基于iKnife智能手术刀质谱技术金枪鱼内脏脂质组学研究

本研究利用iKnife智能手术刀质谱建立了一种金枪鱼内脏组织脂质组学检测新技术,用于对金枪鱼内脏营养价值进行深入研究。iKnife智能手术刀切割组织样品产生含有大量含磷脂离子的气溶胶,经专门的质谱接口装置直接引入质谱进行实时检测。结果显示,金枪鱼内脏中共鉴定出磷脂离子峰41种,质量范围m/z 699.5~911.6,其中信号最强离子峰m/z 790.5经鉴定为[PE 40:6-H]^-(相对丰度10.03%),其次为m/z 745.5([PA 40:7-H]^-,相对丰度9.02%)。该方法选择性强、精密度高、灵敏度好。本研究结果为质谱相关技术发展提供了参考,为金枪鱼副产物中脂质检测与综合利用提供了依据。     

富含EPA/DHA型结构磷脂的酶法合成条件优化及表征

为建立富含二十碳五烯酸(EPA)和二十二碳六烯酸(DHA)型结构磷脂的制备方法,本试验以南极磷虾磷脂为原料,以EPA和DHA总结合率为指标,通过响应面法优化富含EPA/DHA型结构磷脂的酶法合成反应参数,并采用气相色谱(GC)和亲水作用色谱-质谱联用(HILIC-MS)技术对其结构进行表征。结果表明,最优酶催化反应条件为:底物质量比(游离脂肪酸:磷脂, m/m)5.15,反应温度55.22℃,水分添加量(以反应底物总质量计)0.92%,酶添加量(以反应底物总质量计)20%,正己烷3 mL,反应时间24 h,所得结构磷脂中EPA/DHA结合率达64.35%;经GC和HILIC-MS综合分析可知,结构磷脂中磷脂酰胆碱(PC)亚类40:10(20:5/20:5)(18.46%),42:11(20:5/22:6)(12.78%)和38:6(18:1/20:5&16:0/22:6) (7.07%)等分子种含量较高;溶血磷脂酰胆碱LPC亚类20:5(20:5/0:0)(34.40%),22:6(22:6/0:0)(25.99%)和O-16:0(O-16:0/0:0)(10.22%)等分子种含量较高;结构磷脂的PC和LPC组分中脂肪酸单链平均长度≥20的分子种相对含量较原料磷脂分别提高了33.12和37.28个百分点;结构磷脂的PC组分中双键数≥5和双键数≥10的分子种相对含量较原料磷脂分别提高了24.89和27.53个百分点,表明该方法有效提高了结构磷脂中EPA和DHA的含量。本研究结果为进一步探索和丰富酶促合成EPA/DHA型结构磷脂的机理和实践提供了一定的数据支持。     

猪肉提取净化过程中脂质含量变化及其对β-受体激动剂残留分析的影响研究

采用靶向脂质组学方法分析了猪肉提取净化过程中24种磷脂的含量变化,考察了其对β-受体激动剂残留检测基质效应的影响。结果表明,磷脂总量随着提取溶液中水比例的增大而增加,其中磷脂酰胆碱(PC)脂质的增加最为明显。净化吸附剂C_(18)和PSA的使用可以降低磷脂总量,C_(18)的除脂效果优于PSA,且对磷脂酰乙醇胺(PE)、磷脂酰肌醇(PI)和磷脂酰丝氨酸(PS)脂质的去除效果好于PC脂质。随着水的增加,基质效应处于90%～100%范围内的化合物个数有所降低,但对弱基质效应(80%～120%)的化合物个数总体影响不大,当使用50 mg PSA和50 mg C_(18)对样品进行净化时化合物个数达到最大。磷脂含量的降低一定程度上降低了基质效应,减少了对目标分析物的影响,这些结果为明确样品前处理过程中基质动态变化,从而针对性地优化建立残留分析方法提供了重要依据。     

秦川牛腿骨油的脂质组成分析

为了实现畜禽骨副产物的有效利用,本研究采用超声波辅助溶剂浸出法提取秦川牛腿骨油,应用高效液相色谱(HPLC)、气相色谱-质谱联用(GC-MS)法和基于多维质谱“鸟枪法”脂质组学技术(MDMS-SL)综合分析牛腿骨油的脂质组成,并对牛腿骨油的氧化稳定性、酸价和过氧化值等理化性质以及酚类化合物进行测定。结果表明,秦川牛腿骨油中含有丰富的甘油酯,其中甘油三酯(TAG)含量为93.85%,同时含有少量的甘油二酯(DAG)。在秦川牛腿骨油中共检测出24种脂肪酸,单不饱和脂肪酸和多不饱和脂肪酸含量分别为62.85%和2.18%;24种酰基肉碱,总含量为1.18 nmol·g^-1;17种鞘磷脂,总含量为35.36 nmol·g^-1;38种磷脂酰胆碱,总含量为22.16 nmol·g^-1。样品具有较强的氧化稳定性,酸价和过氧化值分别为16.43 mg KOH·g^-1和5.22 mmol·kg^-1,含有α-生育酚、α-三烯酚及γ-三烯酚3种酚类化合物。丰富的TAG、不饱和脂肪酸及鞘磷脂含量使秦川牛腿骨油在促进营养吸收、降血糖血脂及抗炎等方面具有较大的利用潜力。本研究为秦川牛腿骨油相关高附加值产品的开发提供了理论基础。     

炭基肥和竹炭对土壤氮素淋失和微生物的影响

为了研究竹炭和炭基缓释肥添加对毛竹林土壤氮素流失和细菌群落结构的影响,采用室内土柱淋溶试验,测定了淋溶水中NH_4⁺—N和NO_3^-—N浓度,分析了细菌群落多样性和组成。结果表明,与对照相比,炭基缓释肥的添加可以使土柱NH_4⁺—N和NO_3^-—N累积淋溶量分别降低12.5%和13.6%。此外,竹炭的添加可以显著降低NH_4⁺—N累积淋溶量(P<0.05),但是对NO_3^-—N的影响不显著。炭基缓释肥的添加可以增加土壤Nitrospira(硝化螺菌属)、Nitrosospria(亚硝化螺菌属)和Nitrobacter(硝化杆菌属)细菌的相对丰度,提高下层淋溶土壤反硝化优势菌群(丰度>0.1%)Burkholderia(伯克霍尔德菌属)、Cupriavidus(贪铜菌属)和Bradyrhizobium(慢生根瘤菌属)的相对丰度。炭基缓释肥添加对土壤NH_4⁺—N和NO_3^-—N的淋溶影响可能与土壤氮素循环微生物密切相关。     

单甘酯硅烷化衍生及气相色谱-质谱分析

为分析罗氏海盘车性腺的单甘酯组成,本研究对单甘酯的衍生方法和色谱条件进行优化,并对单甘酯硅烷化衍生物的断裂规律和质谱特征进行分析,同时对罗氏海盘车性腺的单甘酯组成进行分析测定。结果表明,三甲基硅烷化法对单甘酯具有理想的衍生效果,且通过弱极性毛细管柱HP-5MS(30 m×0.25 mm×0.25 μm)对单甘酯硅烷化衍生物取得了理想的色谱分离效果。根据断裂规律和质谱特征,1-单甘酯硅烷化衍生物的特征离子为m/z 73、m/z 205、[M-103]⁺(基峰离子)和[M-15]⁺;2-单甘酯硅烷化衍生物的特征离子为m/z 73(基峰离子)、m/z 129、m/z 218、[M+H-162]⁺和[M-15]⁺。单甘酯硅烷化衍生物的色谱保留时间具有一定的规律性,其中2-单甘酯衍生物先于1-单甘酯衍生物出峰。同时,从罗氏海盘车中共鉴定出8种单甘酯,以1-单软脂酸甘油酯(1-C16:0-MG)和1-单硬脂酸甘油酯(1-C18:0-MG)等1-单甘酯为主,而2-单甘酯含量较低。本研究结果为单甘酯的衍生、色谱分析和质谱鉴定提供了一定的理论参考。     

湘中丘陵区不同植被恢复阶段林地土壤可溶性氮组分含量和密度

以湘中丘陵区的檵木—南烛—白栎灌草丛(LVR)、檵木—杉木—白栎灌木林(LCQ)、马尾松—柯—檵木针阔混交林(PLL)、柯—红淡比—青冈常绿阔叶林(LAG)作为1个恢复序列,设置固定样地,采集土壤样品,测定土壤可溶性有机氮(SON)、铵态氮(NH_4⁺—N)、硝态氮(NO_3^-—N)含量及其密度,分析SON、NH_4⁺—N、NO_3^-—N含量与土壤黏粒、全氮(TN)、有机碳(SOC)、微生物生物量的相关性。结果表明:各土层SON、NH_4⁺—N含量随植被恢复而增加,与LVR相比,LAG、PLL、LCQ 0—40 cm土层SON含量分别增加225.78%,121.22%,54.73%,NH_4⁺—N分别增加22.10%,14.74%,7.80%;而各土层NO_3^-—N含量随植被恢复先下降再增加,LAG各土层NO_3^-—N含量最高,LCQ最低;0—40 cm土壤层SON、NH_4⁺—N密度分别为143.82~528.12,55.73~65.57 kg/hm²,与LVR相比,LAG、PLL、LCQ土壤SON密度分别增加267.20%,98.40%,86.30%,NH_4⁺—N密度分别增加17.70%,7.90%和11.60%;0—40 cm土壤层NO_3^-—N密度为22.91~25.87 kg/hm²,与LVR相比,LAG增加13.16%;SON、NH_4⁺—N密度各阶段间的增长速率呈快—慢—快的特征,而NO_3^-—N呈慢—慢—快的特征;土壤理化性质和微生物生物量对SON、NH_4⁺—N的影响大于NO_3^-—N,表明植被恢复有利于土壤N养分积累,提高土壤可溶性氮组分的含量和密度,增加土壤N的可利用性。     

复合生物保鲜剂在南美白对虾防黑变中的应用

为解决南美白对虾易腐易黑变问题,延长其货架期,通过响应面优化方法,利用植酸、壳聚糖和ε-聚赖氨酸进行复配。结果表明,该复配保鲜剂最佳配比为植酸0.07%;壳聚糖1.5%;ε-聚赖氨酸0.1%(质量分数)。利用该保鲜剂处理南美白对虾,在(4±1)℃条件下,通过对虾体进行感官、pH值、挥发性盐基氮(total volatile base nitrogen,TVB-N)、菌落总数和三甲胺(trimethylamine,TMA)指标的测定,表明了复合保鲜剂能够有效地保持南美白对虾的品质,使南美白对虾在(4±1)℃的货架期由原来的3～4d延长至7～8d,细菌总数在第10天才达到5.7lg(CFU/g),为生物保鲜剂的优化及南美白对虾保鲜技术的推广提供一定的技术支持。     

0℃贮藏下南美白对虾品质变化研究

为分析冰藏冷链运输条件下南美白对虾品质和微观结构的变化,本研究测定了0℃贮藏条件下南美白对虾的色差、质构、挥发性盐基氮(TVB-N)及其组分含量和蛋白质图谱的变化情况,并对其肌肉的微观结构进行观察。结果表明,随着0℃贮藏时间的延长,南美白对虾L^*值、质构以及肌原纤维蛋白和肌基质蛋白含量呈下降趋势。0℃贮藏条件下,TVB-N值与贮藏时间呈正相关;贮藏4 d后南美白对虾色泽、质构和肌肉微观结构开始出现明显变化,肌原纤维蛋白和肌基质蛋白含量明显下降,而碱溶性蛋白含量呈增加趋势;贮藏6 d后蛋白条带降解明显。综合考虑,建议0℃贮藏条件下南美白对虾的货架期为4 d。本研究结果为冷藏条件下南美白对虾提供了品质评价的方法。     

涂膜及气调保鲜对南美白对虾品质的影响

为了改善南美白对虾黑变快、货架期短的情况,制备含黑变抑制组分（M）和1%壳聚糖（Chitosan）的复合保鲜剂,用其涂膜保鲜南美白对虾,采用高阻隔复合包装袋（K-PET/PE）,采用40%CO2/30%O2/30%N2（Ⅰ）和85%CO2/ 5%O2/10%N2（Ⅱ）2种气调包装,(4±1)℃冷藏,通过菌落总数、挥发性盐基氮、pH值、多酚氧化酶的活性和感官评定等指标评价不同保鲜工艺对南美白对虾的黑变抑制和防腐保鲜效果。通过正交试验获得的黑变抑制组分（M）最优组合为：0.01%4-己基间苯二酚+1.5%柠檬酸+1.0%抗坏血酸;组合（Chitosan+Ⅰ）、（Chitosan+M+Ⅰ）、（Chitosan+Ⅱ）、（Chitosan+M+Ⅱ）分别在第6天、第8天、第12天和第14天开始出现显著黑变;相比对照组,所有处理组在整个贮藏期内的菌落总数、挥发性盐基氮、pH值、多酚氧化酶的活性的增长速度均有所下降,组合（Chitosan+M+Ⅱ）保鲜效果最佳,使(4±1)℃条件下的货架期延长2倍多。涂膜保鲜剂结合气调包装(MAP)的处理方式是南美白对虾保鲜的有效手段,具有应用推广价值。     

Changes produced in the antioxidative activity of phospholipids as a consequence of their oxidation

The antioxidative activities of native and oxidized soybean phosphatidylcholine (PC), phosphatidylthanolamine (PE), and phosphatidylinositol (PI) in the protection of soybean oil heated in the dark under air at 60 degrees C were studied in an attempt to clarify the consequences that phospholipid oxidation has on antioxidative activities. The three native phospholipids protected the oil when assayed at 200 ppm, and phospholipid oxidation decreased the antioxidative activity of both PC and PI. However, slightly oxidized PE was more antioxidative than native PE, most likely as a consequence of the formation by amino-carbonyl reactions of pyrrolized phospholipids, which were determined and for which antioxidative properties are known. Nevertheless, further increases in PE oxidation produced a decrease in its antioxidative activity. These results suggest that two opposite reactions are competing in the antioxidative activity of amino phospholipids upon oxidation: fatty acid chain oxidation, which decreases phospholipid antioxidative activity, and amino-carbonyl reactions, which produce derivatives with antioxidant properties. This last property may be useful to increase the antioxidative activity of commercial lecithins containing amino phospholipids.     

A new insight into the formation of odor active carbonyls by thermally-induced degradation of phospholipids in self-assembly structures

The role of molecular organization in heated aqueous dispersions of egg phosphatidylcholine (PC) and egg phosphatidylethanolamine (PE) was studied with respect to the formation of key odorants. Evidence was found for the crucial role of self-assembly structures adopted by phospholipid molecules on the quantitative composition of volatile constituents. The concentrations of seven aldehydes and one vinyl ketone were determined by isotope dilution assay in heated aqueous dispersions of PC and PE present in various ratios. Addition of PE to PC drastically decreased the amount of (E,E)-2,4-decadienal formed, which cannot be explained by the differences in the fatty acid composition of PC and PE. The free amino group in PE does not explain this phenomenon either, as replacing PE by phosphatidic acid distearylester also reduced the amounts of (E,E)-2,4-decadienal. We suggest that the type of self-assembly structure adopted by phospholipids in water significantly influences the reaction yields. However, the mechanisms leading to the preferred formation of phospholipid-derived odorants in a lamellar phase, as compared to the reversed hexagonal phase, remain unknown.     

Inorganic mercury interactions with lipid components of biological membranes:31P-NMR study of Hg(II) binding to headgroups of micellar phospholipids

Phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylcholine (PC) in micellar phase in water have been studied by high resolution phosphorus-31 nuclear magnetic resonance (³¹P-NMR), in order to follow inorganic mercury Hg(II) binding to the lipid headgroups. Decrease of the NMR peak area is observed upon HgCl₂ addition, with greater effect on PE and PS compared to PC. This is interpreted by Hg(II) binding to several phospholipid headgroups, linking different micelles together and forming by extension a large “insoluble” phospholipid-mercury network that is undetectable by high-resolution³¹P-NMR. The extent of phospholipid aggregation depends on the mercury-to-lipid molar ratio, and apparent Hg(II) affinities to phospholipid headgroups are in the order PE>PS>>PC. When HgCl₂ is added to mixed micelles prepared with two lipids (PE/PC or PS/PC), co-precipitation is observed for both components in similar proportions.     

A gas chromatography/electron ionization-mass spectrometry-selected ion monitoring method for determining the fatty acid pattern in food after formation of fatty acid methyl esters

A method using gas chromatography/electron ionization-mass spectrometry (GC/EI-MS) in the selected ion monitoring (SIM) mode was developed for the analysis of fatty acids as methyl esters (FAMEs) in order to determine their percentage contribution to the fatty acid profile in food. In the GC/EI-MS-SIM mode, saturated fatty acids were determined with m/z 87, monoenoic fatty acids were determined with m/z 74, and polyenoic fatty acids were determined via the sum of m/z 79 and m/z 81. The ratios of these fragment ions and the GC retention data provided additional information for tentative structural assignments. The 28 FAME standards tested provided similar results for the novel GC/EI-MS-SIM method and GC/EI-MS in the full scan mode, both of which were slightly worse than GC/flame ionization detection (FID). Analysis of sunflower oil, suet, and cod liver oil verified that both major and minor fatty acids (20-60% and down to 0.001% contribution to the fatty acid pattern) were determined with sufficient quality that justifies application of the GC/EI-MS-SIM method for the analysis of food samples. Furthermore, the method was approximately 20- or approximately 10-fold more sensitive than GC/EI-MS in the full scan mode or GC/FID, respectively. The method is suited for both quantitative purposes and fatty acid identification in samples where only low amounts of lipids are available.     

Lipid profiling by electrospray ionization tandem mass spectrometry and the identification of lipid phosphorylation by kinases in potato stolons

There is limited information about the involvement of lipids and esterified fatty acids in signaling pathways during plant development. The purpose of this study was to evaluate the lipid composition and molecular species of potato (Solanum tuberosum L., cv. Spunta) stolons and to identify phosphorylated lipids in the first two developmental stages of tuber formation. Lipid profiling was determined using ESI-MS/MS, a useful method for the determination of the biosynthesis and catabolism of lipids based on their fatty acid composition. The most prevalent compound identified in this study was phosphatidic acid (PA); digalactosyldiacylglycerol (DGDG) was the second most abundant compound. A 34:2 species was identified in PA, phosphatidylcholine (PC), phosphatidylinositol (PI), and phosphatidylethanolamine (PE). The identification of lipid phosphorylation by kinases was revealed by the presence of the phosphorylated lipids. PA was metabolized to diacylglycerol pyrophosphate (DGPP) by phosphatidic acid kinase (PAK). This work establishes a correlation between lipid fatty acid composition and lipid metabolism enzymes at the beginning of tuber formation and is the first report of PAK activity in the early events of potato tuber formation.     

Eicosapentaenoic acid enrichment from sardine oil by argentation chromatography

Eicosapentaenoic acid (EPA) derived from chemically hydrolyzed sardine oil was concentrated by urea fractionation using methanol at different temperatures (2, 4, and 6 degrees C) and urea/fatty acid ratios (2:1, 3:1, and 4:1 w/w) and purified by argentation neutral alumina column chromatography. The individual fatty acids were determined as fatty acid methyl esters (FAME) by gas-liquid chromatography and gas chromatography-mass spectroscopy as FAME and N-acyl pyrrolidides. In the mass fragmentation pattern of FAME, the base peak was assigned to be the 1-methoxyethenol moiety (m/z = 74) obtained by McLafferty rearrangement. Formation of the cyclic tropylium ion (m/z = 91) in fatty acids with four or more double bonds was apparent in FAME-PUFAs. The base peak of N-acyl pyrrolidides was the McLafferty rearrangement ion, 1-(pyrrolidin-1-yl)ethenol (m/z = 113). The highest concentration of EPA (47.78%) was obtained at the crystallization temperature of 4 degrees C with a urea/fatty acid ratio of 4:1 (w/w) with 93.74% yield. After complexation of saturated and less unsaturated fatty acids by urea complexation, argentation chromatography resulted in an EPA of high purity (99.6%) with an overall recovery of 54.09% using 50% diethyl ether/n-hexane as eluting solvent. The peroxide (POV) and thiobarbituric acid (TBS) values were found to be highest (4.0 mequiv of O2/kg and 5.2 mg of malondialdehyde/kg, respectively) during urea fractionation at the higher crystallization temperature (6 degrees C) and higher urea/fatty acid ratio (4:1). Keywords: Sardine oil; eicosapentaenoic acid (EPA); fatty acid methyl esters (FAME); urea fractionation; argentation column chromatography.     

Effect of dietary omega 3 phosphatidylcholine on obesity-related disorders in obese Otsuka Long-Evans Tokushima fatty rats

Recent reports have shown that dietary phosphatidylcholine (PC) has various beneficial biological effects. Omega 3 polyunsaturated fatty acids (omega3 PUFAs) have also been reported to have lipid-lowering effects in animal models and human studies. In the present study, we investigated the effect of omega3 PUFAs containing PC (omega3-PC) on obesity-related disorders in Otsuka Long-Evans Tokushima fatty (OLETF) rats. Rats were fed semisynthetic diets that contained either 5% corn oil and 2% egg-PC or 5% corn oil and 2% omega3-PC for 4 weeks. During this 4 week feeding of the omega3-PC, the OLEFT rats showed a decrease of omental white adipose tissue weight. In addition, the omega3-PC diet significantly decreased liver weight and hepatic lipid levels in OLETF rats. These changes were attributable to the significant suppression of fatty acid synthase activity and significant enhancement in the activities of carnitine palmitoyltransferase and peroxisomal beta-oxidation. Moreover, the omega3-PC diet reduced serum glucose levels concomitant with the increase of serum adiponectin levels. These results show that compared with egg-PC, omega3-PC can prevent or alleviate obesity-related disorders through the suppression of fatty acid synthesis, enhancement of fatty acid beta-oxidation, and increase of the serum adiponectin level in OLETF rats.