卷枝毛霉△6-脂肪酸脱饱和酶基因的敲除及功能研究

γ-亚麻酸(γ-Linolenic acid,GLA)是人体所必需且具有多种生理功能的一种重要多不饱和脂肪酸。研究表明,卷枝毛霉等GLA生产菌株中△6-脂肪酸脱饱和酶(△6-Fatty acid desaturase,FAD6)是合成γ-亚麻酸的关键和限速酶,然而GLA是否惟一通过△6-脂肪酸脱饱和酶途径合成则目前还没有报道。作者以卷枝毛霉Mucor circinelloides EIM10 sp.为研究对象,以其FAD6基因的5'端启动子和3'端终止子为同源臂,潮霉素B抗性基因为筛选标记,构建了M.circinelloides EIM10 sp.△6-脂肪酸脱饱和酶FAD6的敲除载体p UMCD6-Hm B,转化进入制备好的原生质体中成功地敲除了FAD6基因,发现FAD6基因缺陷菌株GLA质量浓度比野生型对照菌株降低了50%以上,但GLA并没有完全消失,说明卷枝毛霉生物体内还有其他代谢途径可以将碳源转化为GLA。     

深黄被孢霉和拉曼被孢霉多不饱和脂肪酸代谢的研究

应用气相色谱-质谱联用技术对国内和国外两株γ-亚麻酸(GLA)的主要生产菌(深黄被孢霉和拉曼被孢霉)的脂肪酸成分进行分析,然后从不饱和脂肪酸的代谢途径和菌体的生理特性对两株菌进行比较。从代谢途径看,两株菌的不饱和脂肪酸代谢有一定的差异,其中△6-和△12-脂肪酸脱氢酶均为阻碍两株菌高产GLA的关键所在,再结合两株菌的生理特性,发现拉曼被孢霉较深黄被孢霉更适合工业化生产。     

气相色谱-质谱法分析北大西洋瓜参脑苷脂中2-羟基脂肪酸

通过酸甲酯化衍生、羟基乙酰化保护和二甲基二硫衍生方法对北大西洋瓜参脑苷脂中的2-羟基脂肪酸进行衍生,采用气相色谱-质谱技术分析其2-羟基脂肪酸组成。通过衍生处理和色谱优化,取得理想的色谱分析效果。根据质谱断裂规律,采用特征离子可对北大西洋瓜参脑苷脂中2-羟基脂肪酸上的羟基和双键位置进行准确判断,其中2-羟基饱和脂肪酸甲酯的特征离子为m/z 57(基峰离子),90,[M-59]+,[M-18]+和M+;2-羟基单烯脂肪酸甲酯的特征离子为m/z 55(基峰离子),90,[M-59]+,[M-18]+和M+。2-羟基单烯脂肪酸DMDS衍生物的特征离子为A+(m/z 75+14n)和B+(m/z 191+14m),根据这两个碎片离子即可快速准确推断2-羟基单烯脂肪酸中双键所处碳链位置。通过气相色谱-质谱分析,从北大西洋瓜参脑苷脂中共鉴定出9种2-羟基脂肪酸,以2-OH C24:1n-9(70.84%)、2-OH C23:1n-9(7.74%)和2-OH C20:0(6.82%)为主。     

反油酸处理对人HepG2细胞代谢组的影响

本研究以人细胞(Hep G2)为模型,通过MTT实验研究反油酸对Hep G2细胞存活率的影响,选择1.2 mmol/L和1.5 mmol/L的反式油酸处理Hep G2细胞6和12 h,利用UPLC-MS/MS和GC-MS对Hep G2细胞的代谢组进行检测,以了解人细胞对反式油酸的代谢反应。结果表明,反油酸处理引起了Hep G2细胞代谢水平的广泛变化。反油酸处理促进了脂肪酸合成,引起细胞磷脂代谢显著变化及细胞膜的重构,也增加了胆固醇和酰基甘油代谢中间产物的水平;同时,反油酸引起糖类和氨基酸代谢途径的增强,为脂肪酸合成提供原料。本研究获得的体外代谢组水平的变化为揭示反式脂肪酸与人类健康的不良关系提供了新的证据。     

毛细管气相色谱法测定精炼和氢化大豆油中的反式脂肪酸

采用CP—Sil88强极性毛细管柱气相色谱法对精炼和氢化大豆油中的顺、反异构和位置异构的脂肪酸进行定性、定量分析。结果表明，精炼和氢化大豆油中主要顺、反式脂肪酸实现了很好地分离，各种位置异构的反式脂肪酸也实现了较好地分离。采用归一化法对各种脂肪酸进行定量分析，结果表明，精炼大豆油中反式脂肪酸为总脂肪酸含量的3．45％，以△9c，12t—C18：2、△9t，12c—C18：2和△9c，12c，15t—C18：3、△9t，12c，15c—C18：3 4种形式为主；氢化大豆油中反式脂肪酸为总脂肪酸含量的38．73％，以△9t—C18：1、△10t—C18：1、△11t—C18：1 3种形式为主的反式十八碳单烯酸（t—C18：1）占反式脂肪酸总量的90．81％。     

霉菌产γ-亚麻酸的研究进展

γ-亚麻酸(GLA)作为人体必需的不饱和脂肪酸,在人体的激素调节以及生理代谢中发挥着重要作用。介绍了GLA生理作用,重点介绍了产GLA霉菌的菌种选育方面的研究情况,并且对霉菌中合成GLA的关键酶——Δ6脂肪酸脱氢酶的基因克隆和表达进行了较为详细的阐述。随着人们对GLA研究的深入,其应用范围会越来越广。     

响应面法优化壳聚糖对混合液镉铬离子吸附条件的研究

采用响应面法分析壳聚糖对混合液Cd2+离子和Cr6+离子的吸附作用.用Box-Behnken Design实验设计考察了壳聚糖添加量、重金属初始浓度、pH范围和吸附时间四个因素对壳聚糖去除Cd2离子和Cr6+离子的影响程度.通过响应面法优化,得到壳聚糖对Cd2离子和Cr6+离子去除的考察因素条件优化组合参数,即壳聚糖去除Cd2+离子的壳聚糖添加量为26g/L、Cd2+离子初始浓度为319mg/L、pH为6和吸附时间为117min;壳聚糖去除Cr6+离子的壳聚糖添加量为4g/L、Cr6+离子初始浓度为61mg/L、pH为1和吸附时间为360min.在此组合条件下:壳聚糖对Cd2离子去除率为93.62％,吸附量为12.46mg/g;壳聚糖对Cr6+离子去除率为99.27％,吸附量为10.55mg/g.红外光谱分析显示:壳聚糖中—CH3在结合Cd2离子中发挥主要作用;—OH、—CH、—NH和—C—O对Cr6+离子结合起主要作用.     

固定化脂肪酶催化醇解天然奶油合成含硫风味酯的研究

以无水奶油为脂肪酸来源,固定化脂肪酶TL IM为催化剂,无溶剂体系下催化3-甲硫基丙醇与无水奶油反应制备3-甲硫基丙酯。采用十七酸甲酯为内标,GC-FID监测反应过程中转化率的变化,并分析脂肪酶对不同碳链脂肪酸的选择性。结果显示:酶解前8h,脂肪酶对C4-C14脂肪酸选择性优势明显,随着反应的进行,对短链脂肪酸选择性逐渐变小,而对C16-C18脂肪酸选择性增加;当反应温度为50℃,脂肪酶添加量为5wt%,3-甲硫基丙醇与无水奶油的摩尔比3∶1,反应时间为8h时,产物中3-甲硫基丙酯的浓度分别为:丁酸3-甲硫基丙酯29.75mg/m L、己酸3-甲硫基丙酯15.32mg/m L、辛酸3-甲硫基丙酯7.22mg/m L、癸酸3-甲硫基丙酯21.32mg/m L、十二酸3-甲硫基丙酯26.64 mg/m L、十四酸3-甲硫基丙酯77.62mg/m L,C4-C14总转化率为71.16%。     

质谱特征离子定性分析海洋鱼油中的脂肪酸组成

研究质谱特征离子对海洋鱼油脂肪酸的快速定性分析。首先,根据脂肪酸甲酯的断裂规律,基峰离子可快速确定脂肪酸的不饱和度,如基峰离子m/z 74为饱和脂肪酸,m/z 55为单烯脂肪酸,m/z 67为二烯脂肪酸,m/z 79为三烯及以上多不饱和脂肪酸;其次,分子离子M+及其特征性碎片丢失而形成的特征离子如[M-29]~+、[M-31]~+、[M-32]~+、[M-43]~+和[M-74]~+等,可有效用于脂肪酸甲酯分子量和脂肪酸碳链长度的分析;而ω离子和α离子可准确对三烯及以上多不饱和脂肪酸中的双键位置进行分析鉴定。通过质谱特征离子的综合分析,从海洋鱼油胶囊中共分析鉴定出24种脂肪酸,以C_(14:0)、C_(16:0)、C_(18:1n-9)、C_(20:5n-3)和C_(22:6n-3)为主,表明质谱特征离子能快速准确用于海洋鱼油脂肪酸的定性分析。同时,海洋鱼油中C_(20:5n-3)和C_(22:6n-3)的总含量高达29.13%,表明其具有很高的营养价值和保健功能。     

恒化培养条件下粪产碱杆菌凝胶多糖的发酵动力学研究

通过恒化培养实验研究了不同稀释速率下粪产碱杆菌(Alcaligenes faecalis)的生理代谢特性变化,利用Monod、Luedeking-Piret、Herbert-pirt模型分析了粪产碱杆菌的生长、多糖合成以及底物葡萄糖消耗的动力学变化。结果表明,在铵离子限制条件下的菌体比生长速率与铵离子浓度之间的动力学模型为μ=0.36S/(2.27+S),凝胶多糖比产率与铵离子浓度间的动力学模型为q_p=0.0612S/(2.27+S)+0.14×(1-S/64.94),以及比底物葡萄糖消耗速率与比产率的动力学模型为:q_s=1.5μ+q_p+0.0625。通过对恒化培养模型及生产菌生理代谢参数的分析,统计分析表明该生产菌最佳限制性铵离子浓度为5.75 mg/L,得到控制最适比生长速率为0.21 h~(-1)时,A.faecalis的最高产率可达到0.85 g/L/h,并且最大比产率高达0.171 g/g/h,产率和比产率与批培养过程相比分别提升了118%和200%,能够显著提升凝胶多糖的生产效率。     

Maximizing the production of γ-linolenic acid in Mortierella ramanniana var. ramanniana as a function of pH,temperature and carbon source,nitrogen source,metal ions and oil supplementation

A locally isolated (Perryvale, Alta., Canada) fungal genus, Mortierella ramanniana var. ramanniana, was evaluated as a potential industrial producer of γ-linolenic acid (GLA). Six growth variables (pH, temperature, carbon source, nitrogen source, and metal ions and oil supplementation) were systematically manipulated. The results indicated that the GLA production for this particular strain, could be maximized by using a basal growth medium consisting of 5% dextrose and 1% yeast extract, supplemented with 5 mg/L Mn²⁺ with incubation at 20 °C. The lipid yield under optimum conditions was 54.2% of the total dry biomass and consisted of 84.3% unsaturated fatty acids. The yield of GLA per gram biomass represented 13.3% of the total lipid content, slightly higher than that of the typical evening primrose oil GLA yield. The study establishes this variety as an effective intermediate fungal source of GLA.     

利用生物技术生产廿碳五烯酸和廿二碳六烯酸

根据现在的了解，能产生有意义含量的廿碳五烯酸（ＥＰＡ）和廿二碳六烯酸（ＤＨＡ）的微生物有真菌和藻类。在微生物中，多不饱和脂肪酸的合成通常是以单不饱和脂肪酸、油酸为底物。合成途径中，有两个主要的反应，即碳链的增长和去饱和作用。ＥＰＡ和ＤＨＡ的生成通过ω－３代谢途径。微生物脂肪酸构成的质量和数量都受培养基的组分，通气、光照强度、温度和培养时间等环境因素的影响。大多数用于提取鱼油中的ＥＰＡ的方法也可用于微生物脂质中的ＥＰＡ的提取。几种方法被提出来生产高含量的ＥＰＡ和ＤＨＡ。它们一般是基于下列技术的结合：溶剂提取、尿素包合法、分子蒸馏、分馏、液相色谱法和超临界二氧化碳提取。     

Medium Optimization of Gamma Linolenic Acid Production in Mucor rouxii CFR -G15 using RSM

Omega-6 polyunsaturated fatty acids has both pharmaceutical and neutraceutical importance. Central composite rotatable design (CCRD), along with response surface methodology (RSM), was employed to optimize the medium components for maximum production of gamma linolenic acid (GLA) from Mucor rouxii CFR-G15. Nutritional parameter such as carbon (glucose) and nitrogen (yeast extract and ammonium nitrate) sources were focused in this study to enhance the GLA production. The optimal conditions for maximizing GLA production (18.55%) were at 65 g glucose per liter; 3.5 g yeast extract per liter, and 0.5 g ammonium nitrate per liter. Thus, by using CCRD with RSM, it is quite possible to determine the accurate values of the fermentation parameters where maximum production of GLA is achieved.     

河川沙塘鳢肌肉营养成分的分析和评价

为了解河川沙塘鳢肌肉的营养价值,采用生化分析手段对生态养殖的2 龄冬季河川沙塘鳢进行肌肉营养成分分析和品质评价。结果显示,冬季河川沙塘鳢的肌肉水分、粗蛋白、粗脂肪和粗灰分含量分别为78.08%、18.26%、1.11%和1.30%。18 种被检出的常见氨基酸中含量最高的3 种氨基酸是谷氨酸、天冬氨酸和赖氨酸;肌肉鲜样中氨基酸总量（total amino acid,TAA）、必需氨基酸（essential amino acids,EAA）、半必需氨基酸（half essential amino acids,HEAA）和鲜味氨基酸（delicious amino acid,DAA）分别为19.47%、7.74%、1.80%和7.09%;EAA/TAA、EAA/非必需氨基酸（nonessential amino acids,NEAA）、DAA/TAA分别为39.75%、77.92%和36.42%;第1、第2限制性氨基酸分别是色氨酸和缬氨酸;必需氨基酸指数和支链氨基酸与芳香族氨基酸的比值（F值）分别为74.86和2.15。肌肉干样中检出6 种饱和脂肪酸（total saturated fatty acids,SFA）、4 种单不饱和脂肪酸（monounsaturated fatty acids,MUFA）和10 种多不饱和脂肪酸（polyunsaturated fatty acids,PUFA）;ΣSFA、ΣMUFA、ΣPUFA、Σn-3 PUFA、二十碳五烯酸＋二十二碳六烯酸和Σn-6 PUFA分别为31.83%、20.91%、47.27%、30.60%、23.58%和15.56%,ΣSFA/ΣUFA和Σn-3 PUFA/Σn-6 PUFA分别为0.47%和1.97%。结果表明,河川沙塘鳢是一种营养价值较高、味道鲜美的优质鱼类,且有一定的保健作用;冬季河川沙塘鳢肌肉营养价值高于繁殖季节的,其粗蛋白和粗脂肪含量较高且氨基酸和脂肪酸组成较优。     

外源油脂的添加促进卷枝毛霉脂肪酸的积累

卷枝毛霉（Mucor circinelloides）可利用多种碳源实现脂质积累,不同的碳源对其脂质积累的影响不同。在不改变其它发酵培养条件和碳氮比的情况下,以100%葡萄糖为对照,添加不同比例的外源油脂（大豆油）作为另一种碳源,对其细胞生长和脂质生成的影响进行了研究。采用氯仿-甲醇法提取脂质,通过气相色谱技术（gas chromatography,GC）测定其脂肪酸组成及含量。同时,采用实时荧光定量PCR法检测了与脂肪酸合成相关的脱饱和酶基因在菌株中的表达情况。结果表明：碳氮比不变的情况下,最佳碳源混合比例为葡萄糖35 g/L,大豆油24.3 g/L,该培养基中生长的卷枝毛霉的生物量、脂质含量和脂质产量在发酵前期迅速增长,在发酵后期的增长趋势逐渐平缓,在发酵72 h之后略有下降,其最高生物量、脂质含量和产量分别为19.60 g/L、42.62%和8.35 g/L,而对照组（以葡萄糖为单一碳源）仅为14.72 g/L、34.41%和5.06 g/L。与对照组相比,经混合碳源培养的卷枝毛霉的生物量、脂质含量和产量分别提高了33.15%、23.8%和65.10%,且可以将大豆油中的α-亚麻酸...     

发酵条件对发酵性丝孢酵母脂肪酸组成的影响

通过摇瓶发酵，研究了温度、摇瓶转速、碳源种类和浓度对发酵性丝孢酵母（Trichosporom fermentans）脂肪酸组成的影响。气相色谱法分析结果表明，较高发酵温度能提高菌体油脂的脂肪酸饱和程度，此条件下饱和脂肪酸含量占脂肪酸总量的49．1％：摇瓶转速高有利于菌体油脂不饱和脂肪酸的生成；木糖浓度为100g／L时菌体油脂中饱和脂肪酸含量最高，占脂肪酸总量的50．4％；木糖比葡萄糖更容易被转化生成饱和脂肪酸含量较多的油脂。菌体脂肪酸组成包括肉豆蔻酸、棕榈酸、棕榈油酸、硬脂酸、油酸和亚油酸，其中棕榈酸和油酸含量较高。     

Fractionation of lipids and purification of γ-linolenic acid (GLA) from Spirulinaplatensis

The microalgae, Spirulinaplatensis, is an excellent source of γ-linolenic acid (GLA), an essential polyunsaturated fatty acid and a potent nutraceutical. The fatty acid composition of S.platensis ARM 740 was determined after transmethylation by gas chromatography (GC). Lipid fractionation was achieved on silica gel column chromatography and preparative TLC. Neutral lipids, glycolipids and phospholipids accounted for 77.0%, 15.6% and 7.4%, respectively, of the total lipid fraction. S.platensis ARM 740 was found to contain 94% of the total GLA in the glycolipid fraction. Attempts were made to purify GLA methyl ester by using urea to form inclusion complexes with the saturated and the less unsaturated FAMEs (fatty acid methyl esters), which enhanced the purity of GLA methyl ester to 84%. A further approach to isolate GLA methyl ester with higher purity involved the use of argentated silica gel chromatography. An initial PUFA concentration step frequently adopted by most researchers to increase GLA purity was not necessary in the isolation of GLA from S.platensis. A GLA methyl ester with a purity of >96% and a recovery of 66% was obtained. Purity of the isolated GLA methyl ester was confirmed by GC and IR analysis with respect to authentic standard.     

Effect of the unsaturation degree and concentration of fatty acids on the properties of WPI-based edible films

The incorporation of lipids into hydrophilic protein films allows the modification of their barrier properties, improving its commercial application as preservation medium on different foods. The main objective of this study was to develop films from Whey Protein Isolate (WPI) together with saturated and unsaturated fatty acids and to determine the effect of concentration and unsaturation degree on surface tension of the coating solution and on water vapor permeability (WVP), mechanical properties (tensile strength and elongation at break), and opacity, of the films. The results obtained showed that surface tension was significantly decreased by adding unsaturated fatty acids (oleic and linoleic acid), whereas the greatest effect on WVP reduction was achieved with stearic acid. The addition of stearic acid resulted in a decrease of elongation and an increase of tension strength; however unsaturated fatty acid content did not modify the elongation and slightly reduce a tensile strength.     

Studies on the metabolism of essential fatty acids in isolated human testicular cells

The essential fatty acid 22:6(n-3) is a minor component of the Western diet, but a major fatty acid in human testis and semen. In mature spermatozoa, the physical and fusogenic properties of the plasma membrane are probably influenced by its particular fatty acid composition. In this study, the synthesis of 22:6(n-3) and 22:5(n-6) was investigated in isolated human testicular cells. [1-(14)C]20:4(n-6), [1-(14)C]20:5(n-3), [1-(14)C]22:4(n-6) and [1-(14)C]22:5(n-3) were incubated in a 'crude' cell suspension (consisting of a mixture of the cells in the seminiferous tubule), and in fractionated pachytene spermatocytes and round spermatids. The esterification of fatty acids in lipid and phospholipid classes and the fatty acid chain elongation and desaturation were measured. The crude cell suspension metabolized the fatty acids more actively than did the fractionated germ cell suspension, indicating that types of cell other than the germ cells are important for fatty acid elongation and desaturation and thus the production of 22:6(n-3). This finding is in agreement with previous results in rats that indicated that the Sertoli cells are the most important type of cell for the metabolism of essential fatty acids in the testis. Some [1-(14)C]20:5(n-3) was elongated to [(14)C]22:5(n-3) in the fractionated germ cells, but very little was elongated further to [(14)C]24:5(n-3),possibly restricting the formation of [(14)C]22:6(n-3). In the fractionated germ cells, the fatty acid substrates were recovered primarily in the phospholipid fraction, indicating an incorporation in the membranes, whereas in the crude cells, more substrates were esterified in the triacylglycerol fraction. In the phospholipids, more radioactivity was recovered in phosphatidylcholine than in phosphatidylethanolamine and more radioactivity was recovered in phosphatidylethanolamine than in phosphatidylinositol or phosphatidylserine.     

抗氧化乳酸菌对发酵腌干带鱼脂肪氧化的影响及其主成分分析

将从传统腌干鱼中筛选出的3株具有抗氧化活性乳酸菌(干酪乳杆菌、植物乳杆菌和戊糖片球菌)作为发酵剂加入到腌干带鱼中,测定pH值、过氧化值、酸值、硫代巴比妥酸值和正己醛含量等指标,以监测发酵腌干带鱼加工过程中脂肪的氧化程度,并分析了乳酸菌发酵对腌干带鱼成品脂肪酸组成的影响。结果表明,在腌干带鱼生产过程中抗氧化乳酸菌对不饱和脂肪酸的氧化有一定的抑制作用,接菌发酵的腌干带鱼过氧化值、硫代巴比妥酸值、正己醛含量和饱和脂肪酸含量显著低于传统腌干带鱼,而酸值和不饱和脂肪酸含量高于传统腌干带鱼。对测定的指标进行主成分分析的结果表明,第1主成分能反映脂肪水解程度,其线性回归函数为:Y_1=0.131X_1+0.208X_2+0.360X_3+0.244X_4+0.083X_5-0.388X_6+0.324X_7+0.343X_8;第2主成分能反映脂肪氧化程度,其线性回归函数为:Y_2=0.330X_1+0.406X_2+0.182X_3+0.440X_4+0.294X_5-0.205X_6+0.135X_7+0.157X_8。本研究为提高发酵腌干鱼的安全性提供理论依据。