Production of highly enantioselective (−)-menthyl butyrate using Candida rugosa lipase immobilized on epoxy-activated supports

Optically active (−)-menthyl butyrate was synthesized by enantioselective esterification of racemic (±)-menthol and butyric anhydride using lipase from Candida rugosa immobilized onto epoxy-activated supports of Eupergit C and Eupergit C 250 L through physical adsorption method. The effects of various temperature, storage condition, stability in organic solvent and lipase recyclability were investigated for their influence on the enzymatic enantioselective formation of (−)-menthyl butyrate. The immobilized lipases retained high catalytic activity of up to 31% yield and 100% enantiomeric excess of the desired product, and showed better stability compared to the native lipase. They also exhibited about 50% retained activity even after incubation at higher temperatures, storage at room temperature and after long incubation in hexane. Immobilized lipases also showed considerably efficient reusability.     

Optimization of lipase-catalyzed enantioselective esterification of (±)-menthol in ionic liquid

Response surface methodology was successfully applied to optimize lipase-catalyzed enantioselective esterification of (±)-menthol. The effects of various reaction conditions, including reaction time, temperature, enzyme loading, substrate molar ratio and water activity, were investigated. A Central Composite Rotatable Design was employed to search for the optimal conversion of (±)-menthol and enantiomeric excess. A quadratic polynomial regression model was used to analyze the experimental data at a 95% confidence level (p < 0.05). The analysis confirmed that reaction temperature, enzyme loading and reaction time were the significant factors affecting the conversion of (±)-menthol. Moreover, reaction temperature, enzyme loading, substrate molar ratio and reaction time were found to affect the enantiomeric excess significantly. The coefficient of determination of these two models was found to be 0.980 and 0.967, respectively. Two sets of optimum reaction conditions were established and the verified experimental trials were performed for validating the optimum points. Under the optimum conditions, the conversion of (±)-menthol and the enantiomeric ratio exceeded 53% and 40%, respectively.     

Isoamylacetate production by entrapped and covalently bound Candida rugosa and porcine pancreatic lipases

Candida rugosa lipase (CRL) and porcine pancreatic lipase (PPL) were immobilised by entrapping and also by covalent binding for use in synthesis of isoamyl acetate (IAAc), which has a typical banana flavour. Lipase entrapment was carried out by dripping sodium alginate (Na-Alg)-chitosan (Chi)-lipase mixture into CaCl₂-glutaraldehyde (GAL) solution to obtain Ca-Alg/Chi_CRL/PPL. Immobilisation conditions were optimised as 1.5% Na-Alg, 1.5% chitosan and 0.15% GAL. Ca-Alg/Chi_CRL/PPL samples showed the highest activity when they were dried upon reaching 27% of their initial weights. Covalent binding was achived with Chi modified with spacerarm via glutaraldehyde to get Chi_CRL/PPL. The highest IAAc production was observed when 1,3-diaminopropane was used as a spacer arm. The best ester yield was achieved in heptane, at 40 and 45 °C reaction temperatures, 50 mM IAA and 50 or 75 mM AA concentrations. The amount of IAAc was nearly 10 times higher for the batch type than for the continuous packed bed column reactor.     

Comparison of lipase-catalyzed enantioselective esterification of (±)-menthol in ionic liquids and organic solvents

Enantioselective esterification of (±)-menthol was studied using Candida rugosa lipase (CRL) in ionic liquids (1-butyl-3-methyl-imidazolium hexafluorophosphate ([BMIM][PF₆]) and 1-butyl-3-methyl-imidazolium tetraflouroborate) and organic solvents of different hydrophobicities. Propionic anhydride was employed as an acylating agent. Because the enzyme showed comparable conversion yield and enantioselectivity in [BMIM][PF₆] and hexane in a 24-h reaction, more work focused on these two reaction media. Comparison of the activity, stability and enantioselectivity of CRL was carried out by examining the effects of the mole ratio of substrates, temperature, incubation time and enzyme recycling. It was found that temperature control was more crucial in the ionic liquid than in hexane to reach high conversion and enantioselectivity. The ionic liquid system showed an advantage of using less acid anhydride to achieve higher (±)-menthol conversion yield and better enantioselectivity. Moreover, during an incubation of 4–60 days in the ionic liquid, CRL activity was 2.5 times higher than its initial value, while that in hexane decreased to less than 60% in 2 days. In addition, the enzyme showed potentiality of recycled use in the ionic liquid. These advantages of the ionic liquid suggest that it would be used as a green alternative to organic solvents for the enantioselective esterification of (±)-menthol.     

Repeated hydrolysis process is effective for enrichment of omega 3 polyunsaturated fatty acids in salmon oil by Candida rugosa lipase

Enrichment of omega 3 polyunsaturated fatty acids (PUFA) in the glyceride fraction of salmon oil was performed by Candida rugosa lipase (CRL)-catalysed hydrolysis. Total omega 3 PUFA content in the product was 38.71% (mol.%), more than double of the initial level. The hydrolysis reaction was scaled up to 1 l in a stirred tank reactor without any decrease in the selectivity of the reaction. Moreover, non-steroselectivity and acyl chain specificity of CRL-catalysed hydrolysis, as well as CRL’s recognition of the whole triacylglycerol molecule, were proved. The product was purified by short path distillation, which was also shown to contribute to the recovery of omega 3 PUFA in residue. After the removal of free fatty acids, the final product was subjected to a second round of hydrolysis to concentrate omega 3 PUFA further to 50.58%. Recoveries of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) decreased slightly after the second round of hydrolysis, while oleic acid (OA) content was not affected.     

响应面法优化皱褶假丝酵母脂肪酶催化合成甾醇共轭亚油酸酯

以皱褶假丝酵母(Candida rugosa)脂肪酶为催化剂,在正己烷体系中催化植物甾醇与共轭亚油酸合成甾醇共轭亚油酸酯.以植物甾醇酯化率为考察指标,通过单因素实验和响应面实验确定最佳工艺参数为:酸醇摩尔比6∶1,反应温度40℃,酶用量9.6％(占底物质量),反应时间87 h.在此条件下,酯化率达97.5％.     

The Influence of Debaryomyces hansenii, Candida deformans and Candida zeylanoides on the aroma formation of dry-cured “lacón”

The volatile profile of dry-cured “lacón” that has been inoculated with three different yeasts were determined and compared with a non-inoculated dry-cured “lacón”. Yeasts (Debaryomyces hansenii, Candida deformans and Candida zeylanoides) that were used as starter cultures in the present study were selected among yeasts that were isolated from native dry-cured “lacón” at different stages of ripening process. These starters were spread on dry-cured “lacón” surface in order to test their capacity to contribute on the generation of volatile compounds. A total of forty two volatile compounds were detected by dynamic headspace sampling followed by gas chromatography–mass spectrometry analysis. Significant differences (P < 0.001) on the volatile profiles of different batches were found in comparison with non-inoculated samples, showing the highest total area values for the inoculated ones. Esters were the most abundant chemical family in all batches studied except for C. zeylanoides batch, which showed greater amount of hydrocarbons than esters. The second more abundant family was hydrocarbons for control and C. deformans batches (147.6 and 445.24 × 10⁶ area units, respectively), alcohols for D. hansenii (363.77 × 10⁶ area units) and esters for C. zeylanoides (248.33 × 10⁶ area units). However, the aldehyde compound group in control batch samples was found to be significantly higher than in the inoculated ones (P < 0.001). Among inoculated batches, D. hansenii batch showed the lowest hexanal content (14.42 × 10⁶ area units) in comparison with non-inoculated batch (105.99 × 10⁶ area units). Among all batches studied, D. hansenii batch presented the highest area values for esters, alcohols, linear hydrocarbons, ketones, acids and furans; control batch for aldehydes and C. zeylanoides batch for branched hydrocarbons. Therefore, the study showed that every yeast strain produced a specific volatile profile which was also different from that of the control dry-cured “lacón”.     

中空纳米粒子负载光催化纤维的制备及其性能

采用溶胶-凝胶法依次在TiO2表面包覆有机物和SiO2,经高温煅烧除去中间的有机物,成功制得以TiO2为核、SiO2为壳的具有核壳中空结构的新型光催化剂(SiO2@@TiO2),并将此催化剂负载到经碱减量处理的涤纶纤维上,制得了新型光催化纤维(SiO2@@TiO2/PET).采用TEM、EDS、SEM对样品进行表征,同时测试了SiO2@@TiO2对载体的保护性能和SiO2@@TiO2/PET对甲醛的催化降解性能.结果表明:核壳中空结构不仅可以有效地防止TiO2直接和有机载体接触,保护有机载体不被腐蚀,同时甲醛气体可有效地被TiO2光催化降解.     

Enzymatic synthesis of rose aromatic ester (2-phenylethyl acetate) by lipase

BACKGROUND: 2‐Phenylethyl acetate (2‐PEAc) is a highly valued natural volatile ester with a rose‐like odour that is widely used to add scent or flavour to cosmetics, soaps, foods and drinks. In this study, 2‐PEAc was synthesised enzymatically by transesterification of vinyl acetate with 2‐phenethyl alcohol catalysed by immobilised lipase (Novozym^® 435) from Candida antarctic RESULTS: Response surface methodology and a three‐level/three‐factor Box–Behnken design were used to evaluate the effects of time, temperature and enzyme amount on the molar conversion % of 2‐PEAc. The results showed that temperature was the most important variable. Based on the ridge max analysis results, optimum enzymatic synthesis conditions were predicted as a reaction time of 79 min, a temperature of 57.8 °C and an enzyme amount of 122.5 mg. The predicted and experimental yields were 86.4 and 85.4% respectively. CONCLUSION: Three immobilised lipases were screened and 15 reaction conditions were tested in order to find the combination for maximum yield. The optimisation of 2‐PEAc synthesis catalysed by Novozym^® 435 was successfully developed. The kinetic study of this transesterification reaction showed that it followed an ordered ping‐pong bi‐bi mechanism without any inhibition by reactants. Copyright © 2012 Society of Chemical Industry     

响应面法优化以纯棉浴巾为载体的脂肪酶固定化工艺

本文系统研究了以纯棉浴巾为载体的脂肪酶固定化条件,通过单因素实验确定了吸附剂聚乙烯亚胺(PEI)浓度及其pH、交联剂戊二醛(GA)浓度及其pH、交联时间和交联温度的优化范围,进而以PEI浓度、GA浓度和交联时间作为优化因素,通过三因素三水平响应面设计,得到固定化酶制备优化条件为:PEI浓度3.37 mg/mL、GA浓度...     

Effect of the size and interface composition of milk fat globules on their in vitro digestion by the human pancreatic lipase: Native versus homogenized milk fat globules

Although the bioavailability of dietary lipids is of primary importance in human nutrition and health, the mechanisms involved in lipid digestion are not fully understood and are of growing interest. The objective of this study was to determine the effect of the size of milk fat globules and of the composition of their interface on the activity of the human pancreatic lipase (PL). Native milk fat globules of various sizes covered by their biological membrane (MFGM) and homogenized fat globules of various sizes covered by milk proteins were prepared from whole milk and underwent lipolysis by the human PL with colipase and bile salts. A lag phase preceding the hydrolysis of milk TAG occurred with all native milk fat globules samples but not with homogenized milk samples. The kinetic parameters of human PL were determined by measuring the enzyme activity either after the lag phase for native milk fat globules samples or immediately after the addition of the enzyme for homogenized milk samples. The catalytic efficiency of human PL is 4.6-fold higher on small (1.8 μm) than large (6.7 μm) native milk fat globules, related to a 3.6-fold larger available surface. Despite the 25-fold larger available surface, milk TAG from homogenized milk are only 2-fold better hydrolyzed compared to native milk fat globules, as a possible result of a less favourable interface covered by milk proteins. The potential mechanisms involved in native vs. homogenized milk fat globules digestion by the human PL are discussed. Our study highlights the crucial role of the MFGM in the efficient digestion of milk fat globules and brings new insight for the design of dairy products and infant formulas.     

Inhibition of lipase from rice (Oryza sativa) by diethyl-p-nitrophenyl phosphate

The inhibition of rice bran lipase (RBL) by diethyl-p-nitrophenyl phosphate was studied with reference to kinetics, nature of inhibition and also elucidate the effect of the inhibitor on the structure—function of the enzyme. Enzyme activity measurements shows that the inhibitor is more effective at 0.050 mM concentration of diethyl-p-nitrophenyl phosphate and the activity is 50% at this level of inhibitor concentration. The affinity of substrate for the enzyme was observed by the increase in the velocity of the reaction with increase in the substrate concentrations and double reciprocal plot indicates that the inhibition followed a competitive in nature and inhibition constant K _i is found to be 0.016 mM at pH 7.0. The decrease in apparent thermal denaturation temperature to 4 °C compared to control indicates the destabilization of enzyme in the presence of inhibitor. Fluorescence spectral measurements suggests that pronounced quenching of fluorescence intensity of RBL occurs at higher concentrations of diethyl-p-nitrophenyl phosphate and ‘K _a’ value was found to be 2.4 × 10⁴ M⁻¹ with free energy change ΔG^o—26 kJ/mol at 30 °C suggesting strong binding between the enzyme and the inhibitor with microenvironmental changes occur at the active site or in the neighbourhood of active site. The far UV-CD data suggest that there is no significant changes in the conformation of the enzyme as a result of binding of diethyl-p-nitrophenyl phosphate. These results indicate that diethyl-p-nitrophenyl phosphate is a inhibitor of RBL and binds to the enzyme in brining about inhibition without any structural alterations.     

Adulterations in Basmati rice detected quantitatively by combined use of microsatellite and fragrance typing with High Resolution Melting (HRM) analysis

The aim of this work was to setup a DNA based method coupled with High Resolution Melting (HRM) analysis for rice products traceability using five different microsatellite markers to genotyping Basmati and non-Basmati varieties. We also exploit the obtained information to detect the presence of Basmati varieties in commercial rice products. Additionally we used the 8 bp deletion in badh2 gene in combination with HRM to both DNA-typing of the Basmati and non-Basmati varieties and to quantitate accurately adulteration of Basmati rice products with non-Basmati rice products. HRM proved to be a very sensitive tool to genotype rice varieties and detect admixtures as well as able to detect a ratio of 1:100 of non-fragrance in fragrance rice. In conclusion HRM analysis can be a higher resolution, cost effective, alternative method compared to other techniques that could be extended to quantify adulterations in rice varieties and commercial rice food products.     

Barcode High Resolution Melting (Bar-HRM) analysis for detection and quantification of PDO “Fava Santorinis” (Lathyrus clymenum) adulterants

Legumes considered as one of the most important crops worldwide. Due to high price as a PDO product, commercial products of “Fava Santorinis” are often subjected to adulterations from other legume products coming from other Lathyrus or Vicia and Pisum species. Using plant DNA barcoding regions (trnL and rpoC) coupled with High Resolution Melting (Bar-HRM) we have developed a method allowing us to detect and authenticate PDO “Fava Santorinis”. Bar-HRM proved to be a very sensitive tool able to genotype Lathyrus and its closed relative species and to detect admixtures, being sensitive enough to as low as 1:100 of non-“Fava Santorinis” in “Fava Santorinis” commercial products. In conclusion, Bar-HRM analysis can be a faster, with higher resolution and cost effectiveness alternative method to authenticate PDO “Fava Santorinis” and to quantitatively detect adulterations in “Fava Santorinis” with other relative commercial “Fava” food products.     

Proximate composition of guariroba (Syagrus oleracea), jerivá (Syagrus romanzoffiana) and macaúba (Acrocomia aculeata) palm fruits

This study aimed to characterize the pulp and kernel of guariroba (Syagrus oleracea), jerivá (Syagrus romanzoffiana) and macaúba (Acrocomia aculeata) palm fruits, through their proximate composition, carotenoids contents and tocopherol composition. The three kernels showed to be composed mainly of lipids and proteins, as the three pulps, of carbohydrate and fiber. In the kernels the levels of lipids ranged from 45.17 to 56.37% and proteins from 15.46 to 28.61%. In the pulps the total fiber content ranged from 20.26 to 26.98%. The pulps also presented a significant amount of ash, which represents a significant mineral content, especially in the guariroba (5.16%). Moreover, the pulp oils showed higher carotenoids and tocopherol contents. The jerivá pulp oil contained carotenoid and tocopherol on average 1219 ??g/g and 323.50 mg/kg, respectively. The consumption of the whole fruit, pulp, and kernel supplies important quantities of many necessary nutrients for human diet, including vitamins A and E.     

淀粉接枝聚醋酸乙烯酯纳米粒的制备及其对虾青素稳定性影响

以二羟基二过碘酸合镍(IV)钾作为引发剂,引发醋酸乙烯酯在淀粉上接枝,生成淀粉接枝共聚物。再用三聚磷酸钠作交联剂制备成具有疏水核心、亲水表面的纳米粒。然后采用透析法,将虾青素与纳米粒混合制成虾青素纳米粒。测定淀粉及接枝共聚物的红外光谱、纳米粒的平均粒径、粒径分布、纳米粒形态,并以虾青素的丙酮溶液作为比较,对虾青素纳米粒的稳定性进行测定。结果表明,在不同条件下,虾青素纳米粒比原料的稳定性均有提高。