Characterization of glucan-producing Leuconostoc strains isolated from sourdough

Sourdough was previously demonstrated to be a fruitful biotope for isolation of lactic acid bacteria producing exopolysaccharides and more accurately diverse glycan polymers which have interesting applications as texturing agents or prebiotics. Characterization of polymers by ¹H and ¹³C NMR spectroscopy analysis demonstrated that these strains could synthesize glucans of high structural variety and containing different amounts of α-(1 → 2), α-(1 → 3) and α-(1 → 6) linkages. In this study, fifteen glucan-producing Leuconostoc mesenteroides and L. citreum strains from sourdoughs were characterized according to carbohydrate fermentation, rep-PCR fingerprinting using (GTG)₅ primers and glycansucrase activity (soluble or cell-associated). Enzyme characterization using SDS-PAGE and in situ polymer production after incubation with sucrose correlated with synthesis of classical or α-(1 → 2) branched dextrans, alternan and levan. In addition, the presence of genes coding for alternansucrase was detected by PCR and partially characterized by sequence analysis. We thus provide new information on the biodiversity of glucan production by sourdough Leuconostoc strains.     

In vitro human digestion models for food applications

In vitro digestion models are widely used to study the structural changes, digestibility, and release of food components under simulated gastrointestinal conditions. However, the results of in vitro digestion models are often different to those found using in vivo models because of the difficulties in accurately simulating the highly complex physicochemical and physiological events occurring in animal and human digestive tracts. This paper provides an overview of current trends in the development and utilisation of in vitro digestion models for foods, as well as information that can be used to develop improved digestion models. Our survey of in vitro digestion models found that the most predominant food samples tested were plants, meats, fish, dairy, and emulsion-based foods. The most frequently used biological molecules included in the digestion models were digestive enzymes (pancreatin, pepsin, trypsin, chymotrypsin, peptidase, α-amylase, and lipase), bile salts, and mucin. In all the in vitro digestion models surveyed, the digestion temperature was 37 °C although varying types and concentrations of enzymes were utilised. With regard to digestion times, 2 h (the stomach, small intestine, and large intestine each) was predominantly employed. This survey enhances the understanding of in vitro digestion models and provides indications for the development of improved in vitro digestion models for foods or pharmaceuticals.     

Grape skin extract inhibits mammalian intestinal α-glucosidase activity and suppresses postprandial glycemic response in streptozocin-treated mice

Intestinal α-glucosidases are the key enzymes responsible for starch digestion and absorption and their inhibition has been proven effective in both preventing and treating diabetes through improvement of postprandial hyperglycaemia. This study, for the first time, identified that a Norton grape skin extract (GSE) significantly inhibited mammalian intestinal α-glucosidases but not other digestive enzymes including structurally relevant pancreatic α-amylase. Norton GSE inhibited rat intestinal α-glucosidases through a competitive mode with an IC₅₀ of 0.384 mg/ml. Further animal study revealed that the oral intake of Norton GSE (400 mg/kg) significantly reduced postprandial blood glucose by 30.9% in the streptozocin-treated male C57BL/6 J mice following starch challenge. These findings suggest that Norton GSE may have a unique property of suppressing postprandial blood glucose through a mechanism involving the inhibition of α-glucosidases, thereby providing a novel dietary opportunity for diabetes management.     

Structure of a galactoarabinoglucuronoxylan from tamarillo (Solanum betaceum), a tropical exotic fruit,and its biological activity

Tamarillo (Solanum betaceum) is a tropical exotic fruit whose polysaccharides were extracted from the ripe pulp. After various purification steps, homogeneous fractions (designated PTW, STK-1000R and PF) were analyzed by sugar composition, HPSEC, methylation and NMR spectroscopy analysis. The results showed that the fraction PTW consisted of a linear arabinan with (1 → 5)-linked α-l-arabinofuranosyl units. Fractions designated as STK-1000R and PF contained galactoarabinoglucuronoxylans, with (1 → 4)-linked β-d-Xylp residues in the backbone, carrying branches exclusively at O-2. The polysaccharide in STK-1000R is less branched than that in the PF fraction (∼20.0% and 36.5%, respectively), with side-chains formed by (1 → 5)-linked α-l-Araf residues and (1 → 4)-linked α-d-GlcpA residues and with non-reducing end units formed by α-l-Araf, β-Arap, β-d-Galp, α-d-GlcpA and 4-O-Me-α-d-GlcpA. Intraperitoneal administration of the STK-1000R fraction in mice significantly reduced the number of abdominal constrictions induced by 0.6% acetic acid and the inflammatory phase of nociception induced by 2.5% formalin, indicating that that fraction has an antinociceptive effect on inflammatory pain models.     

Treatment of soy milk with Debaryomyces hansenii cells immobilised in alginate

Whole cells of Debaryomyces hansenii UFV-1 were permeabilised with ethanol and immobilised in calcium alginate hydrogel. The optimum pH and temperature for α-galactosidase activities of permeabilised free (PFC) and permeabilised immobilised cells (PIC) were 4.5 and 60 °C; and 4.0 and 70 °C, respectively. PIC α-galactosidase was more stable than that of PFC. The incubation of PIC at 60 and 70 °C promoted an increase in α-galactosidase activity. The α-galactosidase activity was maintained when PIC was used in three repeated batches. The K_m values for PIC and PFC α-galactosidases, with ρNPαGal, were 0.82 mM and 0.30 mM, respectively. Soy milk treatment with PIC for 6 h at 60 °C promoted 100% hydrolysis of raffinose oligosaccharides.     

Controlling lipid digestion by encapsulation of protein-stabilized lipid droplets within alginate–chitosan complex coacervates

The control of lipid digestibility within the human gastrointestinal tract is important for the development of many functional food and pharmaceutical products. This article describes the preparation, characterization, and in vitro digestibility of lipid droplets encapsulated within hydrogel beads. Protein-stabilized lipid droplets were first coated with an alginate layer, and then they were trapped within chitosan/calcium alginate coacervates. The filled hydrogel beads were formed using two methods: “direct method” – add a suspension of alginate-coated lipid droplets to a calcium/chitosan solution; “indirect method” – add chitosan solution to a suspension of alginate-coated lipid droplets and calcium. The in vitro digestibility of the encapsulated lipid droplets was then monitored using a pH-stat method to simulate the small intestine. For both methods, the filled hydrogel beads were relatively stable to aggregation/dissociation from pH 1 to 6, but underwent extensive aggregation and sedimentation at higher pH values. Relatively small hydrogel beads (d < 50 μm) caused a moderate delay in the rate of lipid digestion, while large hydrogel beads (d > 100 μm) could delay the digestion rate appreciably. This study has important implications for designing delivery systems that control lipid digestion by encapsulating lipid droplets within hydrogel beads.     

Effects of fatty acid supplements on ruminal and total tract nutrient digestion in lactating dairy cows

Saturated and unsaturated fatty acid supplements (FS) were evaluated for effects on ruminal digestion kinetics, and ruminal and postruminal nutrient digestion. Eight early lactation ruminally and duodenally cannulated cows (77 ± 12 days in milk, mean ± SD) were used in a replicated 4 × 4 Latin square design experiment with 21-d periods. Treatments were control and a linear substitution of 2.5% fatty acids from supplemented saturated FS (SAT; prilled, hydrogenated free fatty acids) for partially unsaturated FS (UNS; calcium soaps of long-chain fatty acids). All rations contained identical forage and concentrate components including 37.2% forage and 13.5% cottonseed. Saturated FS linearly decreased ruminal digestibility of dry matter and organic matter and linearly decreased ruminal neutral detergent fiber (NDF) digestibility. The reduction in ruminal NDF digestibility was because of a linear decrease in digestion rate and a linear increase in passage rate of potentially digestible NDF with increasing saturated FS. Total tract digestibility of NDF was not different between treatments because of compensatory postruminal digestion. Ruminal fatty acid and C18 fatty acid digestibility tended to increase linearly with increasing unsaturated FS, and postruminal C18 fatty acid digestibility decreased with increasing saturated FS. Saturated FS linearly decreased ruminal organic matter digestibility and decreased intestinal long-chain fatty acid digestibility, although differences in fatty acid digestibility may be partially explained by fatty acid intake.     

Effect of soaking and hydrothermal processing methods on the levels of antinutrients and in vitro protein digestibility of Bauhinia purpurea L. seeds

The effects of various domestic processing methods such as soaking, cooking and autoclaving on the levels of certain antinutritional factors and in vitro protein digestibility of seeds of Bauhinia purpurea L., an underutilised legume collected from South India, were investigated. The raw seeds were found to contain antinutritional factors like total free phenolics (2.75 g/100 g), tannins (2.35 g/100 g), phytic acid (692 mg/100 g) and flatulence factors, raffinose (0.54 g/100 g), stachyose (1.17 g/100 g) and verbascose (0.95 g/100 g). Soaking the seeds in distilled water caused maximum reduction in the phytic acid content (37%), whereas soaking in NaHCO₃ solution reduced significant levels of phenolics and tannins (72% and 78%, respectively). A reduction in the levels of oligosaccharides (raffinose by 63%, stachyose by 42% and verbascose by 79%) was observed during cooking. Of the attempted treatments, autoclaving appeared to be most effective in reducing levels of all the investigated antinutrients, except phytic acid, and also improved the in vitro protein digestibility of B. purpurea seeds.     

Antihypertensive,ACE-inhibitory and vasodilator properties of an egg white hydrolysate: Effect of a simulated intestinal digestion

In previous studies we showed that the egg white hydrolysate with pepsin (HEW) and its fraction with molecular mass lower than 3000 Da (HEW < 3000) have ACE-inhibitory activity in vitro and exert antihypertensive effects after single-oral and long-term administrations to spontaneously hypertensive rats (SHR). In this paper we have simulated an intestinal digestion on HEW and HEW < 3000 and addressed its effect on their ACE-inhibitory and vasodilator activities in vitro and antihypertensive activity in vivo. The results showed that the ACE-inhibitory activity of HEW and HEW < 3000 was maintained after the simulated intestinal digestion. HEW also exerted vasodilator activity in isolated aortic rings before and after the digestion. Both activities may explain the antihypertensive effects of these products in SHR. The peptides RADHP and YPI, which were detected by RP–HPLC–MS in the hydrolysates after the action of the pancreatic enzymes, could be responsible for the antihypertensive effects. In conclusion, HEW or HEW < 3000 could be useful in the prevention and/or treatment of hypertension and other associated disorders.     

Type of corn endosperm influences nutrient digestibility in lactating dairy cows

An experiment was conducted to evaluate the effect of type of corn endosperm on nutrient digestibility in lactating dairy cows. Near-isogenic variants of an Oh43 × W64A normal dent endosperm hybrid carrying floury-2 or opaque-2 alleles were grown in spatial isolation in field plots and harvested as dry shelled corn. Six ruminally cannulated, multiparous Holstein cows (67 ± 9 d in milk at trial initiation) were randomly assigned to a replicated 3 × 3 Latin square design with 14-d periods; the first 11 d of each period were for diet adaptation followed by 3 d of sampling and data collection. Treatment diets that contained dry rolled vitreous-, floury-, or opaque-endosperm corn [33% of dry matter (DM)], alfalfa silage (55% of DM) and protein-mineral-vitamin supplement (12% of DM) were fed as a total mixed ration. The percentage vitreous endosperm was zero for floury and opaque endosperm corns and 64 ± 7% for the vitreous corn. Prolamin protein content of floury and opaque endosperm corns was 30% of the content found in vitreous corn. Degree of starch access and in vitro ruminal starch digestibility measurements were 32 and 42% greater on average, respectively, for floury and opaque endosperm corns than for vitreous corn. Dry matter and starch disappearances after 8-h ruminal in situ incubations were, on average, 24 and 32 percentage units greater, respectively, for floury and opaque endosperm corns than for vitreous corn. Ruminal pH and acetate molar percentage were lower, propionate molar percentage was greater, and acetate:propionate ratio was lower for cows fed diets containing floury and opaque endosperm corns than for cows fed vitreous corn. In agreement with laboratory and in situ measurements, total-tract starch digestibility was 6.3 percentage units greater, on average, for cows fed diets containing floury and opaque endosperm corns than vitreous corn. Conversely, apparent total-tract neutral detergent fiber (NDF) digestibility was lower for cows fed diets containing floury and opaque endosperm corns compared with vitreous corn. The type of endosperm in corn fed to dairy cows can have a marked effect on digestion of starch and NDF. Feeding less vitreous corn increased starch digestion but decreased NDF digestion.     

Effect of heat,rutin and disulfide bond reduction on in vitro pepsin digestibility of Chinese tartary buckwheat protein fractions

Protein fractions (albumin, globulin, prolamin and glutelin) were extracted from defatted tartary buckwheat flour. The in vitro pepsin digestibilities of the four protein fractions were different, and albumin was more susceptible to pepsin hydrolysis. The native structure of the four protein fractions may be destroyed by heat treatment, and the digestibilities were all improved significantly (P < 0.05). Adding rutin to the digestion mixture of the four fractions did not cause a decrease in pepsin digestibility, although it did cause a significant increase in certain instances (P < 0.05). Treatment with β-mercaptoethanol (2-ME) only caused a higher initial proteolysis rate and did not increase the final digestibility distinctly except for prolamin. After pepsin digestion, the remaining proteins of unhydrolyzed albumin, globulin, prolamin and glutelin (untreated) shared some similarities. They also exhibited a minor band at 20,000 Da and a broad band at 10,000–14,000 Da.     

Structural characterization and immunostimulatory activity of a novel protein-bound polysaccharide produced by Hirsutella sinensis Liu,Guo, Yu & Zeng

HS002-II, a novel protein-bound polysaccharide with 44 kDa molecular weight, was fractionated from submerged cultures of Hirsutella sinensis Liu, Guo, Yu & Zeng by DEAE-Sepharose and Sephacryl S200 chromatography. Based on the results of infrared spectroscopy, high performance liquid chromatography, methylation, amino acid analysis, NMR spectroscopy and atomic force microscopy, the polysaccharide moieties of HS002-II mainly contained a long backbone of (1 → 3)-linked α-d-ribofuranosyl units (1 → 4)-linked α-d-xylopyranosyl units and (1 → 4)-linked β-d-glucopyranosyl units, which was substituted at C-6. The two branches were β-d-mannopyranosyl residues and β-d-galactopyranosyl residues terminated with α-l-arabinopyranosyl residues, respectively. HS002-II consisted of 57.9% polysaccharide and 42.1% protein with the existence of N-type carbohydrate–protein linkage. In terms of the pro-inflammatory cytokines assay (NO, TNF-α, IL-1β and NF-κB) using murine macrophages cell line (RAW264.7), HS002-II exhibited significant immunomodulatory activity by stimulating the IκB-NF-κB pathway.     

Stability of sunflower 2S albumins and LTP to physiologically relevant in vitro gastrointestinal digestion

In order for a protein to elicit a systemic allergic response it must reach the circulatory system through the intestinal mucosa as a sufficiently large fragment with adequate structural integrity. Sunflower LTP and 2S albumins (SFA8 and three mixed fractions of Alb1 and Alb2) were digested in simulated gastric fluid (SGF) for 2 h and the conditions were then changed to mimic the intestinal environment for a further 2 h digestion. The effects of phosphatidylcholine (PC) and emulsification on the digestibility of the proteins were investigated. PC protected all of the proteins studied against both gastric and intestinal digestive enzymes but to different extents. Emulsification of SFA8 resulted in strong protection against digestion, which was further enhanced by the presence of PC in the SGF. These results highlight the importance of considering real food structures such as emulsified systems and also the gastrointestinal environment that proteins are exposed to once consumed when assessing allergenicity.     

Factors affecting lipase digestibility of emulsified lipids using an in vitro digestion model: Proposal for a standardised pH-stat method

The control of lipid digestibility within the human gastrointestinal tract is important for the development of many functional food and pharmaceutical products. The influence of product composition and microstructure on lipid digestibility is typically studied using in vitro digestion methods. This article focuses on the impact of various experimental factors on lipid digestion in oil-in-water emulsions, using a pH-stat method that simulates the small intestine. The rate and extent of lipid digestion were found to increase with: increasing lipase (from 0 to 4.8 mg/ml), decreasing bile extract (from 20 to 0 mg/ml), increasing CaCl₂ (from 0 to 20 mM), decreasing lipid (from 2.5 to 0.1 wt.%); decreasing droplet diameter (from d = 800 to 200 nm), and decreasing fatty acid molecular weight (medium chain triglycerides versus corn oil). These affects are interpreted in terms of the surface area of lipid exposed to the aqueous phase, and factors affecting the accumulation of reaction products (fatty acids) at the oil–water interface. Based on our own and others’ work, we propose a standardised in vitro digestion model to test emulsified lipids, based on pH-stat titration. This study has important implications for designing and testing delivery systems that control lipid digestion using the pH-stat method.     

Matrix transformation in fiber-added extruded products: Impact of different hydration regimens on texture, microstructure and digestibility

The objective of this work was to study the effect of three hydration regimens on the extent of matrix transformation, texture, microstructure and digestibility of a corn-based extruded product blended with apple pomace. Blends containing 0%, 17%, 22%, and 28% pomace were hydrated to 17.2% by adding water either into the preconditioner (P), the extruder (E) or dividing half in each (PE). Increasing the opportunity for hydration (P > PE > E) promoted more complete starch gelatinization at all pomace levels. Apple pomace promoted milder extrusion conditions, resulting in less starch gelatinization and solubilization, poor expansion and reduced starch digestibility. Digestible starch was inversely correlated to cell wall thickness/cell size ratio (r = 0.91), which increased with pomace addition. Thus, pomace decreased digestibility by promoting changes in structure and gelatinization. Delayed water addition (E) promoted broader cell size and cell wall thickness distributions, attributed to poor mixing and hydration. Therefore, water incorporation (not just total content) is a key factor in defining extrudate microstructure, texture and digestibility patterns.     

Biochemical activities of Brochothrix thermosphacta

Activities of 19 hydrolases were estimated in cell suspensions of 40 Brochothrix thermosphacta strains, isolated from meat and meat products packaged under various conditions, by using API ZYM® test. These strains produced: acidic phosphatase, esterase C4, esterase/lipase C8, α-chymotrypsin, leucine arylamidase, β-glucosidase and α-glucosidase. The predominating biotype produced only the first four of the listed enzymes. The extracellular lipolytic activity for bromo-chloro-indolyl-caprylate was detected only in culture broth supernatants of 4 strains thereby indicating that esterase/lipase C8 is cell-bound. 13 of the strains displayed proteolytic activity for albumin at 4 °C (0.31-2.07 U) while 9 strains showed this activity at 25 °C (0.11-1.21 U). Only 4 strains digested albumin at both temperatures. Thus the meat spoilage potential of B. thermosphacta strains results not only from digestion of carbohydrates but also from their proteolytic activity.     

Galactooligosaccharides derived from lactose and lactulose: influence of structure on Lactobacillus, Streptococcus and Bifidobacterium growth

The effect of structure on the fermentative properties of potential prebiotic trisaccharides derived from lactulose like 6′-galactosyl-lactulose (β-d-galactopyranosyl-(1 → 6)-β-d-galactopyranosyl-(1 → 4)-β-d-fructopyranose), 4′-galactosyl-lactulose (β-d-galactopyranosyl-(1 → 4)-β-d-galactopyranosyl-(1 → 4)-β-d-fructopyranose), and 1-galactosyl-lactulose (β-d-galactopyranosyl-(1 → 4)-β-d-fructopyranosyl-(1 → 1)-β-d-galactopyranose); and from lactose like 4′-galactosyl-lactose (β-d-galactopyranosyl-(1 → 4)-β-d-galactopyranosyl-(1 → 4)-β-d-glucopyranose) and 6′-galactosyl-lactose (β-d-galactopyranosyl-(1 → 6)-β-d-galactopyranosyl-(1 → 4)-β-d-glucopyranose), has been assessed in vitro. Fermentations with twelve pure strains of Lactobacillus, Streptococcus and Bifidobacterium were carried out using the purified trisaccharides as the sole carbon source, and bacteria growth was evaluated at 600 nm by means of a microplate reader during 48 h. Maximum growth rates (μ_max) and lag phase were calculated. In general, all the strains tested were able to utilize lactulose and pure trisaccharides derived from lactulose and lactose when they were used as sole carbon source. Nonetheless, glycosidic linkage and/or the monosaccharide composition of the trisaccharides affected the individual strains lag phase, cell densities and growth rates. A general preference towards β-galactosyl residues β(1-6) and β(1-1) linked over those β(1-4) linked was observed, and some strains showed higher cell densities and speed of growth on 6′-galactosyl-lactulose than on 6′-galactosyl-lactose. This is the first study of the effect of lactulose-derived oligosaccharides on pure culture growth which shows that transglycosylation of lactulose allows for obtaining galactooligosaccharides with new glycosidic structures and would open new routes to the synthesis of compounds with potential prebiotic effects.     

Particle size-starch–protein digestibility relationships in cowpea (Vigna unguiculata)

The kinetics of starch and protein digestion in hammer- and cryo-milled cowpea (70–370 μm) were investigated. The pH during the protein digestion reduced with time, and both the starch and protein digestion exhibited monophasic digestograms, which were suitably (r² > 0.97, p < 0.001) described by a modified first-order kinetic model. The in vitro protein digestibility of the cowpea (>80%) was independent of the milling conditions. The hammer-milled cowpea digested more, but the reciprocal of its rate of protein digestion was independent of the square of the particle size. The rate of protein digestion in the cryo-milled cowpea inversely depended (p < 0.05) on the square of the particle size, with 67 × 10⁻⁷ cm² s⁻¹ as the diffusion coefficient. For the starch digestion, diffusion coefficients (cm² s⁻¹) were 0.6 × 10⁻⁷ (hammer-milled) and 0.3 × 10⁻⁷ (cryo-milled). The protein digestion proceeded at a much faster (100×) rate than the starch digestion.     

Chemical composition,antimicrobial potential against cariogenic bacteria and cytotoxic activity of Tunisian Nigella sativa essential oil and thymoquinone

We investigate in this work the chemical composition by GC–EIMS, the antibacterial and the cytotoxic activities of Tunisian Nigella sativa essential oil and its bioactive compound, thymoquinone, were tested against various clinical cariogenic bacteria (n = 30). Eighty-four compounds were identified in the essential oil. The major one was p-cymene (49.48%) whereas thymoquinone represented only 0.79%. The essential oil (2.43 mg/disc) containing only 3.35 μg of thymoquinone showed pronounced dose dependant antibacterial activity against Streptococcus mitis, Streptococcus mutans, Streptococcus constellatus and Gemella haemolysans (15.5 ± 0.707 mm). However, pure thymoquinone compound (150 μg/disk) was active against all the studied strains especially S. mutans and S. mitis (24.5 ± 0.71 and 22 ± 1.41 mm inhibition zones, respectively).     

Roasting-induced changes in arabinotriose,a model of coffee arabinogalactan side chains

Thermal processing can promote reactions that change the structure of food constituents, often by unknown mechanisms, such as those occurring in arabinose residues of coffee arabinogalactan side chains. Aiming to know more about these modifications, the structurally related α-(1 → 5)-l-arabinotriose was roasted at 200 °C and the products obtained were identified by ESI-MS and MALDI-MS and characterised by ESI-MSⁿ. Depolymerised and polymerised oligosaccharides with up to 16 residues and new types of linkages were formed. Also, products resulting from dehydration, oxidation, and cleavage of a carbon–carbon bond at the reducing end of the corresponding non-modified oligosaccharide were formed, probably promoting the release of formaldehyde, formic acid, glycolaldehyde, glyoxal, acetic acid, glycolic acid, glyceraldehyde, 2-hydroxypropanedialdehyde and lactic acid. As many of these compounds have been reported to occur in roasted coffee beans and/or brews, it can be suggested that the degradation of coffee arabinogalactan side chains can contribute to their formation upon roasting.