磷脂酶A1催化水解大豆油制备甘油二酯研究（Ⅲ）——产品分析与检测

用高效液相色谱(HPLC)分析了大豆油和经过磷脂酶 A1 催化水解、分子蒸馏纯化的甘油二酯产品中甘油酯的含量,用折光指数检测器(RID)测定水解产品甘油二酯含量为42.64%,通过高效液相色谱电喷雾(ESI)质谱(MS)联用分析了甘油酯的组成.用气相色谱分析了大豆油、分子蒸馏轻相脂肪酸及水解产品的脂肪酸组成.结果发现,和原料大豆油相比,磷脂酶 A1 催化水解得到的脂肪酸中棕榈酸含量显著提高.     

牦牛肉品质相关的去饱和脂肪酶1基因表达差异分析

检测牦牛组织中去饱和脂肪酶1(stearoyl-CoA desaturase 1,SCD1)基因表达量,分别选择1月份、4月份、9月份年龄相近,取其肝脏、肾脏、结肠、背最长肌4个组织,采用实时荧光定量聚合酶链式反应(real-time fluorescent quantitative polymerase chain reaction,RT-PCR)技术和酶联免疫反应测定牦牛不同组织中SCD1的表达量和含量。结果表明:以肝脏为对照组,SCD1基因在牦牛肝脏、肾脏、结肠、背最长肌4个组织中均有表达,且SCD1基因表达量和蛋白含量均在牦牛背最长肌中最高,差异显著(P0.05)。不同月份间,1月份牦牛SCD1基因表达量和蛋白含量都低于9月份和4月份。说明SCD1基因在牦牛不同组织均有表达,且在寒冷季节表达量较低。     

油茶脂肪酸代谢途径中关键酶基因调控油脂合成的规律研究

以国审油茶"华硕"和普通油茶"望城1号"不同发育时期种仁为材料,分析种仁含油率、脂肪酸成分和脂肪酸代谢关键酶基因表达规律。结果表明,油茶酰基载体蛋白基因(CoACP)、硬脂酰脱饱和酶基因(CoSAD)和油酸脱氢酶基因(CoFAD2)表达规律与油茶种仁成熟程度有关而与品种关系不大;"华硕"较"望城1号"生殖发育期长,成熟期晚,脂肪酸各成分变化幅度大,采收期可比"望城1号"延迟1个月;10月底采收时虽然"华硕"含油率低于"望城1号",但不饱和脂肪酸含量却高些,若延迟采收"华硕"可保产量质量均优。     

响应面法优化重组谷氨酸棒杆菌G-BC产脂肪酸的培养条件北大核心CSCD

乙酰辅酶A羧化酶是谷氨酸棒杆菌中调控脂肪酸合成的关键酶。为了研究乙酰辅酶A羧化酶α亚基(accBC)基因对脂肪酸合成的影响,以accBC表达的菌株重组谷氨酸棒杆菌G-BC为材料,研究了诱导剂异丙基-β-D-硫代半乳糖苷(IPTG)浓度、培养时间、培养温度对重组谷氨酸棒杆菌G-BC脂肪酸产量的影响,并通过响应面法优化了重组谷氨酸棒杆菌G-BC产脂肪酸的培养条件,同时与原始菌株脂肪酸产量进行了对比。结果表明:重组谷氨酸棒杆菌G-BC产脂肪酸的最佳条件为诱导剂浓度1 mmol/L、培养时间20 h、培养温度32℃,在此条件下脂肪酸产量为34.56 mg/g(以菌体干质量计),比原始菌株提高了1.68倍。说明乙酰辅酶A羧化酶α亚基对于谷氨酸棒杆菌合成脂肪酸有促进作用。     

CRISPRi/ddCpf1促进大肠杆菌中丙二酰辅酶A的积累

丙二酰辅酶A是多种有价值化合物（包括食品、保健品等）合成的重要前体物质，其合成已成为大肠杆菌中生产目标代谢物的潜在瓶颈。为解决其生物合成的瓶颈问题，作者通过CRISPR干扰（CRISPR interference，CRISPRi） 促进丙二酰辅酶A的积累。首先，构建了丙二酰辅酶A的生物传感器，实现了其快速、可视化的胞内测量。随后，构建了CRISPRi/ddCpf1系统，实现了基因转录的抑制，并尝试用ddCpf1（催化失活的Cpf1）和RNA聚合酶抑制蛋白Gp2融合表达（CRISPRi/ddCpf1-Gp2）。结果表明，Gp2虽然有一定的转录抑制效果，但是严重影响了生长。最后，利用CRISPRi/ddCpf1系统进行两组双靶点基因抑制，分别靶向乙醛脱氢酶和3-氧代酰基-酰基载体蛋白合酶II基因，以及3-氧代酰基-酰基载体蛋白合酶I和琥珀酰辅酶A合成酶基因，均实现了丙二酰辅酶A浓度的提高。     

磷脂酶A1对大豆毛油脱胶效果的影响

以大豆毛油为原料,研究磷脂酶A1 添加量、柠檬酸溶液添加量、脱胶温度和脱胶时间对脱胶效果以及中性油脂肪酸和甘油酯组成的影响。结果表明,磷脂酶A1 脱胶的最佳反应条件为20 mg/100 g 油的磷脂酶A1、0.15 mL 45% 柠檬酸溶液、50 ℃脱胶温度、4 h 反应时间。在最佳的脱胶工艺条件下,磷脂酶A1 脱胶中性油中含磷量降至0.47 mg/kg,油脂得率（95.55%）高于酸化脱胶中性油的得率（92.94%）。酶法脱胶中性油与毛油的脂肪酸和甘油酯组成相比,酶法脱胶中性油的脂肪酸组成没有明显变化,甘油酯组成中的甘一酯和甘二酯相对含量减少、甘三酯相对含量增加;与酸化脱胶油脚相比,磷脂酶A1 脱胶油脚中溶血磷脂（溶血磷脂酰胆碱、溶血磷脂酰乙醇胺和溶血磷脂酰肌醇）的相对含量增加至48%。     

酶催化酯交换法制备零反式脂肪酸人造奶油基料油的理化性质研究

以松籽油和棕榈油硬脂为原料,通过酶催化酯交换法生产零反式脂肪酸人造奶油基料油。酯交换反应得到油脂的主要脂肪酸组成为:棕榈酸(37.88%),油酸(28.30%),亚油酸(20.19%),同时含有少量功能性脂肪酸——松籽油酸(4.77%),且没有检测到反式脂肪酸;结构脂质的熔点为37.5℃,其固态脂肪含量在20、25、30℃和40℃时分别是40.28%、32.62%、22.06%、3.40%,并且其主要晶型是β’型。因此,酯交换反应得到的零反式脂肪酸油脂适合作为人造奶油的基料油。     

水相体系酶法制备溶血磷脂酰乙醇胺研究

探索了磷脂酶A1在水相体系中酶解制备溶血磷脂酰乙醇胺(LPE)的可行性,并进行了酶解条件对LPE相对含量的影响研究。得到最佳酶解条件为:磷脂酰乙醇胺(PE)的纯度为80%,液料比10∶1,酶解温度35℃,酶添加量0.04 m L/g,Ca Cl2添加量0.010 g/m L,酶解时间30 min。在最佳酶解条件下,得到酶解产物中的LPE相对含量为42.0%。     

异源表达乙酰辅酶A羧化酶对大肠杆菌产脂肪酸的影响

以谷氨酸棒杆菌Corynebacterium glutamicum ATCC 13032基因组为模板,通过聚合酶链式反应（polymerase chain reaction,PCR）扩增得到DNA分子量大小为1 815 bp和1 700 bp的乙酰辅酶A羧化酶α-亚基以及β-亚基,将目的基因连接到pXMJ19质粒,得到表达载体pXMJ19-accBC、pXMJ19-accD1,将其转化至表达菌株BL21,得到异源表达菌株BL21-accBC、BL21-accD1。使用气相色谱测定发酵液中脂肪酸含量。结果显示：重组菌株BL21-accBC与BL21-accD1经1 mmol/L的异丙基-β-D-硫代半乳糖苷有氧诱导20 h后,脂肪酸含量分别达到45.57 mg/g菌体干重和18.81 mg/g菌体干重,较对照菌株分别提高了266%和51.32%。     

河西肉牛脂肪酸成分比较及主成分分析

通过对河西肉牛西门塔尔和安格斯牛中脂肪酸成分测定,阐明两个品种间牛肉中脂肪酸的组成和特征差异。随机选取相同育肥方式的18个月龄发育健康的西门塔尔和安格斯公牛各9头,禁食12h后进行屠宰,取背最长肌为样本,以气象色谱内标法对西门塔尔牛和安格斯牛肉中的脂肪酸组成及其含量进行测定,最后使用主成分分析法对两个品种牛肉脂肪酸的差异进行评价分析。2种牛肉中共鉴定出17种脂肪酸,包括5种饱和脂肪酸和12种不饱和脂肪酸,饱和脂肪酸中棕榈酸含量最高,分别为21.81%和23.54%,安格斯牛肉的不饱和脂肪酸含量比西门塔尔牛高7.10%,同时肉豆蔻脑酸、二十碳一烯酸、花生四烯酸及DHA 4种不饱和脂肪酸含量显著高于西门塔尔牛;基于主成分分析法提取7个核心脂肪酸为2种牛肉中的特征脂肪酸,建立综合数学模型F=0.590×F1+0.154×F2+0.124×F3,从脂肪酸组成及综合得分上来看,安格斯牛肉要优于西门塔尔牛肉。脂肪酸测定和主成分分析揭示了两种牛肉中脂肪酸组成的特征与差异,为河西优质牛肉产品的合理开发利用提供理论依据。     

Lipid characteristics of subcutaneous adipose tissue and M. longissimus thoracis of Angus and Wagyu steers fed to US and Japanese endpoints

We hypothesized that the concentrations of monounsaturated fatty acids (MUFA) and cholesterol of adipose tissue and M. longissimus thoracis would not differ between Angus and American Wagyu steers when fed to a typical US live weight, but would diverge when fed to a Japanese live weight. To test this, 8 steers of each breed type were assigned to a high-energy, corn-based diet, and another 8 steers of each breed type were fed coastal bermuda grass hay diet, supplemented with the corn-based diet to achieve a daily gain of 0.9 kg/d. Targeted final body weights were 525 kg for steers fed for 8 or 12 mo the corn- or hay-based diets, respectively, and were 650 kg for steers fed for 16 or 20 mo the corn- or hay-based diets. Digesta concentrations of stearic (18:0) and trans-vaccenic acid decreased, whereas linoleic acid (18:2n-6) increased between the US and Japanese endpoints (all P ? 0.03). α-Linolenic acid (18:3n-3) increased in digesta only in the hay-fed steers during this time. Plasma concentrations of palmitic (16:0) and palmitoleic acid (16:1n-7), and the 16:1:18:0 ratio, were higher in Angus steers than in Wagyu steers. Also, the plasma 16:1:18:0 ratio was decreased by hay feeding in Angus steers, but increased in Wagyu steers, when fed to the Japanese endpoint. Concentrations of oleic (18:1n-9), linoleic, α-linolenic, and 18:2trans-10,cis-12 conjugated linoleic acid all were higher in Wagyu than in Angus subcutaneous (s.c.) adipose tissue, whereas myristic (14:0) and palmitic acid were higher in Angus s.c. adipose tissue (P ? 0.05). All MUFA increased, and saturated fatty acids decreased, between the US and Japanese endpoints. Slip points of lipids in s.c. adipose tissue were over 10 °C lower (P = 0.01) in Japanese-endpoint steers than in US endpoint steers, consistent with the overall increase in MUFA with time on feed. The concentration of cholesterol in the M. longissimus thoracis increased with time, which may have been related to the increase in oleic acid. Because the breed × endpoint interaction was not significant for cholesterol or any of the adipose tissue fatty acids, we conclude that our original hypothesis was incorrect. Of the three factors tested (breed type, diet, and slaughter age endpoint), endpoint had the greatest effect on adipose tissue lipid composition.     

Influence of stearoyl-coenzyme A desaturase 1 genotype and stage of lactation on fatty acid composition of Canadian Jersey cows

Bovine milk contains high proportions of saturated fatty acids (SFA) because of the extensive biohydrogenation of dietary fatty acids in the rumen. Stearoyl-coenzyme A desaturase 1 (SCD1) catalyzes the conversion of C10 to C18 SFA into their monounsaturated (MUFA) counterparts in the mammary glands of ruminant animals; and 2 alleles (A and V) have previously been identified at the SCD1 locus. Genotypes at this locus were identified and fatty acid contents of milk were measured for 525 Canadian Jersey cows. Association analysis indicated that allele A is positively associated with higher C10 (C10I), C12 (C12I) and C14 (C14I) indices and, consequently, with greater contents of C10:1 and C12:1, but not C14:1, relative to allele V. Allele A was also positively associated with increased 305-d milk and protein yields. Allele A, however, had no influence on C16 (C16I), C18 (C18I), or conjugated linoleic acid indices (CLAI) compared with the V allele. Stage of lactation had an influence on desaturase indices and consequently on the MUFA contents of milk fat. The indices C10I, C12I, C14I, and CLAI increased from early to mid lactation as did their respective MUFA. Genetic selection for increased unsaturation of the hypercholesterolemic fatty acids in milk fat is feasible and may be accompanied by increased lactation milk and protein yields.     

Endogenous production of n-3 and n-6 fatty acids in mammalian cells

Polyunsaturated fatty acids (PUFA) are important components of mammalian diets, and the beneficial effects of n-3 PUFA on human development and cardiovascular health have been well documented. Caenorhabditis elegans is one of the few animals known to be able to produce linoleic (LA, 18:2n-6) and alpha-linolenic (ALA, 18:3n-3) essential fatty acids. These essential PUFA are generated by the action of desaturases that successively direct the conversion of monounsaturated fatty acids (MUFA) to PUFA. The cDNA coding sequences of the C. elegans Delta(12) and n-3 fatty acid desaturases were each placed under the control of separate constitutive eukaryotic promoters and simultaneously introduced into HC11 mouse mammary epithelial cells by adenoviral transduction. Phospholipids from transduced cells showed a significant decrease in the ratios of both MUFA:PUFA and n-6:n-3 fatty acids relative to control cultures. The fatty acid profile of transduced cellular phospholipids revealed significant decreases in MUFA and arachidonic acid (20:4n-6), and increases in LA, ALA, and eicosapentaenoic acid (20:5n-3). The fatty acid composition of triacylglycerols derived from transduced cells was similarly, but less dramatically, affected. These results demonstrate the functionality of C. elegans fatty acid desaturase enzymes in mammalian cells. Expression of these desaturases in livestock might act to counterbalance the saturating effect that rumen microbial biohydrogenation has on the fatty acid profile of ruminant products, and allow for the development of novel, land-based dietary sources of n-3 PUFA.     

Association analyses of single nucleotide polymorphisms in the LEP and SCD1 genes on the fatty acid profile of muscle fat in Simmental bulls

The aim was to investigate the effect of the genetic polymorphisms of leptin (LEP) and stearoyl-CoA desaturase (SCD1) genes on the fatty acid (FA) composition of the muscle of 103 Simmental bulls. Ten single nucleotide polymorphisms (SNP) were detected in exons 2 and 3 of the LEP gene, two of them encoding non-synonymous mutations. Allelic substitution effects of all the SNP on 28 single fatty acids, monounsaturated (MUFA) and polyunsaturated (PUFA) and desaturation indexes were estimated. Both the SCD1 SNP, as well as three SNP of the leptin gene, affected, to different extents, the desaturation of FA into MUFA. Because it was previously proposed that leptin's metabolic action involves down-regulation of SCD1, it is possible that, beyond the mere additive effect of SCD1 gene on FA desaturation, the non-synonymous mutations in the leptin gene also contribute to the variability of FA composition in muscle fat.     

An influence of cooking on fatty acid composition in three varieties of common beans and in lentil

The fatty acid composition of three raw and cooked freeze-dried common bean varieties (Phaseolus vulgaris), namely enjevec, Semenarna 22 and Cipro, and of the lentil (Lens esculenta), var. Anicia, was determined and the influence of storage on their composition was studied. Analyses of fatty acid composition were conducted by in situ transesterification and capillary column gas-liquid chromatography. In raw milled beans average values of about 16% saturated fatty acids (SAT), 6% monosaturated fatty acids (MUFA) and 78% polyunsaturated fatty acids (PUFA) were found. Somewhat different values of 15% of SAT, 25% MUFA and 60% PUFA were found in lentil. In cooked beans the content of all fatty acids was slightly decreased. In cooked lentil the decrease was almost 50%, but the ratios of SAT, MUFA and PUFA in both cases were practically the same. After two years of storage at 4 °C the fatty acid content in raw milled beans was unchanged, but altered in cooked ones. The amounts of linoleic (18:2, n-6) and -linolenic (18:3, n-3) acid decreased, but myristic (14:0), margaric (17:0) and arachidic (20:0) acids increased. It was found that freeze-dried cooked beans, prepared from raw seed beans, kept 2.5 years at 10 °C, have practically the same fatty acid composition as freeze-dried cooked beans 0.5 year after harvesting.     

The polymorphisms of stearoyl-CoA desaturase (SCD1) and sterol regulatory element binding protein-1 (SREBP-1) genes and their association with the fatty acid profile of muscle and subcutaneous fat in Fleckvieh bulls

The previously reported genetic polymorphisms of the stearoyl-CoA desaturase (SCD1) and sterol regulatory element binding protein-1 (SREBP-1) genes were investigated in Fleckvieh bulls using the PCR-RFLP and AS-PCR methods, respectively. The genomic DNA was obtained from a total of 370 bulls. The frequencies of alleles A and V of the single nucleotide polymorphism in exon 5 of the SCD1 gene (SNP 878C>T) were 0.555 and 0.445, respectively. In the 84-bp Ins/Del polymorphism in intron 5 of the SREBP-1 gene, the frequency of the L allele (insertion) was markedly higher (0.920) than that of the S allele (deletion; 0.080). Fatty acid profile was determined in a total of 367 samples of muscle fat (MSF) and 150 samples of subcutaneous fat (SCF). The AA genotype of SCD1 polymorphism showed a lower content of C18:0 (P < 0.01) and higher contents of C14:1 cis-9 (P < 0.001) and C18:1 cis-9 (P < 0.05) in MSF compared to the VV genotype. As a result, the bulls with genotypes AA or AV had lower SFA (P < 0.01), higher MUFA (P < 0.05) and higher MUFA/SFA (P < 0.01) than VV animals. The results obtained for SCF were similar. The SREBP-1 polymorphism was associated with a higher content of C14:1 cis-9 (P < 0.01) in the LS compared to LL genotype in SCF. The results of this study demonstrated the existence of the polymorphisms in the SCD1 and SREBP-1 genes in the population of Fleckvieh cattle and their associations with the concentrations of several MSF and SCF fatty acids.     

基于脂肪酸指纹的放牧与舍饲牛肉真实性判别模型建立

为论证以脂肪酸指纹建立模型判别牛肉放牧和舍饲来源的可行性,从内蒙古优势肉牛养殖带8个旗县/区采集放牧和舍饲牛股二头肌、背最长肌和肋部皮下脂肪共91份,气相色谱法测定脂肪酸,进行主成分分析(principal component analysis,PCA)和描述性统计,并建立软独立建模分类(soft independen...     

Milk fatty acid unsaturation: genetic parameters and effects of stearoyl-CoA desaturase (SCD1) and acyl CoA: diacylglycerol acyltransferase 1 (DGAT1)

With regard to human health aspects of milk fat, increasing the amount of unsaturated fatty acids in milk is an important selection objective. The cow's diet has an influence on the degree of unsaturation, but literature suggests that genetics also plays a role. To estimate genetic variation in milk fatty acid unsaturation indices, milk fatty acid composition of 1,933 Dutch Holstein Friesian heifers was measured and unsaturation indices were calculated. An unsaturation index represents the concentration of the unsaturated product proportional to the sum of the unsaturated product and the saturated substrate. Intraherd heritabilities were moderate, ranging from 0.23 ± 0.07 for conjugated linoleic acid (CLA) index to 0.46 ± 0.09 for C16 index. We genotyped the cows for the SCD1 A293V and DGAT1 K232A polymorphisms, which are known to alter milk fatty acid composition. Both genes explain part of the genetic variation in unsaturation indices. The SCD1 V allele is associated with lower C10, C12, and C14 indices, and with higher C16, C18, and CLA indices in comparison to the SCD1 A allele, with no differences in total unsaturation index. In comparison to the DGAT1 K allele, the DGAT1 A allele is associated with lower C10, C12, C14, and C16 indices and with higher C18, CLA, and total indices. We conclude that selective breeding can contribute to higher unsaturation indices, and that selective breeding can capitalize on genotypic information of both the SCD1 A293V and the DGAT1 K232A polymorphism.     

Fatty acid elongase 6 plays a role in the synthesis of long-chain fatty acids in goat mammary epithelial cells

In nonruminants, it is well established that elongation of very long-chain fatty acid-like fatty acid elongase 6 (ELOVL6) catalyzes the synthesis of C18:0 from C16:0 in lipogenic tissues like adipose and liver. However, the role of ELOVL6 in regulating lipid metabolism in ruminant mammary gland remains unknown. In the present study, ELOVL6 was overexpressed or knocked down via adenoviral transfection to assess its role in goat mammary epithelial cells. Results revealed that ELOVL6 overexpression had a weak effect on the expression of genes related to triacylglycerol (TAG) synthesis and desaturation. Overexpression of ELOVL6 increased the content of C18:0 at the expense of C16:0, and increased the elongation index of C16:0. Overexpression of ELOVL6 had no significant effect on the elongation index of C16:1n-7 and the desaturation indices of C16:0 and C18:0. Knockdown of ELOVL6 had a negative effect on mRNA expression of the esterification genes GPAM and diacylglycerolacyltransferase 2 (DGAT2) and TAG concentration; however, it increased the concentration of C16:0 and decreased C18:1n-7 and C18:1n-9 in goat mammary epithelial cells. Accordingly, downregulation of ELOVL6 significantly decreased the elongation indices of C16:0 and C16:1n-7. The lack of change in the desaturation indices of C16:0 and C18:0 upon knockdown of ELOVL6 was consistent with the minor change in SCD1 expression. In conclusion, these are the first results highlighting an important role of ELOVL6 in long-chain fatty elongation and TAG synthesis in ruminant mammary cells.