Valorization of Grape Pomace as a Renewable Source of Techno-Functional and Antioxidant Pectins.

The food industry's increasing demand for new functional ingredients that meet both organoleptic and healthy requirements has driven the exploration of new sources of functional ingredients in agro-industrial by-products. The aim of this work was to valorize grape pomace (Vitis vinifera L. garnacha) as a source of pectins using food-grade extracting agents. Obtained pectins were evaluated for monomeric composition, methyl esterification, molecular weight, water retention, oil-holding capacity, and antioxidant properties. The relatively soft extraction conditions used permitted obtaining low methoxyl pectin (10-42%) enriched in homogalacturonan (38-45%) or rhamnogalacturonan (33-41%) with different branching degrees, molecular weight, and fewer impurities than those found in the scarce previous literature. The relationship between structure and functionality was studied. Among the different pectins obtained, the sample derived from the extraction with sodium citrate could resume the best characteristics, such as pectin purity and higher water retention and oil holding capacity. These results underscore the relevance of grape pomace as a viable alternative source of pectin.

Supplementation with the Symbiotic Formulation Prodefen® Increases Neuronal Nitric Oxide Synthase and Decreases Oxidative Stress in Superior Mesenteric Artery from Spontaneously Hypertensive Rats.

In recent years, gut dysbiosis has been related to some peripheral vascular alterations linked to hypertension. In this work, we explore whether gut dysbiosis is related to vascular innervation dysfunction and altered nitric oxide (NO) production in the superior mesenteric artery, one of the main vascular beds involved in peripheral vascular resistance. For this purpose, we used spontaneously hypertensive rats, either treated or not with the commercial synbiotic formulation Prodefen® (108 colony forming units/day, 4 weeks). Prodefen® diminished systolic blood pressure and serum endotoxin, as well as the vasoconstriction elicited by electrical field stimulation (EFS), and enhanced acetic and butyric acid in fecal samples, and the vasodilation induced by the exogenous NO donor DEA-NO. Unspecific nitric oxide synthase (NOS) inhibitor L-NAME increased EFS-induced vasoconstriction more markedly in rats supplemented with Prodefen®. Both neuronal NO release and neuronal NOS activity were enhanced by Prodefen®, through a hyperactivation of protein kinase (PK)A, PKC and phosphatidylinositol 3 kinase-AKT signaling pathways. The superoxide anion scavenger tempol increased both NO release and DEA-NO vasodilation only in control animals. Prodefen® caused an increase in both nuclear erythroid related factor 2 and superoxide dismutase activities, consequently reducing both superoxide anion and peroxynitrite releases. In summary, Prodefen® could be an interesting non-pharmacological approach to ameliorate hypertension.

Production of α-rhamnosidases from Lactobacillus plantarum WCFS1 and their role in deglycosylation of dietary flavonoids naringin and rutin.

This work addresses the amino acid sequence, structural analysis, biochemical characterization and glycosidase activity of two recombinant α-rhamnosidases, Ram1 and Ram2, from Lactobacillus plantarum WCFS1. The substrate specificity of both enzymes towards the disaccharide rutinose and natural dietary flavonoids naringin and rutin was also determined and compared to that of a commercial multienzyme complex (Pectinex Ultra Passover, PPO). Ram1 is a less acidic- and heat-active enzyme than Ram2 and exhibited a high activity towards pNP-α-L-rhamnopyranoside, but it was unable to hydrolyze neither rutinose, naringin or rutin. In contrast, Ram2 enzyme showed a substrate specificity towards α-(1➔6) glycosidic flavonoids, such as rutin, and the disaccharide rutinose. The mechanism of action of Ram2 towards rutin was elucidated and revealed the potential cost-effective and selective production of the monoglycosylated flavonoid isoquercetin (quercetin-3-O-glucoside). PPO efficiently converted both naringin and rutin into their corresponding aglycones. These findings revealed the potential usefulness of PPO for the improvement of sensory properties of beverages through debittering of citrus juices, as well as the potential use of Ram2 to selectively produce isoquercetin, a highly valued and bioactive flavonoid whose production is not currently affordable.

In vitro digestion of polysaccharides: InfoGest protocol and use of small intestinal extract from rat.

Starch, dextran, pectin and modified citrus pectin were subjected to intestinal digestion following InfoGest protocol and a rat small intestine extract (RSIE) treatment. Gastric stage did not show any modification in the structure of the carbohydrates, except for modified pectin. Regarding intestinal phases, starch was hydrolyzed by different ways, resulting in a complementary behavior between InfoGest and RSIE. Contrarily, digestion of dextran was only observed using RSIE. Similar situation occurred in the case of pectins with RSIE, obtaining a partial hydrolysis, especially in the modified citrus pectin. However, citrus pectin was the less prone to hydrolysis by enzymes. The results demonstrated that InfoGest method underestimates the significance of the carbohydrates hydrolysis at the small intestine, thus indicating that RSIE is a very reliable and useful method for a more realistic study of polysaccharides digestion.

Bringing the digestibility of prebiotics into focus: update of carbohydrate digestion models.

Oro-gastrointestinal digestion of dietary carbohydrates involves up to six different carbohydrases in a multistage process. Enzymes from the small intestinal brush border membrane play a major role in the digestibility of these substrates. However, to date, the inclusion of these small intestinal enzymes has been dismissed in most in vitro studies carried out, despite their importance in the degradation of carbohydrates. Several in vitro and in vivo studies have demonstrated the capability of brush border enzymes to degrade certain "non-digestible" carbohydrates to a different extent depending on their structural composition (monomeric composition, glycosidic linkage, etc.). In this sense, considering the available evidence, mucosal disaccharidases embedded in the small intestinal brush border membrane vesicles must be considered in addition to α-amylases; therefore, new approaches for the evaluation of the digestibility of carbohydrates have been recently reported. These new methods based on the utilization of the small intestinal enzymes present in the brush border membrane aim to fulfill the final and key step of the digestion of carbohydrates in the small intestine. Here, rat small intestinal extract enzymes as well as brush border membrane vesicles from pig have emerged as very reliable and useful tools to evaluate carbohydrate digestion. Thus, this review aims to go briefly through the most relevant digestion methods for carbohydrates that are currently available and to highlight the new improved methods, which include mammalian intestinal enzymes, and their current use in the evaluation of the digestibility of prebiotics.

Behaviour of citrus pectin and modified citrus pectin in an azoxymethane/dextran sodium sulfate (AOM/DSS)-induced rat colorectal carcinogenesis model.

Large intestine cancer is one of the most relevant chronic diseases taking place at present. Despite therapies have evolved very positively, this pathology is still under deep investigation. One of the recent approaches is the prevention by natural compounds such as pectin. In this paper, we have assessed the impact of citrus pectin and modified citrus pectin on colorectal cancer in rats (Rattus norvegicus F344) to which azoxymethane and DSS were supplied. The lowest intake of food and body weight were detected in animals fed with citrus pectin, together with an increase in the caecum weight, probably due to the viscosity, water retention capacity and bulking properties of pectin. The most striking feature was that, neither citrus pectin nor modified citrus pectin gave rise to a tumorigenesis prevention. Moreover, in both, more than 50% of rats with cancer died, probably ascribed to a severe dysbiosis state in the gut, as shown by the metabolism and metagenomics studies carried out. This was related to a decrease of pH in caecum lumen and increase in acetate and lactic acid levels together with the absence of propionic and butyric acids. A relevant increase in Proteobacteria (Enterobacteriaceae) were thought to be one of the reasons for enteric infection that could have provoked the death of rats and the lack of cancer prevention. However, a reduction of blood glucose and triacylglycerides level and an increase of Bifidobacterium and Lactobacillaceae were found in animals that intake pectin, as compared to universal and modified citrus pectin feeding.

Evaluation of the impact of a rat small intestinal extract on the digestion of four different functional fibers.

The degree of digestion, modulated by rat small intestinal extract on different functional fibers was investigated. In general, inulin-type fructans and fructooligosaccharides were the most resistant to the enzymatic digestion. Results evidenced the high-resistance of fructosyl-fructose bonds. This fits well with the concept of prebiotic carbohydrates. However, the mixture of melibiose, manninotriose and verbascotetraose (α-GOS) from peas, with a considerably lower molecular weight (0.6 kDa) than the fructans studied, were highly digested (61.2%). Interestingly, the Gal-(1 → 6)-Gal bonds present into the manninotriose and verbascotetraose were more prone to be hydrolyzed than Gal-(1 → 6)-Glc (melibiose). However, when melibiose was the only disaccharide present in the reaction mixture, the hydrolysis was also high (67.7%). The use of small intestinal enzymatic preparations is a realistic approximation to evaluate the digestion of different carbohydrates, thus, showing that recognized non-digestible carbohydrates can also be partially digested.

Kinetic study on the digestibility of lactose and lactulose using small intestinal glycosidases.

Lactose is mostly hydrolysed in the small intestine, whereas lactulose, recognised prebiotic carbohydrate, reaches the colon to be fermented by the intestinal microbiota. Digestibility of these substrates was investigated by an in vitro digestion model using a Rat Small Intestine Extract (RSIE). A kinetic study of lactose digestion showed levels of hydrolysis (82.8%) at 0.2 mg*mL-1 and the highest hydrolysis rate constant (kobt). Considering these conditions, lactulose showed high resistance to intestinal digestion by RSIE, resulting in low hydrolysis degrees (20.4%) after 5 h reaction. These results underline the suitability of these intestinal extracts under the studied conditions, as a reliable tool to evaluate carbohydrate digestion and support the evidences towards the higher resistance of galactosyl-fructose linkages during its intestinal degradation.

Enzymatic Production and Characterization of Pectic Oligosaccharides Derived from Citrus and Apple Pectins: A GC-MS Study Using Random Forests and Association Rule Learning.

Pectic oligosaccharides (POS) from citrus and apple pectin hydrolysis using ViscozymeL and Glucanex200G have been obtained. According to the results, maximum POS formation was achieved from citrus pectin after 30 min of hydrolysis with ViscozymeL, with a yield of 652 mg g-1 and average molecular mass ( Mw) of 0.8-2.5 kDa, while with Glucanex200G, the yield was 518 mg g-1 and Mw was 0.8-7.1 kDa. Digalacturonic and trigalacturonic acids were identified among other low Mw compounds as di- and tri-POS. In addition, differences in GC-MS spectra of all oligosaccharides found in the hydrolysates were studied by employing random forests and other algorithms to identify structural differences between the obtained POS, and high prediction rates were shown for new samples. Chemical structures were proposed for some influential m/ z ions, and 12 association rules that explain differences according to pectin and enzyme origin were built. This information could be used to establish structure-function relationships of POS.

In Vitro Digestibility of Galactooligosaccharides: Effect of the Structural Features on Their Intestinal Degradation.

Small intestinal brush border membrane vesicles from pig were used to digest galactooligosaccharides from lactose (GOS) and from lactulose (OsLu). Dissimilar hydrolysis rates were detected after digestion. Predominant glycosidic linkages and monomeric composition affected the resistance to intestinal digestive enzymes. The ß(1→3) GOS mixture was the most susceptible to hydrolysis (50.2%), followed by ß(1→4) (34.9%), whereas ß(1→6) linkages were highly resistant to digestion (27.1%). Monomeric composition provided a better resistance in ß(1→6) OsLu (22.8%) compared to ß(1→6) GOS (27.1%). This was also observed for ß-galactosyl fructoses and ß-galactosyl glucoses, where the presence of fructose provided higher resistance to digestion. Thus, the resistance to small intestinal digestive enzymes highly depends upon the structure and composition of prebiotics. Increasing knowledge in this regard could contribute to the future synthesis of new mixtures of carbohydrates, highly resistant to digestion and with potential to be tailored prebiotics with specific properties, targeting, for instance, specific probiotic species.

Behaviour of citrus pectin during its gastrointestinal digestion and fermentation in a dynamic simulator (simgi®).

The behaviour of citrus pectin during digestion and its potential prebiotic properties were examined using a Dynamic Gastrointestinal Simulator (simgi®) model for the human gut, which simulates processes in the stomach, small intestine, ascending, transverse and descending colon. A remarkable non-digestibility of pectin in the upper gastrointestinal tract was observed by HPLC-ELSD analysis, where ∼88% of citrus pectin remained intact during its transit through the stomach and small intestine. Fermentation of pectin stimulated the growth of beneficial bacteria such as Bifidobacterium spp, Bacteroides spp and Faecalobacterium prausnitzii. High increases of short-chain fatty acids (SCFA) were observed, especially in acetate and butyrate concentration due to direct fermentation of pectin or by cross-feeding interaction between bacteria. This is the first study on the digestibility and fermentation of pectin carried out in a complex dynamic gastrointestinal simulator, being of special relevance the results obtained for F. prausnitzii.

Application of a commercial digestive supplement formulated with enzymes and probiotics in lactase non-persistence management.

Strategies to avoid lactose malabsorption, which affects 70% of the world's population, are focused on the restriction of milk and dairy products or the use of non-human ß-galactosidases or probiotics endowed with ß-galactosidase activity added at mealtime. Our evaluation of a commercial blend of probiotics and enzymes (protease, lactase, lipase and amylase) and its potential application in lactase non-persistence management is described in this work. Recommended amounts (460-1000 mg) of the commercial probiotics-enzyme blend were shown to be adequate for performing in vitro lactose hydrolysis in standard solutions (0.25-5%) and commercial dairy products, namely milks (5% lactose) and yogurts (3% lactose), reaching hydrolysis values between 44 and 96%. According to these percentages, the use of the enzymatic preparation would guarantee the intake of less than 12 g, the recommendation of the EFSA for lactose intolerance. Furthermore, formation of prebiotic galactooligosaccharides was also detected, increasing the potential benefits of the enzymatic preparation in the gastrointestinal system.

In vitro fermentation properties of pectins and enzymatic-modified pectins obtained from different renewable bioresources.

The suitability of artichoke and sunflower by-products as renewable sources of pectic compounds with prebiotic potential was evaluated by studying their ability to modulate the human faecal microbiota in vitro. Bacterial populations and short-chain fatty acid (SCFA) production were measured. Reduction of the molecular weight of artichoke pectin resulted in greater stimulation of the growth of Bifidobacterium, Lactobacillus and Bacteroides/Prevotella, whilst this effect was observed only in Bacteroides/Prevotella for sunflower samples. In contrast, the degree of methoxylation did not have any impact on fermentability properties or SCFA production, regardless of the origin of pectic compounds. Although further in vivo studies should be conducted, either pectin or enzymatically-modified pectin from sunflower and artichoke by-products might be considered as prebiotic candidates for human consumption showing similar ability to promote the in vitro growth of beneficial gut bacteria as compared to well-recognized prebiotics such as inulin or fructo-oligosaccharides.

Assessment of in Vitro Digestibility of Dietary Carbohydrates Using Rat Small Intestinal Extract.

There are few studies on the assessment of digestibility of nondigestible carbohydrates, despite their increasingly important role in human health. In vitro digestibility of a range of dietary carbohydrates classified as digestible (maltose, sucrose, and lactose), well-recognized (lactulose, fructooligosaccharides (FOS), and two types of galactooligosaccharides (GOS) differing in the predominant glycosidic linkage), and potential (lactosucrose and GOS from lactulose, OsLu) prebiotics using a rat small intestinal extract (RSIE) under physiological conditions of temperature and pH is described. Recognized and potential prebiotics were highly resistant to RSIE digestion although partial hydrolysis at different extents was observed. FOS and lactulose were the most resistant to digestion, followed closely by OsLu and more distantly by both types of GOS and lactosucrose. In GOS, ß(1 → 6) linkages were more resistant to digestion than ß(1 → 4) bonds. The reported in vitro digestion model is a useful, simple, and cost-effective tool to evaluate the digestibility of dietary oligosaccharides.