Purification of hydroperoxide lyase from green bell pepper (Capsicum annuum L.) fruits for the generation of C6-aldehydes in vitro

The aim of this work was to compare the efficiency of different extracts of hydroperoxide lyase from green bell peppers in producing aldehydes: a crude extract, a chloroplastic fraction, and a purified enzyme were investigated. From a crude extract, the HPO lyase was purified by ion-exchange chromatography with a 22.3-fold increase in purification factor. Analysis by SDS-PAGE electrophoresis under denaturating conditions showed only one protein with a molecular weight of 55 kDa, whereas size-exclusion chromatography indicated a molecular weight of 170 kDa. A maximum of 7500 mg of aldehydes per g of protein was obtained with the purified enzyme within 20 min of bioconversion compared to 392 and 88 mg of aldehydes per g of protein within 50 and 60 min, respectively, for the chloroplast fraction and the crude extract.     

Thermal stability of lipoxygenase and hydroperoxide lyase from olive fruit and repercussion on olive oil aroma biosynthesis

Thermal stabilities of main enzymes involved in the biosynthesis of virgin olive oil (VOO) aroma through the lipoxygenase (LOX) pathway were studied in crude enzymatic preparations. Kinetic parameters of thermal inactivation for LOX were determined graphically and were shown to be compatible with the presence of two LOX isoenzymes (LOXlab and LOXres) having different thermal stabilities and displaying relative activities of 88 and 12% each. Data on hydroperoxide lyase (HPL) suggest the existence of just one HPL isoform. Thermal stabilities of LOX and HPL enzymatic activities in crude preparations seem to explain the observed decrease of volatile contents in VOO aroma as a consequence of heat treatments of olive fruit. Moreover, differences in thermal stability of LOXlab and LOXres would justify the distinct pattern of reduction of C6 and C5 compound contents observed in the aroma of these oils.     

A simple and efficient system for green note compound biogenesis by use of certain lipoxygenase and hydroperoxide lyase sources

Six-carbon (C(6)) aldehydes and alcohols are important components of the aroma and flavor of fruits and vegetables. Soybean lipoxygenase (LOX) isozyme LOX 3 was reported not only to produce less 13-hydroperoxides, precursors of C(6) aldehydes, but also to convert them to ketodiene products. Here, we examined the effects of LOX 3 on hexenal formation from linolenic acid homogenized with watermelon 13-hydroperoxide lyase (HL)-overexpressing Nicotiana tabacum leaves and soybean acetone powder. Compared to the wild type, which contains LOXs 1, 2, and 3, the elimination of LOX 3 in LOX 1 + 2 facilitates greater production of hexenals. The use of LOX 2 alone yielded the highest hexenal production, while a two-step conversion was required for LOX 1 to produce hexenals at high levels due to different pH optima of the enzymes involved. These results clearly demonstrate that the soybeans lacking LOX 3 in combination with watermelon HL-overexpressing leaf tissues greatly enhance hexenal formation.     

Impact of the suppression of lipoxygenase and hydroperoxide lyase on the quality of the green odor in green leaves

Most of the volatile compounds responsible for the "green" notes to the aroma of fruits and vegetables are produced by the degradation of polyunsaturated fatty acids through the lipoxygenase pathway. The most determinant steps of this pathway are the peroxidation of free linoleic or linolenic acid by the action of lipoxygenase and then the lysis of the resulting hydroperoxides through a reaction catalyzed by the hydroperoxide lyase. This work analyzes the impact of the depletion of these enzymes on the volatile composition of leaves from potato plants. A characterization of the volatile profiles of the different potato mutants, a study of the metabolism of radiolabeled linoleic acid, and a determination of lipoxygenase activity have been carried out. The depletion of hydroperoxide lyase induced an increase in the lipoxygenase activity and the content of C5 volatiles, whereas the lipoxygenase silencing caused a severe decrease in the amount of volatiles produced by the leaves and always in the intensity of their aroma. The changes in the sensory evaluation of leaf aroma, as correlated to depletion of the two enzymes, have been investigated. The perspectives of producing vegetable products with a modified aroma by genetic engineering are discussed in light of the statistical results.     

Characterization of the aroma of a meatlike process flavoring from soybean-based enzyme-hydrolyzed vegetable protein

Defatted soybean meal was converted into enzyme-hydrolyzed vegetable protein (E-HVP) using the proteolytic enzyme Flavorzyme. Total free amino acids increased by 40-fold after enzyme hydrolysis, with leucine being the most abundant, followed by phenylalanine, lysine, glutamine/glutamic acid, and alanine. Volatile components from a meatlike process flavoring made from E-HVP were isolated by direct solvent extraction (DSE)-high vacuum transfer (HVT), dynamic headspace sampling and static headspace sampling and analyzed by gas chromatography (GC)-mass spectrometry and GC-olfactometry. Aroma extract dilution analysis was used to establish a flavor dilution chromatogram of the DSE-HVT extract. Results of these complementary techniques indicated the importance of odorants of high (hydrogen sulfide and methanethiol), intermediate (2-methyl-3-furanthiol, 3-mercapto-2-pentanone, 2-furanmethanethiol, and 3-(methylthiol)propanal) and low volatility (maltol and Furaneol) in the overall aroma of the meatlike process flavoring.     

Improved amperometric method for the rapid and quantitative measurement of lipoxygenase activity in vegetable tissue crude homogenates

An improved amperometric method for rapid (2 min) quantitative determination of lipoxygenase (LOX) activity in vegetable tissue crude homogenates is presented. Measured LOX activity was linear (R(2) > 0.99) throughout the entire activity range for green bean and for corn below 70% activity. The resolution was 0.4% or 1.11 micromol L(-1) s(-1) of oxygen. The limit of detection was 3.43 micromol L(-1) s(-1) of oxygen. The amperometric method was improved by encapsulating linoleic acid (LA) in beta-cyclodextrin (CD) resulting in a stable substrate-buffer solution at a pH below 8.0. Ethanol and Tween 20 were not effective in solubilizing high LA concentrations required by the assay. A prototype benchtop instrument with the potential for use in an industrial environment is also presented.     

Determination of nonprotein amino acids and betaines in vegetable oils by flow injection triple-quadrupole tandem mass spectrometry: a screening method for the detection of adulterations of olive oils

A novel screening method using an automated flow injection electrospray ionization tandem mass spectrometry system is proposed for the simultaneous determination of five nonprotein amino acids (β-alanine, alloisoleucine, ornithine, citrulline, pyroglutamic acid) and three betaines (glycine betaine, trigonelline, proline betaine) after derivatization with butanolic HCl. MS/MS experiments were carried out in a triple-quadrupole instrument using multiple reaction monitoring mode in <2 min. The proposed method provided high fingerprinting power to identify the presence of five of the studied compounds in different types of vegetable oils (soybean, sunflower, corn, olive) with LODs at parts per billion levels. The method was validated, and different mixtures of extra virgin olive oil with seed oils were analyzed, achieving the typification for the detection of adulterations in extra virgin olive oils up to 2% w/w. The nonprotein amino acid ornithine was confirmed as a marker for adulteration in the olive oils analyzed.     

红酵母发酵豆腐渣产类胡萝卜素工艺优化

豆腐渣虽含有多种营养成分,但大约50%的干物质是难以消化的纤维素及半纤维素,直接用作饲料,饲喂效果并不理想。为了提高豆腐渣的饲用价值,进行了红酵母发酵豆腐渣产类胡萝卜素工艺优化研究。首先,在单因素试验基础上进行正交试验的结果显示,优化的豆腐渣酸解产还原糖工艺参数为:盐酸浓度1.0mol/L,料液比(g/mL)1∶10,酸解温度100℃,酸解时间3.0h,该条件下还原糖得率达29.06%±0.07%。然后,选用嗜还原糖红酵母发酵富含还原糖的豆腐渣酸解产物,以产生具有抗氧化、增强机体免疫等多种生物学功能的类胡萝卜素。通过单因素试验、Plackett-Burman试验回归分析、Box-Behnken试验及响应面分析,获得优化的发酵工艺参数:发酵底物pH值6.0,装液量80 mL/(500 mL),接种龄48 h,接种量11%(种子液浓度为8.5×10~9 CFU/mL),转速60 r/min,发酵温度31℃,发酵时间128 h;该条件下类胡萝卜素产量达(2.65±0.02)mg/L,比工艺优化前产量提高了67.7%,研究结果可为利用廉价豆腐渣开发高附加值类胡萝卜素功能饲料提供参考。     

Development of multiresidue analysis for twenty phthalate esters in edible vegetable oils by microwave-assisted extraction-gel permeation chromatography-solid phase extraction-gas chromatography-tandem mass spectrometry

A novel multiresidue analysis method is developed for the determination of twenty phthalate esters at the μg/kg level in edible vegetable oils by microwave-assisted extraction-gel permeation chromatography-solid phase extraction-high resolution gas chromatography-tandem mass spectrometry (MAE-GPC-SPE-HRGC-MS/MS). The samples were extracted with methanol under microwave incubation. Cleanup was carried out with GPC followed by a further C18 SPE column and then separated by the HP-5MS capillary column under a temperature program. The eluents were qualitatively and quantitatively determined by tandem mass analyzer with selected reaction monitoring (SRM) type and positive ion mode. The calibration curves showed good linearity in the range 5 μg/kg to 2.50 mg/kg with correlation coefficients larger than 0.999. Low detection limits (LODs) of 0.218-1.367 μg/kg and quantification limits (LOQ) of 0.72-4.51 μg/kg were achieved. The mean recoveries were in the range from 93.04% to 104.6% at 5, 15, and 40 μg/kg spiked levels, and the relative standard deviations (RSDs) were in the range of 1.01% and 5.26% (n = 7). This method could potentially overcome the interference from large amounts of lipids and pigment. The real sample test showed this method can be used for sensitive and accurate determination and confirmation of phthalate ester residues in high-fat and complex samples.     

Low-quality vegetable oils as feedstock for biodiesel production using k-pumice as solid catalyst. Tolerance of water and free Fatty acids contents

Waste oils are a promising alternative feedstock for biodiesel production due to the decrease of the industrial production costs. However, feedstock with high free fatty acids (FFA) content presents several drawbacks when alkaline-catalyzed transesterification reaction is employed in biodiesel production process. Nowadays, to develop suitable processes capable of treating oils with high free fatty acids content, a two-step process for biodiesel production is being investigated. The major problem that it presents is that two catalysts are needed to carry out the whole process: an acidic catalyst for free fatty acids esterification (first step) and a basic catalyst for pretreated product transesterification (second step). The use of a bifunctional catalyst, which allows both reactions to take place simultaneously, could minimize the production costs and time. In the present study, the behavior of pumice, a natural volcanic material used as a heterogeneous catalyst, was tested using oils with several FFA and water contents as feedstock in the transesterification reaction to produce biodiesel. Pumice as a bifunctional solid catalyst, which can catalyze simultaneously the esterification of FFA and the transesterification of fatty acid glycerides into biodiesel, was shown to be an efficient catalyst for the conversion of low-grade, nonedible oil feedstock into biodiesel product. Using this solid catalyst for the transesterification reaction, high FAME yields were achieved when feedstock oils presented a FFA content until approximately 2% wt/wt and a water content until 2% wt/wt.     

荷兰蔬菜种苗生产装备系统发展现状及对中国的启示

为提高生产率、降低劳动强度和减少生产成本,荷兰蔬菜育苗生产中广泛使用精量播种生产线、嫁接作业生产线、岩棉块种苗生产线和相应配套机具,并借鉴花卉物流化生产模式,开发出穴盘输送装备、岩棉块搬运铺放专用车等生产物料搬运装备,实现了高效、省力、自动化生产。该文结合生产实例分析了利用岩棉块种苗生产线直接播种生产蔬菜种苗和利用播种生产线、嫁接作业生产线及岩棉块种苗生产线生产蔬菜嫁接苗的作业流程,并讨论了各生产流程的作业特性。综合以上论述,总结出荷兰温室种苗生产具有以下特点：由于岩棉具有保水透气、病菌易控等优点,潮汐灌溉具有操作方便可自控、节约水资源等优点,在温室蔬菜生产中广泛采用岩棉块栽培和潮汐灌溉;蔬菜生产效益低于花卉生产效益,造成蔬菜种植企业在自动化生产方面的资金投入相对较低,与花卉生产相比自动化程度也相对较低;精量播种生产线、嫁接作业生产线和岩棉块种苗生产线构成蔬菜种苗生产装备系统的主体,生产线将装备作业环节与人工作业环节按作业流程有机结合,快速提高蔬菜生产效率;针对蔬菜种苗生产中作业量大、劳动强度大的生产物料搬运环节,采取多种途径,提高自动化作业水平,减轻劳动强度,提高生产率。针对中国温室蔬菜生产所面临的劳动力短缺、劳动成本逐年提高、自动化生产水平低下和作业生产率不高等问题,借鉴荷兰设施园艺自动化生产技术的发展经验,中国蔬菜种苗生产应以提高生产率和降低劳动强度为核心目标,注重集成开发,利用现有成熟技术,重点集中开发自动化装备的关键技术,提高开发效率;在构建设施农业装备生产系统方面,应在提高生产自动化水平的基础上,综合考虑装备性价比,引入温室内部物流化生产模式,构建物流化生产系统,提高种苗生产的总体生产效率,促进中国温室园艺生产模式的现代化转型。     

Development of a screening method for genetically modified soybean by plasmid-based quantitative competitive polymerase chain reaction

A novel type of quantitative competitive polymerase chain reaction (QC-PCR) system for the detection and quantification of the Roundup Ready soybean (RRS) was developed. This system was designed based on the advantage of a fully validated real-time PCR method used for the quantification of RRS in Japan. A plasmid was constructed as a competitor plasmid for the detection and quantification of genetically modified soy, RRS. The plasmid contained the construct-specific sequence of RRS and the taxon-specific sequence of lectin1 (Le1), and both had 21 bp oligonucleotide insertion in the sequences. The plasmid DNA was used as a reference molecule instead of ground seeds, which enabled us to precisely and stably adjust the copy number of targets. The present study demonstrated that the novel plasmid-based QC-PCR method could be a simple and feasible alternative to the real-time PCR method used for the quantification of genetically modified organism contents.     

Solid-liquid transfer of biophenols from olive leaves for the enrichment of edible oils by a dynamic ultrasound-assisted approach

A continuous approach assisted by ultrasound for direct enrichment of edible oils (olive, sunflower, and soya) with the main phenols in olive leaves (i.e., oleuropein, verbascoside, apigenin-7-glucoside, and luteolin-7-glucoside) has been developed. Multivariate methodology was used to carry out a detailed optimization of the enrichment, and quantitation of the transferred compounds was based on LC-MS-MS in multiple reaction monitoring optimizing the most sensitive transition for each biophenol. Under the optimal working conditions, only 20 min is necessary to enrich the edible oils with 14.45-9.92 microg/mL oleuropein, 2.29-2.12 microg/mL verbascoside, 1.91-1.51 microg/mL apigenin-7-glucoside, and 1.60-1.42 microg/mL luteolin-7-glucoside. The enrichment method is carried out at room temperature and is organic-solvent-free; thus, the healthy properties of the edible oils improve as does their quality. Also, the low acquisition and maintenance costs of an ultrasound source and its application in a dynamic system make advisable the industrial implementation of the proposed method.     

Modifications of stearidonic acid soybean oil by enzymatic acidolysis for the production of human milk fat analogues

Structured lipids (SLs) from stearidonic acid (SDA) soybean oil pre-enriched with palmitic acid (PA) at the sn-2 position with Novozym 435 (NSL) or Lipozyme TL IM (LSL) from previous research were further enriched with γ-linolenic acid (GLA) or docosahexaenoic acid (DHA). Small-scale acidolysis reactions with Lipozyme TL IM were performed to determine the optimal reaction conditions as 1:1 substrate mole ratio of NSL or LSL to free DHA at 65 °C for 24 h and a 1:0.5 substrate mole ratio of NSL or LSL to free GLA at 65 °C for 12 h. Optimized SL products were scaled up in a 1 L stir-batch reactor, and the resulting SLs of NSL:DHA (NDHA), LSL:DHA (LDHA), NSL:GLA (NGLA), and LSL:GLA (LGLA) were chemically and physically characterized. The SLs contained >54% PA at the sn-2 position with GLA >8% for the GLA SLs and DHA >10% for the DHA SLs. The oxidative stabilities of the SLs were increased by the addition of 200 ppm TBHQ, with NGLA being more stable due to higher tocopherol content than the other SLs. The melting and crystallization profiles did not differ between the DHA SLs or the GLA SLs. The triacylglycerol (TAG) species were similar for the GLA SLs but differed between the DHA SLs, with tripalmitin being the major TAG species in all SLs.     

果蔬类多孔介质干燥传质过程的分子动力学模拟

果蔬类多孔介质内部水中溶解有大量的营养物质（溶质）,在干燥过程中溶质的迁移与湿分的传递同时进行,其内部微孔内的干燥传质机理尚不明确。为了揭示果蔬类多孔介质干燥过程中内部溶液的迁移机理,确定果蔬微孔结构特性对干燥传质过程的影响规律。该研究采用分子动力学方法模拟研究了果蔬类多孔介质微孔道中的干燥传质过程,构建了光滑壁面溶液扩散过程模型与粗糙壁面溶液扩散过程模型。模拟过程采用SPC/E水分子模型,选取OPLS-AA全原子力场和正则系综,溶液势函数选用静电库伦相互作用与Lennard-Jones相互作用,中心水分子的初始速度由高斯分布给出,采用Velocity-Verlet算法,用SHAKE算法固定水分子,x、y方向施加周期性边界条件,z方向上施加固定壁面边界条件。从分子水平模拟分析了果蔬类多孔介质内部溶液的扩散过程,并以马铃薯的热风干燥试验结果进行模型的验证。得出试验值与KCl溶液粗糙壁面模型的模拟值最为接近,其最大相对误差为17.39%;与纯水模型的模拟值相差最大,说明溶质的存在对水分扩散系数的影响不可忽略,且粗糙壁面模型更接近于真实孔道结构。从径向分布函数分布可以看出K+、Cl-对水分子的氢键结构有破坏作用。K+、Cl-均存在两层水化层,H2O分子以O原子靠近K+,以H原子靠近Cl-。溶质浓度、孔道直径、壁面粗糙度因子和相面积分数均对孔隙中的水分扩散系数有重要影响。随着孔道内KCl溶液质量分数的增大,其水分扩散系数逐渐减小。孔道直径变大、粗糙壁面粗糙度因子减小和粗糙壁面相面积分数增大,均会导致干燥过程中水分扩散系数增大。研究结果为果蔬干燥品质及工艺优化分析提供了理论依据。     

我国大豆最佳施肥量和种植密度评价

施肥量和种植密度是影响大豆高产的重要因素。在收集了大量的大豆试验数据(1998～ 2017年)基础上,通过拟合氮、磷、钾肥用量和种植密度与产量之间的二次函数,得出最佳的施肥量和种植密度,通过逐步回归分析了施肥量和种植密度对大豆产量的影响。结果表明,我国春大豆和夏大豆的产量逐年增加,平均产量分别为 2 610和 2 724 kg/hm²。夏大豆最高产量下的氮、磷、钾肥用量分别为 N 96 kg/hm²、P2O5 80 kg/hm²和K2O 126 kg/hm²;春大豆最高产量下的氮、磷、钾肥用量分别为 N 71 kg/hm²、P2O5 108 kg/hm²和K2O 74 kg/hm²;实现夏、春大豆高产的最佳密度分别为 27万和 34万株/hm²。逐步回归分析显示,磷用量对春大豆产量影响最大,其次为钾肥和密度;在夏大豆产区,密度对产量影响最大,其次为磷肥用量。种植密度是大豆高产的关键因素,春、夏大豆需要提高种植密度获得高产,同时均应注重磷肥施用。     

Development of a seven-target multiplex PCR for the simultaneous detection of transgenic soybean and maize in feeds and foods

The detection of genetically modified organisms (GMOs) in food and feed is an important issue for all the subjects involved in raw material control, food industry, and distribution. Because the number of GMOs authorized in the EU increased during the past few years, there is a need for methods that allow a rapid screening of products. In this paper, we propose a method for the simultaneous detection of four transgenic maize (MON810, Bt11, Bt 176, and GA21) and one transgenic soybean (Roundup Ready), which allows routine control analyses to be sped up. DNA was extracted either from maize and soybean seeds and leaves or reference materials, and the recombinant DNA target sequences were detected with 7 primer pairs, accurately designed to be highly specific for each investigated transgene. Cross and negative controls were performed to ensure the specificity of each primer pair. The method was validated on an interlaboratory ring test and good analytical parameters were obtained (LOD = 0.25%, Repeatability, (r) = 1; Reproducibility, (R) = 0.9). The method was then applied to a model biscuit made of transgenic materials baked for the purpose and to real samples such as feed and foodstuffs. On account of the high recognition specificity and the good detection limits, this multiplex PCR represents a fast and reliable screening method directly applicable in all the laboratories involved in raw material and food control.     

Dynamic relationships between soil properties and foliar disease as affected by annual additions of organic amendment to a sandy-soil vegetable production system

Additions of organic amendments to agricultural soils can lead to improved soil quality and reduced severity of crop diseases. However, the relationship between disease severity and soil properties as affected by repeated additions of these amendments is poorly understood. The primary objectives of this study were to (i) resolve multivariate relationships between soil properties and foliar disease severity and (ii) identify soil properties that contribute to disease severity in an intensive irrigated vegetable production system receiving annual additions of fresh and composted paper mill residuals (PMR). Foliar diseases caused by Pseudomonas syringae pv. syringae on snap bean (bacterial brown spot) and P. s. pv. lachrymans on cucumber (angular leaf spot) are the focus of this report. The experiment consisted of a 3-year crop rotation of potato (1998 and 2001), snap bean (1999 and 2002), and cucumber (2000). Treatments included a non-amended fertilizer control and two rates of fresh PMR, PMR composted alone (PMRC), and PMR composted with bark (PMRB). Soil measures included total soil carbon (TC) and nitrogen (TN), particulate organic matter carbon (POMC) and nitrogen (POMN), volumetric soil moisture (VM) and in situ NO₃-N. Multiple regression (MR) and principal component analyses (PCA) were conducted to identify key soil properties that influenced the amount of disease. On average, the amount of TC in plots amended with PMR composts increased 77-178% from 1999 to 2002 compared to the non-amended soils. In 1999, a year in which compost additions reduced the amount of bacterial brown spot of bean, TC explained 42% of the total variation in disease severity in the best MR model. Midseason TN alone was inversely related to angular leaf spot incidence in 2000, while POMN explained 51% of the variation in the best MR model for that year. In 2002, a year in which PMRC-amended soils exacerbated brown spot symptoms, midseason quantities of TN explained 80% of the variation in disease severity. Unique to 2002, NO₃-N alone positively correlated with disease severity. Overall, the influence of soil carbon on disease severity was displaced by the increasing importance of TN and NO₃-N, indicating a transition from a C-dependent to an N-dependent system.