Francisco Javier Moreno

Effect of prebiotic carbohydrates on the growth and tolerance of Lactobacillus

Resistance to gastrointestinal conditions is a requirement for bacteria to be considered probiotics. In this work, we tested the resistance of six different Lactobacillus strains and the effect of carbon source to four different gastrointestinal conditions: presence of α-amylase, pancreatin, bile extract and low pH. Novel galactooligosaccharides synthesized from lactulose (GOS-Lu) as well as commercial galactooligosaccharides synthesized from lactose (GOS-La) and lactulose were used as carbon sources and compared with glucose. In general, all strains grew in all carbon sources, although after 24 h of fermentation the population of all Lactobacillus strains was higher for both types of GOS than for glucose and lactulose. No differences were found among GOS-Lu and GOS-La. α-amylase and pancreatin resistance was retained at all times for all strains. However, a dependence on carbon source and Lactobacillus strain was observed for bile extract and low pH resistance. High hydrophobicity was found for all strains with GOS-Lu when compared with other carbon sources. However, concentrations of lactic and acetic acids were higher in glucose and lactulose than GOS-Lu and GOS-La. These results show that the resistance to gastrointestinal conditions and hydrophobicity is directly related with the carbon source and Lactobacillus strains. In this sense, the use of prebiotics as GOS and lactulose could be an excellent alternative to monosaccharides to support growth of probiotic Lactobacillus strains and improve their survival through the gastrointestinal tract.

The cytotoxic effect of Bowman–Birk isoinhibitors, IBB1 and IBBD2, from soybean (Glycine max) on HT29 human colorectal cancer cells is related to their intrinsic ability to inhibit serine proteases

Bowman-Birk inhibitors (BBI) from soybean and related proteins are naturally occurring protease inhibitors with potential health-promoting properties within the gastrointestinal tract. In this work, we have investigated the effects of soybean BBI proteins on HT29 colon adenocarcinoma cells, compared with non-malignant colonic fibroblast CCD-18Co cells. Two major soybean isoinhibitors, IBB1 and IBBD2, showing considerable amino acid sequence divergence within their inhibitory domains, were purified in order to examine their functional properties, including their individual effects on the proliferation of HT29 colon cancer cells. IBB1 inhibited both trypsin and chymotrypsin whereas IBBD2 inhibited trypsin only. Despite showing significant differences in their enzyme inhibitory properties, the median inhibitory concentration values determined for IBB1 and IBBD2 on HT29 cell growth were not significantly different (39.9+/-2.3 and 48.3+/-3.5 microM, respectively). The cell cycle distribution pattern of HT29 colon cancer cells was affected by BBI treatment in a dose-dependent manner, with cells becoming blocked in the G0-G1 phase. Chemically inactive soybean BBI had a weak but non-significant effect on the proliferation of HT29 cells. The anti-proliferative properties of BBI isoinhibitors from soybean reveal that both trypsin- and chymotrypsin-like proteases involved in carcinogenesis should be considered as potential targets of BBI-like proteins.

Protein Quality, Antigenicity, and Antioxidant Activity of Soy-Based Foodstuffs

Commercial soy-based foodstuffs, including beverages ( n = 15), cows milk supplemented with soy isoflavones ( n = 1), snacks ( n = 1), and biscuits ( n = 2), were analyzed to find any link between alterations in protein quality, safety (antigenicity), functionality (antioxidant activity), and food processing. Protein content was analyzed by the Kjeldhal method and available lysine by OPA assay. Chromatographic (RP-HPLC) and electrophoretic (SDS-PAGE) protein profiles were obtained to monitor modifications in the structure of soy allergens. The antigenicity was estimated by immunoblotting against soy total antibodies. Total phenol content was measured by Folin-Ciocalteu, while peroxyl radical scavenging activity of the sample was determined by ORAC FL assay. Protein content did not differ of those declared by the producers. Lysine availability was higher in liquid soy beverages compared to that in other soy foodstuffs studied here. 7S and 11S soy allergens were detected by RP-HPLC and SDS-PAGE, respectively. Both data indicated changes in soy protein patterns due to processing of instant powdered soymilk, soy snacks, and biscuits. Immunoblotting assay showed modifications in the antigenic response of these foodstuffs based on soy, suggesting that their processing had altered the structure of soy allergens. RP-HPLC, SDS-PAGE, and immunoblotting resulted in adequate analytical approaches for detecting changes in protein structure due to processing and adulteration. Protein quality, antigenicity, and antioxidant activity of soy products can be affected as a function of the intensity of the thermal processing.

Development of a new method using HILIC-tandem mass spectrometry for the characterization of O-sialoglycopeptides from proteolytically digested caseinomacropeptide

This work addresses the optimization of HILIC (hydrophilic interaction liquid chromatography)‐ESI‐MSn conditions for the comprehensive characterization of O‐glycopeptides from proteolytically digested caseinomacropeptide. O‐Glycopeptides were satisfactorily analysed on a zwitterionic HILIC column based on their glycan structure and amino acid sequence. The contribution of ionic interactions to the retention of charged glycopeptides was found to be substantial. Thus, O‐glycopeptides carrying neutral glycans were more retained than O‐sialoglycopeptides because negatively charged sialic acid residues were electrostatically repelled by the stationary phase. In addition, glycopeptides differing only in the position of the linkage of the sialic acid moiety could be separated. The same chromatographic behaviour was observed for model systems constituted by a synthetic tetrapeptide covalently conjugated to neutral and sialylated carbohydrates. Subsequent detection of caseinomacropeptide O‐glycopeptides was carried out on an electrospray ion trap tandem mass spectrometer at both positive and negative ionization modes. MS2 fragmentation at positive ionization mode was valid for determining the glycan structure as the resulting main fragments corresponded to Yn‐type ions derived from sequential glycosidic bond fragmentation, whilst the fragmentation of the peptide structure was preferably obtained through the formation of bn‐type ions at the MS3 stage, allowing the complete structure elucidation of the peptidic chain. Overall, the developed method allowed the identification and characterization of 41 O‐glycopeptides covering all the known glycosylation sites without any previous enrichment step. These results point out that HILIC coupled to multistage MS procedures can be a powerful technique for future glycoproteomic applications.

Determination of maltodextrins in enteral formulations by three different chromatographic methods

SummaryMaltodextrins (G1 to G11) present in enteral formulations have been determined by three analytical methods —thin-layer chromatography (TLC), high-performance anion-exchange chromatography with pulsed electrochemical detection (HPAEC-PED), and high-performance liquid chromatography with refractive-index detection (HPLC-RI). The repeatability of the three methods was similar. Although HPAEC-PED was more sensitive than HPLC-RI and TLC, the relatively high maltodextrin content of the enteral formulations resulted in reasonable agreement among results from quantitative determination of G1 to G7 by the three methods studied. Differences between methods were higher for compounds G8 to G11.

Selective linkage detection of O-sialoglycan isomers by negative electrospray ionization ion trap tandem mass spectrometry.

Sialylated O-linked oligosaccharides are involved in many biological processes, such as cell-cell interactions, cell-substance adhesion, and virus-host interactions. These activities depend on their structure, which is frequently determined by tandem mass spectrometry. However, these spectra are frequently analyzer-dependent, which makes it difficult to develop widely applicable analytical methods. In order to deepen the origin of this behavior, two couples of isomers of sialylated O-linked oligosaccharides, NeuAc alpha2-3Gal beta1-3GalNAc-ol/Gal beta1-3(NeuAc alpha2-6)GalNAc-ol and NeuGc alpha2-3Gal beta1-3GalNAc-ol/Gal beta1-3(NeuGc alpha2-6)GalNAc-ol, were analyzed by liquid chromatography/negative electrospray ionization ion trap tandem mass spectrometry (LC/ESI(-)-MS(n)) using both an ion trap and a triple quadrupole mass spectrometer. Results clearly showed that while ions obtained in the triple quadrupole instrument fitted very well with the standard fragmentation routes, in the ion trap several intense ions could not be explained by these rules, specially a fragment at m/z 597. Furthermore, this ion was observed in the mass spectrum of those isomers that sialic acid binds to GalNAc by an alpha2-6 linkage. From the MS(3) spectrum of this ion an unexpected structure was deduced, and it led to propose alternative fragmentation pathways. Molecular mechanics calculations suggested that the found atypical route could be promoted by a hydrogen bond located only in alpha2-6-linked oligosaccharides. It has also been demonstrated that this process follows a slow kinetic, explaining why it cannot be observed using an ion beam-type mass analyzer. In conclusion, ion traps seem to be more appropriate than triple quadrupoles to develop a reliable analytical method to distinguish between isomeric O-linked glycans.

Analysis of monosaccharides in bovine, caprine and ovine κ-casein macropeptide by gas chromatography

SummaryMonosaccharides present in κ-casein macropeptide (CMP) from bovine, ovine and caprine milks have been determined by gas chromatography after acid hydrolysis with trifluoroacetic acid (TFA) or methanolysis with HCL-methanol. The method has been evaluated for repeatability and recovery usingmyo-inositol as internal standard with satisfactory results. Galactose, N-acetyl-galactosamine and neuraminic acid were found in the κ-casein macropeptides from the three species and similar levels of galactose andN-acetyl-galactosamine were found after treatment with TFA and HCl-methanol. Bovine CMP exhibited the highest level of glycosylation. In general, the main component of the carbohydrate fraction was neuraminic acid. The method is suitable for the analysis of monosaccharides in CMP.