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.     

Lipase‐catalysed enrichment of DHA and EPA in acylglycerols resulting from squid oil ethanolysis

The fatty acid specificity of four lipases towards eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) was evaluated when performing ethanolysis of squid oil. During the first part of ethanolysis, no DHA ethyl esters were detected when using the lipases from Thermomyces lanuginosus, Pseudomonas cepacia or Pseudomonas fluorescens (in the case of the second and third lipases, no EPA ethyl esters were detected either). This indicates that these three lipases could not catalyse the conversion of DHA located in a triacylglycerol to ethyl ester, and that the Pseudomonas lipases could not catalyse the conversion of EPA either. This pattern was not found for the lipase from Rhizomucor miehei. The lipase from Thermomyces lanuginosus showed the lowest specificity towards DHA and the highest DHA recovery during DHA enrichment in the acylglycerol fraction. It was thus used to catalyse the ethanolysis of squid oil on a larger scale. The ethyl esters formed were removed using short‐path distillation, resulting in a product containing mainly mono‐ and diacylglycerols. The product contained 34 mol‐% DHA and 17 mol‐% EPA, compared with 19 mol‐% DHA and 12 mol‐% EPA in the original squid oil.     

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.     

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.     

Effects of Lipase Immobilization Conditions and Support Materials for the Production of Structured Triacylglycerols

Structured triacylglycerols (STAG) with desired properties can be synthesized by transesterification using immobilized lipases. Herein, the effect of immobilization conditions and support material on the immobilization yield, specific activity and regioselectivity of lipases from Rhizomucor miehei (RML) and Rhizopus oryzae (ROL) in the production of STAG, are evaluated. Four different support materials utilizing adsorption and one with covalent binding are investigated. Ammonium sulfate is found to significantly increase the activity-based immobilization yield (12% and 38% for RML and ROL, respectively) and specific activity on Accurel MP1000 (MP1000) when used as the immobilization buffer. Furthermore, the immobilization principle and support material influenced both the activity and regiospecificity. Immobilization by adsorption is found to result in higher catalytic activity, while covalent binding resulted in lipase inactivation. For RML, the highest specific activity of 43 µmol STAG min⁻¹ g⁻¹ (U) is obtained on MP1000, while ROL, which exhibited higher activities in general, results in a maximum activity of 120 U on Lifetech ECR8806. The obtained specific activites are comparable to the commercial preparations Novozym 40086 (45 U) and Lipase DF “Amano” IM (147 U) while the regiospecificity of the developed preparations is even higher, forming at least 64% less byproduct. Practical applications: There is an increasing need for lipids with specific nutritional and physical properties in health, nutrition, and food applications. In this context, STAG are highly promising products due to the possibility to tailor the composition to obtain the desired properties. Immobilized lipases are the catalyst of choice for the production of STAG, in which activity and regioselectivity are particularly important parameters for the process performance. In this study, it is shown that these parameters can be affected by the immobilization conditions and support material. Immobilized preparations with high activity and excellent regiospecificity are created on commercially available supports. This shows the possibility of STAG synthesis with high purity and beneficial properties.     

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.     

Estrogen metabolism and excretion in Oriental and Caucasian women

BACKGROUND: Caucasian and Oriental women have different incidence rates of breast cancer. Among the underlying risk factors for the development of breast cancer in the women of these two groups may be their different diets and patterns of estrogen metabolism and excretion. The absolute levels and relative ratios of 16 alpha-hydroxylated estrogens and 2-hydroxylated estrogens (catechol estrogens) in the body may have a role in the etiology of breast cancer, but studies so far have provided only conflicting results. PURPOSE: Our goal was to study estrogen metabolism, in particular, the extent of 2-hydroxylation and 16 alpha-hydroxylation of estrogens in two groups of women, one Caucasian and one Oriental, with inherently different breast cancer risks. METHODS: Dietary records were analyzed over 3-day periods in the mid-follicular phase, twice, at 6-month intervals for 13 premenopausal Oriental women, recent immigrant arrivals in Hawaii with presumed low risk of breast cancer, and for 12 premenopausal Finnish women with presumed higher risk. The urinary estrogen profile was measured by gas chromatography-mass spectrometry and plasma and fecal estrogens were assayed by chromatographic radioimmunoassays. RESULTS: Mean fat intake per 1000 kcal was 73% higher (P < .001) in the Finnish women, but the mean fiber intake and fecal weights were similar to those of the Oriental women. Compared with Oriental women, Finnish women had 46% higher plasma estradiol (P < .01) and 124% higher plasma estrone sulfate (P < .01); however, after adjustment for differences in age and body mass index, only the difference in estrone sulfate remained statistically significant (P < .05). Mean plasma levels of estrone and estradiol correlated with height after adjustment for body mass index (P < .05). Mean plasma levels of estrone and sex hormone-binding globulin were similar. The Finns had higher mean urinary estrone (193%), estradiol (166%), various catechol estrogens (130%-439%), and total estrogen excretion (123%) (all P < .001), but similar 16 alpha-hydroxylated estrogen excretion. As calculated, 16 alpha-hydroxylation of estrone was significantly increased (P < .01) in the Oriental women, but 2-hydroxylation, 4-hydroxylation, and 16 beta-hydroxylation of estrone were similar in both groups. The ratio of catechol estrogen to 16 alpha-hydroxylated estrogen was four to five times higher (P < .001) in the Finnish women. The Oriental women had two to three times higher fecal excretion of estrogens than the Finnish women (P < .01). CONCLUSIONS: Our results indicate that high catechol estrogen formation may be a greater risk factor for breast cancer than high 16 alpha-hydroxylation of estrogens. However, the main risk factor for the Finnish women, as opposed to the Oriental women, may be their higher estrogen levels that result from a higher fat diet, higher estrogen production related to their greater height, and lower fecal estrogen excretion.     

Strategies for the enzymatic enrichment of PUFA from fish oil

PUFA from oil extracted from Nile perch viscera were enriched by selective enzymatic esterification of the free fatty acids (FFA) or by hydrolysis of ethyl esters of the fatty acids from the oil (FA‐EE). Quantitative analysis was performed using RP‐HPLC coupled to an evaporative light scattering detector (RP‐HPLC‐ELSD). The lipase from Thermomyces lanuginosus discriminated against docosahexaenoic acid (DHA) most, resulting in the highest DHA/DHA‐EE enrichment while lipase from Pseudomonas cepacia discriminated against eicosapentaenoic acid (EPA) most, resulting in the highest EPA/EPA‐EE enrichment. The lipases discriminated between DHA and EPA with a higher selectivity when present as ethyl esters (EE) than when in FFA form. Thus when DHA/EPA were enriched to the same level during esterification and hydrolysis reactions, the DHA‐EE/EPA‐EE recoveries were higher than those of DHA/EPA‐FFA. In reactions catalysed by lipase from T. lanuginosus, at 26 mol% DHA/DHA‐EE, DHA recovery was 76% while that of DHA‐EE was 84%. In reactions catalysed by lipase from P. cepacia, at 11 mol% EPA/EPA‐EE, EPA recovery was 79% while that of EPA‐EE was 92%. Both esterification of FFA and hydrolysis of FA‐EE were more effective for enriching PUFA compared to hydrolysis of the natural oil and are thus attractive process alternatives for the production of products highly enriched in DHA and/or EPA. When there is only one fatty acid residue in each substrate molecule, the full fatty acid selectivity of the lipase can be expressed, which is not the case with triglycerides as substrates.