Fucus vesiculosus-Rich Extracts as Potential Functional Food Ingredients: A Holistic Extraction Approach.

Brown macroalgae are rich sources of nutrients and health-promoting compounds. Nevertheless, their consumption is still limited by their strong organoleptic characteristics, thus requiring the development of extraction strategies to profit from their nutritional value. To fulfil this, two sequential extraction approaches were developed, differing in the solvent used in the first extraction step, water in approach 1 or food-grade ethanol in approach 2, to obtain economic and affordable extracts rich in specific compounds from Fucus vesiculosus. The use of water in the first step of extraction allowed us to recover water-soluble phlorotannins, laminarans and mannuronic-rich alginates, making the subsequent 70% ethanol extract richest in fucoxanthin (0.07% algae DW), and the hot water fractions purest in fucoidans and alginates with a lower mannuronic-to-guluronic (M/G) ratio (2.91). Conversely, when beginning extraction procedures with 96% ethanol, the recovered yields of phlorotannins increased (0.43 g PGE/100 g algae DW), but there was a concomitant seven-fold decrease in the recovery of fucoxanthin in the subsequent 70% ethanol extract. This approach also led to less pure hot water fractions containing fucoidans, laminarans and alginates with a higher M/G ratio (5.50). Overall, this work unveiled the potential of the first extraction steps in sustainable and holistic cascade strategies to modulate the composition of food-grade extracts, creating prospects of their application as tailored functional ingredients in food products.

Water-Soluble Saccharina latissima Polysaccharides and Relation of Their Structural Characteristics with In Vitro Immunostimulatory and Hypocholesterolemic Activities.

Brown macroalgae are an important source of polysaccharides, mainly fucose-containing sulphated polysaccharides (FCSPs), associated with several biological activities. However, the structural diversity and structure-function relationships for their bioactivities are still undisclosed. Thus, the aim of this work was to characterize the chemical structure of water-soluble Saccharina latissima polysaccharides and evaluate their immunostimulatory and hypocholesterolemic activities, helping to pinpoint a structure-activity relationship. Alginate, laminarans (F1, neutral glucose-rich polysaccharides), and two fractions (F2 and F3) of FCSPs (negatively charged) were studied. Whereas F2 is rich in uronic acids (45 mol%) and fucose (29 mol%), F3 is rich in fucose (59 mol%) and galactose (21 mol%). These two fractions of FCSPs showed immunostimulatory activity on B lymphocytes, which could be associated with the presence of sulphate groups. Only F2 exhibited a significant effect in reductions in in vitro cholesterol's bioaccessibility attributed to the sequestration of bile salts. Therefore, S. latissima FCSPs were shown to have potential as immunostimulatory and hypocholesterolemic functional ingredients, where their content in uronic acids and sulphation seem to be relevant for the bioactive and healthy properties.

Structure-function relationships of pectic polysaccharides from broccoli by-products with in vitro B lymphocyte stimulatory activity.

To study structure-function relationships of pectic polysaccharides with their immunostimulatory activity, broccoli by-products were used. Pectic polysaccharides composed by 64 mol% uronic acids, 18 mol% Ara, and 10 mol% Gal, obtained by hot water extraction, activated B lymphocytes in vitro (25-250 µg/mL). To disclose active structural features, combinations of ethanol and chromatographic fractionation and modification of the polysaccharides were performed. Polysaccharides insoluble in 80 % ethanol (Et80) showed higher immunostimulatory activity than the pristine mixture, which was independent of molecular weight range (12-400 kDa) and removal of terminal or short Ara side chains. Chemical sulfation did not promote B lymphocyte activation. However, the action of pectin methylesterase and endo-polygalacturonase on hot water extracted polysaccharides produced an acidic fraction with a high immunostimulatory activity. The de-esterified homogalacturonan region seem to be an important core to confer pectic polysaccharides immunostimulatory activity. Therefore, agri-food by-products are a source of pectic polysaccharide functional food ingredients.

Rheological characterization of the exopolysaccharide produced by Alteromonas macleodii Mo 169.

Rheology modifiers are essential additives in numerous products in a variety of industries. Due to environmental awareness, consumer-oriented industries are interested in novel natural rheological agents that can replace synthetic chemicals. In this study, the chemical composition and rheological properties of a novel exopolysaccharide (EPS) produced by Alteromonas macleodii Mo 169 were investigated. It was mainly composed of uronic acids (50 mol%) and total carbohydrates were 17 % sulfated. The EPS viscosity increased with concentration, and a non-Newtonian shear thinning behavior was found for concentrations above 0.1 wt%. The elastic and viscous moduli indicated a weak gel-like structure above 0.4 wt%. It maintained its shear thinning behavior and viscoelastic properties in the presence of NaCl and CaCl2 for pH range 5-7 and temperatures up to 55 °C. Though the apparent viscosity decreased at pH 3 and 9 and temperatures above 65 °C, the shear thinning behavior was retained. The viscous and viscoelastic properties were recovered after heating (95 °C) and cooling (0 °C), indicating a good thermal stability and recoverability. After high shear force, the solution recovered original rheological properties within few seconds, demonstrating self-healing properties.

Isolation and identification of an arabinogalactan extracted from pistachio external hull: Assessment of immunostimulatory activity.

This work studies the extraction and purification of a novel arabinogalactan from pistachio external hull. It was extracted with a simple method from pistachio hull which is considered as unexploited waste. Based on the results of sugar analysis by GC-FID, glycosidic linkage by GC-MS, NMR spectroscopy, and molecular weight by Size Exclusion Chromatography, pistachio hull water soluble polysaccharides (PHWSP) were identified as a type II arabinogalactan (AG), with characteristic terminally linked α-Araf, (α1 â†’ 5)-Araf, (α1 â†’ 3,5)-Araf, terminally linked ß-Galp, (ß1 â†’ 6)-Galp, and (ß1 â†’ 3,6)-Galp. DEPT-135, HSQC, HMBC and COSY NMR data suggested the presence of (ß1 â†’ 3)-Galp mainly branched at O-6 with (ß1 â†’ 6)-Galp chains, α-Araf chains, and terminally linked α-Araf. These AG from pistachio external hulls showed in vitro stimulatory activity for B cells, suggesting their possible use as an immunological stimulant in nutraceutical and biomedical applications.

Role of Coffee Caffeine and Chlorogenic Acids Adsorption to Polysaccharides with Impact on Brew Immunomodulation Effects.

Coffee brews have High Molecular Weight (HMW) compounds with described immunostimulatory activity, namely polysaccharides and melanoidins. Melanoidins are formed during roasting and are modified during brews technological processing. In addition, brews have Low Molecular Weight (LMW) compounds, namely free chlorogenic acids and caffeine, with well-known anti-inflammatory properties. However, this study shows that both espresso and instant coffee brews did not present immunostimulatory neither anti-inflammatory in vitro activities. It is possible that the simultaneous existence of compounds with antagonistic effects can mitigate their individual effects. To test this hypothesis, an ultrafiltration separation process was applied, studying the behavior of coffee brews' HMW on retention of LMW compounds. Several ultrafiltration sequential cycles were required to separate retentates from LMW compounds, suggesting their retention. This effect was higher in instant coffee, attributed to its initial higher carbohydrate content when compared to espresso. Separation of HMW and LMW compounds boosted their immunostimulatory (6.2-7.8 µM nitrites) and anti-inflammatory (LPS induced nitrite production decrease by 36-31%) in vitro activities, respectively. As coffee anti-inflammatory compounds are expected to be first absorbed during digestion, a potential in vivo fractionation of LMW and HMW compounds can promote health relevant effects after coffee intake.

Blanching impact on pigments, glucosinolates, and phenolics of dehydrated broccoli by-products.

Because of high water content, the valorisation of broccoli by-products requires dehydration that can preserve bioactive compounds. Blanching pre-treatment has been reported to improve the drying rate of broccoli. As a thermal treatment, it promotes also enzyme inactivation. Therefore, in this study, the impact of pre-dehydration blanching step, freeze-drying, air-drying at 40 °C, and microwave hydrodiffusion and gravity (MHG) dehydration on the levels of pigments, glucosinolates, and phenolics, was evaluated by UHPLC-DAD-ESI/MSn. When compared to freeze-drying, a technique known to preserve compounds, a pre-blanching step increased the extractability of both pigments and phenolics, while air-drying only retained 49% of the pigments and 70% of phenolics, both without affecting glucosinolates. However, when air-drying was preceded by blanching, less than 50% of compounds were retained. On the other hand, MHG dehydration increased the phenolics extractability by 26%, particularly that of kaempferol derivatives while also retaining the amount of the glucosinolate glucoraphanin, when compared to freeze-drying. Nevertheless, only 23% of indole glucosinolates were recovered and pigments were severely reduced, with lutein accounting only for 32% and only chlorophyll b was observed in trace amounts after MHG dehydration. Therefore, to valorise broccoli by-products as ingredients, different drying technologies may be used when targeting different composition richness: freeze-drying is suitable for pigments and glucosinolates, air-drying is suitable for glucosinolates, while MHG promotes the extractability of phenolic compounds.

Structural analysis and potential immunostimulatory activity of Nannochloropsis oculata polysaccharides.

The relevance of microalgae biotechnology for producing high-value compounds with biomedical application, such as polysaccharides, has been increasing. Despite this, the knowledge about the composition and structure of microalgae polysaccharides is still scarce. In this work, water-soluble polysaccharides from Nannochloropsis oculata were extracted, fractionated, structurally analysed, and subsequently tested in terms of immunostimulatory activity. A combination of sugar and methylation analysis with interaction data of carbohydrate-binding proteins using carbohydrate microarrays disclosed the complex structural features of the different polysaccharides. These analyses showed that the water-soluble polysaccharides fractions from N. oculata were rich in (ß1→3, ß1→4)-glucans, (α1→3)-, (α1→4)-mannans, and anionic sulphated heterorhamnans. The immunostimulatory assay highlighted that these fractions could also stimulate murine B-lymphocytes. Thus, the N. oculata water-soluble polysaccharides show potential to be further explored for immune-mediated biomedical applications.

Apple Pomace Extract as a Sustainable Food Ingredient.

Apple pomace is a by-product of apple processing industries with low value and thus frequent disposal, although with valuable compounds. Acidified hot water extraction has been suggested as a clean, feasible, and easy approach for the recovery of polyphenols. This type of extraction allowed us to obtain 296 g of extract per kg of dry apple pomace, including 3.3 g of polyphenols and 281 g of carbohydrates. Ultrafiltration and solid-phase extraction using C18 cartridges of the hot water extract suggested that, in addition to the apple native polyphenols detected by ultra-high-pressure liquid chromatography coupled to a diode-array detector and mass spectrometry UHPLC-DAD-ESI-MSn, polyphenols could also be present as complexes with carbohydrates. For the water-soluble polyphenols, antioxidant and anti-inflammatory effects were observed by inhibiting chemically generated hydroxyl radicals (OH•) and nitrogen monoxide radicals (NO•) produced in lipopolysaccharide-stimulated macrophages. The water-soluble polyphenols, when incorporated into yogurt formulations, were not affected by fermentation and improved the antioxidant properties of the final product. This in vitro research paves the way for agro-food industries to achieve more diversified and sustainable solutions towards their main by-products.

Salt pan brine water as a sustainable source of sulphated polysaccharides with immunostimulatory activity.

Marine environments are an enormous source of materials with biological interest, such as sulphated polysaccharides, which have relevant biological activities. In this study the potential of salt pan brine water as an easily accessible source of sulphated polysaccharides was evaluated. This water revealed to have a high quantity of polymeric material, five times more than sea water, mainly composed by highly sulphated polysaccharides. Structural analysis identified a diversity of polysaccharides, namely rhamnans, fucans, mannans, xylomannans, glucuronomannans, galactans, and glucans. All these structures seem to form complexes that are resistant to the salt pan conditions along salt production. These polysaccharides showed in vitro stimulatory activity for B cells, suggesting their potential application in nutraceutical and biomedical fields. Salt pan brine water is a valuable source of environmentally friendly and low-cost available bioactive compounds prone to be exploited.

Microwave assisted dehydration of broccoli by-products and simultaneous extraction of bioactive compounds.

Broccoli by-products from frozen-food industry account for 45% of the initial broccoli heads. They consist on stalks, inflorescences, and leaves, blanched and non-blanched, sharing the nutritional value and bioactive compounds of commercial broccoli heads. However, their high perishability prevents further valorisation. Therefore, in this study microwave hydrodiffusion and gravity (MHG) technology was used to dehydrate broccoli by-products and simultaneously recover the water-soluble diffused compounds for food ingredients use. The hydrodiffusion allowed to obtain a dried material with 12% moisture in 43 min when 550 g of broccoli by-products were used, preserving polysaccharides and proteins. Diffused water contained up to 317 µg/mL gallic acid equivalents of phenolic compounds, 11 mg/mL free sugars, 9 mg/mL amino acids, and 356 µg/mL glucosinolates, depending on the type of by-product used. These results show the potential of MHG technology for valorisation of broccoli by-products by its simultaneous stabilization by dehydration and extraction of bioactive compounds.

Structural polymeric features that contribute to in vitro immunostimulatory activity of instant coffee.

An instant coffee fraction, rich in arabinogalactans, obtained by ultrafiltration, using 1 and 5kDa membranes, has previously shown in vitro stimulatory activity on BALB/c mice spleen B lymphocytes. The compounds inducing lymphocytic activation were shown to elute, mainly near the void volume by size-exclusion chromatography, using Bio-Gel P6 (1-6kDa). Treatment of the compounds with chymotrypsin, a digestive protease, did not affect the induced B lymphocyte activation. On the contrary, hydrolysis with an α-l-arabinofuranosidase, removing single terminally-linked arabinose residues, decreased the extent of B cell activation. The immunostimulatory activity of these compounds was also observed by in vitro experiments, using bone marrow-derived macrophages and dendritic cells as responders. Altogether, these results show the relevance of single arabinose residues, present at the non-reducing end of polymeric compounds, to the coffee stimulatory activity in cells mediating innate and acquired immunity.

Structure-function relationships of immunostimulatory polysaccharides: A review.

Immunostimulatory polysaccharides are compounds capable of interacting with the immune system and enhance specific mechanisms of the host response. Glucans, mannans, pectic polysaccharides, arabinogalactans, fucoidans, galactans, hyaluronans, fructans, and xylans are polysaccharides with reported immunostimulatory activity. The structural features that have been related with such activity are the monosaccharide and glycosidic-linkage composition, conformation, molecular weight, functional groups, and branching characteristics. However, the establishment of structure-function relationships is possible only if purified and characterized polysaccharides are used and selective structural modifications performed. Aiming at contributing to the definition of the structure-function relationships necessary to design immunostimulatory polysaccharides with potential for preventive or therapeutical purposes or to be recognized as health-improving ingredients in functional foods, this review introduces basic immunological concepts required to understand the mechanisms that rule the potential claimed immunostimulatory activity of polysaccharides and critically presents a literature survey on the structural features of the polysaccharides and reported immunostimulatory activity.

Influence of molecular weight on in vitro immunostimulatory properties of instant coffee.

Instant coffee was prepared and fractionated into higher (>100kDa), medium (5-10, 10-30, 30-100kDa) and lower (1-5, <1kDa) molecular weight fractions. Sugars and linkage composition characteristics of arabinogalactans and galactomannans were recovered in all fractions. Also, amino acid analysis performed after hydrolysis showed similar compositions in all fractions. On the contrary, free chlorogenic acids and caffeine were only detected in the lowest molecular weight fraction (<1kDa). A direct relationship between the melanoidins browning index and the molecular weight was observed. The fractions obtained were incubated in vitro with murine spleen lymphocytes in order to evaluate their possible immunostimulatory abilities. The surface expression of CD69 (early activation marker) on different lymphocyte sub-populations showed that the fraction with 1-5kDa was able to induce activation of B-lymphocytes. This was the only fraction to induce B-lymphocyte activation, since all the other fractions failed, even when higher concentrations were used.