Emilia M. Guadix

Nondigestible oligosaccharides exert nonprebiotic effects on intestinal epithelial cells enhancing the immune response via activation of TLR4‐NFκB

SCOPE Prebiotic effects of non absorbable glucids depend mainly on digestion by the colonic microbiota. Our aim was to assess nonprebiotic, direct effects of 4 prebiotics, namely fructooligosaccharides, inulin, galactooligosaccharides, and goats milk oligosaccharides on intestinal epithelial cells. METHODS AND RESULTS Prebiotics were tested in intestinal epithelial cell 18 (IEC18), HT29, and Caco-2 cells. Cytokine secretion was measured by ELISA and modulated with pharmacological probes and gene silencing. Prebiotics induced the production of growth-related oncogene, (GROα), monocyte chemoattractant protein 1 (MCP-1), and macrophage inflammatory protein 2 (MIP2) in IEC18 cells, with an efficacy that was 50-80% that of LPS. Prebiotics did not change RANTES expression, which was robustly induced by LPS in IEC18 cells. Cytokine secretion was suppressed by Bay11-7082, an inhibitor of IκB-α phosphorylation. The response was markedly decreased by Myd88 or TLR4 gene knockdown. Prebiotics also elicited cytokine production in HT29 but not in Caco-2 cells, consistent with reduced and vestigial expression of TLR4 in these cell lines, respectively. Prebiotic-induced MCP-1 secretion was reduced also in colonic explants from TLR4 KO mice compared with the controls. CONCLUSIONS We conclude that prebiotics are TLR4 ligands in intestinal epithelial cells and that this may be a relevant mechanism for their in vivo effects.

Functional and antioxidant properties of hydrolysates of sardine (S. pilchardus) and horse mackerel (T. mediterraneus) for the microencapsulation of fish oil by spray-drying.

The functionality of fish protein hydrolysates (FPH) for the microencapsulation of fish oil was investigated. Muscle protein from sardine (Sardina pilchardus) and horse mackerel (Trachurus mediterraneus) was hydrolysed using Alcalase or trypsin. Physically stable emulsions suitable for spray-drying were obtained when using FPH with a degree of hydrolysis of 5%. Microencapsulation efficiency amounted to 98±0.1% and oxidative stability of the encapsulated oil over a period of twelve weeks was in a similar range as it is reported for other matrix systems. Therefore, the suitability of FPH for use in spray-dried emulsions has been shown for the first time. Since no clear correlation between the antioxidative activity of the FPH and the course of lipid oxidation could be established future research is required to more specifically characterise the molecular structure of the peptides and its impact on protein alteration and role in lipid oxidation.

Prebiotic oligosaccharides directly modulate proinflammatory cytokine production in monocytes via activation of TLR4.

SCOPE Prebiotic oligosaccharides are currently used in a variety of clinical settings for their effects on intestinal microbiota. Here, we have examined the direct, microbiota independent, effects of prebiotics on monocytes and T lymphocytes in vitro. METHODS AND RESULTS Prebiotics generally evoked cytokine secretion (TNF-α, IL-6, and IL-10) by mouse splenocytes but inhibited LPS -induced IFN-γ and IL-17 release. Inulin was found to enhance LPS-induced IL-10 secretion. Splenocytes from TLR4(-/-) (where TLR is Toll-like receptor) mice showed a markedly depressed response. Conversely, in both basal and LPS-stimulated conditions, prebiotic inhibition of IFN-γ levels was preserved. These results suggested a predominant effect on monocytes via TLR4 ligation and possible inhibition of T cells. Hence, we studied the modulation of primary rat monocytes and T lymphocytes, focusing on fructooligosaccharides (FOS) and inulin. In monocytes, FOS and inulin induced TNF-α, growth-regulated oncogene α, and IL-10, but not IL-1β release. The NF-κB inhibitor Bay 11-7082 fully prevented these effects. Pharmacological evidence also indicated a significant involvement of mitogen-activated protein kinase and phosphatidylinositol-3-kinase. There was little effect on T cells. FOS and inulin also generally increased TNF-α, IL-1β, and IL-10, but not IL-8, in human peripheral blood monocytes. CONCLUSION We conclude that prebiotics may act as TLR4 ligands or as indirect TLR4 modulators to upregulate cytokine secretion in monocytes.

Bovine glycomacropeptide induces cytokine production in human monocytes through the stimulation of the MAPK and the NF-κB signal transduction pathways

Background and purpose:  Bovine glycomacropeptide (BGMP) is a natural milk peptide that is produced naturally in the gastrointestinal tract during digestion. Glycomacropepide has intestinal anti‐inflammatory activity, but the mechanism of action is unknown. Here we have characterized the effects of BGMP on monocytes.

Physical and oxidative stability of fish oil-in-water emulsions stabilized with fish protein hydrolysates.

The emulsifying and antioxidant properties of fish protein hydrolysates (FPH) for the physical and oxidative stabilization of 5% (by weight) fish oil-in-water emulsions were investigated. Muscle proteins from sardine (Sardina pilchardus) and small-spotted catshark (Scyliorhinus canicula) were hydrolyzed to degrees of hydrolysis (DH) of 3-4-5-6% with subtilisin. Sardine hydrolysates with low DH, 3% and 4%, presented the most effective peptides to physically stabilize emulsions with smaller droplet size. This implied more protein adsorbed at the interface to act as physical barrier against prooxidants. This fact might also be responsible for the higher oxidative stability of these emulsions, as shown by their lowest peroxide value and concentration of volatiles such as 1-penten-3-one and 1-penten-3-ol. Among the hydrolysates prepared from small-spotted catshark only the hydrolysate with DH 3% yielded a physically stable emulsion with low concentration of unsaturated aldehydes. These results show the potential of FPH as alternative protein emulsifiers for the production of oxidatively stable fish oil-in-water emulsions.

Production of resistant starch by enzymatic debranching in legume flours.

Resistant starch (RS) was produced by enzymatic hydrolysis of flours from five different legumes: lentil, chickpea, faba bean, kidney bean and red kidney bean. Each legume was firstly treated thermally, then hydrolyzed with pullulanase for 24h at 50°C and pH 5 and lyophilized. At the end of each hydrolysis reaction, the RS amount ranged from 4.7% for red kidney beans to 7.5% for chickpeas. With respect to the curves of RS against hydrolysis time, a linear increase was observed initially and a plateau was generally achieved by the end of reaction. These curves were successfully modeled by a kinetic equation including three parameters: initial RS, RS at long operation time and a kinetic constant (k). Furthermore, the relative increase in hydrolysis, calculated using the kinetic parameters, was successfully correlated to the percentage of amylose.

Lipid characterization and properties of protein hydrolysates obtained from discarded Mediterranean fish species

BACKGROUND Discards are an important fraction of the by-products produced by the fishing industry. As a consequence of their low commercial acceptance, it is necessary to provide added value to these underutilized materials. In this study the lipid fraction of three discarded fish species in the western Mediterranean Sea, namely sardine (Sardina pilchardus), mackerel (Scomber colias) and horse mackerel (Trachurus trachurus), was characterized and the angiotensin I-converting enzyme (ACE)-inhibitory and antioxidative activities of their protein hydrolysates were evaluated. RESULTS Processing of these biomaterials led to oils with a high content of omega-3 polyunsaturated fatty acids (PUFAs), ranging from 220.5 g kg(-1) for horse mackerel to 306.0 g kg(-1) for sardine. Regarding the protein fraction, most of the hydrolysates presented ACE inhibition values higher than 60%, corresponding to IC50 values varying from 345 µg protein mL(-1) for mackerel to 400 µg protein mL(-1) for sardine. Moreover, most of the hydrolysates exhibited acceptable antioxidative activity, namely 35-45% inhibition of 1,1-diphenyl-2-picrylhydrazyl (DPPH). CONCLUSION This study suggests that the three discarded species evaluated are valuable raw materials for the production of bioactive ingredients such as omega-3 PUFAs and protein hydrolysates exhibiting antihypertensive and antioxidative activities.

Influence of enzymes, pH and temperature on the kinetics of whey protein hydrolysis / Influencia de los enzimas, pH y temperatura en la cinética de la hidrólisis de las proteínas del lactosuero

The influence of pH, temperature and the mixture of enzymes (MKC Protease 660 L and PEM 2500 S) on the enzymatic hydrolysis of whey proteins was studied. The experiments show that all results were reproducible via a kinetic model that supposes the rapid and irreversible binding of part of the proteases to an inhibitor in the substrate, followed by a zero-order hydrolysis with respect to the substrate which occurs simultaneously with a second order enzymatic denatural ization produced by an attack of the free proteases upon those bound to the substrate-enzyme complex. Use of the optimum operating temperature of 60 °C and pH 8-10 led to a greater degree of hydrolysis. However, increasing the pH to these levels means that the salt content, on neutral izing the hydrolysate, is somewhat high and this is often unsuitable for the preparation of special diets. In the experiments performed with mixtures of enzymes, two contrasting phenomena occurred; there appears to be synergism between the proteases, which is preceded by a loss in enzymatic activity greater than that which can be accounted for by the presence of the inhibitor in the whey proteins.

Serum amino acid concentrations in growing rats fed intact protein versus enzymatic protein hydrolysate-based diets.

The aim of this study was to evaluate the effect of the molecular form of dietary protein (native or enzymatically hydrolyzed) on the total serum protein concentrations and the serum amino acid profile of growing rats at weaning. Wistar male rats at weaning were randomly assigned to one of the four isocaloric and isonitrogenous (12% protein equivalent content) diets and fed for 7 days. The protein sources of the diets were: whey protein, casein and their respective hydrolysates. Differences in the serum amino acid profiles exclusively related to the amino acid composition of the protein (casein or whey proteins) were observed, but differences due to their molecular form were not observed. It is concluded that the use of enzymatic hydrolysates of whey proteins and casein has the same effects as their native proteins on nitrogen intake, body weight gain and serum amino acid profile of growing rats at weaning.

A simple method for obtaining kinetic equations to describe the enzymatic hydrolysis of biopolymers

A procedure to obtain the overall rate of hydrolysis of biopolymers is proposed, based on the fitting of the experimental data x = f(t) to cubic spline functions and from these, by differentiation, to obtain dx/dt. The values of these dx/dt slopes are an exclusive function of the conversion, x, when E 0 , S 0 , pH and temperature are constant. The fitting of dx/dt versus x leads to equations of the type dx / dt = a.x exp(-b. x) for the glucoamylase-starch system, where b = 8.75 and a = f(E 0 , T).