【作者】 夏冬华；

【导师】 阮榕生；

【作者基本信息】 南昌大学 ， 食品科学与工程， 2023， 博士

【摘要】 小球藻（Chlorella vulgaris）是绿藻门的微藻,具有生长繁殖快、固碳能力强等优点,其在不同的环境条件下富集油脂、蛋白质和功能性色素等的能力会发生显著的变化,为人类利用小球藻开发生物质燃油和功能性食品提供了无限潜力。但是在大规模应用方面还存在如下两方面的问题:一方面小球藻生物燃料的商业化应用受到油脂产率低的制约难以广泛应用;另一方面小球藻营养物质被坚韧的细胞壁包裹导致消化吸收率低,造成小球藻在食品中的应用推广受限。本文利用植物生长调节剂,通过优化小球藻的培养方式,来实现小球藻生物量和油脂含量同步提升,解决小球藻油脂产率低的问题,并在生物能源生产中应用,制备高品质生物燃油;采用低温超高压均质细胞破壁技术,解决小球藻营养消化吸收率低的困难,并在食品加工中应用,制作营养健康食品,进一步拓展小球藻资源综合开发利用。主要研究结果如下:（1）研究了2,4-表油菜素内酯（EBR）对高温诱导普通小球藻培养应激阶段油脂的抗逆性,基于转录组学技术阐明了EBR增强小球藻对高温诱导抗逆性的外源性机制。与传统方法相比,EBR应用于小球藻培养可提高其干重、叶绿素含量、生长率、油脂含量和油脂生产力,添加5×10^-8g·L^-1EBR,在35°C条件下,分别增加了19.9%、145.1%、7.5%、9.7%和36.7%。高温35°C各项增长率比25°C和30°C的增长率高,叶绿素含量的增幅特别明显,这揭示了EBR处理可以帮助小球藻细胞获得对高温引起胁迫的抗逆性,从而提高油脂生产力。基于转录组学的代谢通路分析表明,与BG对照组对比,EBR组的光合作用-天线蛋白通路中的相关基因上调,小球藻细胞合成了更多天线蛋白来收集、传递和转化光能,提高了小球藻的光合作用;谷胱甘肽（GSH）代谢通路中的相关基因上调,促进了谷胱甘肽合成,有助于清除过量的活性氧自由基以防止细胞损伤;三羧酸循环（TCA循环）通路中的相关基因上调,产生了更多的乙酰辅酶A和草酰乙酸,促进了油脂的合成。（2）研究了吲哚-3-丙酸（IPA）与铵盐对小球藻油脂产力的影响,基于转录组学技术阐明了IPA提高小球藻油脂产力的机制。通过响应面Box-Behnken设计实验,得到最佳优化培养条件:温度30°C、铵60 mg·L^-1和IPA 252.5 mg·L^-1,无硝酸钠的BG11培养基以及最佳小球藻油脂生产力0.31 g·L^-1·d^-1,且饱和脂肪酸含量为4.21%,单不饱和脂肪酸含量为4.71%,多不饱和脂肪酸含量为7.18%和总油脂含量为16.10%。转录组学分析表明,与BG对照组对比,IPA组的生物素代谢通路中的相关基因上调,小球藻细胞合成了更多乙酰辅酶A羧化酶（ACC）来促进油脂的合成,提高了小球藻油脂产力。（3）研究了HZSM-5/CeO₂协同催化藻粉皂脚混合物与玉米芯微波热解制备生物燃油相关机制。着重探讨了催化温度、HZSM-5/CeO₂比例、混合物料与催化剂比例、物料混合比例对热解产物分布和生物油化学组成的影响。实验结果表明,在最优条件（热解温度为500°C、催化温度为400°C、HZSM-5/CeO₂比例为3:1、原料/催化剂比例为2:1、藻粉皂脚混合物与玉米芯比例为2:1）下,高H/C_eff的富氢藻粉皂脚混合物和低H/C_eff的缺氢玉米芯在HZSM-5/CeO₂的催化下微波热解,热解产物相互作用,生成大量的芳烃。FTIR分析与GC-MS分析结果一致,生物油总烃含量为82.63%（脂肪烃含量为16.39%和芳香烃含量为66.24%）。研究结果表明,HZSM-5有利于芳烃的形成,两种催化剂的串联有效地提高了生物油的烃含量,降低了含氧化合物的含量。（4）采用低温超高压均质技术对食品级小球藻细胞进行均质破壁,保持样品营养活性,并将小球藻匀浆添加到焙烤食品中制备了小球藻面包。通过响应面中心组合设计（CCD）优化了工艺配方:小球藻用量1.2%、白糖用量15.4%、酵母用量1.2%和黄油用量4.7%,并基于此构建了多元线性回归方程预测模型。与普通对照组面包对比,小球藻面包的比容提高9.4%,弹性（7天储存期后）提高59.63%,硬度（7天储存期后）降低27.41%,老化度（3天储存期后）降低37.2%,添加小球藻可以改善面包的口感,延缓面包的老化,延长面包的货架期。更多还原

【Abstract】 Chlorella vulgaris（C.vulgaris）,a kind of microalga of the Chlorophyta phylum,has several advantages,such as fast growth and reproduction,and strong carbon fixation ability.Its abilities to enrich oil,protein,and functional pigment can change significantly under different environmental conditions,which provides infinite potentials for humans to use C.vulgaris to develop bio-fuel and functional food.However,there are still some issues for large-scale applications:on the one hand,the commercial application of C.vulgaris biofuel is limited by low oil yield,on the other hand,the application and promotion of C.vulgaris in food is limited as the nutrients are wrapped in tough cell walls,resulting in low digestion and absorption rate.In this study,we employed plant growth regulators and optimized the cultivation methods of C.vulgaris to achieve simultaneous enhancement of biomass and lipid content.This approach effectively addresses the issue of low lipid productivity and enables the production of high-quality biofuels for bioenergy applications.Furthermore,we applied low-temperature,high-pressure homogenization cell disruption technology to overcome the low nutrient digestibility and absorption challenges of C.vulgaris.This innovative technique allows for the production of nutritious and healthy food products,thereby expanding the comprehensive utilization of C.vulgaris resources.The main research results are as follows:（1）The effect of 2,4-epibrassinolide（EBR）on the lipid stress resistance of C.vulgaris during high-temperature induction was studied.The exogenous mechanisms underlying EBR-enhanced thermotolerance in C.vulgaris were elucidated using transcriptomic analysis.Compared to traditional methods,the application of EBR in C.vulgaris cultivation resulted in increased dry weight,chlorophyll content,growth rate,lipid content,and lipid productivity.Specifically,the addition of 5×10^-8g·L^-1EBR under 35°C conditions led to respective increases of 19.9%,145.1%,7.5%, 9.7%,and 36.7%in these parameters.The growth rates at 35°C were significantly higher than those at 25°C and 30°C,with a particularly notable increase in chlorophyll content.These findings suggest that EBR treatment can enhance C.vulgaris cells’thermotolerance to mitigate the stress induced by high temperatures and consequently improve lipid productivity.Metabolic pathway analysis based on transcriptomics revealed that,compared to the control group,the EBR-treated group exhibited upregulation of genes related to the photosynthesis-antenna protein pathway.This indicates that C.vulgaris cells synthesized more antenna proteins to capture,transfer,and convert light energy,thereby enhancing photosynthesis.Additionally,genes associated with glutathione（GSH）metabolism were upregulated,promoting GSH synthesis and facilitating the elimination of excess reactive oxygen species to prevent cellular damage.Moreover,genes involved in the tricarboxylic acid（TCA）cycle were upregulated,leading to increased production of acetyl-Co A and oxaloacetate,which promote lipid synthesis.（2）The impact of indole-3-propionic acid（IPA）in combination with ammonium salts on lipid productivity in C.vulgaris was studied.The underlying mechanisms by which IPA enhances lipid productivity in C.vulgaris were elucidated using transcriptomic analysis.Through a response surface Box-Behnken design experiment,the optimal cultivation conditions were determined as follows:temperature of 30°C,ammonium concentration of 60 mg·L^-1,IPA concentration of 252.5 mg·L^-1,BG11medium without sodium nitrate.Under these conditions,the highest lipid productivity of C.vulgaris was achieved at 0.31 g·L^-1·d^-1,with a saturated fatty acid content of4.21%,monounsaturated fatty acid content of 4.71%,polyunsaturated fatty acid content of 7.18%,and total lipid content of 16.10%.Transcriptomic analysis revealed that,compared to the control group,the IPA-treated group exhibited upregulation of genes associated with biotin metabolism.This indicates that C.vulgaris cells synthesized more acetyl-Co A carboxylase（ACC）to facilitate lipid synthesis,thereby enhancing lipid productivity in C.vulgaris.（3）The mechanisms of HZSM-5/CeO₂catalytic co-pyrolysis of a mixture of algal soapstock and corncob with microwave heating for the production of biofuel were investigated.The focus was on exploring the effects of catalytic temperature,HZSM-5/CeO₂ratio,mixture ratio of the feedstock and catalyst,and feedstock blending ratio on the product distribution and chemical composition of the bio-oil.The results showed that under optimal conditions（pyrolysis temperature of 500°C,catalytic temperature of 400°C,HZSM-5/CeO₂ratio of 3:1,feedstock/catalyst ratio of2:1,algal soapstock and corncob ratio of 2:1）,the high H/C_effalgal soapstock rich in hydrogen and the low H/C_effhydrogen-deficient corncob underwent microwave pyrolysis under the catalysis of HZSM-5/CeO₂,resulting in a significant interaction between the pyrolysis products and the generation of a large amount of aromatic hydrocarbons.FTIR analysis and GC-MS analysis were consistent with the results,showing a total hydrocarbon content of 82.63%in the bio-oil（comprising 16.39%of fatty hydrocarbons and 66.24%of aromatic hydrocarbons）.The study demonstrated that HZSM-5 facilitated the formation of aromatic hydrocarbons,and the tandem use of the two catalysts effectively increased the hydrocarbon content of the bio-oil while reducing the content of oxygenated compounds.（4）Food-grade Chlorella vulgaris cells were homogenized using low-temperature ultra-high pressure homogenization technology to disrupt the cell walls while preserving the nutritional activity of the sample.The homogenized C.vulgaris slurry was then added to baked goods to prepare C.vulgaris bread.A response surface central composite design（CCD）was employed to optimize the process formulation,which included 1.2%C.vulgaris,15.4%sugar,1.2%yeast,and4.7%butter.Based on this,a multiple linear regression equation prediction model was constructed.The specific volume and the elasticity（after a 7-day storage period）of chlorella bread were 9.4%and 59.63%higher than those of ordinary control bread,while the hardness（after a 7-day storage period）and the aging degree（after a 3-day storage period）were 27.41%and 37.2%lower than those of ordinary control bread.The supplementation of C.vulgaris in bread could improve the taste,delay the aging,and prolong the shelf life.更多还原