Partial hydrolysis of soybean oil by phospholipase A1 (Lecitase Ultra)

The partial hydrolysis of soybean oil, as catalysed by phospholipase A₁ (Lecitase Ultra) in a solvent-free system, was investigated in this study. The optimal pH and temperature for the partial hydrolysis of soybean oil by phospholipase A₁ (Lecitase Ultra) were 6.8 and 40 °C, respectively. Phospholipase A₁ (Lecitase Ultra) displayed good stability over a range of pH values from 4.7 to 7.4, and at temperatures below 60 °C. Phospholipase A₁ (Lecitase Ultra) is postulated to possess sn-1,3-position regiospecificity towards triacylglycerols (TAGs), based on the identification of the fatty acids released from TAGs following partial hydrolysis by swine pancreatic lipase (SPL) and phospholipase A₁ (Lecitase Ultra). Alternative TAG hydrolysis routes for phospholipase A₁ (Lecitase Ultra) are postulated, and several reaction equilibrium and rate constants were determined.     

Production of palm oil-based diacylglycerol using Lecitase Ultra-catalyzed glycerolysis and molecular distillation

Diacylglycerol (DAG) was prepared via glycerolysis of palm oil catalyzed by Lecitase Ultra (LU), a novel phospholipase from the fusion of lipase genes from Thermomyces lanuginose and phospholipase genes from Fusarium oxysporum. Glycerolysis was performed in a solvent-free system. The optimized reaction conditions were: a glycerol/palm oil mole ratio of 7.5:1, initial substrate water content of 5%, substrate enzyme load of 2%, reaction temperature of 40°C, and reaction time of 8 h. In a scale-up reaction, a DAG content of 59.5% in the lipid layer was achieved. Through a two-step molecular distillation, the composition of the target product was 88.1% DAG, 2.8% TAG, 9.0% MAG, and 0.1% FFA. The fatty acid composition of the DAG oil, determined using GC-MS, was enriched compared with the original palm oil.     

Changes in rheological behaviour and functional properties of hen's egg yolk induced by processing and fermentation with phospholipases

Functional changes of hen's egg yolk rheological behaviour as a result of processing (pasteurisation, freeze-drying) and modifying its phospholipids using phospholipase A₁ (PLA₁) and phospholipase A₂ (PLA₂) were studied. It could be shown how fermentation with phospholipases affects the different steps of heat-induced gelation of egg yolk. As a consequence of fermentation of native yolk with 0.1% PLA₁, the first viscosity maximum was shifted from 79 to 97 °C. In fermented pasteurised yolk, the homogenous gel-like consistency from the first viscosity maximum was even stabilised up to 100 °C. The second viscosity ascent, which indicates solid gel structures (caused by aggregation of denatured proteins), was prevented entirely. In contrast to pancreatic PLA₂, fermentation of egg yolk with microbial PLA₁ resulted in a hydrolysis of approximately 78% of phosphatidylethanolamine and a significant change in flow behaviour (of yolk solutions).     

磷脂酶Lecitase Ultra和磷脂酶C复合对冷榨菜籽油脱胶的影响北大核心CSCD

为了提高脱胶效率,以冷榨菜籽原油为原料,磷脂含量为指标,采用磷脂酶Lecitase Ultra和磷脂酶C复合酶法对冷榨菜籽油进行脱胶。采用单因素试验考察磷脂酶Lecitase Ultra反应时间、磷脂酶C反应时间、加水量、磷脂酶Lecitase Ultra添加量、磷脂酶C添加量、柠檬酸溶液添加量对脱胶油磷脂含量的影响,并通过响应面法优化脱胶条件。对优化的脱胶条件下所得到的脱胶油的理化指标进行了检测,并与国标一级压榨菜籽油进行了比较。结果表明:磷脂酶Lecitase Ultra和磷脂酶C对冷榨菜籽油进行酶法脱胶的最佳工艺条件为磷脂酶Lecitase Ultra添加量33 mg/kg,磷酯酶Lectase Ultra反应时间90 min,磷脂酶C添加量65 mg/kg,磷脂酶C反应时间60 min,加水量33 mL/kg,柠檬酸溶液添加量1.2 mL/kg;在优化条件下脱胶,脱胶油中磷脂含量为2.3 mg/kg,脱胶油的过氧化值和酸值均达到一级压榨菜籽油的国家标准。综上,磷脂酶Lecitase Ultra和磷脂酶C复合脱胶效果较好,所优化的工艺条件可用于菜籽油的脱胶。     

磷脂酶A1(Lecitase Ultra)催化水解油脂机理研究(Ⅰ)——磷脂酶A1(Lecitase Ultra)组成及酶学特性

采用凝胶色谱、凝胶电泳和生物质谱分析了磷脂酶A1(Lecitase Ultra)(PLA1)的主要组成,结果表明PLA1主要由4种同工酶组成,这4种同工酶在氨基酸序列上与棉状嗜热丝孢菌(Thermomyces lanuginosus)的脂肪酶高度吻合.测得PLA1水解大豆油的最适pH为6.8,最适温度为40℃.PLA1在pH 4～8之间,温度低于45℃下具有良好的稳定性,在pH 3以下和pH 10以上不稳定.     

PURIFICATION AND PROPERTIES OF PHOSPHOLIPASE A2 ISOZYMES FROM PYLORIC CECA OF THE STARFISH (ASTERINA PECTINIFERA)

Phospholipase A₂ isozyme II (PLA₂ II), which showed different mobility on native PAGE from that of the PLA₂ isozyme I (PLA₂ 1) isolated previously, was purified from pyloric ceca of the starfish (Asterina pectinifera). The PLA₂ II mainly released oleic acid from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. N-terminal amino acid sequence of the PLA₂ II was SVYQF. Temperature and pH optima of the PLA₂ II were at around 50C and pH 9.0, respectively, and the enzyme activity was enhanced by sodium deoxycholate and 1 mM or higher concentration of Ca²⁺. The PLA₂ II did not show fatty acid specificity for hydrolysis of phosphatidylcholine (PC). Specific activity of the PLA² II was about 10 times higher than that of commercially available porcine pancreatic PLA₂. The PLA₂ II hydrolyzed PC more effectively than phosphatidylethanolamine. These characteristics of the PLA₂ II were the same as those of the PLA₂ I.     

磷脂酶A1(Lecitase Ultra)水解磷脂酰胆碱的动力学和热力学研究

本论文研究了磷脂酶A1(Lecitase Ultra)在水相体系中水解磷脂酰胆碱(PC)的动力学和热力学性质。依据Arrhenius经验方程式,计算出了催化水解反应的活化能为5.96 kJ/mol;并进行了Lecitase Ultra水解PC的Briggs-Haldane稳态法酶催化模型动力学模拟,利用底物抑制原理进行了模型修正;采用Lineweaver-Burk作图法,计算出反应的动力学参数Km,Vmax和kI分别为4.02 × 10-2 mol/L、10.05 mol/(L.min)和1.33 × 102 mol/L。研究结果为进一步探讨Lecitase Ultra催化机理及其变性机理提供了基础数据和理论支撑。     

磷脂酶A1(Lecitase Ultra)大孔树脂固定化研究

选用9种树脂对磷脂酶A1(Lecitase Ultra)进行固定化,比较得出最适合的固定化载体为D101型号的大孔树脂.对其固定化条件进行优化,得到了固定化最优的条件为:酶液和树脂液料比1∶1(mL/g),磷酸盐缓冲液pH为6.5,室温下固定化时间60 min.在此条件下的得到的固定化酶的酶活力为750.0 U/g.通过扫描电子显微镜观测了固定化前后,树脂表面特征的变化.     

In situ measurement of white wine absorbance in clear and in coloured bottles using a modified laboratory spectrophotometer

In situ absorbance of visible light by white wine in bottles was determined with a modified spectrophotometer. A linear correlation was obtained between absorbance at 420 nm (A₄₂₀) measured in a cuvette (10 mm pathlength) and absorbance of that same wine measured in a 750 mL Flint bottle (colourless transparent glass). In addition, 5 types of coloured bottles, Emerald Green, Classic Green, French Green, Georgia Green and Cobalt Blue , also yielded strong correlations. A₄₂₀ of white wine in bottles of Antique Green or Amber glass could not be measured directly due to a strong absorbance of that wavelength by the glass. However, a strong correlation was established between measurement of A₄₂₀ (cuvette) and A₅₄₀ for white wine in Antique Green bottles and between A₄₂₀ (cuvette) and either A₅₄₀ or A₆₀₀ for white wine in Amber bottles. The method employed a standard UV-Visible spectrophotometer with an inexpensive modified sample port, and proved applicable to bottles with diameters ranging from 62 mm (375 mL capacity half bottles) up to 81 mm (750 mL capacity Burgundy bottles). In situ absorbance measurements plus subsequent chemical analysis and sensory assessment of a Chardonnay wine in Antique Green bottles showed that the method provided an estimate of oxidation in this white wine. Wine absorbance spectra over the full visible range were obtained in the Flint bottle. Measurements of the absorbance of visible light by the glass of empty bottles could also be obtained and used as a form of quality control for glass bottle manufacture.     

Phospholipase A2-mediated synthesis of phosphatidylethanolamine containing highly unsaturated fatty acids

Phosphatidylethanolamine (PE), which contains highly unsaturated fatty acid (HUFA) specifically in sn -2 position, was synthesized by porcine pancreatic phospholipase A₂-catalysed esterification of lysophosphatidylethanolamine with HUFA. the use of glycerol containing only a little water as solvent was the important key to this reaction. A 27% yield of up to 94.5% sn -2 eicosapentaenoic acid (EPA)-containing PE was obtained using >99% EPA as a substrate HUFA.     

磷脂酶A1(Lecitase Ultra)的纯化及氨基酸序列分析

本论文研究了超滤法和RP-HPLC对磷脂酶A1(Lecitase Ultra)的纯化,利用MALDI SYNAPT Q-TOF MS和MS/MS对其氨基酸序列进行分析及推测。纯化去除了Lecitase Ultra中的山梨醇和山梨醇盐,纯度97.7%,酶蛋白和亚基的分子量为30838.0和16536.0。通过MALDI SYNAPT Q-TOF MS和MS/MS对酶蛋白进行分析,确定了Lecitase Ultra的氨基酸序列组成;进行数据库(http: //www.matrixscience.com)比对,发现其氨基酸序列信息与棉状嗜热丝孢菌(T. lanuginosus)脂肪酶和尖孢镰刀菌(F. oxysporum)脂肪酶高度吻合,序列中1-284是T. lanuginosus脂肪酶的氨基酸序列,序列中285-339是脂肪酶F. oxysporum脂肪酶的氨基酸序列,其中113、118和121可能是被基因突变成113 G-A、118 D-W和121 E-K。研究结果为进一步探讨Lecitase Ultra的三维空间结构及其活性中心催化机理提供了数据支撑。     

酶法制备n-3多不饱和脂肪酸型磷脂的工艺

研究了无溶剂体系中固定化磷脂酶A1催化大豆磷脂与乙酯型鱼油进行酯交换反应,制备n-3多不饱和脂肪酸型磷脂的工艺。考察了反应温度、反应时间、底物比(乙酯型鱼油与磷脂的质量比)以及加水量的影响,得到了较优的反应条件。实验结果表明,加酶量(按大豆磷脂的质量计算)20%,底物比8∶1,底物总质量5.0g,加水量55μL,55℃反应12h时,所得产物中EPA和DHA的含量分别为8.0%和17.8%。     

Extraction of Poppy Seed Oil Using Supercritical CO2

ABSTRACT: Extraction of poppy seed oil with supercritical carbon dioxide (SC-CO₂) was performed and the effect of extraction conditions on oil solubility and yield as well as oil composition was evaluated. Within the temperature (50 to 70 °C) and pressure (21 to 55 MPa) ranges studied, 55 MPa/70 °C gave the highest oil solubility (24.1 mg oil/g CO₂) and oil yield (38.7 g oil/100g seed). Fatty acid composition of the oil obtained with SC-CO₂ at 55 MPa/70 °C was similar to that of petroleum ether-extracted oil ( p > 0.05) with linoleic acid making up 69.0 to 73.7% of fatty acids. Tocol content of the SC-CO₂-extracted oils varied from 22.37 to 33.35 mg/100 g oil, which was higher than that of petroleum ether-extracted oil (15.28 mg/100 g oil). Poppy seed oil may have potential in the rapidly growing specialty oil market.     

THE EFFECT OF IRRIGATION WITH TREATED WASTEWATER ON "CHEMLALI" OLIVE OIL QUALITY

The aim of this work was to study the effect of the irrigation with treated wastewater (TWW) on the quality parameters of "Chemlali" olive oil through two crop seasons (2004/2005 and 2006/2007). The results showed that irrigation with TWW did not affect the examined oil quality indices (free acidity, specific ultraviolet absorbance K₂₃₂ and K₂₇₀). During the two crop seasons, the mean values of these parameters are lower than the upper limits established for the best commercial olive oil quality designated as "Extra Virgin." However, a significant increase of palmitic, palmitoleic, linoleic, linolenic and stearic acid contents was found in the 2006/2007 crop season in the plot irrigated with TWW as compared to the one treated with well water. In addition, a decrease of oleic acid and polyphenol content was observed at the end of experimental period.

PRACTICAL APPLICATIONS

The availability of fresh water is very scarce in the Middle East and North Africa. Yet consequently, good quality water is used to irrigate crops more sensitive to water shortage than olive trees. Brackish water or recycled wastewater can be used for irrigation, minimizing the exploitation of natural water resources. In Tunisia, treated wastewater (TWW) is commonly used to irrigate olive trees. The aim of the present study was to assess changes in quality characteristics (free acidity, specific ultraviolet absorbance K₂₃₂ and K₂₇₀, fatty acid composition and total phenolic compounds) of "Chemlali" olive oil obtained from trees irrigated with TWW.     

The preparation of protein hydrolysate from defatted coconut and soybean meals

This study was conducted to determine the effects of temperature, time and concentration of acids on the hydrolysis of coconut and soybean meals. Using 6∼ hydrochloric acid (HCl), the complete hydrolysis of soybean meal was reached after 36 hr at 95°C, while it took only 24 hr to complete the process when 18 N sulphuric acid (H₂SO₂) was used at the same temperature. Coconut protein exhibited some degree of resistance to hydrolysis. Using 10 N HC1 and 18 N H₂SO₂ in two separate tests, it took 48 hr to complete hydrolysis at 95°C. Sulphuric acid caused a considerably greater decomposition of amino acids than HCl during longer periods of reaction with high acid concentration and temperature. Flavour development is a function of the free amino acids released which in turn is a function of acid concentration, reaction time and temperature.     

Detecting vegetable oil adulteration in hazelnut paste (Corylus avellana L.)

The possibilities of detecting hazelnut paste adulterated with refined and non-refined vegetable oils have been studied. Research was focussed mainly on peanut, high oleic-acid sunflower, corn and soybean oils which have a similar composition to hazelnut oil. The analytical procedures to detect fatty acid (FA), triacylglycerol (TAG) and tocopherol profiles as indicators of adulteration were determined. The better indicators experimentally determined were seven FA (palmitic, stearic, linoleic, linolenic, arachidonic, behenic and lignoceric acids) and different TAG with three unsaturated FA (the code letters used for FA are: P = C_16:0; S = C_18:0; O = C_18:1; L = C_18:2;; L_n = C_18:3) (LLL_n, LLL and OOO), two unsaturated FA (POL, PLL and SOO), and one unsaturated FA (PPL). As expected, when refined vegetable oils were added to hazelnut paste, the increment of stigmasta-3,5-diene allowed detection at levels of 2% oil added. Limits of detection were measured using standard and adulterated hazelnut with different amounts of non-refined vegetable oils added (5%, 10%, 20% and 30%). The distribution of tocopherols and tocotrienols is highly useful, except in the case of added sunflower oil. The differences between the experimental and theoretical values of the TAG with equivalent carbon number (ΔECN) of 42 does not improve the detection limit of hazelnut paste adulterated with peanut or sunflower oils. Similarly, tocopherols usually added to refined vegetable oils as an antioxidant were also determined.     

Specificity in Thermal Hydrolysis of Triglycerides

SUMMARY— Samples of triglycerides and triglyceride mixtures were heated in the presence of water under controlled conditions and the released fatty acids quantitatively analyzed by gas chromatography. Experiments with both a mixture of monoacyl-triglycerides and glycerides with equimolar amounts of randomly distributed fatty acids showed a preference for the hydrolysis of the shorter chain and the unsaturated fatty acids. The C₄, C₈, C₁₂, and C_18:1, fatty acids were used in the above mixtures. A trilaurin, in which the fatty acid in the P-position is labelled with C¹⁴, was synthesized. When the free acids released by heat were analyzed by a combination gas chromatographic-radioactivity detector system, no evidence for a positional specificity was apparent.     

CHANGES IN FREE FATTY ACID AND DIACYLGLYCEROL COMPOUNDS IN SHORT-RIPENING DRY-CURED SAUSAGE

A study on lipolysis of Southern Italy short-ripened dry-cured sausages manufactured without and with starters was carried out. Samples were submitted to microbiological and chemical analysis during drying and ripening, whereas sensory analysis was performed at the end of the ripening. The starters, addition caused the inhibition of the Enterobacteriaceae. An increase of free fatty acids (FFAs) and diacylglycerols (DAGs) during the ripening was observed. The addition of starter did not affect FFA and DAG release. The DAG profile including 1,2- and 1,3-isomers was analyzed for the first time in dry-cured sausages. Oleic, linoleic and palmitic acids, and 1,2-, 1,3-palmitinolein, 1,2- and 1,3-diolein were the most abundant compounds in FFA and DAG, respectively. A decrease in the concentration of some 1,2-DAG and an increase of 1,3-OO and 1,3-OP species during the process was observed. Finally, the addition of starter cultures slightly influenced the sensory properties of sausages.

PRACTICAL APPLICATIONS

In this work the development and application of an analytical method for the determination of free fatty acids (FFAs) and diacylglycerols (DAGs) in sausages was achieved. DAGs are intermediate products of hydrolysis and also constitute a substrate for hydrolysis reactions leading to FFA release. These latter compounds undergo a series of reactions leading to the formation of low-molecular-weight products responsible for sensory properties of dry-cured sausages. Therefore, the assessment of these lipolysis products is a useful tool for evaluating the ripening process of dry-cured sausages.     

ADSORPTION EFFICIENCY OF SELECTED ADSORBENTS IN SESAME OIL MISCELLAS

In a sesame oil miscella (40% and 60%) system the adsorption efficiency for free fatty acids (Q_FFA.) and carotenoids (Q_c) provided by pine wood carbon (also known as vegetable carbon), two commercial synthetic silica adsorbents, and rice hull ash were compared at different adsorbent dosages (1.0, 2.5 and 5.0%).
At all the adsorbent dosages investigated the pine wood carbon achieved a higher Q_FFA than synthetic silicas and rice hull (P < 0.05). This effect was more evident as the carbon concentration decreased (P < 0.05). However, in the case of carotenoids none of the adsorbents exhibited a significant advantage in Q_c (P < 0.50) at any of the adsorbents dosages utilized. Rice hull ash had the highest oil retention, while silicas exhibited an oil retention profile with lower values than that obtained with pine wood carbon (P < 0.10). The results suggest that pine wood carbon might have the same capability as silicas when used with sequential addition of clay in sesame oil miscella refining, with the advantage of higher adsorption efficiency than silicas for free fatty acid adsorption.     

Enzyme-Catalyzed Production and Chemical Composition of Diacylglycerols from Corn Oil Deodorizer Distillate

Diacylglycerols (DAG) were enzymatically synthesized by lipase-catalyzed esterification of glycerol with fatty acids from corn oil deodorizer distillate (CrODD). Effects of reaction parameters such as reaction time, temperature, enzyme type, enzyme load, substrate mole ratio, and water content, as well as the effect of molecular sieves as a water adsorbent were investigated. Rhizomucor miehei lipase (Lipozyme RM IM) was found to be most effective among the lipases screened. The following conditions yielded 70.0% (w/w) DAG: 5 h reaction time, 65°C reaction temperature, 10% (w/w) Lipozyme RM IM, 2.5:1 fatty acid to glycerol molar ratio, and 30% (w/w) molecular sieves. DAG synthesis of 12.4% (w/w) was still observed at 10% (w/w) water content. 84.2% (w/w) of DAG was obtained after purification. The DAG oil comprised predominantly of 1-oleoyl-3-linoleoyl-glycerol (28.5%), 1,3-diolein (22.7%), 1-oleoyl-2-linoleoyl-glycerol (17.9%), and 1,2-diolein (10.9%). Fatty acid profile was similar to that of refined, bleached and deodorised (RBD) corn oil. The ratio of 1,3- to 1,2-positional isomers of DAG was at 1.82:1.