Fenghong Huang

Immobilization of Candida rugosa lipase on hydrophobic/strong cation-exchange functional silica particles for biocatalytic synthesis of phytosterol esters

In this work, mixed-mode silica particles functionalized with octyl and sulfonic acid groups was conveniently prepared by co-bonding a mixture of n-octyltriethoxysilane and 3-mercaptopropyltriethoxysilane and then oxidized with hydrogen peroxide. Candida rugosa lipase (CRL) was immobilized on the mixed-mode silica particles via hydrophobic and strong cation-exchange interaction. The resulting immobilized CRL increased remarkably its stability at high temperature in comparison to free CRL. The immobilized CRL was used as biocatalysts for enzymatic esterification of phytosterols with free fatty acids (FFAs) to produce phytosterol esters. The phytosterols linolenate esterification degree of 95.3% was obtained under the optimized condition. Phytosterols esters could also been converted in high yields to the corresponding long-chain acyl esters via transesterification with methyl esters of fatty acids (80.5%) or triacylglycerols (above 95.5%) using mixed-mode silica particles immobilized CRL as biocatalyst. Furthermore, the immobilized CRL by absorption retained 78.6% of their initial activity after 7 recycles.

Ultrasonic pretreatment for lipase-catalyed synthesis of phytosterol esters with different acyl donors

This study is focused on the enzymatic esterification of phytosterols with different acyl donors to produce the corresponding phytosterol esters catalyzed by Canadia sp. 99-125 lipase under ultrasound irradiation. An ultrasonic frequency of 35 kHz, power of 200 W and time of 1h was determined to guarantee satisfactory degree of esterification and lipase activity. The influence of temperature, substrates concentration and molar ratio was investigated subsequently. The optimum production was achieved in isooctane system at 60°C with phytosterol concentration of 150 μmol/mL and phytosterol to fatty acid molar ratio of 1:1.5, resulting in a phytosterol esters conversion of above 85.7% in short reaction time (8h). Phytosterols esters could also be converted in high yields to the corresponding long-chain acyl esters via transesterification with triacylglycerols (above 90.3%) under ultrasound irradiation. In optimum conditions, the overall esterification reaction rate using the ultrasonic pretreatment process was above 2-fold than that of mechanical stirring process without damage the lipase activity.

Influence of microwaves treatment of rapeseed on phenolic compounds and canolol content.

Rapeseeds were treated with microwaves under 800 W for 0, 1, 2, 3, 4, 5, 6, 7, and 8 min at a frequency of 2450 MHz, and oil was extracted with a press to investigate the influence on phenolic compounds, including sinapine, the main free phenolic acids, and canolol content in the rapeseeds and oil from them. The results indicated that sinapine and sinapic acid was the main phenolic compound and free phenolic acid in the rapeseed, respectively, and canolol was the main phenolic compound in the oil from rapeseed by cold press. Microwave treatment significantly influenced phenolic compounds content in the rapeseeds and oil from them. The sinapine, sinapic acid, and canolol content in rapeseed first increased and then decreased depending on the period of microwave radiation (p < 0.05). The canolol content of 7 min microwave pretreatment rapeseed increased to the maximum and was approximately six times greater than that of the unroasted rapeseed. The amount of canolol formed was significantly correlated with the content of sinapic acid and sinapine (for sinapic acid, r = -0.950, p < 0.001, for sinapine, r = -0.828, p < 0.05) and also the loss of sinapic acid and sinapine (for sinapic acid, r = 0.997, p < 0.001, for sinapine, r = 0.952, p < 0.05) during roasting. There were differences in the transfer rate of difference phenolic compounds to the oil extracted by press. Almost all of the sinapine remained in the cold-pressed cake and only 1.4-2.7% of the sinapic acid, whereas approximately 56-83% of the canolol was transferred to the oil. The transfer ratio of canolol significantly increased with microwave radiation time (p < 0.001). Microwave pretreatment of rapeseed benefited improving the oxidative stability of oil.

Lipase Immobilization on Hyper-Cross-Linked Polymer-Coated Silica for Biocatalytic Synthesis of Phytosterol Esters with Controllable Fatty Acid Composition

In this study, a novel mixed-mode composite material, SiO(2)@P(MAA-co-VBC-co-DVB), was prepared via the hyper-cross-linking of its precursor, which was produced via suspension polymerization in the presence of SiO(2) particles. Candida rugosa lipase (CRL) was immobilized on the SiO(2)@P(MAA-co-VBC-co-DVB) particles via hydrophobic and weak cation-exchange interaction. The resulting immobilized CRL showed much better thermal stability and reusability in comparison to free CRL. On the basis of the excellent biocatalyst prepared, a method for high-efficiency enzymatic esterification of phytosterols with different fatty acids to produce the corresponding phytosterol esters was developed. Six phytosterol esters with conversions above 92.1% and controllable fatty acid composition were obtained under the optimized conditions: 80 μmol/mL phytosterols, 160 μmol/mL linolenic acid, and 15 mg/mL CRL@HPCS at 300 rpm and 50 °C for 7 h in 30 mL of isooctane. The prepared phytosterol esters possessed a low acid value (≤0.86 mg of KOH/g), peroxide value (≤3.3 mequiv/kg), and conjugated diene value (≤1.74 mmol/kg) and high purity (≥97.8%) and fatty solubility (≥28.9 g/100 mL). All the characteristics favored the wide application of phytosterol esters with controllable fatty acid composition in different fields of functional food.

A mixed-function-grafted magnetic mesoporous hollow silica microsphere immobilized lipase strategy for ultrafast transesterification in a solvent-free system

Although enzymatic catalysis is an attractive approach for the green synthesis of chemicals, it often suffers from low reactivity and poor stability during the reaction. In this study, lipase from Candida rugosa (CRL) was immobilized and stabilized on magnetically-separable, mixed-function-grafted, large pore mesostructured magnetic hollow mesoporous silica microspheres (MHMSS) by means of multiple-mode adsorption based on both hydrophobic and strong cation-exchange interactions. Benefiting from the hollow large mesoporous structure, ultrafast enzyme immobilization could be realized in 5 min, with a high loading of CRL (95.2 mg g−1). Stabilized CRL@MHMSS was successfully used for the ultrafast transesterification of phytosterol with fatty acids and triglycerides in a solvent-free system, which reached high conversions (≧90.9%) within 15 min at 55 °C. Magnetic separation of MHMSS facilitated the repeated usage of CRL@MHMSS for more than 50 successive reactions without damaging its catalytic activity. Its high activity and stability make the MHMSS immobilized enzyme an attractive catalyst for green synthesis in a solvent-free system.

Rapid and sensitive determination of Sudan dyes in hot chilli products by solid-phase extraction directly combined with time-of-flight mass spectrometry

A novel method based on combination of a solid-phase extraction (SPE) with humic acid-bonded silica (HAS) as sorbent with electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QTOF MS) was developed for rapid and sensitive determination of four Sudan dyes in hot chilli products. A SPE procedure was optimized for the improvement of extraction efficiency and the removal of the matrix interferences such as the pigments and lipophilic compounds from hot chilli products. Under the optimum conditions, the desorbed analytes from the SPE sorbent allowed direct analysis by ESI-QTOF MS without time-consuming chromatographic separation. In the positive ion mode and MS/MS mode, linearities for concentration ranges of two orders of magnitude were obtained with the correlation coefficients (R2) greater than 0.9990; the limit of detection (LOD) and limit of quantification (LOQ) for four Sudan dyes were in the ranges of 1.1–4.3 and 3.8–14.3 ng g−1 depending on matrices, respectively. The recoveries of four Sudan dyes in two matrices ranged from 85.6 to 119.7%, with relative standard deviations (n = 4, 3) less than 11.3% and 11.6%. The developed method was successfully applied for the determination of Sudan dyes in six hot chilli products and the positive samples were confirmed with a high number of identification points (IPs) according to IPs system defined by Commission Decision 2002/657/EC.

Enzymatic deacidification of the rice bran oil and simultaneous preparation of phytosterol esters-enriched functional oil catalyzed by immobilized lipase arrays

Rice bran oil (RBO) is a great and important vegetable oil resource, but usually contain high content of free fatty acids (FFAs). Phytosterol esters are functional lipids possessing hypocholesterolemic activity. In this study, Candida rugosa lipase (CRL) was immobilized on the octyl-functionalized ordered mesoporous silica (C8-OMMs) to obtain immobilized CRL arrays (CRL@C8-OMMs). The resulting CRL@C8-OMMs arrays showed much better thermal stability and reusability in comparison to free CRL. The deacidification and simultaneous preparation of functional oil rich in phytosterol esters (PEs) was successfully achieved by enzymatic esterification of high-acid RBO with phytosterol. The contents of FFAs and PEs were below 2.3% and above 84.7%, respectively. Repeated reaction tests indicated that CRL@C8-OMSs arrays could be used 11 times with 80.7% of its original catalytic activity still retained. The strategy could not only take advantage of the high-acid oils resources, but also product PEs-enriched functional oils.

Production of Novel “Functional Oil” Rich in Diglycerides and Phytosterol Esters with “One-Pot” Enzymatic Transesterification

Diglycerides and phytosterol esters are two important functional lipids. Phytosterol esters mixed with dietary diglyceride could not only influence body weight but also prevent or reverse insulin resistance and hyperlipidemia. In this study, a kind of novel functional oil rich in both diglycerides and phytosterol esters was prepared with one-pot enzymatic transesterification. First, lipase AYS (Candida rugosa) was immobilized on the porous cross-linked polystyrene resin beads (NKA) via hydrophobic interaction. The resulting immobilized AYS showed much better transesterification activity and thermal stability to freeways. On the basis of the excellent biocatalyst prepared, a method for high-efficiency enzymatic esterification of phytosterols with different triglycerides to produce corresponding functional oils rich in both diglycerides and phytosterol esters was developed. Four functional oils rich in both diglycerides and phytosterol esters with conversions >92.1% and controllable fatty acid composition were obtained under the optimized conditions: 80 mmol/L phytosterols, 160 mmol/L triglycerides, and 25 mg/mL AYS@NKA at 180 rpm and 50 °C for 12 h in hexane. The prepared functional oil possessed low acid value (≤1.0 mgKOH/g), peroxide value (≤2.1 mmol/kg), and conjugated diene value (≤1.96 mmol/kg) and high diglyceride and phytosterol ester contents (≥10.4 and ≥20.2%, respectively). All of the characteristics favored the wide application of the functional oil in different fields of functional food.