Synthesis of β‐Galactooligosaccharides from Lactose Using Microbial β‐Galactosidases

Abstract: Galactooligosaccharides (GOSs) are nondigestible oligosaccharides and are comprised of 2 to 20 molecules of galactose and 1 molecule of glucose. They are recognized as important prebiotics for their stimulation of the proliferation of intestinal lactic acid bacteria and bifidobacteria. Therefore, they beneficially affect the host by selectively stimulating the growth and/or activity of a limited number of gastrointestinal microbes (probiotics) that confer health benefits. Prebiotics and probiotics have only recently been recognized as contributors to human health. A GOS can be produced by a series of enzymatic reactions catalyzed by β‐galactosidase, where the glycosyl group of one or more D‐galactosyl units is transferred onto the D‐galactose moiety of lactose, in a process known as transgalactosylation. Microbes can be used as a source for the β‐galactosidase enzyme or as agents to produce GOS molecules. Commercial β‐galactosidase enzymes also do have a great potential for their use in GOS synthesis. These transgalactosyl reactions, which could find useful application in the dairy as well as the larger food industry, have not been fully exploited. A better understanding of the enzyme reaction as well as improved analytical techniques for GOS measurements are important in achieving this worthwhile objective.     

Purification and physicochemical characterization of ovine β‐lactoglobulin and α‐lactalbumin

Ovine whey proteins were fractionated and studied by using different analytical techniques. Anion‐exchange chromatography and reversed‐phase high‐performance liquid chromatography (HPLC) showed the presence of two fractions of β‐lactoglobulin but only one of α‐lactalbumin. Gel permeation and sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis allowed the calculation of the apparent molecular mass of each component, while HPLC coupled to electrospray ionisation‐mass spectrometry (ESI‐MS) technique, giving the exact molecular masses, demonstrated the presence of two variants A and B of ovine β‐lactoglobulin. Amino acid compositions of the two variants of β‐lactoglobulin differed only in their His and Tyr contents. Circular dichroism spectroscopy profiles showed pH conformation changes of each component. The thermograms of the different whey protein components showed a higher heat resistance of β‐lactoglobulin A compared to β‐lactoglobulin B at pH 2, and indicated high instability of ovine α‐lactalbumin at this pH.     

α 1,4 — α 1,6 Glucopolysaccharides Contained in Developing Maize Kernels

Corn grains with different filling times were fractionated in order to obtain their α 1,4 — α 1,6 glucopolysaccharide composition and structure. Sugary, waxy, flint and several hybrids were the varieties analyzed. There is an increase in the total content of polysaccharides, as the grain is completed. But, no differences in composition and structure were detected between the first sample analyzed (15th day after pollination) and the last one, which corresponds to the complete grain filling period. The methodology developed by us, allowed us to observe that the introduction of su character on the hybrids (F x S and S x F) has influence on the final polysaccharide structure. Also, in the F x W hybridα 1,4 — α 1,6 glucans, their structural characteristics were different from those of F or W pure.     

Naturally occurring ω‐Hydroxyacids

ω‐Hydroxyacids are fatty acids bearing a hydroxyl group on the terminal carbon. They are found in mammals and higher plants and are often involved in providing a permeability barrier, the primary purpose of which is to reduce water loss. Some ω‐hydroxyacid derivatives may be involved in waterproofing and signalling. The purpose of this review was to survey the known natural sources of ω‐hydroxyacids. ω‐Hydroxyacids are produced by two different P450‐dependent mechanisms. The longer (30–34 carbons) ω‐hydroxyacids are produced by chain extension from palmitic acid until the chain extends across the membrane in which the extension is taking place, and then the terminal carbon is hydroxylated. Shorter fatty acids can be hydroxylated directly to produce C16 and C18 ω‐hydroxyacids found in plants and 20‐eicosatetraenoic acid (20‐HETE) by a different P450. The C16 and C18 ω‐hydroxyacids are components of polymers in plants. The long‐chain ω‐hydroxyacids are found in epidermal sphingolipids, in giant‐ring lactones from the sebum of members of the equidae, as a component of meibum and in carnauba wax and wool wax.     

β-GLUCAN AND β-GLUCANASE IN BREWING

The development of endo β-glucanase during the micro-malting of barley, with and without the addition of gibberellic acid, was compared. Results indicated that the stimulative effect of gibberellic acid on the enzyme system was of only marginal practical importance. From the assessment of the enzyme activity in a number of commercial malts it would appear that the germination time used for some malts is too short to take full advantage of the critical phase of very rapid enzyme development. The viscosity-reducing power of β-glucanase was demonstrated in miniature brewery mashing experiments and details of the progressive degradation of β-glucan by the enzyme system were analysed by gel filtration methods. The β-glucan content of a number of varieties of barley was established.     

Comparative study of enzymatic hydrolysis of α/β- and γ-gliadins

Enzymatic hydrolysis of proteins and fractionation of hydrolysates is a route of diversifying their functional properties. Chymotryptic hydrolysis of different sulphur-rich gliadins (α/β- and γ-types), major wheat storage proteins, was studied. The peptides formed in the course of digestion were characterised by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate (SDS-PAGE) and reversed-phase high performance liquid chromatography (RP-HPLC). With reference to previous work, a general scheme of degradation was assessed for γ-gliadins. Limited hydrolysis released two types of polypeptides, comprising respectively the repetitive and the non-repetitive moieties of the protein. In spite of strong sequence homologies between the two groups of sulphur-rich gliadins, it was not possible to prepare similar peptide fractions from α/β-gliadins. They were more resistant to hydrolysis and the region where the two domains merge appeared inaccessible to chymotrypsin. Restricted accessibility of cleavage sites was attributed to the less expanded conformation of α/β-type than γ-type gliadins. A first step of scaling-up was performed. This offers opportunities to prepare functional peptides from wheat storage proteins.     

Investigation of the interaction between (−)‐epigallocatechin‐3‐gallate with trypsin and α‐chymotrypsin

Tea polyphenol (TP) inhibits digestive enzymes and reduces food digestibility. To explore the interaction between TP with digestive enzymes, bindings of ‐epigallocatechin‐3‐gallate (EGCG) to trypsin and α‐chymotrypsin were studied in detail using fluorescence, resonance light‐scattering, circular dichroism, fourier transform infrared spectroscopy methods and protein‐ligand docking. The binding parameters were calculated according to Stern–Volmer equation, and the thermodynamic parameters were determined by the van't Hoff equation. The results indicated that EGCG was capable of binding trypsin and α‐chymotrypsin with high affinity, resulting in a change of native conformation of these enzymes. EGCG had a greater influence on the structure of α‐chymotrypsin than trypsin. This study can be used to explain the binding interaction mechanism between TP and digestive enzymes.     

Investigation of selected toxicological parameters of γ-pentachlorocyclohexene (γ-PCCH)

The toxic effects of the γ-hexachlorocyclohexane metabolite γ-pentachlorocyclohexene (γ-PCCH) were studied by acute and subacute (6 weeks) experiments. The investigations included cerebral convulsibility with chemoshock (Tetrazolium), reactivity with hot plate method, the learning ability with learning tests, and peripheral nervous activity (EMG). Nociceptive reaction time was not influenced, the learning process (6 weeks) was inhibited by γ-PCCH. The conduction velocity of the peripheral nerve was decreased. At the end of the 6th week liver enlargement was found.     

Adsorption of Shiga Toxin to Poly‐γ‐Glutamate Precipitated

We screened foods containing indigestible ingredients in the ability to adsorb Shiga toxin (Stx). When 5 mg of foods and dietary fibers such as dry vegetables and inulin were mixed and incubated with 0.5 mL of Stx solution (100 ng/mL) containing 0.5% bovine serum albumin, both Stx1 and Stx2 seemed to be adsorbed by only a fermented food, natto (a traditional Japanese food prepared from steamed soybeans by the biological action of Bacillus subtilis). We purified the Stx‐adsorbing substance from natto by extraction with H₂O, acid treatment, Proteinase K treatment, and an ion exchange chromatography. The purified substance showed an average molecular mass of about 600 kDa. We identified it as poly‐γ‐glutamate (PGA) by amino acid analysis of its hydrolysate and peptide analysis after its treatment with Proteinase K. Purified PGA (MW: molecular weight = about 600 kDa) was considered to adsorb both Stx1 and Stx2 when we separated adsorbed and unadsorbed Stxs (MW = about 72 kDa) by an ultrafiltration method with a centrifugal filter unit (MWCO: molecular weight cut‐off = 100 K). However, PGA with the ability to adsorb Stx was an insoluble form precipitated in the filter unit during centrifugation. PGA precipitated beyond the saturated density was also confirmed to well adsorb both Stx1 and Stx2 by an equilibrated dialysis method. To the best of our knowledge, this is the 1st report on food‐adsorbing Stx.