This study aims to investigate the impact of high-pressure homogenization (HPH) on the fine structure of pectin, and further investigate the relationships between the fine structure of pectin and carotenoid bioaccessibility in peach, carrot and combined juices. Results showed that HPH affected the yield and structure of water-soluble solids (WSS) pectins. HPH increased the amount of homogalacturonan (HG) extracted, decreased molecular weight, degree of methyl-esterification and degree of acetylation. Furthermore, the distribution of the methyl esters over the HG backbone was modified towards fewer blocks of non-methyl-esterified GalA residues and more segments with a moderate level of methyl esterification. Carotenoid bioaccessibility was found to have negative correlations with certain descriptive parameters of methyl esterification related to methyl ester level and distribution. These descriptive parameters include degree of blockiness (DB), absolute degree of blockiness (DBabs) and the degree of blockiness of methyl-esterified oligomers by PG (DBPGme). Our findings showed pectin structure is associated with carotenoid bioaccessibility, and HPH has the potential to enhance carotenoid bioaccessibility.

The restrictions on excessive use of antimicrobials in the poultry industry have led to the search for alternative strategies including nutritional interventions to enhance gut health with the ultimate aim to prevent gut infections. Pectins as prebiotics have shown beneficial effects on gut health in humans and mice by improving the gut barrier function, altering the gut microbiota, and by modulating the gut immune response. However, little is known about immunomodulatory properties of pectins in chickens. The present in vitro study assessed the effect of three pectins (SPE6, SPE7, SPE8) differing in methyl esterification, on responsiveness of the chicken macrophage cell line HD11 cells and primary monocyte derived macrophage from the blood, through interaction with chicken TLRs. All three pectins increased gene expression of iNOS and IL10 in chicken macrophages. Differences in immunomodulatory activity between the three pectins were observed in other assays. The low methoxyl pectin (SPE8) interacted with TLR4 leading to the production of NO, but also to increased phagocytosis of E. coli, while high methoxyl pectins SPE6 and SPE7 did not activate TLR4. All three pectins were able to attenuate PAM3CSK4 induced activation of chicken macrophages as measured by decreased NO production and phagocytosis. Additional studies using ITC and flow cytometry suggest that the inhibiting properties of pectins (SPE6, SPE7) on macrophages are due to pectins occupying TLR2 and blocking PAM3CSK4 to activate chicken macrophages, whereas SPE8 actually binds to the TLR2 ligand and that way attenuates the PAM3CSK4 induced activation. Based on these immunomodulatory properties observed in this study, these pectins may in the future be suitable as feed additive for the treatment and prevention of inflammatory disorders in poultry.

Mucin glycan degradation and utilization by microbes colonizing the human intestine is an essential host-microbe interaction. In this study, degradation and utilization of porcine gastric mucin glycans by Akkermansia muciniphila, Ruminococcus torques, Bacteroides thetaiotaomicron, co-cultures, and a synthetic bacterial community were investigated over time. Liquid chromatography-tandem mass spectrometry O-glycan patterns revealed that all three monocultures removed sialic acid residues. Furthermore, R. torques first targeted fucosylated O-glycans, while A. muciniphila and B. thetaiotaomicron equally favoured fucosylated and non-fucosylated O-glycans. A. muciniphila, R. torques, and B. thetaiotaomicron favoured degradation of first core 2 O-glycan structures relative to core 1 O-glycan structures. Co-cultures, compared to monocultures, demonstrated different O-glycan degradation patterns suggesting distinct ecological interactions between the bacteria. Although extensive O-glycan degradation was observed by the monocultures and co-cultures, only the synthetic community completely degraded all O-glycans within 24 h. Regarding degradation of the constituent N-glycans, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry showed that A. muciniphila and R. torques can partly degrade N-glycans, B. thetaiotaomicron can completely degrade high-mannose N-glycans, and the synthetic community can degrade all N-glycans. The utilization of mucin glycans was observed by production of different metabolites among the bacteria. These results indicate that degradation of mucin glycans depends on microbial interactions and ecological networks.

This study investigated bioactive carbohydrate-related compounds in rye grain overgrown with Agaricus subrufescens mycelia (ROM), using cytokine induction in chicken immune cells. We focused on polysaccharides, as they have been suggested to mediate immunomodulatory effects of rye and Agaricus subrufescens. Fractionation of rye and ROM and subsequent testing fractions on macrophage cell line HD11 showed that IL-12p40 production was mainly induced by hot water-soluble solids (HWSS) with ROM-HWSS more active than rye-HWSS. The bioactive HWSS consisted of polymers >10 kDa. Stimulation of chicken splenocytes further confirmed their immunomodulatory capacity. Rye- and ROM-HWSS >10 kDa mainly consisted of arabinoxylan and glucan with smaller and variable amounts of protein and ferulic acid. Polymeric arabinoxylan structures were confirmed to be present by enzymatic fingerprinting using pure xylanase. NaOH treatment (0.1 M) substantially reduced IL-12p40 induction by HWSS fractions and removed some ferulic ester groups, suggesting that feruloylated arabinoxylan structures contribute to IL-12p40 induction. Degradation of arabinoxylan and small amounts of glucan and fructan in the HWSS fractions with specific enzymes did not affect IL-12p40 induction. Protease, chitosanase and Driselase treatments reduced IL-12p40 induction in ROM-HWSS, suggesting that mycelial compounds (e.g., protein, chitosan and galactan/mannan) may (also) contribute to the bioactivity.

In recent years, immunomodulation by pectin and pectin-derived galacturonic acid oligosaccharides has been the subject of wide-spread scientific research due to the potential of different pectin structures as bioactive biomolecules. Yet, gaps remain in understanding the structure-dependent immunomodulation of galacturonic acid. This study describes in vitro immunomodulatory effects of well-characterized galacturonic acid oligosaccharides. Both methyl-esterified and non-methyl-esterified galacturonic acid oligosaccharides with a saturated non-reducing end (degree of polymerization 1-10) significantly induced cytokine production by THP-1 macrophages and directly activated TLR2 and TLR4 in transfected HEK-293 cells, even when accounting for minor endotoxin contamination. In contrast, both methyl-esterified and non-methyl-esterified galacturonic acid oligosaccharides with a Delta 4,5-unsaturated non-reducing end (degree of polymerization 1-7) did not activate TLR2 and TLR4 and led to significantly reduced cytokine production (p < 0.05), suggesting Delta 4,5-(un)saturation as a pivotal factor for immunomodulation by galacturonic acid oligosaccharides. Exposure to non-methyl-esterified saturated galacturonic acid oligosaccharides resulted in significantly lower TNF-alpha production, IL-1 beta production and TLR4 activation (p < 0.05) compared to methyl-esterified saturated galacturonic acid oligosaccharides, while IL-10 production and TLR2 activation remained unchanged. These findings establish galacturonic acid oligosaccharides as versatile immunomodulators with TLR2 and TLR4 binding capacity, fit for different immunomodulatory applications depending on their structural characteristics.

Passion fruit mesocarp is rich in pectin, and high-temperature/pressure modification of this pectin has been shown to yield bioactive fragments with anticancer potential. To clarify the structure-function relationship of passion fruit pectins, we purified native and modified pectins using two fractionation methods. Comprehensive chemical characterization revealed molecular weight as the primary difference between fractions, along with varying proportions of homogalacturonan (HG) and rhamnogalacturonan-I (RG-I). All samples activated TLR2, such as specific agonists (Pam3CSK4, HKLM, FSL-1). Notably, only native and lower-molecular-weight fractions inhibited TLR2/1 activation by the specific agonist Pam3CSK4. Higher methyl esterification correlated with TLR2/1 inhibition at lower doses, whereas RG-I content showed a negative correlation; however, the galacturonic acid-to-rhamnose ratio positively influenced heterodimer inhibition. A highly methyl esterified galacturonic acid heptamer demonstrated a strong affinity for the TLR2/1 binding pocket, as evidenced by molecular dynamics simulations. This study elucidates how modified passion fruit pectin structures interact with TLR2, reinforcing the link between plant polysaccharides and human immune responses.

A vast variety of bacterial, fungal, and plant-derived pectin methylesterases (PMEs) have been characterized in literature for their ability to deesterify pectins. However, when compared to fungal PMEs, the availability, characterisation and application of commercial enzyme preparations comprising plant PMEs is still lacking. Here, we characterized the PME activity in commercially available crude plant extracts originating from papaya (papain), pineapple (bromelain), and kiwi (actinidin). The highest PME activity towards pectin was determined in papain preparations, which did not comprise pectin-backbone degrading side activities. The pH and temperature optimum of the salt-dependent papain PMEs ranged from 7.0 to 8.0 and 50-70 degrees C, respectively. Using enzymatic fingerprinting, it was shown that papain PMEs exhibited a processive mode of action towards lemon pectin. Papain PMEs had a broad substrate specificity, as 61, 83, and 58% of the methylesters were released from lemon, apple and sugar beet pectin, respectively. The release of acetic acid from sugar beet pectin indicated the presence of acetyl esterases in the papain preparations. Both the determined processive mode of action and broad substrate specificity allows to consider papain preparations as an alternative to commercial fungal-derived PME preparations to modify the methylester distribution pattern of pectin for food applications.

This study explored the conversion of citrus juice side streams into fermentable oligosaccharides for potential gut health benefits. Alcohol washed, insoluble lemon peel waste was enzymatically treated using technical pectinolytic enzyme preparations, yielding mixtures of galactose- arabinose- and either methyl-esterified or non-methyl-esterified galacturonic acid oligosaccharides (OS) with a Δ4,5-unsaturated non-reducing end resulting in mixtures of pectin-derived OS: POS and POSNME. Both mixtures were completely fermented during in vitro batch fermentation by proximal and distal microbiota of three healthy adult donors. Fermentation by distal and proximal microbiota resulted in similar methyl-ester-dependent mechanisms of POS utilization, yielding health beneficial acetate, propionate and butyrate in significant amounts. Arabinose-, galactose- and non-methyl-esterified Δ4,5-unsaturated galacturonic acid OS were utilized significantly faster by the distal and proximal microbiota of donors 1 and 2 compared to methyl-esterified Δ4,5-unsaturated galacturonic acid OS, suggesting methyl-esterification of Δ4,5-unsaturated galacturonic acid oligosaccharides as a substantial regulator of POS fermentability. The findings presented in this manuscript suggest that carbohydrate molecular structure and availability, rather than microbiota composition, determine carbohydrate fermentation patterns along the colon, emphasizing that the consumption of differently fermentable fiber is essential to promote gut health along the colon.

The physicochemical properties of apple juice from 13 apple varieties were correlated with the structural features of isolated apple pectin populations. Mathematical models explaining these structural properties, revealed variations in pectins as present in alcohol insoluble solids (AIS), water-soluble solids (WSS), and chelating soluble solids (ChSS). Significant differences in turbidity (89.00-4129 NTU), particle size (59.75-218.01 μm in D[4, 3]), and titratable acid (0.13 to 0.90 %) were observed, while pH, total soluble solids, and ζ-potential showed less variation. Juice yield (54.85-63.35 %) was inversely correlated with AIS, WSS, and ChSS content. Juice ζ-potential was positively correlated with the degree of methyl-esterification, methyl-ester distribution patterns, and the side chain sugar ratios of WSS pectin fractions. Juice quality as represented by juice clarity, texture and stability was significantly influenced by the diverse structure's features like sugar composition, methyl-esterification level and distribution of endogenous pectin. A partial least squares (PLS) regression model effectively predicted juice properties based on pectin characteristics. These findings underscore the importance of pectin structure in determining apple juice quality and provide a valuable framework for the juice industry to enhance quality control and inform cultivar selection.

Soluble dietary fibers (SDFs) are recognized for their health benefits through their fermentation and gut microbiota modulation. Previous studies focused on individual SDFs without sufficient structural information and a comparative analysis using different SDFs on microbiota composition and function is lacking. The present study aimed to determine key structural features of different SDFs, including soluble resistant starch (SRS), inulin (INU), four structurally diverse pectins (PS1 to PS4), one pectic derivative (PS5) and larch arabinogalactan (AG). Their effects on gut microbiota composition and function were investigated by 72 h experiments in TIM-2 in vitro colon system with pooled feces, upon constant feeding (2.5 mL/h, total of 7.5 g/day) of test compounds. The tested SDFs were structurally different, inducing distinct effects on the relative abundance of specific bacterial genera and overall microbiota composition. AG, PS2, PS3, PS5, and SRS demonstrated marked changes compared to control in the overall community structure over time. SCFA production increased over time for all SDFs, but only PS1 to PS4 resulted in significantly higher SCFA levels compared to control. These findings demonstrate that structurally different SDFs exhibit different effects on the gut microbiota composition and function, however this could not be solely explained by Mw and monosaccharide composition.

This study explored potential underlying mechanisms (e.g. chyme characteristics, fermentation, and intestinal morphology), that contribute to the negative effect of high dietary starch on faecal integrity in yellowtail kingfish (Seriola lalandi) (experiment 1), and whether amylase supplementation can mitigate the apparent negative effects (experiment 2). Fish were fed LS (4 % starch) or HS (19 % starch) diets in experiment 1. Experiment 2 tested 21 % starch diets without amylase (NA diet) and with amylase supplementation (AS diet). Both experiments examined faeces waste production, faeces characteristics (e.g. removal efficiency and particle size distribution), distal intestine chyme composition, viscosity, osmolality, dry matter, fermentation processes (volatile fatty acids and lactic acid), and intestinal morphology. In experiment 1, the HS diet increased faecal waste production and decreased the faecal integrity. Only minor differences were observed in regard to chyme characteristics and fermentation products. Intestinal morphology appeared unaffected by the treatments. In experiment 2, the AS diet reduced faecal waste production and numerically increased removal efficiency, reducing non-removed feces compared to the NA diet. Amylase supplementation did not affect intestinal morphology or chyme characteristics. The present study showed that high dietary starch inclusion does not affect chyme dry matter content, osmolality at egestion, fermentation processes, or intestinal morphology. The exact mechanism by which high dietary starch harms yellowtail kingfish feces is unknown. The apparent negative effects on faeces may be due to variations in starch content. Amylase supplementation reduced faecal waste, partially mitigating the negative effects of high dietary starch inclusion on waste management in yellowtail kingfish.

Pectin's physicochemical, structural, and functional characteristics vary widely depending on the source of extraction. In this study, pectins were extracted from seedless quince and pomegranate peel, and their physicochemical, structural, and functional properties were investigated. A Box-Behnken Design with three factors and three levels was applied to optimize the pectin extraction yield from each matrix. As a result, the best extraction yields for quince pectin (QP) and pomegranate peel pectin (PPP) were 11.44 and 12.08 % (w/w), respectively. Both extracted pectins exhibit a linear structure, with the homogalacturonan domain dominating the rhamnogalacturonan I. Both pectins are highly methyl-esterified (DM > 69 %) with a higher degree of acetylation for PPP than QP, with 12 and 8 %, respectively. Unlike QP, PPP has a narrow, homogenous distribution and greater molecular weight (120 kDa). Regarding functionality, 1 g of QP could retain 4.92 g of water, and both pectin emulsions were more stable at room temperature than at 4 °C. When the concentration of QP is increased, rheological measurements demonstrate that it exhibits pseudoplastic behavior. Finally, QP can be used as a thickener, whereas PPP can be utilized as starting material for chemical changes to create multifunctional pectins.

Olive oil production generates substantial quantities of pomace, which are often disposed of in soil, leading to adverse effects on agriculture and the environment. Furthermore, climate change exacerbates plant diseases and promotes the use of toxic phytochemicals in agriculture. However, olive mill wastes can have high potential as reusable and valuable bioresources. Using diluted ethanol, an environmentally friendly solvent, we extracted a fraction containing short and long oligogalacturonides, short arabino-oligosaccharides and polysaccharides. The obtained extract elicited key features of plant innate immunity in Arabidopsis seedlings, including the phosphorylation of mitogen-activated protein kinases MPK3 and MPK6 and the upregulation of defence genes such as CYP81F2, WRKY33, WRKY53, and FRK1. Notably, pretreatment of adult Arabidopsis and tomato plants with the olive pomace extract primed defence responses and enhanced their resistance to the phytopathogens Botrytis cinerea and Pseudomonas syringae. Our results highlight the opportunity to upcycle the two-phase olive pomace collected at the late stage of olive oil campaign, in low-cost and sustainable glycan elicitors, contributing to reducing the use of chemically synthesized pesticides.

In this study, an in vitro co-culture model using an electric cell-substrate impedance sensing system (ECIS) for testing the impact of real-time fermentation of non-digestible carbohydrates (NDCs) by the intestinal microbiota on gut barrier function was established. We applied Lactobacillus plantarum WCFS1 as a model intestinal bacterium and alginate-pectin as immobilization polymers as well as a source of NDCs to determine the impact of pectin fermentation on the barrier function of T84 gut epithelial cells. In the first design, L. plantarum WCFS1 was encapsulated in an alginate capsule followed by embedding in an agar layer to mimic a firm mucus layer that might be present in the colon. In this experimental design, the presence of the agar layer interfered with the transepithelial electrical resistance (TEER) measurement of T84 cells. Subsequently, we removed the agar layer and used encapsulated bacteria in an alginate gel and found that the TEER measurement was adequate. The encapsulation of the L. plantarum WCFS1 does avoid direct contact with cells. Also, the encapsulation system allows higher amounts of packing densities of L. plantarum WCFS1 in a limited space which can limit the oxygen concentration within the capsule and therefore create anaerobic conditions. To test this design, T84 cells were co-incubated with L. plantarum alginate-capsules supplemented with graded loads of fermentable pectin (0, 4, and 8 mg/ml per capsule) to investigate the effect of pectin fermentation on gut barrier function. We observed that as the pectin content in the L. plantarum capsules increased, pectin showed a gradually stronger protective effect on the TEER of the gut epithelium. This could partly be explained by enhanced SCFA production as both lactate and acetate were enhanced in L. plantarum containing alginate capsules with 8 mg/ml pectin. Overall, this newly designed in vitro co-culture model allows for studying the impact of bacteria-derived fermentation products but also for studying the direct effects of NDCs on gut barrier function in a relatively high-throughput way.

Consumption of fructo- (FOS) and galacto-oligosaccharides (GOS) has health benefits which have been linked in part to short-chain fatty acids (SCFA) production by the gut microbiota. However, detailed knowledge of this process in the human intestine is lacking. We aimed to determine the acute fermentation kinetics of a FOS:GOS mixture in healthy males using a naso-intestinal catheter for sampling directly in the ileum or colon. We studied the fate of SCFA as substrates for glucose and lipid metabolism by the host after infusion of 13C-SCFA. In the human distal ileum, no fermentation of FOS:GOS, nor SCFA production, or bacterial cross-feeding was observed. The relative composition of intestinal microbiota changed rapidly during the test day, which demonstrates the relevance of postprandial intestinal sampling to track acute responses of the microbial community toward interventions. SCFA were vividly taken up and metabolized by the host as shown by incorporation of 13C in various host metabolites.

Awareness is required for the presence of digestible starch when studying in vitro fermentation of resistant starch (RS). Such starch may steer and overrule the fermentation of RS, thereby affecting the evaluation of the prebiotic potential of RS.

Synbiotics combine the concepts of probiotics and prebiotics to synergistically enhance the health-associated effects of both components. Previously, we have shown that the intestinal persistence of inulin-utilizing L. plantarum Lp900 is significantly increased in rats fed an inulin-supplemented, high-calcium diet. Here we employed a competitive population dynamics approach to demonstrate that inulin and GOS can selectively enrich L. plantarum strains that utilize these substrates for growth during in vitro cultivation, but that such enrichment did not occur during intestinal transit in rats fed a GOS or inulin-supplemented diet. The intestinal persistence of all L. plantarum strains increased irrespective of their prebiotic utilization phenotype, which was dependent on the calcium level of the diet. Analysis of fecal microbiota and intestinal persistence decline rates indicated that prebiotic utilization capacity did not selectively stimulate intestinal persistence in prebiotic supplemented diets. Moreover, microbiota and organic acid profile analyses indicate that the prebiotic utilizing probiotic strains are vastly outcompeted by the endogenous prebiotic-utilizing microbiota, and that the collective enhanced persistence of all L. plantarum strains is most likely explained by their well-established tolerance to organic acids.

This study investigated the impact of enzymatic hydrolyzation on the characteristics, composition, and in vitro fermentation patterns of pectins extracted from pomelo (Citrus maxima) by thermal treatments. Pectins were obtained using subcritical water extraction (SWE) and hot water extraction with chelator assistance, and subsequently, enzymatically modified using pectinase. The investigation covered constituent monosaccharide composition, molecular weight (Mw) distribution, and other structural parameters of the pectins. The primary monosaccharide present in the extracted pomelo pectins was uronic acid (UA), accompanied by relatively low levels of other neutral monosaccharides. The findings indicate that chelating agents coupled with thermal treatment allowed for the successful extraction of low degree of methylation (DM) pectins from pomelo peel. Enzymatic hydrolyzation reduced the DM and Mw of pectin, while maintaining a nearly unchanged monosaccharide mole ratio compared to the parental pectins. In vitro fermentation models were employed to compare the fermentation patterns of pectin before and after enzymatic modification. Enzymatic modification not only heightened the fermentability of pectins but also altered UA utilization patterns, expediting the production of short-chain fatty acids (SCFAs) during in vitro fermentation. Moreover, enzymatic hydrolyzation induced changes in microbial composition, elevating the relative abundance of potential probiotics such as Bacteroides while suppressing potential pathogens like Escherichia-Shigella. In conclusion, the study posits that enzymatic modification of pectins into low Mw and low DM fragments enhances fermentability and beneficially modifies the microbiota of fermented digesta into a potentially healthy pattern.

The differences in maximum Maillard glycation extent of proteins incubated with saccharides of different size/charge might be explained by differences in saccharide reactivity towards each protein modification site. Mixtures of α-lactalbumin-glucose (AG), α-lactalbumin-maltotriose (AMTT) or α-lactalbumin-galacturonic acid (AGalA) were incubated for 10 h, reaching a glycation extent of 42, 19 and 30 %, respectively. Glycated protein samples were enzymatically hydrolyzed followed by UPLC-UV-MS determination and quantification of glycation in individual modification sites. On average, around 80 % of protein glycation was recovered as glycated peptides. For all protein-saccharide mixtures, the N-terminal amino group, K58, K62 and K114 were never glycated, whilst K5 and K122 were always the most glycated sites. The glycated sites R10, K79, K93, K94 and K108 found in AGalA, were absent from AG and AMtt. Differences in glycated/non-glycated sites could not explain the differences in total protein glycation between the various samples.

Soybean tempeh contains bioactive carbohydrate that can reduce the severity of diarrhea by inhibiting en-terotoxigenic Escherichia coli (ETEC) adhesion to mammalian epithelial cells. Lactic acid bacteria (LAB) are known to be present abundantly in soybean tempeh. Some LAB species can produce exopolysaccharides (EPS) with anti-adhesion bioactivity against ETEC but there has been no report of anti-adhesion bioactive EPS from tempeh-associated LAB. We isolated EPS-producing LAB from tempeh-related sources, identified them, unambiguously elucidated their EPS structure and assessed the bioactivity of their EPS against ETEC. Pediococcus pentosaceus TL, Leuconostoc mesenteroides WA and L. mesenteroides WN produced both dextran (alpha-1,6 linked glucan; >1000 kDa) and levan (beta-2,6 linked fructan; 650-760 kDa) in varying amounts and Leuconostoc citreum TR produced gel-forming alpha-1,6-mixed linkage dextran (829 kDa). All four isolates produced EPS that could adhere to ETEC cells and inhibit auto-aggregation of ETEC. EPS-PpTL, EPS-LmWA and EPS-LmWN were more bioactive towards pig-associated ETEC K88 while EPS-LcTR was more bioactive against human-associated ETEC H10407. Our finding is the first to report on the bioactivity of dextran against ETEC. Tempeh is a promising source of LAB isolates that can produce bioactive EPS against ETEC adhesion and aggregation.