Glyceride synthesis in a solvent-free system

Synthesis of partial glycerides in a solvent-free system has been investigated with various acyl donors and glycerol as substrates and a 1,3-specific immobilized lipase to catalyze the reaction. Capric acid was the most efficient acyl donor, compared with ethyl caprate and tricaprin. However, to obtain a high yield of dicaprin and a low amount of tricaprin, ethyl caprate was the acyl donor of choice. The composition of the product mixture was determined by the ratio of ethyl caprate to glycerol; a molar ratio of 3∶1 was optimum for dicaprin synthesis. The water content in glycerol did not influence the final yield of dicaprin, but initial production of capric acid increased with increasing water content. The reaction was found to be controlled entirely by external mass transfer. The yield of diglyceride could be increased from 70 to 90% by lowering the reaction temperature, so that the diglyceride precipitated during the reaction.     

Enterolignans enterolactone and enterodiol formation from their precursors by the action of intestinal microflora and their relationship with non-starch polysaccharides in various berries and vegetables

The aim of this work was to investigate the production of enterolactone (ENL) and enterodiol (END) both enterolignans, from their precursors by the action of intestinal microflora and their relationship with non-starch polysaccharides (NSP) in common plant foods such as berries and vegetables. For the investigation of the bioconversion of plant lignans the technique of in vitro fermentation was used and the quantitative analysis of their metabolites ENL and END was performed by HPLC with coulometric electrode array detection. The enterolignan production from various berries ranged from 7.8 to 382.8 nmol/g as well as from vegetables - from 10.5 till 91.2 nmol/g. By comparing different kind of berries, the cloudberry, raspberry, and strawberry were the best enterolignan producers. Considering vegetables, potatoes produced the highest quantity of total enterolignans. Garlic, zucchini and broccoli were the other good producers of enterolignans in this product group. The quantitative relationship between NSP components and their associated lignan metabolites were determined. The results showed that there is a correlation between the particularities of fermented food matrices and the production of enterolignans. For berries, an intermediate correlation was found between the total NSP and ENL values. For vegetables, higher correlations between NSP and END were found.     

Intake of rye bread ileostomists increases ileal excretion of fiber polysaccharide components and organic acids but does not increase plasma or urine lignans and isoflavonoids

The excretion of starch, enzyme-resistant starch, dietary fiber components and organic acids (short-chain fatty acids plus lactic acid) as well as plasma and urine lignans and isoflavonoids was studied in eight ileostomists consuming mixed diets with wheat bread (low fiber diet) or rye bread (high fiber diet) in a crossover design. Average ileal excretions of enzyme-available starch were 3.5 g/d during the low fiber period and 4.1 g/d during the high fiber period. The excretion of enzyme-resistant starch was approximately the same (2.3 g/d) in both periods. In comparison with intake, similar amounts of total fiber residues were excreted both by subjects receiving the low fiber diet (3.4 g/d) and by those receiving the high fiber diet (2.7 g/d). However, subjects excreted significantly more of certain polysaccharide residues (fucose, galactose, and uronic acids) than they ingested. On average, the excretion of organic acids was 18.6 mmol/d during the low fiber period and 30.2 mmol/d during the high fiber period. No significant differences in plasma lignans were observed between the high fiber and the low fiber dietary periods. The present findings indicate that enzyme-available starch is highly digested and that a microbial breakdown of dietary fibers and probably other carbohydrates occurs in the small intestine. However, the bacterial activity in the ileostomists was not sufficient to cause an increased level in plasma lignans even when subjects consumed the high fiber rye diet.     

Factors governing the activity of lyophilised and immobilised lipase preparations in organic solvents

Active site titration and activity measurements were performed in hexane on lyophilised lipase preparations containing different amounts of phosphate buffer and lipase immobilised on porous polypropylene. Lyophilisation of Thermomyces lanuginosus lipase with large quantities of phosphate salts (200 mM) increased the specific activity fourfold, and the number of rapidly titratable active sites increased to 50 % from the 13 % observed when smaller amounts of phosphate buffer were used (20 mM) during lyophilisation. The phosphate buffer worked as an immobilisation matrix for the lipase, and the increase in specific activity was at least partly due to decreased mass transfer limitations. When lipase was immobilised on porous polypropylene, the specific activity was 770 times higher than that of the best freeze-dried preparation. At optimal enzyme loading, 93 % of the enzyme molecules were titrated at a high rate; this indicates that this adsorption on a hydrophobic surface was a very efficient means of reducing mass transfer limitations and of immobilising the enzyme in its active conformation for use in organic solvents. The variation in specific activity with water activity was found to correlate very well with the variation in titratable active sites when lipases from Burkholderia cepacia and Thermomyces lanuginosus were immobilised on porous polypropylene. The catalytic activity per competent active site was thus constant over the whole range of water activities.     

Effects of Regioselectivity and Lipid Class Specificity of Lipases on Transesterification,Exemplified by Biodiesel Production

Lipase-catalyzed ethanolysis of triolein was studied as a model for biodiesel production. Four lipases were immobilized on porous polypropylene, and ethanolysis reactions were carried out in methyl t-butyl ether. The reaction products were analyzed using gas chromatography. Three of the four lipases studied were efficient in the conversion of triolein to 2-monoolein, but slow in the final step of producing glycerol. However, Candida antarctica lipase B was slow in the conversion of triolein, but more efficient in the subsequent two steps than the other lipases. The 1,3-selectivity of the lipases was less pronounced for the monooleins than for triolein. Silica gel was investigated as a catalyst for acyl migration, showing an increase in biodiesel yield with three of the lipases, but a reduction in yield when C. antarctica lipase B was used. The highest biodiesel yield (96 %) was obtained with a combination of Rhizopus arrhizus lipase and C. antarctica lipase B.     

Continuous lipase-catalyzed production of wax ester using silicone tubing

Enzymatic synthesis of cetyl palmitate was performed in a solvent-free system at 65°C using immobilized Candida antarctica lipase. Batch reactions at controlled water activity showed that the yield could be increased from 88.8 to 99.1% by decreasing the water activity from 1 to 0.05. A continuous reactor configuration was constructed, where two tubular reactors were run in sequence with a separation container in between, in which the water phase was separated from the wax ester phase. The reactor was run for 1 wk at low flow rate (0.005 g/min) with very good operational stability and a productivity of 7.2 g d⁻¹ using 0.4 g of biocatalyst. The activity of the individual preparations decreased during operation. The first reactor had only 30% activity left after 1 wk of operation whereas the second reactor showed only a 10% decrease. This difference in enzyme stability is a direct result of the different water activity in the two reactors.     

Radioimmunoassay of free genistein in human serum

Two radioimmunoassay (RIA) systems for genistein have been established, based on polyclonal antibodies against genistein-4'-O-(carboxymethyl)ether-bovine serum albumin and genistein-7-O-(carboxymethyl)ether-bovine serum albumin conjugates. The sensitivities of assays were 4.44 and 10.4 fmol (1.2 and 2.8 pg)/tube, respectively, the intraassay coefficients of variation ranged from 3.54 to 9.30%, the interassay C.V. varied from 6.72 to 19.7%, depending on the type of method and on genistein concentration. The cross-reactivities with other chemically related compounds (with exception of genistein derivatives at the position used for construction of the immunogen) were 5.5 and 6.1% for daidzein and 3.9 and 0.04% for formononetin in RIAs using reagents prepared through positions 4'- and 7- of genistein, respectively. The method was used for measurement of genistein levels in 26 omnivore subjects and in three volunteers after consumption of a meal prepared from 125 g of cooked whole soybeans. The values obtained in ether extracts from human sera were almost identical for both RIA systems, indicating that both RIAs measure the same entity.     

Evaluation of radioimmunological methods for assay of plasma and urinary aldosterone

Two radioimmunological methods for assay of plasma and urinary aldosterone were carefully evaluated. In the plasma method a radioimmunoassay is preceded by chromatography on a Sephadex LH-20 column. The method for urine includes a preextraction, hydrolysis of the acid-labile conjugates of aldosterone, and a radioimmunoassay. Both methods fulfill the criteria of reliability and are suitable for both routine and demanding research assays. The plasma method, using columns of double length, is also applicable to analysis of aldosterone in plasma of newborn children, and pregnant females and in cord plasma. The concentration of plasma aldosterone in healthy subjects on an ad lib salt diet was 162 +/- 93 (S.D.) pmol/1 in the supine position and 312 +/- 217 (S.D.) pmol/1 upright. The urinary excretion of aldosterone in healthy subjects was 28.3 +/- 16.7 (S.D.) nmol/24 h.