Agnieszka Wikiera

Development of complete hydrolysis of pectins from apple pomace.

Enzymatically extracted pectins have a more complex structure than those obtained by conventional methods. As a result, they are less susceptible to hydrolysis, which makes the precise determination of their composition difficult. The aim of the study was to develop a method of complete hydrolysis of enzymatically extracted apple pectins. Substrates were pectins isolated from apple pomace by the use of xylanase and multicatalytic preparation Celluclast and apple pomace. Hydrolysis was performed by a chemical method with 2M TFA at 100 °C and 120 °C and a combined acidic/enzymatic method. After hydrolysis, the contents of galacturonic acid and neutral sugars were measured by HPLC. Complete hydrolysis of polygalacturonic acid occurred after 2.5h incubation with 2M TFA at 120 °C. The efficient hydrolysis of neutral sugars in pectins was performed with 2M TFA at 100 °C for 2.5h. Monomers most susceptible to concentrated acid were rhamnose, mannose and arabinose.

Endo-xylanase and endo-cellulase-assisted extraction of pectin from apple pomace.

Pectins were extracted from apple pomace with monoactive preparation of endo-xylanase and endo-cellulase. The process was conducted for 10 h in conditions of pH 5.0 at 40 °C, with constant shaking. Endo-xylanase application resulted in the highest extraction efficiency of pectins (19.8%). The obtained polymer was characterised by a very high molecular mass, high level of neutral sugars - mainly arabinose, galactose and glucose, and very high DM (73.4). It also contained the highest level of protein and phenols. Pectin extracted with endo-cellulase had 1.5 fold lower molecular mass but contained significantly more GalA (70.5%) of a high degree of methylation (66.3%). The simultaneous application of both enzymatic preparations resulted in their cooperation, leading to a decrease of both the extraction efficiency and the molecular mass of pectin. However, this pectin was distinguished by the highest GalA (74.7%) and rhamnose contents.

Application of Celluclast 1.5L in apple pectin extraction.

Pectins were extracted from apple pomace with Celluclast 1.5L at a dose of 25, 50 and 75 μl per 1g of material. In obtained pectin, the galacturonic acid (GalA) content, the neutral sugars (NS) profile, the degree of methylation (DM) and acetylation (DAc), the molecular mass, protein, ash and polyphenol levels as well as antioxidant and antitumor activity were determined. The lowest dose of enzymatic preparation resulted in the yield of pectin isolation comparable with acidic treatment (15.3%). Application of higher dose caused further, almost 4% increase in polymer recovery. Enzymatically isolated pectin was characterised by larger molecular mass and contained more GalA of higher DM and DAc than polymer extracted with acid. It was also richer in protein and polyphenols, and had different NS profile, which resulted in higher antiradical activity as well as the ability to inhibit the proliferation and invasion of Caco-2 adenocarcinoma cells.

Methylxanthine Drugs Are Human Pancreatic Lipase Inhibitors

Methylxanthine Drugs Are Human Pancreatic Lipase Inhibitors Methylxanthines such as caffeine, theobromine and theophylline are intensively consumed as food components by large proportion of human population all over the world. This class of compounds show various biological activities and have been found to act as broad specificity inhibitors towards numerous enzymes. However, their action on digestive enzymes have not been yet investigated. In this paper we aimed to evaluate the effects of methylxanthines on the human pancreatic lipase activity in vitro. Emulsions of short- and long-chain triglycerides (tributyrin and tripalmitate, respectively) were used as substrates. The concentrations of methylxanthines in the reaction mixtures covered the range between 0.015 mmol/L to 15 mmol/L. We demonstrated that all three tested substances, caffeine, theophylline and theobromine inhibited the hydrolysis of tributyrin and tripalmitate catalysed by human pancreatic lipase in dose-dependent fashion. The highest lipase inhibition ratios during tripalmitate and tributyrin hydrolysis were 25.74% and 79.54% respectively in the presence of caffeine, 29.89% and 62.79% respectively with theophylline and 21.08% and 67.74% respectively in the presence of theobromine. All the tested methylxanthines exert stronger inhibition in the short-chain triglyceride lipolysis comparing to long-chain substrates. Their mechanism of action involves most likely the interaction with enzyme protein but not substrate emulgation. In case of tripalmitate lipolysis all the methylxanthines showed mixed type of inhibition. Interestingly, during tributyrin lipolysis theophylline behaved as classical noncompetitive inhibitor.

[Health-promoting properties of pectin].

Pectin, a heteropolysaccharide commercially derived from the cell wall of higher plants, is mainly used in food as a gelling agent in jams and jellies as well as a stabilizer in fruit juice and milk drinks. It has also received great interest as a source of dietary fiber. Furthermore, pectin is proved to have diverse biological activities including lipid and cholesterol level lowering effects, serum glucose and insulin content lowering effects, gastric emptying delay, and anti-cancer activities. Pectin and pectic oligosaccharides have been shown to induce apoptosis in human colonic adenocarcinoma cells and to have anti-metastatic properties. Dietary pectin can bind metal ions, particularly lead ions, thus reducing their retention in the body and diminishing their toxic effects. On the other hand, pectin enhances intestinal solubility and absorption of ferric iron. Pectin with a low degree of esterification or having a large volume of linear oligogalacturonide segments shows significant mucoadhesion capacity in the gastrointestinal tract. In this way pectin forms a physical barrier protecting epithelium against opportunistic microbial invasion during stress.

Food Stabilizing Antioxidants Increase Nutrient Bioavailability in the in Vitro Model

ABSTRACT Objective: We investigated whether antioxidants may enhance bioavailability of lipids and carbohydrates and therefore increase the risk of obesity development. Methods: We tested how supplementation with antioxidants (0.01% butylated hydroxytoluene [BHT], α-tocopherol, and green tea catechins) of a diet containing butter and wheat bread affects bioavailability of fats and carbohydrates. The absorption of the in vitro digested diet was estimated in the intestinal epithelia model of the Caco-2 cells cultured in Transwell chambers. Results: In the case of the antioxidant-supplemented diets, we observed increased bioavailability of glucose, cholesterol, and lipids, as well as elevated secretion of the main chylomicron protein apoB-48 to the basal compartment. Importantly, we did not detect any rise in the concentrations of lipid peroxidation products (malondialdehyde, MDA) in the control samples prepared without antioxidants. Conclusions: Addition of antioxidants (in particular BHT) to the diet increases bioavailability of lipids and carbohydrates, which consequently may increase the risk of obesity development. The dose of antioxidants is a factor of fundamental importance, particularly for catechins: low doses increase absorption of lipids, whereas high doses exert the opposite effect.

Proteolysis in tempeh-type products obtained with Rhizopus and Aspergillus strains from grass pea (Lathyrus sativus) seeds.

BACKGROUND Tempeh is a food product obtained from legumes by means of solid-state fermentation with Rhizopus sp. Our previous research proved that mixed-culture inoculum may also be successfully applied. The objective of present research was to study the proteolytic activity of R. microsporus var. chinensis and A. oryzae during tempeh-type fermentation of grass pea seeds, and the effect of inoculum composition on the protein level and in vitro protein bioavailability in products. METHODS Fermentation substrate were soaked and cooked grass pea seeds. Material was mixed with single- or mixed-culture inoculum, and incubated in perforated plastic bags at 30°C for 32 hrs. In the products, the proteolytic activity (pH 3, 5 and 7), glucosamine, total protein and free amino acids levels, as well as protein in vitro bioavailability and degree of protein hydrolysis were obtained. RESULTS The significant correlation was found between glucosamine content and proteolytic activity in grass pea seeds fermented with Rhizopus or Aspergillus. The activities of Rhizopus proteases were higher than Aspergillus ones, which corresponded with the degree of seed protein hydrolysis. Both strains showed the highest activity of protease at pH 3. Tempeh made with pure culture of Rhizopus had 37% protein of 69% in-vitro bioavailability. Mixed-culture fermentation improved nutritional parameters of products only when the dose of Aspergillus spores in the inoculum was equal and lower than that of Rhizopus. This process resulted in higher in-vitro bioavailability of protein, slightly more efficient protein hydrolysis and higher level of free amino acids, as compared to standard tempeh. CONCLUSIONS The activity of A. oryzae in tempeh-type fermentation is beneficial as long as it does not dominate the activity and/or growth of Rhizopus strain.