Marta Corzo-Martínez

Structural Characterization of Bovine β-Lactoglobulin−Galactose/Tagatose Maillard Complexes by Electrophoretic, Chromatographic, and Spectroscopic Methods

To investigate the influence of the type of carbonyl group of the sugar on the structural changes of proteins during glycation, an exhaustive structural characterization of glycated beta-lactoglobulin with galactose (aldose) and tagatose (ketose) has been carried out. Conjugates were prepared via Maillard reaction at 40 and 50 degrees C, pH 7, and a w = 0.44. The progress of the Maillard reaction was followed by indirect formation of Amadori and Heyns compounds, advanced glycation end products, and brown polymers. The structural characterization of glycoconjugates was conducted by using a number of analytical techniques such as RP-HPLC, isoelectric focusing, MALDI-ToF, SDS-PAGE, size exclusion chromatography, and spectrofluorimetry (tryptophan fluorescence). In addition, the surface hydrophobicity of the beta-lactoglobulin glycoconjugates was also assessed. The results showed a higher reactivity of galactose than tagatose to form the glycoconjugates, probably due to the higher electrophilicity of the aldehyde group. At 40 degrees C, more aggregation was produced when beta-lactoglobulin was conjugated with tagatose as compared to galactose. However, at 50 degrees C hardly any difference was observed in the aggregation produced by galactose and tagatose. These results afford more insight into the importance of the functional group of the carbohydrate moiety during the formation of protein-carbohydrate conjugates via Maillard reaction.

Synthesis of prebiotic carbohydrates derived from cheese whey permeate by a combined process of isomerisation and transgalactosylation.

BACKGROUND Lactose from cheese whey permeate (WP) was efficiently isomerised to lactulose using egg shell, a food-grade catalyst, and the subsequent transgalactosylation reaction of this mixture with β-galactosidase from Bacillus circulans gave rise to a wide array of prebiotic carbohydrates derived from lactose and lactulose. RESULTS Lactulose obtained by efficient isomerisation of WP (16.1% by weight with respect to the initial amount of lactose) showed great resistance to the hydrolytic action of β-galactosidase from B. circulans, which preferentially hydrolysed lactose, acting as a galactosyl donor and acceptor. Lactulose had capacity as an acceptor, leading to the formation of lactulose-derived oligosaccharides. The enzymatic synthesis was optimised by studying reaction conditions such as pH, temperature, time, enzyme concentration and carbohydrate concentration. The maximum formation of galactooligosaccharides with degrees of polymerisation from 2 to 4 was achieved after 5 h of reaction at pH 6.5 and 50 °C with 300 g kg(-1) carbohydrates and 3 U mL(-1) β-galactosidase. CONCLUSION These findings indicate that the transgalactosylation of isomerised WP with β-galactosidase from B. circulans could be a new and efficient method to obtain a mixture with 50% of potentially prebiotic carbohydrates composed of lactulose, and galactooligosaccharides derived from lactose and lactulose.

Chemical and physicochemical quality parameters in carrots dehydrated by power ultrasound.

Preservation of the quality and bioactivity of carrots dehydrated by power ultrasound (US) under different experimental conditions including prior blanching has been evaluated for the first time by measuring the evolution of the Maillard reaction and the changes in soluble sugars, proteins, total polyphenols, antioxidant activity, and rehydration ability. This study also includes a comparison with a freeze-dried sample and data of commercial dehydrated carrots. The synergic effect of US and temperature (60 degrees C) increased the dehydration rate of carrots (90% moisture loss in only 75 min) while still providing carrots with a level of 2-furoylmethyl-amino acids significantly lower than that of dehydrated commercial samples. Whereas a decrease in the content of reducing soluble sugars was observed with processing temperature, minor carbohydrates (scyllo- and myo-inositol and sedoheptulose) were rather stable, irrespective of the US dehydration parameters. Blanching significantly improved the rehydration ability of US-dehydrated carrots without increasing the loss of soluble sugars by leaching. As supported by the similarity of most quality indicators studied in both US-treated and freeze-dried carrots, the mild processing conditions employed in US dehydration gave rise to premium quality dehydrated carrots.

Effect of milk protein glycation and gastrointestinal digestion on the growth of bifidobacteria and lactic acid bacteria.

In this paper, β-lactoglobulin (β-Lg) and sodium caseinate (SC) have been glycated via Maillard reaction with galactose and lactose and, subsequently, the effect of glycoconjugates hydrolyzed under simulated gastrointestinal digestion on the growth of pure culture of Lactobacillus, Streptococcus and Bifidobacterium has been investigated. Glycopeptides were added to the growth media as the sole carbon source. None of the bacterial strains was able to grow in hydrolysates of native and control heated β-Lg and SC. However, glycopeptides were fermented, in different degree, by Lactobacillus and Bifidobacterium and hardly any effect was detected on the growth of Streptococcus. Digested β-Lg glycoconjugates showed a strain-dependent effect whereas growth profiles of bacteria when hydrolysates of SC glycoconjugates were used as substrates were very similar, regardless of the strain. A general preference towards peptides from β-Lg/SC glycated with galactose, particularly at the state of the reaction in which the highest content in the Amadori compound tagatosyl-lysine is present, was observed. SC glycoconjugates were quickly fermented by some strains, promoting their growth in a greater extent than β-Lg complexes or even glucose. Therefore, from the results obtained in this work it can be concluded that conjugation of both milk proteins with galactose and lactose via the Maillard reaction could be an efficient method to obtain novel food ingredients with a potential prebiotic character.

Effect of glycation on sodium caseinate-stabilized emulsions obtained by ultrasound.

This work explores the potential of high-intensity ultrasound to produce fine-dispersion, long-time-stable, oil-in-water emulsions prepared with native and glycated bovine sodium caseinate (SC). Regardless the ultrasound amplitude and time assayed, the sonicated emulsions of native SC at 0.5 mg/mL had much higher emulsifying activity indexes compared with those emulsions formed by Ultra-Turrax (IKA Werke GmbH & Co., Staufen, Germany) homogenization. Nevertheless, the native SC emulsions were very unstable despite the optimization of parameters such as protein concentration, amplitude of ultrasound wave, and sonication time by using a Box-Behnken design. Early glycation of SC with either galactose, lactose, or 10 kDa dextran substantially improved both emulsifying activity and the stability, whereas at advanced stages of glycation, SC emulsions showed notably reduced emulsifying properties, likely because extensive glycation of SC promoted its polymerization mainly through covalent cross-linking, as was demonstrated by particle size measurements. The increase in particle diameter of glycoconjugates likely affected the diffusion of SC from bulk to the oil-water interface and slowed the reorientation process of the protein at the interface. These findings show that the combined effect of early-stage glycation of SC and high-intensity ultrasound as an emergent technique to form emulsions has the potential to provide improved emulsions that could be used in several food applications.

Application of liquid chromatography-tandem mass spectrometry for the characterization of galactosylated and tagatosylated β-lactoglobulin peptides derived from in vitro gastrointestinal digestion.

This article describes a comprehensive characterization of bovine beta-lactoglobulin peptides glycated with an aldohexose (galactose) or a ketohexose (tagatose), derived from in vitro gastrointestinal digestion, by liquid chromatography coupled to positive electrospray ion trap tandem mass spectrometry. In addition to the dissociation pathway previously described for aldohexoses-derived Amadori compounds, i.e. formation of the pyrylium ([M+H](+)-54) and furylium ions ([M+H](+)-84) via the oxonium ion ([M+H](+)-18), another and more direct fragmentation route involving the formation of the imminium ion ([M+H](+)-150) is also reported following extensive glycation rates of beta-lactoglobulin with both carbohydrates. These results indicated that the analysis of digested proteins by LC-ESI-MS(2) on a three-dimensional ion trap monitoring neutral losses is an efficient and direct method to identify peptides glycated not only through the Amadori rearrangement but also via the Heyns rearrangement. Nevertheless, as the predominating MS(2) fragmentation pattern of the glycated peptides is derived from the sugar moiety, the sequence-informative b- and y-ions resulting from peptide backbone cleavage were undetected. To overcome this drawback, and taking advantage of multi-stage fragmentation capabilities of ion traps, the indicative Amadori and Heyns-derived imminium ions were successfully used in MS(3) analyses to identify the peptide backbone, as well as the specific glycation site. In addition, further MS(4) analyses were needed to carry out the characterization of doubly glycated peptides.

Impact of high-intensity ultrasound on the formation of lactulose and Maillard reaction glycoconjugates

The impact of high-intensity ultrasound (US) on the formation of lactulose during lactose isomerization and on the obtention of lysine-glucose glycoconjugates during Maillard reaction (MR) has been studied, respectively, in basic and neutral media. As compared to equivalent conventional heat treatments, a higher formation of furosine, as indicator of initial steps of MR, was observed together with more advance of the reaction in US treated samples, this effect being more pronounced with the increase of US amplitude (50-70%) and temperature (25-40 °C). Regarding the influence of US on lactulose formation, in general, in a buffered system (pH 10.0), US at 70% of amplitude and 60 °C increased the rate of lactose isomerization, higher values of lactulose, epilactose and galactose being observed in comparison to conventional heating. The results of this work showed an acceleration of both reactions by US, indicating its usefulness to promote the formation of functional ingredients.

Role of Pyridoxamine in the Formation of the Amadori/Heyns Compounds and Aggregates during the Glycation of β-Lactoglobulin with Galactose and Tagatose

The effect of pyridoxamine on the Maillard reaction during the formation of conjugates of beta-lactoglobulin with galactose and tagatose under controlled conditions (pH 7, 0.44 aw, 40 and 50 degrees C, for 6 days) has been studied, for the first time, by means of the changes in reducing carbohydrates, formation of Amadori or Heyns compounds, and aggregates and browning development. The results showed the formation of interaction products between pyridoxamine and galactose or tagatose either in the presence or in the absence of beta-lactoglobulin, indicating that pyridoxamine competes with the free amino groups of beta-lactoglobulin for the carbonyl group of both carbohydrates. Thus, a small inhibitory effect of pyridoxamine on the initial stages of the Maillard reaction was pointed out. Furthermore, much lower aggregation and color formation rates were observed in the conjugates of beta-lactoglobulin galactose/tagatose with pyridoxamine than without this compound, supporting its potent inhibitory effect on the advanced and final stages of the Maillard reaction. These findings reveal the usefulness of food-grade inhibitors of the advanced stages of the Maillard reaction, such as pyridoxamine, that, in combination with mild storage conditions, could lead to the formation of safer neoglycoconjugates without impairing their nutritional quality.

Tofu Whey Permeate Is an Efficient Source To Enzymatically Produce Prebiotic Fructooligosaccharides and Novel Fructosylated α-Galactosides.

This work addresses a novel and efficient bioconversion method for the utilization of tofu whey permeate (TWP), an important byproduct from the soybean industry, as a precursor of high value-added ingredients such as prebiotic fructooligosaccharides and novel fructosylated α-galactosides. This process is based on the high capacity of the commercial enzyme preparation Pectinex Ultra SP-L to transfructosylate the main carbohydrates present in TWP as sucrose, raffinose, and stachyose to produce up to a maximum of 164.2 g L(-1) (equivalent to 57% with respect to initial sucrose, raffinose, and stachyose contents in TWP) of fructooligosaccharides and fructosylated α-galactosides in a balanced proportion. Raffinose- and stachyose-derived oligosaccharides were formed by elongation from the nonreducing terminal fructose residue up to three fructosyl groups bound by β-(2→1) linkages. These results could provide new findings on the valorization and upgrading of the management of TWP and an alternative use of raw material for the production of FOS and derivatives.

Synthesis of Oligosaccharides Derived from Lactulose (OsLu) Using Soluble and Immobilized Aspergillus oryzae β-Galactosidase

β-Galactosidase from Aspergillus oryzae offers a high yield for the synthesis of oligosaccharides derived from lactulose (OsLu) by transgalactosylation. Oligosaccharides with degree of polymerization (DP) ≥ 3 have shown to possess higher in vitro bifidogenic effect than di- and tetrasaccharides. Thus, in this work, an optimization of reaction conditions affecting the specific selectivity of A. oryzae β-galactosidase for synthesis of OsLu has been carried out to enhance OsLu with DP ≥ 3 production. Assays with β-galactosidase immobilized onto a glutaraldehyde–agarose support were also carried out with the aim of making the process cost-effective and industrially viable. Optimal conditions with both soluble and immobilized enzyme for the synthesis of OsLu with DP ≥ 3 were 50 °C, pH 6.5, 450 g/L of lactulose, and 8 U/mL of enzyme, reaching yields of ca. 50% (w/v) of total OsLu and ca. 20% (w/v) of OsLu with DP 3, being 6′-galactosyl-lactulose the major one, after a short reaction time. Selective formation of disaccharides, however, was favored at 60 °C, pH 4.5, 450 g/L of lactulose and 8 U/mL of enzyme. Immobilization increased the enzymatic stability to temperature changes and allowed to reuse the enzyme. We can conclude that the use, under determined optimal conditions, of the A. oryzae β-galactosidase immobilized on a support of glutaraldehyde–agarose constitutes an efficient and cost-effective alternative to the use of soluble β-galactosidases for the synthesis of prebiotic OsLu mixtures.