油料作物中有效成分分离制备及其检测方法研究
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摘要
油脂和蛋白质是现代食品工业重要的原料。油料作物是植物油脂和蛋白质的最重要来源,在保证油脂和蛋白质的有效供给、改善食物结构、促进养殖业和加工业发展等诸方面均居重要地位。随着食品加工产业的发展,充分合理地利用油料资源,提高产品附加值,降低加工消耗成为食品加工产业发展的主要方向和科技发展的需求。油料种子是蛋白、油脂和众多次生代谢产物组成的复杂生物体系,充分研究和利用油料作物的各个有效组分是进行油料资源开发的基础。高效的分离技术为油料作物全资源利用提供了必需的手段和方法。而现代分离检测技术为提高油料产品的质量提供了技术支撑。本文将现代分离与检测技术应用于油料加工,用于满足人类对食物,特别是对油脂和蛋白不断增长的需要。主要研究工作如下：
(1)超声和超滤技术的发展为食品科学提供了高效的分离富集手段。本研究将超声和超滤技术应用于菜籽蛋白的提取制备,以提高蛋白的提取率。菜籽蛋白是一种理想的全价蛋白,但等电点和分子量分布比较复杂,完全提取比较困难,限制了菜籽蛋白的应用。通过单因素实验和响应面分析优化了超声波辅助提取菜籽蛋白的工艺,最优工艺参数为超声波辅助功率为450W,超声时间为84min,料液比为1：24,pH=11.5,温度35℃,此工艺条件下菜籽蛋白的提取率为76.83+2.53%,较传统的水浴提取法提高了35.43+1.84%。因此,超声辅助提取是提取菜籽蛋白的快速有效的方法。采用超滤法对菜籽蛋白提取液进行浓缩纯化,超滤得到的粗蛋白RPs含量为863.4±5.1g/kg干重,高于碱溶酸沉蛋白RP5.8和RP3.6,吸油性、乳化性、起泡性和泡沫稳定性优于蛋白RP5.8和RP3.6。菜籽超滤蛋白的溶解性实验表明沉淀法分离菜籽蛋白时应采取二级或多级沉淀。超滤得到的菜籽蛋白可以作为大豆蛋白的潜在替代资源用于食品加工。
(2)纳米包埋技术已被广泛应用于食品工业领域,在色、香、味、型、营养保健以及安全等方面发挥积极作用。本研究利用植物甾醇脂质体包埋纳豆激酶,为功能产品开发奠定基础。脂质体是由类似细胞膜的双层膜构成,利用植物甾醇生产纳豆激酶脂质体能够兼具植物甾醇和纳豆激酶的功效。实验采用正交试验优化了植物甾醇脂质体的制备过程,并测定了脂质体的性质。结果表明：利用纳豆激酶生产植物甾醇脂质体的最优条件为：NK活性为2500 U/mL,卵磷脂与甾醇摩尔比为2：1和甘露醇与卵磷脂的质量比为3：1。在此条件下得到NK脂质体的包封率为65.25%。植物甾醇脂质体的Z电位为-(51+3)mV,平均粒径为194.1+4.98nm,为圆形、形状规则、无粘性的颗粒。4。C储存实验表明脂质体比较稳定。
(3)固相微萃取作为样品前处理技术广泛应用于食品工业中风味的研究。本实验利用固相微萃取技术研究了高低油酸花生中的主要风味成分。花生风味是影响消费者的接受性的重要特征,花生改良后至少应该保持原有的风味和香气。而且,食品的不同加工方式影响花生油的风味,实验采用固相微萃取-气质联用(SPME-GC-MS)分析和鉴定不同加工方式的高低油酸花生挥发性成分,优化了花生油挥发性成分的固相微萃取条件,比较了不同加工处理方式(焙烤、蒸煮和焙烤、微波焙烤)对花生油挥发性成分的影响。结果表明：花生油挥发性成分固相微萃取的最佳条件为：DVB/CAR/PDMS萃取头,萃取温度70。C,顶空萃取时间40min,质谱解析时间5min。主成分分析(PCA)表明,不同加工方式的高油酸花生油具有相似的挥发性成分,同种基因型的花生有类似的风味特征；不同加工处理下高低油酸花生的挥发性成分有明显不同,相同加工处理的花生油具有类似的挥发性成分,特别是同种油酸型的花生油,挥发性成分更接近。
(4)质谱技术是目前脂质及其代谢产物分析检测中应用较多的方法,本研究利用质谱技术对油脂氧化的产物-氢过氧化物进行了分析。油脂氧化是油基食品品质劣变最主要的原因,氢过氧化物是脂质氧化早期的主要产物。实验构建了多重反应模式(MRM)高效液相色谱-三重四级杆-离子阱串联质谱(HPLC-ESI-MS-MS)快速分离检测氢过氧化物的方法,研究了高低油酸花生脂肪酸组成和处理方式对脂肪酶酶活和氢过氧化物产物的影响。结果表明,运用HPLC-ESI-MS-MS方法快速分离检测氢过氧化物的方法,在5.0-200 ng/mL时峰面积比与质量浓度的线性关系良好(r>0.99),检测灵敏度高。利用典型相关分析研究了花生中油酸、亚油酸和亚麻酸含量与脂肪氧合酶酶活、氢过氧化物含量的关系。油酸含量和碱液浸泡决定脂肪酸组成与脂肪氧合酶酶活之间的关系,成负相关；亚油酸含量直接决定产物中氢过氧化物的量,成正相关；油酸含量与氢过氧化物HPOT的含量成负相关。以上与高油酸含量的花生抗氧化性增强的理论一致。
油料作物成分复杂,既有含量较高的蛋白和油脂,又有含量极低的功能成分,而且含有多种结构和性质相近的物质。进行有效成分的提取、分离、鉴定及功能产品开发是目前国内外研究的热点和难点。通过研究现代分离检测技术在油料加工中的应用,对于我国的资源优势和科技优势转化为产业优势意义重大,并能为农业的可持续发展,饮食结构的改善和人民生活水平的提高带来积极影响。
Fats and proteins are the kinds of important raw materials of modern food industry. It is the most importantof oil-crop source for vegetable oil and protein, to ensure the efficient supply of vegetable oil and protein, to improve food structure and promote the development of aquaculture and processing. With the development of food industry, comprehensive utilization of oil crops resources are becoming the main direction and the development requirements, which are favorable to increase the added value of products, and reduce energy consumption of processing. Oilseed is the very complex biological material system including protein, fats and many kinds of secondary metabolites. It is necessary to improve efficient separation and detection technologies for the utilization of oil crops resources. In this thesis, it is mainly focused on the application of modern separation and detection technologies in oil processing in order to meet the demands of food, especially fats and protein. The main contents and results are summarized as follows:
(1) Ultrasonication and ultrafiltration technology provides an efficient means of separation and enrichment for the food science. In this study, these technologies are applied in the extraction of rapeseed protein to improve the protein extraction efficiency. The presence of complex protein constituents and difficulties in extracting protein from rapeseed meal limit the application of rapeseed protein in food processing. However, double-low rapeseed (low erucic acid, low glucosinolate) protein is a type of complete protein that is of potential use in the food industry. Single factor experiments and analysis of the regression equation indicated that the optimal conditions for ultrasonic-assisted extraction were an ultrasonic power of 450 W, an ultrasonic treatment time of 84 min, a solid/liquid ratio of 1:24, a pH of 11.5 and an ultrasonic temperature of 35℃. under these optimized condition, the protein extraction efficiency was 76.83±2.53%, which increased by 35.43±1.84% comparising with the traditional water bath.The functional properties (oil adsorption ability, emulsifying capacity, foaming capacity and foam stability) of RPs, RP5.8 and RP3.6 were found to be better than those of soybean protein. The solubility of rapeseed protein indicated that a multistage precipitation method for extracting rapeseed protein was necessary. The results of this study provide useful indicators for rapeseed protein as a potential replacement for other proteins.
(2) Nano-encapsulation technology has been widely used in food industry, especially in color, smell, taste, type, nutrition and health and safety. Phytosterol liposomes were prepared using the thin film method and used to encapsulate nattokinase (NK). In order to obtain a high encapsulation efficiency within the liposome, an orthogonal experiment (L9 (3)4) was applied to optimise the preparation conditions. The molar ratio of lecithin to phytosterols, NK activity and mass ratio of mannite to lecithin were the main factors that influenced the encapsulation efficiency of the liposomes. Based on the results of a single-factor test, these three factors were chosen for this study. We determined the optimum extraction conditions to be as follows:a molar ratio of lecithin to phytosterol of 2:1, NK activity of 2500U/mL and a mass ratio of mannite to lecithin of 3:1. Under these optimised conditions, an encapsulation efficiency of 65.25% was achieved, which agreed closely with the predicted result. Moreover, the zeta potential, size distribution and microstructure of the liposomes prepared were measured, and we found that the zeta potential was-(51±3) mV and the mean diameter was 194.1 nm. From the results of the scanning electron microscopy, we observed that the phytosterol liposomes were round and regular in shape and showed no aggregation.
(3) Solid-phase microextraction (SPME), as a sample preparation technique, is widely applied in the flavor of the food industry. With the improvement of living quality, high-oleic peanuts have drawn people's attention. Increasing oleic acid content of peanut oil should be desired for both improved shelf life and potential health benefits. The flavor of peanut products is important to the overall acceptance of these products by consumers. Volatile components of peanut oils were analyzed and identified by solid-phase microextraction/gas chromatograph/mass spectrometer (SPME-GC-MS). First, influence of SPME extraction, such as, fiber coating, extraction temperature and extraction time, was optimized. Then volatile compounds in high-oleic and normal peanut oils was profiled following three different processing method, such as roasting, boiling and roasting, and roasting by microwave-assisted. Principal components analysis (PCA) was used to analyze volatile components in peanut oils of different cultivars. The results showed that there were obviously differences about the volatile components between high-oleic and normal peanuts. Among different processing method of peanuts, clear separation on volatile components was observed. The same kind of genotype had the similar flavor characteristics. Moreover, different processing methods had significant effect on volatile components in peanut oils.
(4) Mass spectrometry (MS) has been widely applied in the analysis and detection of lipid and its metabolites. In this study, hydroperoxides of lipid oxidation are analyzed by MS. Lipid oxidation is the main cause of quality deterioration of oil-based food. Hydroperoxides are the original products of lipid oxidation, which was separated and analyzed by high performance liquid chromatography-ion trap tandem mass spectrometry (HPLC-ESI-MS-/MS) with the multiple reaction mode (MRM). Effection of fatty acids compositions and treatment methods of high-oleic and normal peanuts on lipoxygenase and hydroperoxides were investigated. Results Linearity was found in the range of 5.0-200 ng/mL (r> 0.99) with the rapid separation and detection methods of hydroperoxides using HPLC-ESI-MS/MS. Relationship of fatty acids compositions (oleic acid, linoleic acid and linolenic acid content), lipoxygenase activity and hydroperoxide content was clarified by using canonical correlation analysis. Oleic acid content had a negative correlation with lipoxygenase activity soaked by alkaline solution. The amounts of peroxides had a positive correlation with linoleic acid content. And the content of 13(s)-HPOT had negative correlation with oleic acid content. These results were consistent with the antioxidation of high-oleic peanuts.
Oilseeds contain complex compositions, not only including high content protein and fats, but also having low content functional components. Moreover, many compositions have similar structure and properties, and these lead to the difficulties of extraction and purification technologies. Extraction, separation, identification and functional components and product development technologies of effective components are domestic and overseas hot spots and difficult points. Application of modern separation and detection technology in the processing of oil crops has a great signality to insure the whole agricultural sustainable development, increase farmers income, and improve people's living standard.
(1)超声和超滤技术的发展为食品科学提供了高效的分离富集手段。本研究将超声和超滤技术应用于菜籽蛋白的提取制备,以提高蛋白的提取率。菜籽蛋白是一种理想的全价蛋白,但等电点和分子量分布比较复杂,完全提取比较困难,限制了菜籽蛋白的应用。通过单因素实验和响应面分析优化了超声波辅助提取菜籽蛋白的工艺,最优工艺参数为超声波辅助功率为450W,超声时间为84min,料液比为1：24,pH=11.5,温度35℃,此工艺条件下菜籽蛋白的提取率为76.83+2.53%,较传统的水浴提取法提高了35.43+1.84%。因此,超声辅助提取是提取菜籽蛋白的快速有效的方法。采用超滤法对菜籽蛋白提取液进行浓缩纯化,超滤得到的粗蛋白RPs含量为863.4±5.1g/kg干重,高于碱溶酸沉蛋白RP5.8和RP3.6,吸油性、乳化性、起泡性和泡沫稳定性优于蛋白RP5.8和RP3.6。菜籽超滤蛋白的溶解性实验表明沉淀法分离菜籽蛋白时应采取二级或多级沉淀。超滤得到的菜籽蛋白可以作为大豆蛋白的潜在替代资源用于食品加工。
(2)纳米包埋技术已被广泛应用于食品工业领域,在色、香、味、型、营养保健以及安全等方面发挥积极作用。本研究利用植物甾醇脂质体包埋纳豆激酶,为功能产品开发奠定基础。脂质体是由类似细胞膜的双层膜构成,利用植物甾醇生产纳豆激酶脂质体能够兼具植物甾醇和纳豆激酶的功效。实验采用正交试验优化了植物甾醇脂质体的制备过程,并测定了脂质体的性质。结果表明：利用纳豆激酶生产植物甾醇脂质体的最优条件为：NK活性为2500 U/mL,卵磷脂与甾醇摩尔比为2：1和甘露醇与卵磷脂的质量比为3：1。在此条件下得到NK脂质体的包封率为65.25%。植物甾醇脂质体的Z电位为-(51+3)mV,平均粒径为194.1+4.98nm,为圆形、形状规则、无粘性的颗粒。4。C储存实验表明脂质体比较稳定。
(3)固相微萃取作为样品前处理技术广泛应用于食品工业中风味的研究。本实验利用固相微萃取技术研究了高低油酸花生中的主要风味成分。花生风味是影响消费者的接受性的重要特征,花生改良后至少应该保持原有的风味和香气。而且,食品的不同加工方式影响花生油的风味,实验采用固相微萃取-气质联用(SPME-GC-MS)分析和鉴定不同加工方式的高低油酸花生挥发性成分,优化了花生油挥发性成分的固相微萃取条件,比较了不同加工处理方式(焙烤、蒸煮和焙烤、微波焙烤)对花生油挥发性成分的影响。结果表明：花生油挥发性成分固相微萃取的最佳条件为：DVB/CAR/PDMS萃取头,萃取温度70。C,顶空萃取时间40min,质谱解析时间5min。主成分分析(PCA)表明,不同加工方式的高油酸花生油具有相似的挥发性成分,同种基因型的花生有类似的风味特征；不同加工处理下高低油酸花生的挥发性成分有明显不同,相同加工处理的花生油具有类似的挥发性成分,特别是同种油酸型的花生油,挥发性成分更接近。
(4)质谱技术是目前脂质及其代谢产物分析检测中应用较多的方法,本研究利用质谱技术对油脂氧化的产物-氢过氧化物进行了分析。油脂氧化是油基食品品质劣变最主要的原因,氢过氧化物是脂质氧化早期的主要产物。实验构建了多重反应模式(MRM)高效液相色谱-三重四级杆-离子阱串联质谱(HPLC-ESI-MS-MS)快速分离检测氢过氧化物的方法,研究了高低油酸花生脂肪酸组成和处理方式对脂肪酶酶活和氢过氧化物产物的影响。结果表明,运用HPLC-ESI-MS-MS方法快速分离检测氢过氧化物的方法,在5.0-200 ng/mL时峰面积比与质量浓度的线性关系良好(r>0.99),检测灵敏度高。利用典型相关分析研究了花生中油酸、亚油酸和亚麻酸含量与脂肪氧合酶酶活、氢过氧化物含量的关系。油酸含量和碱液浸泡决定脂肪酸组成与脂肪氧合酶酶活之间的关系,成负相关；亚油酸含量直接决定产物中氢过氧化物的量,成正相关；油酸含量与氢过氧化物HPOT的含量成负相关。以上与高油酸含量的花生抗氧化性增强的理论一致。
油料作物成分复杂,既有含量较高的蛋白和油脂,又有含量极低的功能成分,而且含有多种结构和性质相近的物质。进行有效成分的提取、分离、鉴定及功能产品开发是目前国内外研究的热点和难点。通过研究现代分离检测技术在油料加工中的应用,对于我国的资源优势和科技优势转化为产业优势意义重大,并能为农业的可持续发展,饮食结构的改善和人民生活水平的提高带来积极影响。
Fats and proteins are the kinds of important raw materials of modern food industry. It is the most importantof oil-crop source for vegetable oil and protein, to ensure the efficient supply of vegetable oil and protein, to improve food structure and promote the development of aquaculture and processing. With the development of food industry, comprehensive utilization of oil crops resources are becoming the main direction and the development requirements, which are favorable to increase the added value of products, and reduce energy consumption of processing. Oilseed is the very complex biological material system including protein, fats and many kinds of secondary metabolites. It is necessary to improve efficient separation and detection technologies for the utilization of oil crops resources. In this thesis, it is mainly focused on the application of modern separation and detection technologies in oil processing in order to meet the demands of food, especially fats and protein. The main contents and results are summarized as follows:
(1) Ultrasonication and ultrafiltration technology provides an efficient means of separation and enrichment for the food science. In this study, these technologies are applied in the extraction of rapeseed protein to improve the protein extraction efficiency. The presence of complex protein constituents and difficulties in extracting protein from rapeseed meal limit the application of rapeseed protein in food processing. However, double-low rapeseed (low erucic acid, low glucosinolate) protein is a type of complete protein that is of potential use in the food industry. Single factor experiments and analysis of the regression equation indicated that the optimal conditions for ultrasonic-assisted extraction were an ultrasonic power of 450 W, an ultrasonic treatment time of 84 min, a solid/liquid ratio of 1:24, a pH of 11.5 and an ultrasonic temperature of 35℃. under these optimized condition, the protein extraction efficiency was 76.83±2.53%, which increased by 35.43±1.84% comparising with the traditional water bath.The functional properties (oil adsorption ability, emulsifying capacity, foaming capacity and foam stability) of RPs, RP5.8 and RP3.6 were found to be better than those of soybean protein. The solubility of rapeseed protein indicated that a multistage precipitation method for extracting rapeseed protein was necessary. The results of this study provide useful indicators for rapeseed protein as a potential replacement for other proteins.
(2) Nano-encapsulation technology has been widely used in food industry, especially in color, smell, taste, type, nutrition and health and safety. Phytosterol liposomes were prepared using the thin film method and used to encapsulate nattokinase (NK). In order to obtain a high encapsulation efficiency within the liposome, an orthogonal experiment (L9 (3)4) was applied to optimise the preparation conditions. The molar ratio of lecithin to phytosterols, NK activity and mass ratio of mannite to lecithin were the main factors that influenced the encapsulation efficiency of the liposomes. Based on the results of a single-factor test, these three factors were chosen for this study. We determined the optimum extraction conditions to be as follows:a molar ratio of lecithin to phytosterol of 2:1, NK activity of 2500U/mL and a mass ratio of mannite to lecithin of 3:1. Under these optimised conditions, an encapsulation efficiency of 65.25% was achieved, which agreed closely with the predicted result. Moreover, the zeta potential, size distribution and microstructure of the liposomes prepared were measured, and we found that the zeta potential was-(51±3) mV and the mean diameter was 194.1 nm. From the results of the scanning electron microscopy, we observed that the phytosterol liposomes were round and regular in shape and showed no aggregation.
(3) Solid-phase microextraction (SPME), as a sample preparation technique, is widely applied in the flavor of the food industry. With the improvement of living quality, high-oleic peanuts have drawn people's attention. Increasing oleic acid content of peanut oil should be desired for both improved shelf life and potential health benefits. The flavor of peanut products is important to the overall acceptance of these products by consumers. Volatile components of peanut oils were analyzed and identified by solid-phase microextraction/gas chromatograph/mass spectrometer (SPME-GC-MS). First, influence of SPME extraction, such as, fiber coating, extraction temperature and extraction time, was optimized. Then volatile compounds in high-oleic and normal peanut oils was profiled following three different processing method, such as roasting, boiling and roasting, and roasting by microwave-assisted. Principal components analysis (PCA) was used to analyze volatile components in peanut oils of different cultivars. The results showed that there were obviously differences about the volatile components between high-oleic and normal peanuts. Among different processing method of peanuts, clear separation on volatile components was observed. The same kind of genotype had the similar flavor characteristics. Moreover, different processing methods had significant effect on volatile components in peanut oils.
(4) Mass spectrometry (MS) has been widely applied in the analysis and detection of lipid and its metabolites. In this study, hydroperoxides of lipid oxidation are analyzed by MS. Lipid oxidation is the main cause of quality deterioration of oil-based food. Hydroperoxides are the original products of lipid oxidation, which was separated and analyzed by high performance liquid chromatography-ion trap tandem mass spectrometry (HPLC-ESI-MS-/MS) with the multiple reaction mode (MRM). Effection of fatty acids compositions and treatment methods of high-oleic and normal peanuts on lipoxygenase and hydroperoxides were investigated. Results Linearity was found in the range of 5.0-200 ng/mL (r> 0.99) with the rapid separation and detection methods of hydroperoxides using HPLC-ESI-MS/MS. Relationship of fatty acids compositions (oleic acid, linoleic acid and linolenic acid content), lipoxygenase activity and hydroperoxide content was clarified by using canonical correlation analysis. Oleic acid content had a negative correlation with lipoxygenase activity soaked by alkaline solution. The amounts of peroxides had a positive correlation with linoleic acid content. And the content of 13(s)-HPOT had negative correlation with oleic acid content. These results were consistent with the antioxidation of high-oleic peanuts.
Oilseeds contain complex compositions, not only including high content protein and fats, but also having low content functional components. Moreover, many compositions have similar structure and properties, and these lead to the difficulties of extraction and purification technologies. Extraction, separation, identification and functional components and product development technologies of effective components are domestic and overseas hot spots and difficult points. Application of modern separation and detection technology in the processing of oil crops has a great signality to insure the whole agricultural sustainable development, increase farmers income, and improve people's living standard.
引文
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