Antimicrobial activity of thermophilin 110 against the opportunistic pathogen Cutibacterium acnes.

OBJECTIVE: Thermophilin 110, a bacteriocin produced by Streptococcus thermophilus B59671, inhibited planktonic growth and biofilm formation of Cutibacterium acnes, a commensal skin bacterium associated with the inflammatory disease, acne vulgaris, and more invasive deep tissue infections. RESULTS: Thermophilin 110 prevented planktonic growth of C. acnes at a concentration ≥ 160 AU mL-1; while concentrations ≥ 640 AU mL-1 resulted in a > 5 log reduction in viable planktonic cell counts and inhibited biofilm formation. Arabinoxylan (AX) and sodium alginate (SA) hydrogels were shown to encapsulate thermophilin 110, but as currently formulated, the encapsulated bacteriocin was unable to diffuse out of the gel and inhibit the growth of C. acnes. Hydrogels were also used to encapsulate S. thermophilus B59671, and inhibition zones were observed against C. acnes around intact SA gels, or S. thermophilus colonies that were released from AX gels. CONCLUSIONS: Thermophilin 110 has potential as an antimicrobial for preventing C. acnes infections and further optimization of SA and AX gel formulations could allow them to serve as delivery systems for bacteriocins or bacteriocin-producing probiotics.

Recovery of pectic hydrocolloids and phenolics from huanglongbing related dropped citrus fruit.

BACKGROUND: Citrus pre-harvest fruit drop, caused by huanglongbing infection, has increased dramatically concomitant with declining tree health and crop harvest size. This loss of harvestable fruit is damaging to both growers and juice processors. Recovering and converting this fruit to alternative value added products would benefit the citrus industry. Therefore, we have explored the potential of using this fruit as a feedstock in our newly developed pilot scale continuous steam explosion process. RESULTS: Whole fruits were converted to steam-exploded biomass using a continuous pilot scale process. The sugar composition of raw fruit and steam-exploded biomass was determined. Recovered pectic hydrocolloids and phenolic compounds were characterized. Pectic hydrocolloids comprised 78 g kg-1 of the dry material in the dropped fruit. Following the steam explosion process almost all of the pectic hydrocolloids were recoverable with a water wash. They could be functionalized in situ or separated from the milieu. Additionally, approximately 40% of the polymethoxylated flavones, 10% of the flavanone glycosides, 85% of the limonoids and almost 100% of hydroxycinnamates were simultaneously recovered. CONCLUSION: The continuous steam explosion of pre-harvest dropped citrus fruit provides an enhanced, environmentally friendly method for the release and recovery of valuable coproducts from wasted biomass. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

Structural characterization of strawberry pomace.

Strawberries are a nutrient dense food rich in vitamins, minerals, non-nutrient antioxidant phenolics, and fibers. Strawberry fiber bioactive structures are not well characterized and limited information is available about the interaction between strawberry fiber and phenolics. Therefore, we analyzed commercial strawberry pomace in order to provide a detailed carbohydrate structural characterization, and to associate structures with functions. The pomace fraction, which remained after strawberry commercial juice extraction, contained mostly insoluble (49.1 % vs. 5.6 % soluble dietary fiber) dietary fiber, with pectin, xyloglucan, xylan, ß-glucan and glucomannan polysaccharides; glucose, fructose, xylose, arabinose, galactose, fucose and galacturonic acid free carbohydrates; protein (15.6 %), fat (8.34 %), and pelargonidin 3-glucoside (562 µg/g). Oligosaccharides from fucogalacto-xyloglucan, methyl-esterified rhamnogalacturonan I with branched arabinogalacto-side chains, rhamnogalacturonan II, homogalacturonan and ß-glucan were detected by MALDI-TOF MS, NMR and glycosyl-linkage analysis. Previous reports suggest that these oligosaccharide and polysaccharide structures have prebiotic, bacterial pathogen anti-adhesion, and cholesterol-lowering activity, while anthocyanins are well-known antioxidants. A strawberry pomace microwave acid-extracted (10 min, 80 °C) fraction had high molar mass (2376 kDa) and viscosity (3.75 dL/g), with an extended rod shape. A random coil shape, that was reported previously to bind to phenolic compounds, was observed for other strawberry microwave-extracted fractions. These strawberry fiber structural details suggest that they can thicken foods, while the polysaccharide and polyphenol interaction indicates great potential as a multiple-function bioactive food ingredient important for gut and metabolic health.

1D and 2D NMR datasets, resonance assignments and coupling constant analysis of red beet fiber and pectin.

The datasets presented in this article represent detailed NMR spectral analyses on red beet fiber, including the pomace, water-soluble and water-insoluble fractions, as well as the acid-extracted pectin. The samples were solvated in deuterium oxide and investigated by 1D-1H, 1D-13C NMR, and multiple 2D-NMR experiments, including gCOSY, zTOCSY, HSQC, HMBC, HSQCTOCSY, and H2BC. The NMR chemical shifts, coupling constants and spin-systems were identified for the major carbohydrate residues in each sample. This article provides additional data related to the research article "Structural characterization of red beet fiber and pectin" published in Food Hydrocolloids [1].

Cranberry Arabino-Xyloglucan and Pectic Oligosaccharides Induce Lactobacillus Growth and Short-Chain Fatty Acid Production.

Numerous health benefits have been reported from the consumption of cranberry-derived products, and recent studies have identified bioactive polysaccharides and oligosaccharides from cranberry pomace. This study aimed to further characterize xyloglucan and pectic oligosaccharide structures from pectinase-treated cranberry pomace and measure the growth and short-chain fatty acid production of 86 Lactobacillus strains using a cranberry oligosaccharide fraction as the carbon source. In addition to arabino-xyloglucan structures, cranberry oligosaccharides included pectic rhamnogalacturonan I which was methyl-esterified, acetylated and contained arabino-galacto-oligosaccharide side chains and a 4,5-unsaturated function at the non-reducing end. When grown on cranberry oligosaccharides, ten Lactobacillus strains reached a final culture density (ΔOD) ≥ 0.50 after 24 h incubation at 32 °C, which was comparable to L. plantarum ATCC BAA 793. All strains produced lactic, acetic, and propionic acids, and all but three strains produced butyric acid. This study demonstrated that the ability to metabolize cranberry oligosaccharides is Lactobacillus strain specific, with some strains having the potential to be probiotics, and for the first time showed these ten strains were capable of growth on this carbon source. The novel cranberry pectic and arabino-xyloglucan oligosaccharide structures reported here combined with the Lactobacillus strains that can metabolize cranberry oligosaccharides and produce short-chain fatty acids, have excellent potential as health-promoting synbiotics.

Identification of Lactobacillus Strains Capable of Fermenting Fructo-Oligosaccharides and Inulin.

Novel probiotic strains that can ferment prebiotics are important for functional foods. The utilization of prebiotics is strain specific, so we screened 86 Lactobacillus strains and compared them to Bifidobacterium breve 2141 for the ability to grow and produce SCFA when 1% inulin or fructo-oligosaccharides (FOS) were provided as the carbon source in batch fermentations. When grown anaerobically at 32 °C, ten Lactobacillus strains grew on both prebiotic substrates (OD600 ≥ 1.2); while Lactobacillus coryniformis subsp. torquens B4390 grew only in the presence of inulin. When the growth temperature was increased to 37 °C to simulate the human body temperature, four of these strains were no longer able to grow on either prebiotic. Additionally, L. casei strains 4646 and B441, and L. helveticus strains B1842 and B1929 did not require anaerobic conditions for growth on both prebiotics. Short-chain fatty acid analysis was performed on cell-free supernatants. The concentration of lactic acid produced by the ten Lactobacillus strains in the presence of prebiotics ranged from 73-205 mM. L. helveticus B1929 produced the highest concentration of acetic acid ~19 mM, while L. paraplantarum B23115 and L. paracasei ssp. paracasei B4564 produced the highest concentrations of propionic (1.8-4.0 mM) and butyric (0.9 and 1.1 mM) acids from prebiotic fermentation. L. mali B4563, L. paraplantarum B23115 and L. paracasei ssp. paracasei B4564 were identified as butyrate producers for the first time. These strains hold potential as synbiotics with FOS or inulin in the development of functional foods, including infant formula.

Global structure of microwave-assisted flash-extracted sugar beet pectin.

The global structure of microwave-assisted flash-extracted pectins isolated from fresh sugar beet pulp has been studied. The objective was to minimize the disassembly and possibly the degradation of pectin molecules during extraction. These pectins have been characterized by high-performance size exclusion chromatography with light scattering, viscometric detection, and atomic force microscopy (AFM). Analysis of molecular parameters was performed on 15 and 8 microm size column packings. Samples analyzed with 15 microm packing gave weight-average molar masses that ranged from 532,000 to 1.2 million Da, radii of gyration from about 35 to 51 nm, polydispersities from 1.78 to 2.58, intrinsic viscosities from about 3.00 to 4.30 dL/g, and recoveries from 8.40 to 14.81% of dry weight. Chromatography revealed that a bimodal distribution of high molar mass spherical particles and lower molar mass coils was obtained. AFM images of pectin corroborated this conclusion and further revealed that these strands and spherical particles were integrated into networks. It is demonstrated that microwave-assisted extraction of sugar beet pulp under moderate pressure and at relatively low temperature could extract under acid conditions high molar mass, moderate-viscosity pectin in minutes rather than hours as required by conventional heating.

The role of modified citrus pectin as an effective chelator of lead in children hospitalized with toxic lead levels.

CONTEXT: Lead toxicity is an ongoing concern worldwide, and children, the most vulnerable to the long-lasting effects of lead exposure, are in urgent need of a safe and effective heavy metal chelating agent to overcome the heavy metals and lead exposure challenges they face day to day. OBJECTIVE: This clinical study was performed to determine if the oral administration of modified citrus pectin (MCP) is effective at lowering lead toxicity in the blood of children between the ages of 5 and 12 years. METHOD: Hospitalized children with a blood serum level greater than 20 microg/dL, as measured by graphite furnace atomic absorption spectrometry (GFAAS), who had not received any form of chelating and/or detoxification medication for 3 months prior were given 15 g of MCP (PectaSol) in 3 divided dosages a day. Blood serum and 24-hour urine excretion collection GFAAS analysis were performed on day 0, day 14, day 21, and day 28. RESULT: This study showed a dramatic decrease in blood serum levels of lead (P = .0016; 161% average change) and a dramatic increase in 24-hour urine collection (P = .0007; 132% average change). CONCLUSION: The need for a gentle, safe heavy metal-chelating agent, especially for children with high environmental chronic exposure, is great. The dramatic results and no observed adverse effects in this pilot study along with previous reports of the safe and effective use of MCP in adults indicate that MCP could be such an agent. Further studies to confirm its benefits are justified.

Molecular and functional properties of a xylanase hydrolysate of corn bran arabinoxylan.

Enzymatic hydrolysis of arabinoxylans to prepare arabinoxylo-oligosaccharides has been of high interest from the commercial point of view. However, some arabinoxylans, such as those extracted from corn bran, tend to be difficult to hydrolyze into oligosaccharides due to their highly branched structure which limits the action of xylanases. This research presents a new arabinoxylo-oligosaccharide preparation by enzymatic treatment of corn bran with an endoxylanase enzyme. The native arabinoxylan had a molecular weight of 253kDa and the hydrolysate polymers ranged from 51.6 to 132kDa. The hydrolyzates showed improved solubility in contrast to the original sample. The molecular properties of the hydrolyzates were related to the enzyme concentration used in the hydrolysis process, with increasing enzyme concentration leading to decreasing molecular weight and size. Solution viscosity of the samples also decreased with increasing enzyme concentration. All of the hydrolyzates showed emulsifying ability that was comparable to the original arabinoxylan.

Electrosprayed Core⁻Shell Composite Microbeads Based on Pectin-Arabinoxylans for Insulin Carrying: Aggregation and Size Dispersion Control.

Aggregation and coalescence are major drawbacks that contribute to polydispersity in microparticles and nanoparticles fabricated from diverse biopolymers. This study presents the evaluation of a novel method for the direct, electrospray-induced fabrication of small, CaCl2/ethanol-hardened low methoxy pectin/arabinoxylans composite microbeads. The electrospray method was evaluated to control particle size by adjusting voltage, flux, and crosslinking solution content of CaCl2/ethanol. A bead diameter of 1µm was set as reference to test the capability of this method. Insulin was chosen as a model carried molecule. Statistical analysis was a central composite rotatable design (CCRD) with a factorial arrangement of 24. The variables studied were magnitude and particle size dispersion. For the determination of these variables, light diffraction techniques, scanning electron microscopy, transmission electron microscopy, and confocal laser scanning microscopy were used. Major interaction was found for ethanol and CaCl2 as well as flow and voltage. Stable spherical structures of core⁻shell beads were obtained with neither aggregation nor coalescence for all treatments where ethanol was included in the crosslinking solution, and the average diameter within 1 ± 0.024 µm for 11 KV, 75% ethanol with 11% CaCl2, and flow of 0.97 mL/h.

Pectic oligosaccharide structure-function relationships: Prebiotics, inhibitors of Escherichia coli O157:H7 adhesion and reduction of Shiga toxin cytotoxicity in HT29 cells.

Shiga toxin (Stx)-producing, food-contaminating Escherichia coli (STEC) is a major health concern. Plant-derived pectin and pectic-oligosaccharides (POS) have been considered as prebiotics and for the protection of humans from Stx. Of five structurally different citrus pectic samples, POS1, POS2 and modified citrus pectin 1 (MCP1) were bifidogenic with similar fermentabilities in human faecal cultures and arabinose-rich POS2 had the greatest prebiotic potential. Pectic oligosaccharides also enhanced lactobacilli growth during mixed batch faecal fermentation. We demonstrated that all pectic substrates were anti-adhesive for E. coli O157:H7 binding to human HT29 cells. Lower molecular weight and deesterification enhanced the anti-adhesive activity. We showed that all pectic samples reduced Stx2 cytotoxicity in HT29 cells, as measured by the reduction of human rRNA depurination detected by our novel TaqMan-based RT-qPCR assay, with POS1 performing the best. POS1 competes with Stx2 binding to the Gb3 receptor based on ELISA results, underlining the POS anti-STEC properties.

Modified sugar beet pectin induces apoptosis of colon cancer cells via an interaction with the neutral sugar side-chains.

Pectins extracted from a variety of sources and modified with heat and/or pH have previously been shown to exhibit activity towards several cancer cell lines. However, the structural basis for the anti-cancer activity of modified pectin requires clarification. Sugar beet and citrus pectin extracts have been compared. Pectin extracted from sugar beet pulp only weakly affected the viability of colon cancer cells. Alkali treatment increased the anti-cancer effect of sugar beet pectin via an induction of apoptosis. Alkali treatment decreased the degree of esterification (DE) and increased the ratio of rhamnogalacturonan I (RGI) to homogalacturonan. Low DE per se did not play a significant role in the anti-cancer activity. However, the enzymatic removal of galactose and, to a lesser extent, arabinose from the pectin decreased the effect on cancer cells indicating that the neutral sugar-containing RGI regions are important for pectin bioactivity.

Isolation, characterization, and pectin-modifying properties of a thermally tolerant pectin methylesterase from Citrus sinensis var. Valencia.

The thermally tolerant pectin methylesterase (TT-PME) was isolated as a monocomponent enzyme from sweet orange fruit (Citrus sinensis var. Valencia). It was also isolated from flower and vegetative tissue. The apparent molecular weight of fruit TT-PME was 40800 by SDS-PAGE and the isoelectric point estimated as pI 9.31 by IEF-PAGE. MALDI-TOF MS identified no tryptic-peptide ions from TT-PME characteristic of previously described citrus PMEs. TT-PME did not absolutely require supplemented salt for activity, but salt activation and pH-dependent activity patterns were intermediate to those of thermolabile PMEs. Treatment of non-calcium-sensitive pectin with TT-PME (reducing the degree of methylesterification by 6%) increased the calcium-sensitive pectin ratio from 0.01 to 0.90, indicating a blockwise mode of action. TT-PME produced a significantly lower end-point degree of methylesterification at pH 7.5 than at pH 4.5. Extensive de-esterification with TT-PME did not reduce the pectin molecular weight or z-average radius of gyration, as determined by HPSEC.

Rhamnogalacturonan I containing homogalacturonan inhibits colon cancer cell proliferation by decreasing ICAM1 expression.

Pectin modified with pH, heat or enzymes, has previously been shown to exhibit anti-cancer activity. However, the structural requirements for modified pectin bioactivity have rarely been addressed. In this study several pectin extracts representing different structural components of pectin were assessed for effects against colon cancer cells. Rhamnogalacturonan I (RGI) extracts reduced proliferation of DLD1 and HCT116 colon cancer cells in a dose- and time-dependent manner. RGI reduced ICAM1 gene expression and siRNA-mediated knockdown of ICAM1 expression decreased cell proliferation providing a potential novel mechanism for the anti-cancer activity of pectin. Structural analysis of bioactive and non-bioactive RGIs suggested that a homogalacturonan component is maybe essential for the anti-proliferative activity, furthering the understanding of the structural requirements for pectin bioactivity.

Cranberry Xyloglucan Structure and Inhibition of Escherichia coli Adhesion to Epithelial Cells.

Cranberry juice has been recognized as a treatment for urinary tract infections on the basis of scientific reports of proanthocyanidin anti-adhesion activity against Escherichia coli as well as from folklore. Xyloglucan oligosaccharides were detected in cranberry juice and the residue remaining following commercial juice extraction that included pectinase maceration of the pulp. A novel xyloglucan was detected through tandem mass spectrometry analysis of an ion at m/z 1055 that was determined to be a branched, three hexose, four pentose oligosaccharide consistent with an arabino-xyloglucan structure. Two-dimensional nuclear magnetic resonance spectroscopy analysis provided through-bond correlations for the α-L-Araf (1→2) α-D-Xylp (1→6) ß-D-Glcp sequence, proving the S-type cranberry xyloglucan structure. Cranberry xyloglucan-rich fractions inhibited the adhesion of E. coli CFT073 and UTI89 strains to T24 human bladder epithelial cells and that of E. coli O157:H7 to HT29 human colonic epithelial cells. SSGG xyloglucan oligosaccharides represent a new cranberry bioactive component with E. coli anti-adhesion activity and high affinity for type 1 fimbriae.

Utilization of an evaporative light scattering detector for high-performance size-exclusion chromatography of galacturonic acid oligomers.

A high-performance size-exclusion chromatography-evaporative light scattering detector method was used to separate, detect and quantify galacturonic acid (GA) oligomers. In 40 mM acetic acid GA monomer, dimer and trimer could be separated with baseline resolution but polygalacturonic acid (PGA) precipitated and could not be eluted from the column. An NH4OAc, pH 3.7, buffer was developed as the eluent which separated GA oligomers as well as PGA and pectin without precipitation. Linear calibration curves for mono-, di- and tri-GA were produced with this buffer which could be used to estimate masses of tetra-, penta- and hexa-GA, as well as 19mer and 20mer.

Characterization of a salt-independent pectin methylesterase purified from valencia orange peel.

The pectin methylesterase (PME; EC 3.1.1.11) present in a commercial orange peel enzyme preparation was characterized to establish its identity among the multiple PME isozymes present in Valencia orange (Citrus sinensis L.) peel. We show the commercial enzyme corresponds to the major peak 2 PME previously separated by heparin-Sepharose chromatography (Cameron et al., J. Food Sci. 1998, 63, 253). Both PMEs have comparable elution profiles on cation-exchange and hydrophobic-interaction perfusion chromatography columns, molecular weights (ca. 34 kDa) and pI (pH 9.2), and biochemical properties, including a broad pH activity range and activity in the absence of added cations. An identical partial amino terminal peptide sequence was also obtained for the PMEs, which further demonstrated a structural identity with other plant PMEs. The biochemical and structural properties readily distinguish this Valencia orange PME from salt-dependent isozymes and further suggest that it is an ortholog to the salt-independent fruit-specific isozyme of tomato. This work provides a well-defined, enzymatically homogeneous, salt-independent (type 1) plant PME isozyme that is suitable for studying details of the enzyme's mode of action and for use in modifying methylester patterns for studying the structure-functional property relationships in pectin.

Monovalent salt-induced gelation of enzymatically deesterified pectin.

Pectin gels were induced by monovalent salts (0.2 M) concurrently with deesterification of high methoxy pectin using a salt-independent orange pectin methylesterase (PME). Constant pH was maintained during deesterification and gelation. If salt or PME was absent, the pectin did not form a gel. The gel strength was influenced by both pH and species of monovalent cation. At pH 5.0, the pectin gel induced by KCl was significantly stronger than the NaCl-induced gel. In contrast, a much stronger gel was produced in the presence of NaCl as compared to KCl at pH 7.0. LiCl did not induce pectin gelation at either pH. Molecular weights of pectins increased from 1.38 x 10(5) to 2.26 x 10(5) during NaCl-induced gelation at pH 7. One proposal to explain these pectin molecular weight changes is a hypothetical PME transacylation mechanism. However, these pectin molecular weight changes can also be explained by metastable aggregation of the enzymatically deesterified low methoxy pectin. We postulate that gelation was induced by a slow deesterification of pectin under conditions that would normally salt out (precipitate) low methoxy pectin in the absence of PME.

Enzymatic modification of pectin to increase its calcium sensitivity while preserving its molecular weight.

A commercial high-methoxy citrus pectin was treated with a purified salt-independent pectin methylesterase (PME) isozyme isolated from Valencia orange peel to prepare a series of deesterified pectins. A series of alkali-deesterified pectins was also prepared at pH 10 under conditions permitting beta-elimination. Analysis of these pectins using high-performance size exclusion chromatography (HPSEC) with on-line multiangle laser light-scattering, differential viscometer, and refractive index (RI) detectors revealed no reduction in weight-average molecular weight (M(w); 150000) in the PME-treated pectin series, whereas a 16% reduction in intrinsic viscosity (IV) occurred below a degree of esterification (DE) of 47%. In contrast, alkali deesterification rapidly reduced both M(w) and IV to less than half of that observed for untreated pectin. PME treatment of a non-calcium-sensitive citrus pectin introduced calcium sensitivity with only a 6% reduction in the DE. Triad blocks of unesterified galacturonic acid were observed in (1)H nuclear magnetic resonance spectra of this calcium-sensitive pectin (CSP). These results demonstrate that the orange salt-independent PME isozyme utilizes a blockwise mode of action. This is the first report of the preparation of a CSP by PME treatment without significant loss of the pectin's M(w) due to depolymerization.

Separation and characterization of a salt-dependent pectin methylesterase from Citrus sinensis var. Valencia fruit tissue.

A pectin methylesterase (PME) from sweet orange fruit rag tissue, which does not destabilize citrus juice cloud, has been characterized. It is a salt-dependent PME (type II) and exhibits optimal activity between 0.1 and 0.2 M NaCl at pH 7.5. The pH optimum shifted to a more alkaline range as the salt molarity decreased (pH 8.5-9.5 at 50 mM NaCl). It has an apparent molecular mass of 32.4 kDa as determined by gel filtration chromatography, an apparent molecular mass of 33.5 kDa as determined by denaturing electrophoresis, and a pI of 10.1 and exhibits a single activity band after isoelectric focusing (IEF). It has a K(m) of 0.0487 mg/mL and a V(max) of 4.2378 nkat/mg of protein on 59% DE citrus pectin. Deblocking the N-terminus revealed a partial peptide composed of SVTPNV. De-esterification of non-calcium-sensitive pectin by 6.5% increased the calcium-sensitive pectin ratio (CSPR) from 0.045 +/- 0.011 to 0.829 +/- 0.033 but had little, if any, effect on pectin molecular weight. These properties indicate this enzyme will be useful for studying the PME mode of action as it relates to juice cloud destabilization.