed pellets supplemented with 1 mg/g of mice weight of AX s and AX h, respectively.
Table 1 Composition of WE-AX extracts from hard and soft wheat. The mean of the value found with its respective standard deviation is indicated. Different letters in the same line indicate significance differences with a p < 0,05. WE-AX (mg/g) Proteins (mg/g) Arabinose (mg/g) Xylose (mg/g) Glucose (mg/g) A/X
AX soft
AX hard
331 � 8 a 148 � 7 a 115 � 9 a 228 � 9 a 41 � 5 a 0.50 � 0.01 a
456 � 6 a 168 � 1 a 135 � 4 a 285 � 1 a 34 � 3 a 0.47 � 0.01 a
the results were compared by the Fisher Method, at a significance level of 0.05. The values were reported as the arithmetic mean and each letter indicates significant differences. The correlations between the measured parameters were evaluated by the Pearson Method and the reported correlation coefficient values were considered significant with p < 0.05 and/or p < 0.01. These analyses were carried out using the INFOSTAT �rdoba, Argentina). software (School of Agricultural Sciences, UNC, Co
2.5.1. Analysis of the intestinal microbiota profile On day 0 and 21 of the treatment, fecal samples were taken and kept at 80 � C until the moment they were processed. The DNA extraction was performed using the commercial Kit Stool DNA Extraction Kit AccuPrep (Bioneer K-3036) and total DNA was quantified through UVspectrophotometry using the equipment and software Gen5, Take 3 (BioTec SRL, Argentina). Real-time PCR was carried out on an iCycler (Bio-Rad) using a reaction mixture with SYBR Green as the fluorescent dye (Applied Biosystems). A 1/10 vol of the DNA preparation was the template, and 250 nM of each primer (sequences available in Table 2, € et al., 2011; Shukla et al., supplementary material) were used (Rinttila 2015). The bacterial groups evaluated were: total Bacteria (16S rDNA), Bifidobacterium, Lactobacillus, Enterococcus, Bacteroidetes (Bacteroide s-Prevotella-Porphyromonas) and Clostridium Cluster I. The PCR protocol was: 95 � C for 180 s (1 time); 95 � C for 30 s; 52–58 � C for 30 s (as appropriate for each primer); 72 � C for 30 s (40 times), and a final step of 95 � C for 60 s (once). Samples were subjected to a melting-curve analysis to confirm the amplification specificity. The change in fluorescence of SYBR Green dye was monitored in every cycle and the threshold cycle (Ct) was calculated above the background for each reaction. The cali bration curves were carried out by amplifying cDNA from pure bacterial cultures of the different genera using regular PCR (Castillo et al., 2006). Serial dilutions of cDNA were made and amplified under the same conditions as the samples. The same procedure was done with the samples of caecal content extracted at the time of mice sacrifice. The concentration found was normalized with the amplification of 16S rDNA gen. Bacterial groups were expressed as number of copies per gram of fecal matter or caecal content. Differences for bacterial groups at the beginning and at the end of the treatment were calculated.
3. Results and discussion 3.1. Characterization of WE-AX extracts from hard and soft whole wheat flour WE-AX fractions were extracted from pools of three varieties of hard (AX h) and four of soft (AX s) whole wheat flour. For each pool, proteins, sugars and WE-AX were quantified (Table 1). The final content of WE-AX in the extracts from hard wheat was significantly higher (46%) than that from soft wheat (33%), as reported by Buksa et al. (2010). This result could be due to the higher AX content and the lower average particle size of hard wheat with respect to the soft ones (Table 1, supplementary material). Considering the evaluated pa rameters, no significant difference was observed in the general compo sition of WE-AX fractions obtained from hard and soft wheats. Arabinose and xylose values, as well as the A/X ratio were similar to those reported by Saulnier et al. (2007). Maes and Delcour, (2002), observed that WE-AX extracted from wheat bran had an A/X ratio of 0.45, but the gradual precipitation of AX with ethanol changed the ratio significantly from 0.31 to 0.85, depending on the percentage of ethanol used. Therefore, the type of extraction can influence A/X ratio. Lempereur et al. (1997) reported that arabinoxylans from hard wheats were char acterized by higher levels of arabinose (indicative of higher substitution) and therefore higher molecular size. In the present work, no significant differences were found in the A/X ratio between the two types of wheat. Saulnier et al. (2007) have shown structural and functional differ ences in the WE-AX extracted from the different layers of the wheat grain. The variations in the structure of the AX are related to the tissue of origin and it is different in each variety. In the present work, however, WE-AX was obtained from all the layers of the grain, including the endosperm, so the heterogeneity of the analyzed AX is greater. Regarding the molecular size (MS) profile, AX h showed a peak of higher MS (>50 KDa) that corresponds to 25% of the total molecules that is not observed in AX s. Between 50 and 60% of the molecules correspond to a MS ranging from 29 to 32 KDa in hard wheat extracts. This is lower than the MS of soft wheat, whereas a third peak of 6 - 4 KDa was observed in both extracts, which comprises 18% of the total (Fig. 1). Dervilly et al. (2000) reported smaller molecular sizes for wheat AX extracted with another method. Maes and Delcour (2002) found a 50 KDa molecular weight AX population that precipitated with different ethanol concentrations less than 40% and another with a lower molec ular size which precipitated with higher ethanol concentrations (be tween 50 and 80%). They also reported that WE-AX precipitated with 40% ethanol showed a molecular weight of approximately 20 KDa, similar to that found in this work. Rumpagaporn et al. (2015) observed a wide-range of molecular size distribution when analyzing extracts of AX
2.5.2. Analysis of short chain fatty acids (SCFA) in caecal content At the end of the treatment (day 22) the animals were anesthetized by intraperitoneal administration of ketamine (100 mg/kg of animal) and xylazine (10 mg/kg) followed by euthanasia. Caeca from individual mice were extracted and preserved at 80 � C until use. Aliquots of 200 mg from caecal content were resuspended in 1.5 mL of 0.05% 2-methylvaleric acid in acetonitrile and homogenized in a vortex for 90 s. Sam ples were centrifuged for 20 min at 10,000 x g at 4 � C and the super natant was filtered using a 0.22 μm filter. The quantification was performed by gas chromatography (CG Agilent Technologies 7890B GC System, USA) with flame ionizer detector (FID) using a fused silica column CP 7747 (WCTO, Varian, Palo Alto, CA, USA). The temperature of the injector was 250 � C at a constant pressure of 2.8 KPa. The initial temperature was 110 � C for 2 min followed by an increase of 2 � C/min up to 140 � C and then 40 � C/min up to the final temperature of 200 � C. A calibration curve was made with the standard mix of SCFA (Volatile Free Acid Mix, COD: CRM46975, Supelco, Bellefonte, PA, USA) in concen trations of 2–8 mM (adapted from Menezes et al., 2010). 2.6. Statistical analysis All results were obtained by duplicate or triplicate. The data ob tained were treated statistically by analysis of variance (ANOVA) and 3
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Fig. 1. Molecular size distribution of WE- AXs extracted from two types of wheat Vo is the dead volume time, and Ve the elution volume. The points on the curve indicate the molecular size of the standards used for the calibration curve (y ¼ 0.1876 x þ 9.5449, R2 ¼ 0.998). 1, 2 and 3 indicate the main peaks observed for each extract.
L. reuteri and B. breve (probiotic strains) cultured in the presence of inulin generated RG values > 1 and PA values > 0 (Table 2), as expected for this compound which has been shown to have prebiotic effect (Huebner et al., 2007). Both extract AX h and AX s showed RG values greater than one, that is, higher than inulin for both probiotic strains. The AX s induced significant differences in the RG and PA of L. reuteri, but no difference was observed for B. breve. It should be noted that both PA and RG of C. perfringes were significantly lower, referring to a lower growth of possible opportunistic pathogen strains and, therefore, beneficial effects on health (Table 2). In the case of B. fragilis, both RG and PA were high (Table 2), possibly due to a great capacity of this strain to metabolize the AX (De Filippo et al., 2010). The increase in Bacter oides is associated with the production of SCFA (Huebner et al., 2007) at the gut level. In addition, other authors have shown that commensal non-toxigenic Bacteroides fragilis confers powerful health benefits to the host and is a promising probiotic candidate (Xu et al., 2018). The growth of cell wall-degrading Bacteroides strains increases the presence of oli gosaccharides available for fermentation by Bifidobacterium strains. Although the PI found for AX h was the highest (Table 2), it was not statistically significant with respect to inulin and AX s. PI for AX s and AX h were 2.66 and 2.96, respectively. These are higher than results reported for AX by Palframan et al. (2003). The values obtained in our study were similar to those reported by Gong et al. (2019), who calcu lated the PI with the same formula used in our work. They showed that the PI values using AX extracts obtained from the whole grain flours were significantly higher than the PI obtained with refined flours extract from the same wheat cultivar. Vardakou et al. (2008) obtained PI of 1.15 and 2.42 for xylanase treated and untreated wheat AX, respectively. WE-AX that showed different molecular size and crosslinking levels may affect the growth selectivity of different bacterial groups, as observed in this work. In this regard, Shin et al. (2003) have charac terized a β-xylosidase and an arabinofuranosidase in Bifidobacterium breve; thus, it would be interesting to confirm the presence of these or related enzymes in the strain used in our work. Our results confirmed the efficacy of WE-AX as potential prebiotics because they promoted the growth of probiotic strains (L. reuteri and B. breve) but did not affect the growth of possible pathogens such as C. perfringes.
Table 2 Relative Growth¼(log CFU/mL x AX24 – log CFU/mL x AX0)/(log CFU/mL x G24 – log CFU/mL x G0); Prebiotic Activity¼(RG x bacteria)- (RG E. coli); Pre biotic Index¼(CFU/mL Lb/total CFU)-(CFU/mL Bac/total CFU)þ(CFU/mL Bif/ total CFU)-(CFU/mL Cl/total CFU). The mean of the value found with its respective standard deviation is indicated. Different letters in the same line indicate significant differences with p < 0.05 (Fisher). Relative Growth
L. reuteri B. breve B. fragilis Cl. perfringes
Prebiotic Activity
L. reuteri B. breve B. fragilis Cl. perfringes
Prebiotic Index
Inulin
AX soft
AX hard
0.99 a 1.47 a 1.28 a 0.99 c
� 0.03
1.27 � 0.03 c 1.70 � 0.03 b 1.68 � 0.03 b 0.81 � 0.02 b
1,04 � 0,01 b
0.08 a 0.65 a 0.46 a 0.17 c
� 0.01
0.51 � 0.01 c 0.94 � 0.01 b 0.91 � 0.07 b 0.05 � 0.01 b
0,34 � 0,03 b
2.66 � 0.09 a
2.96 � 0.29 a
� 0.01 � 0.03 � 0.07
� 0.06 � 0.08 � 0.02
2,58 � 0,06 a
1,68 � 0,05 b 1,70 � 0,01 b 0,32 � 0,01 a
0,98 � 0,09 b 1,00 � 0,05 b a
0,38 � 0,04
from different grains. The different substitution patterns of the side chain present within the set of molecules (Adams et al., 2004) could account for these differences. Thus, the structural differences found in our work between AX are possibly due to intrinsic differences in the AX structures between hard and soft wheats, although other factors, such as flour particle size, could be involved in differential extractability. 3.2. In vitro prebiotic effect In order to evaluate whether the WE-AX extracted from hard and soft wheats have the ability to induce a prebiotic effect, we performed cul tures from gut-representative bacterial strains in the presence or absence of AX h, AX s and inulin as a positive control. Using the rate of growth under different conditions, we calculated indices of Relative Growth (RG) and Prebiotic Activity (PA).
3.3. In vivo prebiotic effect Considering that AX h and AX s showed prebiotic effect in vitro, we analyzed the potential in vivo prebiotic effect, using C57BL6 mice. For 4
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Fig. 2. Profile of intestinal microorganisms Differences between the final and initial amount (Tf-Ti) of each bacterial group after treatment. Bifidobacterium (A), Lactobacillus (B), Clostridium (C), Enterococcus (D) and Bacteroidetes-Porphyromonas (E). Different letters indicate significant differences between groups of treatment with p < 0.05.
5
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of arabinose or ferulic acid and fermentability (Wang et al., 2019). In the present work, the extraction of the AX was made from whole grain flours; therefore, the WE-AX used may present a structure and molecular size distribution resulting from all the grain layers. The intestinal microbial profile was also analyzed in the caecal content of the same mice. The results obtained are shown in Table 3. It could be observed that animals fed with WE-AX extracts showed sig nificant differences in relation to the negative control. In both cases we observed a higher proportion of Bifidobacterium and Lactobacillus, similar to the profile shown by inulin fed mice. Although the increase measured during the experiment in fecal matter was higher in AX h fed mice, the caecal samples from AX s fed mice showed a final amount of both beneficial bacterial groups significantly higher than inulin (Fig. 2A and B). Hughes et al. (2007) reported a bifidogenic effect of AX in vitro, while Neyrinck et al. (2011) showed the supplementation with AX in a quantitative analysis of caecal content induced an increase in the number of bifidobacteria. However, the number of lactobacilli decreased with this diet, while the number of Bacteroides-Prevotella spp. was not significantly modified. In our work, Bacteroidetes did not show a significant variation between treatments. Different parts of AX appear to be a relatively selective substrate because they can be used by some Bifidobacterium and Lactobacillus species, but not by E. coli or Clostridium species (Moura et al., 2007; van Laere et al., 2000).
Table 3 Number of copies per gram of caecal content of the different group of bacteria. Bifidobacterium (A), Lactobacillus (B), Clostridium (C), Enterococcus (D) and Bac teroidetes-Porphyromonas (E). The mean of the value found with its respective standard deviation is indicated. Different letters indicate significant differences between groups with p < 0.05. Bacteria
Group
Copy numbers/g
Bifidobacterium
control inulin AX s AX h
3.2 6.3 2.4 5.0
Lactobacillus
control inulin AX s AX h
3.7 � 0.9 E 03 a 0.10 � 0.03 b 0.29 � 0.07 bc 0.14 � 0.03 b
Clostridium
control inulin AX s AX h
0.020 � 0.010 � 0.010 � 0.010 �
Enterococcus
control inulin AX s AX h
8.1 4.7 1.8 2.3
Bacteroidetes
control inulin AX s AX h
0.15 � 0.17 � 0.43 � 1.35 �
� 0.9 � 1.1 � 0.8 � 1.0
E E E E
04 04 03 04
a ab c ab
0.005 b 0.004 a 0.003 a 0.002 a
� 0.8 � 0.6 � 0.5 � 0.4
E E E E
05 04 03 03
a ab ab ab
0.03 a 0.05 a 0.09 ab 0.14 ab
3.3.2. Short chain fatty acid (SCFA) quantification in the caecal content of mice Gut microbiota-derived metabolites, such as SCFAs, are important mediators of beneficial effects in the host (Wong et al., 2006). Therefore, the production of acetic, propionic, and butyric acids in the caecal content of the mice was determined. The concentration of acetic acid was significantly higher in mice fed with inulin or WE-AX. Acetic acid was the SCFA found in the highest concentration (Fig. 3A). Rumpaga porn et al. (2015) reported that acetate was the most abundant SCFA produced during fermentation of AX extracted from different cereals (corn, wheat, rice, and sorghum). Similarly, the concentration of butyric acid was higher in the caecal content of mice fed with WE-AX compared to the control group (Fig. 3C). Mice fed with AX s showed the highest levels of this SCFA. This effect can be related to the higher final level of bifidobacteria in the caecal content of mice fed with AX s (Table 3). It has been shown that cross-feeding interactions between bifidobacteria and butyrate-producing colon bac teria, such as Faecalibacterium prausnitzii (clostridial cluster IV), Anae rostipes, Eubacterium, and Roseburia species (clostridial cluster XIVa), result in an enhancement of butyrate production (Rivi� ere et al., 2016). Symbiotic combinations of probiotic strains of Lactobacillus acidophilus and Bifidobacterium animalis with prebiotics has shown also significant increase of SCFA levels, especially acetic and butyric acid (van Zanten et al., 2012). The concentration of propionic acid (7.05 mM) produced by the control group was higher than all the other treated groups (Fig. 3B). A decrease in propionic concentration with long-chain AX intake was also reported by Abbeele et al. (2011); this is a very interesting result since the accumulation of propionic is related to a negative effect at gut-brain axis levels. In previous reports, in vivo assays of structurally different AXOS showed that polysaccharide structures have a strong influence in the prebiotic potential and in the SCFA profile. In general, smaller AXOS resulted in a greater increase of butyrate concentrations and a signifi cant bifidogenic effect (Van Craeyveld et al., 2008). The larger com pounds led mainly to the formation of SCFA at low concentrations and the influence of the A/X ratio seemed to be limited. In the present work, we described how the extracts obtained from the two wheat genotypes showed differences in molecular sizes and in biological functionality. A positive correlation was observed between the final concentration of the metabolites of the probiotic strains (SCFA) and the amount of
this, we established 4 experimental groups; we used a feeding protocol consisting on adding minced standard feed pellet hydrated with either sterile water (control) or with suspensions of the different supplements (inulin, AX s or AX h). During the treatment, we found no significant differences in body weight nor in food or water consumption among the different groups (Table 3, supplementary material). 3.3.1. Intestinal microbiota profile The probiotic strains grew efficiently in the presence of the WE-AX as carbon source in the mice intestines. Mice feeding with diet supple mented with WE-AX increase the number of probiotic strains in gut microbiota (Fig. 2A and B). The growth promotion was significantly higher in mice that received AX h supplemented diet. Interestingly, the molecular size distribution of this extract, showed a higher molecule size peak, but also the highest percentage of smaller size molecules, in comparison with AX s (Figs. 1 and Fig. 2A and B). In addition, while the percentage of Clostridium decreased in the intestinal microbiota from mice with AX s, AX h and inulin supple mented diet (Fig. 2C), Bacteroidetes and Enterococcus did not show sig nificant variations during the treatment (Fig. 2D and E). The increase in bifidobacteria and lactobacilli as well as the decrease in clostridia when animals were fed with WE-AX confirm a prebiotic effect of WE-AX in the mice gut microbiota. Yang (2012) also observed an increase in Bifidobacterium and a reduction of Clostridium in fermentation studies using different dietary fiber as substrates (pectin, inulin and β-glucan). The differences in the structure and molecular size of the WE-AX extracted from different wheat genotypes would also explain the variations induced in the profile of the intestinal microbiota from each mouse group. It has been shown that the structural charac teristics of AX lead to different patterns of degradation by colonic bac teria. Rumpagaporn et al. (2015) observed that wheat and corn WE-AX showing higher amount of terminal xylose were fermented more slowly. It was also reported that the fermentability of highly branched-AX from wheat bran was lower than that of a slightly branched-AX from aleurone cells (Arrigoni et al., 2003). However, the distribution of the arabinose substitution or the esterification thereof with feluric acid could result in a larger molecular size and could explain the AX h result. Thus, branching pattern and ferulic acid substitution play crucial roles in AX fermentability. There is a negative correlation between the substitution 6
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Fig. 3. Concentration of SCFA in caecal content of mice Concentrations of acetic (A), propionic (B) and butyric acid (C) in the caecal content of mice at the end of treatments. Different letters indicate significant differences with p < 0.05 between the columns.
these bacteria (the three SCFA showed correlations with r > 0.95; p < 0.01 with lactobacillus and bifidobacterial amount). In turn, a negative correlation was observed between SCFA levels and the bacterial groups that are not considered probiotic, such as Enterococcus and Bacteroides (r < 0.94; p < 0.05). This result suggests that the increase of these compounds is in part due to the metabolism of the increasing bacterial groups considered traditional probiotics in the intestine of mice. Gong et al. (2019), also found positive correlations between both Lactobacillus and Bifidobacterium and the SCFA levels, as well as a negative correlation with Clostridium counts, as van Zanten et al. (2012) did. The fact that the groups of mice fed with WE-AX from hard and soft Argentine wheat presented higher amounts of Lactobacillus and Bifido bacterium, and a lower percentage of Clostridium confirm the potential prebiotic effect of these polysaccharide extracts. Treated groups also presented an increased concentration of acetic and butyric acids in caecal content. These results demonstrate that AX have the capacity to modulate the intestinal microbiota in a beneficial way. The hypothesis of this work was correct, since the arabinoxylans of both wheats had prebiotic effects and they were even greater than the effect observed for
the WE-AX extracted from the hard wheats. 3.3.3. Correlations between the in vitro and in vivo prebiotic effect After having analyzed the in vitro and in vivo prebiotic effect with the same WE-AX extracts, we evaluated whether a correlation between them exists. A positive correlation was found between the RG and the PA of Lactobacillus in relation to the final amount of this strain measured in caeca at the end of the in vivo treatment (r 0.99 and 0.98 with a p < 0.01, respectively). Also, we found a positive correlation between RG of Bifidobacteria and its concentration in vivo (r 0.95 with a p < 0.01). This result reflects a high predictive capacity of the in vitro prebiotic assay performed here, since the result had a similar effect as the one obtained from the in vivo study. In turn, negative correlations were observed between RG and PA of Lactobacillus, and the amount of the Bacteroides and Enterococcus in vivo (r - 0.98 and - 0.97 with a p < 0.01, respec tively). These analyses showed that the increase in the metabolism of the Lactobacillus strain was related to the lower development of strains that are expected not to increase their metabolism with a prebiotic com pound such as WE-AX. 7
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To our knowledge, this is the first study that reports a strong corre lation between the prebiotic capacity in vitro and in vivo induced by the same WE-AX samples. Considering that both in vivo and in vitro tests were performed without any sample modification, we can establish the efficiency of the in vitro analysis to estimate and predict the potential in vivo prebiotic effect for a given compound.
References Abbeele, P. Van Den, G� erard, P., Rabot, S., Bruneau, A., Aidy, S. El, Derrien, M., Kleerebezem, M., Zoetendal, E.G., Smidt, H., Verstraete, W., Wiele, T. Van De, Possemiers, S., 2011. Arabinoxylans and inulin differentially modulate the mucosal and luminal gut microbiota and mucin-degradation in humanized rats. Environ. Microbiol. 13, 2667–2680. https://doi.org/10.1111/j.1462-2920.2011.02533.x. Adams, E.L., Kroon, P.A., Williamson, G., Gilbert, H.J., Morris, V.J., 2004. Inactivated enzymes as probes of the structure of arabinoxylans as observed by atomic force microscopy. Carbohydr. Res. 339, 579–590. https://doi.org/10.1016/j. carres.2003.11.023. Al-sheraji, S.H., Ismail, A., Yazid, M., Mustafa, S., 2013. Prebiotics as functional foods : a review. J. Funct. Foods 5, 1542–1553. https://doi.org/10.1016/j.jff.2013.08.009. Arrigoni, E., Amad, R., Amrein, T.M., Gr, P., 2003. In Vitro Digestibility and Colonic Fermentability of Aleurone Isolated from Wheat Bran, vol. 36, pp. 451–460. https:// doi.org/10.1016/S0023-6438(03)00036-7. Buksa, K., Nowotna, A., Praznik, W., Gambu�s, H., Ziobro, R., Krawontka, J., 2010. The role of pentosans and starch in baking of wholemeal rye bread. Food Res. Int. 43, 2045–2051. https://doi.org/10.1016/j.foodres.2010.06.005. Campbell, G.M., Bunn, P.J., Webb, C., 2001. On predicting roller milling performance Part II. The breakage function. Power Technol. 115, 243–255. https://doi.org/ 10.1016/S0032-5910(00)00349-1. Castillo, M., Martín-Orúe, S.M., Manzanilla, E.G., Badiola, I., Martín, M., Gasa, J., 2006. Quantification of total bacteria, enterobacteria and lactobacilli populations in pig digesta by real-time PCR. Vet. Microbiol. 114, 165–170. https://doi.org/10.1016/j. vetmic.2005.11.055. Crittenden, R., Karppinen, S., Ojanen, S., Tenkanen, M., Ma, J., Poutanen, K., 2002. In Vitro Fermentation of Cereal Dietary Fibre Carbohydrates by Probiotic and Intestinal Bacteria, vol. 789, pp. 781–789. https://doi.org/10.1002/jsfa.1095. Damen, B., Verspreet, J., Pollet, A., Broekaert, W.F., Delcour, J.A., Courtin, C.M., 2011. Prebiotic effects and intestinal fermentation of cereal arabinoxylans and arabinoxylan oligosaccharides in rats depend strongly on their structural properties and joint presence. Mol. Nutr. Food Res. 55, 1862–1874. https://doi.org/10.1002/ mnfr.201100377. De Filippo, C., Cavalieri, D., Di Paola, M., Ramazzotti, M., Poullet, J.B., Massart, S., Collini, S., Pieraccini, G., Lionetti, P., 2010. Impact of diet in shaping gut microbiota revealed by a comparative study in children from Europe and rural Africa. Proc. Natl. Acad. Sci. U.S.A. 107, 14691–14696. https://doi.org/10.1073/ pnas.1005963107. Dervilly, G., Saulnier, L., Roger, P., Thibault, J.F., 2000. Isolation of homogeneous fractions from wheat water-soluble arabinoxylans. Influence of the structure on their macromolecular characteristics. J. Agric. Food Chem. 48, 270–278. https://doi.org/ 10.1021/jf990222k. Gibson, G.R., Fuller, R., 2000. Aspects of in vitro and in vivo research approaches directed toward identifying probiotics and prebiotics for human use. J. Nutr. 130, 391S–395S. https://doi.org/10.1093/jn/130.2.391s. Gibson, G.R., Hutkins, R., Sanders, M.E., Prescott, S.L., Reimer, R.A., Salminen, S.J., Scott, K., Stanton, C., Swanson, K.S., Cani, P.D., Verbeke, K., Reid, G., 2017. Expert consensus document: the International Scientific Association for Probiotics and Prebiotics (ISAPP) consensus statement on the definition and scope of prebiotics. Nat. Rev. Gastroenterol. Hepatol. 14, 491–502. https://doi.org/10.1038/ nrgastro.2017.75. G� omez, M., Oliete, B., Rosell, C.M., Pando, V., Fern� andez, E., 2008. Studies on cake quality made of wheat-chickpea flour blends. LWT - Food Sci. Technol. (Lebensmittel-Wissenschaft -Technol.) 41, 1701–1709. https://doi.org/10.1016/j. lwt.2007.11.024. Gong, L., Chi, H., Wang, J., Zhang, H., Sun, B., 2019. In vitro fermentabilities of whole wheat as compared with refined wheat in different cultivars. J. Funct. Foods 52, 505–515. https://doi.org/10.1016/j.jff.2018.11.027. ~ 2007. Functional activity of commercial Huebner, J., Wehling, R.L., Hutkins, R.W.A., prebiotics. Int. Dairy J. 17, 770–775. https://doi.org/10.1016/j. idairyj.2006.10.006. Hughes, S.A., Shewry, P.R., Li, L., Gibson, G.R., Sanz, M.L., Rastall, R.A., 2007. In vitro fermentation by human fecal microflora of wheat arabinoxylans. J. Agric. Food Chem. 55, 4589–4595. https://doi.org/10.1021/jf070293g. Lagaert, S., Pollet, A., Delcour, J.A., Lavigne, R., Courtin, C.M., Volckaert, G., 2010. Substrate specificity of three recombinant α-l-arabinofuranosidases from Bifidobacterium adolescentis and their divergent action on arabinoxylan and arabinoxylan oligosaccharides. Biochem. Biophys. Res. Commun. 402, 644–650. https://doi.org/10.1016/j.bbrc.2010.10.075. Lempereur, I., Rouau, X., Abecassis, J., 1997. Genetic and agronomic variation in arabinoxylan and ferulic acid contents of durum wheat (Triticum durum l.) grain and its milling fractions. J. Cereal. Sci. 25, 103–110. https://doi.org/10.1006/ jcrs.1996.0090. Maes, C., Delcour, J.A., 2002. Structural characterisation of water-extractable and waterunextractable arabinoxylans in wheat bran. J. Cereal. Sci. 35, 315–326. https://doi. org/10.1006/jcrs.2001.0439. Mendis, M., Simsek, S., 2014. Arabinoxylans and human health. Food Hydrocolloids 42, 239–243. https://doi.org/10.1016/j.foodhyd.2013.07.022. Menezes, E.W., Dan, M.C.T., Cardenette, G.H.L., Go~ ni, I., Bello-p�erez, L.A., Lajolo, F.M., 2010. Vitro Colonic Fermentation and Glycemic Response of Different Kinds of Unripe Banana Flour 379–385. https://doi.org/10.1007/s11130-010-0190-4. Moiraghi, M., Vanzetti, L., Pflüger, L., Helguera, M., P� erez, G.T., 2013. Effect of high molecular weight glutenins and rye translocations on soft wheat flour cookie quality. J. Cereal. Sci. 58, 424–430. https://doi.org/10.1016/j.jcs.2013.08.007.
4. Conclusions Although information on soluble AX fermentation extracted from different layers of wheat grain and AXOS is found in the literature, no studies have been found on WE-AX extracted from the hard and soft wheat whole grain flours, the composition and prebiotic activity of which have been studied in this work. The WE-AX extracted from whole wheat flour of two pools of ge notypes (soft and hard) showed similar A/X ratio but an important heterogeneity in molecular size profile. The WE-AX of hard wheat pre sented a fraction of large molecular size molecules not observed in soft wheat. The extracts of WE-AX showed, in both in vitro and in vivo tests, the ability to increase the number of beneficial bacteria (Lactobacillus and Bifidobacterium) in the profile of intestinal microorganisms and to decrease the number of possible pathogens (Clostridium). They also proved to be able to increase the amount of short chain fatty acids (SCFA) in the intestine of mice. No other studies have evaluated the prebiotic effect in vitro and in vivo using the same extracts of WE-AX. The correlations found in the present work suggest that in vitro indices allow predicting the potential prebiotic effect in vivo. The consumption of WE-AX could maintain the balance and/or modulate more favorable profiles in the intestinal microbiota. The WEAX extracted from Argentine hard wheats, which is the type grown and most consumed in Argentina, showed more important beneficial effects than the AX derived from soft wheats. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. CRediT authorship contribution statement Candela Paesani: Methodology, Validation, Formal analysis, Investigation, Writing - original draft, Visualization. Alicia Laura Degano: Conceptualization, Methodology, Resources, Writing - review & editing, Supervision. Emiliano Salvucci: Conceptualization, Meth odology, Writing - review & editing, Funding acquisition. Maria Ines ~o Paulo Fabi: Resources, Su Zalosnik: Resources, Methodology. Joa pervision, Project administration. Lorena Susana Sciarini: Resources, Writing - review & editing, Funding acquisition. Gabriela Teresa Perez: Conceptualization, Methodology, Writing - review & editing, Supervi sion, Project administration, Funding acquisition. Acknowledgements The authors acknowledge Gabriela Díaz Cort� ez for providing useful suggestions to improve the English language manuscript. This work was supported by CONICET (Consejo Nacional de Investigaciones Científicas �n Científica y Tec y T�ecnicas) and the Agencia Nacional de Promocio nol� ogica (Grant PICT 2015-0606) of Argentina. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.jcs.2020.102956. 8
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Journal of Cereal Science 93 (2020) 102956 polymerase chain reaction: an evidence of dysbiosis. Dig. Dis. Sci. 60, 2953–2962. https://doi.org/10.1007/s10620-015-3607-y. Van Craeyveld, V., Swennen, K., Dornez, E., Van de Wiele, T., Marzorati, M., Verstraete, W., Delaedt, Y., Onagbesan, O., Decuypere, E., Buyse, J., De Ketelaere, B., Broekaert, W.F., Delcour, J.A., Courtin, C.M., 2008. Structurally different wheatderived arabinoxylooligosaccharides have different prebiotic and fermentation properties in rats. J. Nutr. 138, 2348–2355. https://doi.org/10.3945/ jn.108.094367. van Laere, K.M.J., Hartemink, R., Bosveld, M., Schols, H.A., Voragen, A.G.J., 2000. Fermentation of plant cell wall derived polysaccharides and their corresponding oligosaccharides by intestinal bacteria. J. Agric. Food Chem. 48, 1644–1652. https://doi.org/10.1021/jf990519i. van Zanten, G.c., Knudsen, A., Roytio, H., Forssten, S., Lawther, M., Zanten, G.C. Van, Knudsen, A., Ro, H., Blennow, A., Lahtinen, S.J., Jakobsen, M., Svensson, B., Jespersen, L., 2012. The effect of selected synbiotics on microbial composition and short-chain fatty acid production in a model System of the human colon. PloS One 7. https://doi.org/10.1371/journal.pone.0047212. Vardakou, M., Palop, C.N., Christakopoulos, P., Faulds, C.B., Gasson, M.A., Narbad, A., 2008. Evaluation of the prebiotic properties of wheat arabinoxylan fractions and induction of hydrolase activity in gut microflora. Int. J. Food Microbiol. 123, 166–170. https://doi.org/10.1016/j.ijfoodmicro.2007.11.007. Vignola, M.B., Moiraghi, M., Salvucci, E., Baroni, V., P�erez, G.T., 2016. Whole meal and white flour from Argentine wheat genotypes: mineral and arabinoxylan differences. J. Cereal. Sci. 71, 217–223. https://doi.org/10.1016/j.jcs.2016.09.002. Wang, M., Wichienchot, S., He, X., Fu, X., Huang, Q., Zhang, B., 2019. In vitro colonic fermentation of dietary fibers: fermentation rate, short-chain fatty acid production and changes in microbiota. Trends Food Sci. Technol. 88, 1–9. https://doi.org/ 10.1016/j.tifs.2019.03.005. Wong, J.M.W., Souza, R. De, Kendall, C.W.C., Emam, A., Jenkins, D.J.A., 2006. Colonic Health : fermentation and short chain Fatty Acids. J. Clin. Gastroenterol. 40, 235–243. Xu, W., Su, P., Zheng, L., Fan, H., Wang, Y., Liu, Y., 2018. In Vivo Imaging of a Novel Strain of Bacteroides Fragilis via Metabolic Labeling, vol. 9, pp. 1–7. https://doi.org/ 10.3389/fmicb.2018.02298. Yang, J., 2012. Influence of Dietary Fibers and Whole Grains on Fecal Microbiota during in Vitro Fermentation. Zach, J., Hroudov� a, J., Sedlmajer, M., Korjenic, A., 2014. Study of heat transfer process in structure of thermal insulating materials based on natural fibers. Adv. Mater. Res. 1000, 227–230. https://doi.org/10.4028/www.scientific.net/AMR.1000.227.
Moura, P., Barata, R., Carvalheiro, F., Gírio, F., Loureiro-Dias, M.C., Esteves, M.P., 2007. In vitro fermentation of xylo-oligosaccharides from corn cobs autohydrolysis by Bifidobacterium and Lactobacillus strains. LWT - Food Sci. Technol. (LebensmittelWissenschaft -Technol.) 40, 963–972. https://doi.org/10.1016/j.lwt.2006.07.013. Neyrinck, A.M., Possemiers, S., Druart, C., van de Wiele, T., de Backer, F., Cani, P.D., Larondelle, Y., Delzenne, N.M., 2011. Prebiotic effects of wheat Arabinoxylan related to the increase in bifidobacteria, roseburia and bacteroides/prevotella in diet-induced obese mice. PloS One 6. https://doi.org/10.1371/journal. pone.0020944. Paesani, C., Salvucci, E., Moiraghi, M., Fernandez Canigia, L., P� erez, G.T., 2019. Arabinoxylan from Argentinian whole wheat flour promote the growth of Lactobacillus reuteri and Bifidobacterium breve. Lett. Appl. Microbiol. 68, 142–148. https://doi.org/10.1111/lam.13097. Palframan, R., Gibson, G.R., Rastall, R.A., 2003. Development of a quantitative tool for the comparison of the prebiotic effect of dietary oligosaccharides. Lett. Appl. Microbiol. 37, 281–284. https://doi.org/10.1046/j.1472-765X.2003.01398.x. Prado, S.B.R. Do, Ferreira, G.F., Harazono, Y., Shiga, T.M., Raz, A., Carpita, N.C., Fabi, J. P., 2017. Ripening-induced chemical modifications of papaya pectin inhibit cancer cell proliferation. Sci. Rep. 7, 1–17. https://doi.org/10.1038/s41598-017-16709-3. Rinttil€ a, T., Lyra, A., Krogius-Kurikka, L., Palva, A., 2011. Real-time PCR analysis of enteric pathogens from fecal samples of irritable bowel syndrome subjects. Gut Pathog. 3, 1–9. https://doi.org/10.1186/1757-4749-3-6. Rivi� ere, A., Selak, M., Lantin, D., Leroy, F., De Vuyst, L., 2016. Bifidobacteria and butyrate-producing colon bacteria: importance and strategies for their stimulation in the human gut. Front. Microbiol. 7 https://doi.org/10.3389/fmicb.2016.00979. Rumpagaporn, P., Reuhs, B.L., Kaur, A., Patterson, J.A., Keshavarzian, A., Hamaker, B.R., 2015. Structural features of soluble cereal arabinoxylan fibers associated with a slow rate of in vitro fermentation by human fecal microbiota. Carbohydr. Polym. 130, 191–197. https://doi.org/10.1016/j.carbpol.2015.04.041. Saulnier, L., Sado, P.E., Branlard, G., Charmet, G., Guillon, F., 2007. Wheat arabinoxylans: exploiting variation in amount and composition to develop enhanced varieties. J. Cereal. Sci. 46, 261–281. https://doi.org/10.1016/j.jcs.2007.06.014. Shin, H., Park, S., Sung, J.H., Kim, D., 2003. Purification and characterization of ␣ - L -arabinopyranosidase and ␣ - L -arabinofuranosidase from Bifidobacterium breve K110 , a human intestinal anaerobic bacterium Metabolizing Ginsenoside Rb2 and Rc, 69, 7116-7123. https://doi.org/10.1128/AEM.69.12.7116. Shukla, R., Ghoshal, U., Dhole, T.N., Ghoshal, U.C., 2015. Fecal microbiota in patients with irritable bowel syndrome compared with healthy controls using real-time
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