Analytical Method Assessment for the Determination of DHA and Fatty Acids Present in Unextracted <i>Aurantiochytrium limacinum</i>Biomass

Research into long-chain polyunsaturated fatty acids (LC-PUFA), such as docosahexaenoic acid (DHA C22:6 n-3), has shown that their inclusion in the human diet is linked with many health benefits. This has led to an increased interest in the enrichment of certain foodstuffs with DHA by supplementing animal fed with DHA-rich ingredients which can lead to an increased uptake in the meat, milk and eggs animal by-products. The microalgae Aurantiochytrium limacinum has been found to be especially useful in this pursuit. It is subsequently desirable to availably have a simple and robust method for the routine analysis of DHA and other fatty acids in the algal biomass. The AOAC method 996.06 is often followed for the analysis of fatty acids in foods and demonstrating that its fitness for purpose in the analysis of DHA and additional fatty acids in Aurantiochytrium limacinum is therefore the objective of this paper. A validation of the method for the determination of DHA and three other fatty acids in Aurantiochytrium limacinum is presented. The method was found to be linear over the following ranges for each fatty acid methyl ester (FAME) analyte;50 to 15,000 μg/ml (C14:0), 300 to 95,000 (C16:0), 25 to 15,000 (C18:0) and 300 to 59,375 (C22:6). The accuracy, precision and LOD and LOQ of the method were confirmed and its robustness tested. The application of the method to assess the stability of Aurantiochytrium limacinum containing two alternative antioxidants was further examined. The investigation showed that DHA was stable over six months with the inclusion of either Duralox? or ethoxyquin as an antioxidant and ethoxyquin could additionally stabilize DHA in Aurantiochytrium limacinum up to 24 months.

高产DHA裂殖壶菌的诱变选育及转录组分析

裂殖壶菌(Aurantiochytrium limacinum)可以积累大量的Ω-3多不饱和脂肪酸,特别是高含量的二十二碳六烯酸(DHA),在食品工业中发挥着重要作用。为了获得高产DHA的裂殖壶菌,通过对原始菌株进行电子束(EB)辐射诱变,并结合丙二酸和尼罗红染色法筛选,得到了一个DHA产量提高11.42%的突变体M2-11。转录组结果表明,与原始菌株相比较,M2-11中与脂质合成相关的基因绝大多数被上调,还存在多个差异显著的其他功能基因。本研究为裂殖壶菌的诱变选育和遗传改造提供了参考。

Identity and Safety of a Novel <i>Aurantiochytrium sp.</i>for Terrestrial Heterotrophic Docosahexaenoic Acid Production

The objective of the studies in this paper was to expand on the published toxicological assessment of Aurantiochytrium limacinum (AURA) with further strain characterization and to investigate the potential for the biomass or extracted oil to have antimicrobial properties or undesirable substances. AURA is being investigated as a novel source of the omega-3 long-chain polyunsaturated fatty acid docosahexaenoic acid (DHA) for enriching foods of animal origin by means of feed supplementation. In the first studies, we provided the 18S rRNA identification of the novel marine isolated thraustochytrid, established the nutritional composition of AURA biomass for application as a food or feed ingredient including proximate analysis and fatty acid profiling, and confirmed the DHA production potential of the strain. We determined through minimum inhibitory concentration (MIC) analysis that the unextracted AURA biomass was safe, showing no antimicrobial influence and no evidence of any deleterious effects of this product or its extracts at concentrations up to 1% w/w on the reference human intestinal bacteria tested. This would indicate that AURA should not stimulate selective pressure on the commensal microbiota and is therefore unlikely to aid development of antimicrobial resistance and the concomitant harm to humans and animals. Further analysis revealed that the AURA biomass produced through industrial heterotrophic fermentation was free from undesirables;toxic marine microalgal metabolites, heavy metals, pesticides, microbial contaminants, and mycotoxins. Including heterotrophically-grown AURA in food or feed, up to 1% w/w, is a safe and environmentally beneficial strategy for DHA supplementation.

裂殖壶菌pks2基因启动子序列扩增及其活性分析

针对目前裂殖壶菌(Aurantiochytrium limacinum)基因工程相关研究中,对内源启动子的了解和利用仍处在初步阶段的现状,本研究从裂殖壶菌中产多不饱和脂肪酸的主要途径聚酮合酶(PKS)途径出发,利用热不对称交错PCR技术(TAIL-PCR),针对pks2基因扩增出了未知的5’端侧翼序列,初步分析得到pks2基因启动子序列,并分别在原核宿主大肠杆菌(Escherichia coli)与真核宿主酿酒酵母(Saccharomyces cerevisiae)中对启动子活性做进一步研究,发现了pks2基因启动子可以在大肠杆菌和酿酒酵母中有效启动绿色荧光蛋白的表达,而且pks2基因启动子相较于pks1、pks3基因启动子具有更高的启动活性,显现出了其作为启动子用于基因工程研究的可操作性。

裂殖壶菌的双碳源发酵生产二十二碳六烯酸控制策略

研究了葡萄糖、甘油双碳源发酵对裂殖壶菌发酵二十二碳六烯酸(docosahexaenoic acid,DHA)的影响。首先以葡萄糖和甘油为单一碳源,发现以葡萄糖为碳源,菌体增殖速度和底物消耗速度都明显高于以甘油为碳源,以甘油为碳源细胞油脂中DHA的比例显著高于葡萄糖。为了发挥2种碳源各自优势,对葡萄糖和甘油双碳源发酵进行研究,结果显示2种碳源混合或葡萄糖耗尽后补加甘油结果都不如单一碳源好。但若先用葡萄糖为碳源进行培养,当发酵液中的葡萄糖质量浓度降至10~5 g/L时补加甘油既可以缩短发酵周期,又提高了油脂中的DHA比例。采用初始葡萄糖质量浓度为20 g/L,过程补加100 g/L甘油的发酵控制策略,5 L罐发酵,发酵周期可缩短至56 h,细胞油脂中DHA比例最高可达45.50%,充分发挥了2种碳源的优势。该研究得到了一种裂殖壶菌生产DHA葡萄糖、甘油分段发酵控制策略,大幅提高菌体细胞油脂中DHA的比例,同时有效缩短了发酵周期,具有非常好的工业应用价值。

Matrix Extension with Fitness for Purpose and Stability Assessment of DHA and Additional Fatty Acids in Individual Whole Chicken Eggs

The consumption of long chain polyunsaturated fatty acids (LC-PUFA) is associated with several human health benefits. Most notable of these LC-PUFA is docosahexaenoic acid (DHA C22:6) whose inclusion is considered essential for optimum human health. Biofortification of common foods such as eggs with DHA has emerged as a specific approach to increase the intake of DHA in human populations. This can be achieved by supplementing poultry rations with feeds like microalgae or fish oil that are rich in DHA, which results in an increased uptake in the egg. Gas chromatography with flame ionization detection (GC-FID) is the method of choice when analyzing food such as eggs for DHA and other fatty acids. For regulatory studies it is desirable to demonstrate that the method is specifically suitable for the analysis of DHA and fatty acids in eggs. The purpose of this paper is to further extend the scope of the AOAC 996.06 methodology examined in the paper by Dillon et al., and to demonstrate the fitness for purpose of the method by examining specific validation parameters. It is a further objective to investigate the stability of DHA and other fatty acids of short and long timepoints. A validation of the method for the determination of DHA and three other fatty acids in eggs is thus presented.

二氧化碳超临界流体萃取在裂殖壶菌DHA提取中的应用

采用大容量二氧化碳超临界流体萃取设备对裂殖壶菌OUC168菌粉进行油脂萃取,并以四种油料作物黑芝麻、大豆、花生、核桃进行比较,通过气相色谱技术检测了萃取物中的脂肪酸组成及含量,并比较和分析了不同萃取条件下超临界流体对不同脂肪酸的萃取效果。结果表明,四种油料作物中的脂肪酸均以16-18C为主,裂殖壶菌中含有丰富的长链脂肪酸DHA(22C:6,44.34%),是非常好的不饱和脂肪酸来源。二氧化碳超临界流体萃取技术可以有效萃取四种油料作物中的脂肪酸,萃取率均在75%以上。对于裂殖壶菌中脂肪酸的萃取,在萃取温度30-40℃,萃取压力30MPa,分离温度35-55℃条件下,长链脂肪酸的萃取率总体低于短链脂肪酸;同种脂肪酸在萃取温度为30℃时的萃取率高于40℃时的萃取率。在萃取温度30℃,萃取压力30MPa,分离温度55℃,分离压力8~10MPa,CO2流速14L/h的条件下,二氧化碳超临界流体萃取裂殖壶菌总脂肪酸的萃取率为56.03%,DHA萃取率为22.16%。