In vitro digestibility and bioaccessibility of lipid-based delivery systems obtained via enzymatic glycerolysis: a case study of rosemary extract bioaccessibility.

This work studies the effect of enzymatic glycerolysis on digestibility and bioaccessibility of ratfish liver oil (RLO) rich in alkylglycerols (AKGs), as well as the capability of the glycerolysis product (GP) to act as lipid-based delivery system (LBDS) for a supercritical rosemary extract. For comparison purposes, digestibility and bioaccessibility of two additional lipid systems i.e. original RLO and RLO with addition of GRAS monoolein (MO) as emulsifier agent (RLO + MO), have been evaluated. We have studied the efficiency of the GP and RLO + MO lipid systems as LBDS by combining them with a supercritical rosemary extract (RE), i.e. RE lipid-based formulations. In vitro digestibility and bioaccessibility of un-loaded lipid systems, RE lipid-based formulations and un-carried RE have been determined. The results show a higher digestibility and bioaccessibility of the GP as compared to those of original RLO and RLO + MO. Likewise, a substantial improvement of RE bioaccessibility has been observed when GP is used as lipid carrier of RE. The present work demonstrates that enzymatic glycerolysis is an efficient strategy to obtain highly bioaccessible and potentially bioactive alkylglycerol-based delivery systems, which can be used to increase the bioaccessibility of low water-soluble bioactive compounds.

Stearidonic Acid Concentration by Urea Complexation from Echium Oil.

Concentration of polyunsaturated fatty acids ethyl esters (FAEE) by urea complexation from Echium oil was studied. Different variables involved in the process were investigated: amount and particle size of urea, solvent volume and ratio (hexane/ethanol), load of FAEE and reaction time. Hence, the main goal was to optimize SDA concentration (%) and yield (%) of stearidonic acid (SDA, 18:4 ω-3) and other bioactive FAEE. Similar behaviors were observed in fractionation between α-linolenic (ALA)-linoleic (LA), and γ-linolenic (GLA)-stearidonic (SDA) acids, attributed to similarities on their chemical structures, due to the position of the double bonds. At laboratory scale, the optimal conditions were 3 g urea (powder), 3.6 mL of hexane, 0.54 mL of ethanol and 800 mg of FAEE, during 20 h at 25°C. A scaling-up at pilot plant was carried out twice, obtaining more than 100 g of a final product, with ~29% SDA concentration and ~78% yield. Besides, after two washings with water, ethyl carbamates (urethanes) were not detected in the final product. Thus, a mixture of FAEE with about 85% of bioactive fatty acids with anti-inflammatory properties was obtained, which can be a high added-value product with great potential for the synthesis of functional lipids and nutraceuticals.

Influence of thermal processing on the phase behavior of a novel acylglyceride-alkylglyceride lipid-based delivery system.

Lipid-based delivery systems have been broadly utilized to enhance bioavailability and bioaccessibility properties of drugs. Loading capacity and drug stability within the lipid matrix is influenced by the phase behavior of the lipid components that make up the vehicle. In this study we have investigated the influence of thermal processing at 50 °C on the subsequent phase behavior of a lipid-based delivery system comprised of a blend of monoacylglycerols, diacylglycerols, monoalkylglycerols and dialkylglycerols obtained by enzymatic glycerolysis of ratfish liver oil. Differential scanning calorimetry revealed that crystallization and melting points decreased, while powder X-ray diffraction suggested changes in the crystal structure of the product when heated to 50 °C and held for longer than 180 min. Samples heated for a short time, rapidly underwent a polymorphic transition from the α to the ß polymorphic phase, whereas samples heated for long times remained in the α phase for 3 days. Polarized light microscopy also showed changes in the microstructure of the material due to the thermal treatment. These results suggest that the thermal history of lipidic encapsulation materials, above their melting point, can affect eventual encapsulation efficiency and product performance.

Improving In Vivo Efficacy of Bioactive Molecules: An Overview of Potentially Antitumor Phytochemicals and Currently Available Lipid-Based Delivery Systems.

Cancer is among the leading causes of morbidity and mortality worldwide. Many of the chemotherapeutic agents used in cancer treatment exhibit cell toxicity and display teratogenic effect on nontumor cells. Therefore, the search for alternative compounds which are effective against tumor cells but reduce toxicity against nontumor ones is of great importance in the progress or development of cancer treatments. In this sense, scientific knowledge about relevant aspects of nutrition intimately involved in the development and progression of cancer progresses rapidly. Phytochemicals, considered as bioactive ingredients present in plant products, have shown promising effects as potential therapeutic/preventive agents on cancer in several in vitro and in vivo assays. However, despite their bioactive properties, phytochemicals are still not commonly used in clinical practice due to several reasons, mainly attributed to their poor bioavailability. In this sense, new formulation strategies are proposed as carriers to improve their bioefficacy, highlighting the use of lipid-based delivery systems. Here, we review the potential antitumoral activity of the bioactive compounds derived from plants and the current studies carried out in animal and human models. Furthermore, their association with lipids as a formulation strategy to enhance their efficacy in vivo is also reported. The development of high effective bioactive supplements for cancer treatment based on the improvement of their bioavailability goes through this association.

Assessment of Maillard reaction evolution, prebiotic carbohydrates, antioxidant activity and α-amylase inhibition in pulse flours.

In this paper, the quality of bean, chickpea, fava beans, lentil and pea flours from Algeria has been evaluated. Maillard reaction (MR) indicators, modifications in the carbohydrate and protein fractions, antioxidant activity and α-amylase inhibitor of raw, toasted and stored samples were evaluated. Fava beans, beans and peas showed higher content of raffinose family oligosaccharides while chickpeas and lentils showed higher polyol content. Toasting and storage caused slightly change in pulse quality; MR showed slight losses of lysine but increased antioxidant activity. Moreover, inhibition of α-amylase was slightly augmented during processing; this could increase the undigested carbohydrates that reach the colon, modulating the glycemic response. These results point out the suitability of these flours for preparing high-quality foodstuffs intended for a wide spectrum of the population, including hyperglycemic and gluten intolerant individuals.

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 ß-(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 ß-Galactosidase.

ß-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 ß-galactosidase for synthesis of OsLu has been carried out to enhance OsLu with DP ≥ 3 production. Assays with ß-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 ß-galactosidase immobilized on a support of glutaraldehyde-agarose constitutes an efficient and cost-effective alternative to the use of soluble ß-galactosidases for the synthesis of prebiotic OsLu mixtures.

Valorization of Cheese and Tofu Whey through Enzymatic Synthesis of Lactosucrose.

This work deals with the development of a new bioprocess for the efficient synthesis of lactosucrose, a potential prebiotic oligosaccharide with a high value-added, from two important and inexpensive agro-industrial by-products such as tofu whey and cheese whey permeate. The bioconversion is driven by the ability of the enzyme levansucrase SacB from Bacillus subtilis CECT 39 to transfructosylate lactose contained in the cheese whey permeate by using not only sucrose but also raffinose and stachyose, which are present in considerable amounts in the tofu whey, as suitable donors of fructosyl moieties. The maximum lactosucrose concentration obtained from both by-products was 80.1 g L-1 after a short reaction time 120 min at 37°C, leading to productivity and specific productivity values of 40.1 g lactosucrose L-1 h-1 and 80.1 mg lactosucrose U enzyme-1 h-1, respectively. Findings contained in this work could provide a new strategy to valorize agro-industrial by-products as cheese whey permeate and, specially, tofu whey by means of their use as renewable resources in the enzymatic synthesis of bioactive oligosaccharides.

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.

Acute oral safety study of sodium caseinate glycosylated via maillard reaction with galactose in rats.

In order to potentially use sodium caseinate (SC) glycated with galactose (Gal) in the food industry as a new functional ingredient with proved technological and biological properties, an evaluation of oral acute toxicity has been carried out. An acute safety study with SC-Gal glycoconjugates in the Wistar rat with a single oral gavage dose of 2,000 mg/kg of body weight was conducted. The SC-Gal glycoconjugates were well tolerated; no adverse effects or mortality was observed during the 2-week observation period. No abnormal signs, behavioral changes, body weight changes, or alterations in food and water consumption occurred. After this period, no changes in hematological and serum chemistry parameters, organ weights, or gross pathology or histopathology were detected. It was concluded that SC-Gal glycoconjugates obtained via the Maillard reaction were well tolerated in rats at an acute oral dose of 2,000 mg/kg of body weight. The SC-Gal glycoconjugates have a low order of acute toxicity, and the oral 50 % lethal dose for male and female rats is in excess of 2,000 mg/kg of body weight.

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 ß-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 ß-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) ß-galactosidase. CONCLUSION: These findings indicate that the transgalactosylation of isomerised WP with ß-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.

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

In this paper, ß-lactoglobulin (ß-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 ß-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 ß-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 ß-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 ß-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.

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.

Role of pyridoxamine in the formation of the Amadori/Heyns compounds and aggregates during the glycation of beta-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.

Application of liquid chromatography-tandem mass spectrometry for the characterization of galactosylated and tagatosylated beta-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.

Structural characterization of bovine beta-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.

Characterization and in vitro digestibility of bovine beta-lactoglobulin glycated with galactooligosaccharides.

Galactooligosaccharides (GOS) are well-known prebiotic ingredients which can form the basis of new functional dairy products. In this work, the production and characterization of glycated beta-lactoglobulin (beta-LG) with prebiotic GOS through the Maillard reaction under controlled conditions ( a(w) = 0.44, 40 degrees C for 23 days) have been studied. The extent of glycation of beta-LG was evaluated by formation of furosine which progressively increased with storage for up to 16 days, suggesting that the formation of Amadori compounds prevailed over their degradation. RP-HPLC-UV, SDS-PAGE, and IEF profiles of beta-LG were modified as a consequence of its glycation. MALDI-ToF mass spectra of glycated beta-LG showed an increase of up to approximately 21% in its average molecular mass after storage for 23 days. Moreover, a decrease in unconjugated GOS (one tri-, two tetra-, and one pentasaccharide) was observed by HPAEC-PAD upon glycation. These results were confirmed by ESI MS. The stability of the glycated beta-LG to in vitro simulated gastrointestinal digestion was also described and compared with that of the unglycated protein. The yield of digestion products of glycated beta-LG was lower than that observed for the unglycated protein. The conjugation of prebiotic carbohydrates to stable proteins and peptides could open new routes of research in the study of functional ingredients.