CN115308078B - A method for determining the mass fraction of fatty acids in different tissues of soft fish - Google Patents
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- 241000251468 Actinopterygii Species 0.000 title claims abstract description 27
- 238000000034 method Methods 0.000 title claims abstract description 22
- 210000001519 tissue Anatomy 0.000 claims abstract description 138
- 210000000514 hepatopancreas Anatomy 0.000 claims abstract description 43
- 210000003205 muscle Anatomy 0.000 claims abstract description 37
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- 230000001850 reproductive effect Effects 0.000 claims abstract description 8
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- 238000001035 drying Methods 0.000 claims description 6
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- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 5
- 238000005238 degreasing Methods 0.000 claims description 3
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- 241000238366 Cephalopoda Species 0.000 description 10
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Abstract
本发明公开了一种柔鱼类不同组织脂肪酸质量分数的测定方法，属于生物化学检测领域。该测定方法包括采集柔鱼类胴体肌肉、消化腺和性腺组织鲜样冷冻保存，分别冷冻干燥至恒重，研磨粉碎，用组织脂肪索氏萃取法对上述肌肉、消化腺和性腺组织样品中脂肪分两个阶段萃取，得出不同组织的脂肪酸质量分数，根据脂肪萃取用的样品干燥重量以及脂肪酸脂肪占比，得出每个组织脂肪酸脂肪所对应的样品干燥重量，根据测定的脂肪酸浓度得到每个组织的脂肪酸质量分数，解决柔鱼类不同组织脂肪萃取时间差异而导致的组织脂肪萃取不完全以及组织脂肪萃取过程中被氧化等问题，可应用于海洋生态学和繁殖生物生态学等领域开展脂肪酸生物标志物的测定与分析。
The invention discloses a method for measuring the fatty acid mass fraction of different tissues of soft fish, and belongs to the field of biochemical detection. The determination method includes collecting and freezing fresh samples of soft fish carcass muscles, digestive glands and gonad tissues, freeze-drying to constant weight respectively, grinding and pulverizing, and using tissue fat Soxhlet extraction method to analyze the fat in the muscle, digestive gland and gonad tissue samples. Extraction is carried out in two stages to obtain the fatty acid mass fraction of different tissues. According to the dry weight of the sample used for fat extraction and the proportion of fatty acids and fats, the dry weight of the sample corresponding to the fatty acids and fats of each tissue is obtained. According to the measured fatty acid concentration, the dry weight of each sample is obtained. The fatty acid mass fraction of each tissue can solve the problems of incomplete tissue fat extraction and oxidation during tissue fat extraction caused by the difference in fat extraction time of different tissues of soft fish. It can be used in fields such as marine ecology and reproductive ecology. Determination and analysis of fatty acid biomarkers.
Description
技术领域Technical field
本发明属于生物化学检测领域，具体涉及一种柔鱼类不同组织脂肪酸质量分数的测定方法。The invention belongs to the field of biochemical detection, and specifically relates to a method for determining the mass fraction of fatty acids in different tissues of soft fish.
背景技术Background technique
头足类是重要的海洋经济动物，被联合国粮农组织认为未来最有开发潜力的渔业资源种类之一，其中柔鱼类是世界头足类渔业和我国远洋鱿钓渔业的重要捕捞对象，根据联合国粮农组织的最新数据显示，近十年来世界商业捕捞的柔鱼类总渔获量约为300万吨/年，同时这些种类在海洋生态系统中占有重要的生态学地位，在海洋生态食物链中处于中间位置，有些种类甚至是其栖息海域的顶级捕食者。Cephalopods are important marine economic animals and are considered by the Food and Agriculture Organization of the United Nations to be one of the fishery resource species with the greatest potential for development in the future. Among them, soft fish are important targets for the world's cephalopod fishery and my country's offshore squid fishery. According to the United Nations The latest data from the FAO show that the total catch of soft fish caught commercially in the world in the past ten years is about 3 million tons per year. At the same time, these species occupy an important ecological position in the marine ecosystem and are at the forefront of the marine ecological food chain. In the middle, some species are even the top predators in the oceans in which they live.
脂肪酸在食物链传递过程中相对保守，可以作为标志物示踪研究对象的营养关系。同时，脂肪酸是动物个体的重要营养物质和能量物质，影响胚胎发育及正常的生长。目前，脂肪酸作为生物标志物已经成为生态学领域的重要内容，已经被证实为探究生物生态学、摄食生态学等方面的重要技术手段。最近，脂肪作为生物标志物也被应用于大洋性柔鱼类生殖能量积累及其生殖投入策略的研究中，示踪生殖能量的来源方式及其与栖息海域环境的选择适应性。然而，有别于在摄食生态学上的应用研究，生殖投入策略研究不仅仅需要比较分析组织之间、消费者与被捕食者之间的脂肪酸组成相似性及其变化模式，还需要进行脂肪酸质量含量的变化趋势比较。因此，在利用气相色谱-质谱联用仪(GC-MS)测定研究对象的脂肪酸浓度后，需要进行脂肪酸质量分数的转化。Fatty acids are relatively conserved in the food chain and can be used as markers to trace the nutritional relationship of research objects. At the same time, fatty acids are important nutrients and energy substances for individual animals, affecting embryonic development and normal growth. At present, fatty acids as biomarkers have become an important part of the field of ecology and have been proven to be an important technical means for exploring bioecology, feeding ecology and other aspects. Recently, fat as a biomarker has also been used in the study of reproductive energy accumulation and reproductive investment strategies in oceanic soft fish, tracing the source of reproductive energy and its selective adaptability to the habitat of the sea environment. However, different from applied research in feeding ecology, research on reproductive investment strategies not only requires comparative analysis of the similarity and change patterns of fatty acid compositions between tissues, consumers and prey, but also requires the analysis of fatty acid quality. Comparison of changing trends in content. Therefore, after measuring the fatty acid concentration of the research object using gas chromatography-mass spectrometry (GC-MS), it is necessary to convert the fatty acid mass fraction.
值得注意的是，脂肪酸测定之前需要萃取测试组织的脂肪。为了获得准确的脂肪酸数据，组织脂肪需要尽可能地萃取完全，并且过程中要最大可能地降低萃取过程中脂肪氧化现象的发生。然而，柔鱼类不同组织的脂肪含量存在较大的差异，比如肌肉组织的脂肪偏低，平均为湿重的1％–2％；而消化腺组织的脂肪含量很高，平均为湿重的30％–50％。因此，在萃取这些种类的组织脂肪时，需要针对不同组织设定不同的萃取方案，将给实验带来较多的时间成本和系统误差。特别地，在进行消化腺组织脂肪萃取时，时间会相对很长而导致脂肪被氧化严重。为此，在进行柔鱼类组织脂肪酸测定时，需要同时考虑组织脂肪的萃取完整和被氧化，以测定获得准确的脂肪酸质量分数数据。It is worth noting that fatty acid determination requires extraction of fat from the test tissue. In order to obtain accurate fatty acid data, tissue fat needs to be extracted as completely as possible, and the occurrence of fat oxidation during the extraction process must be minimized. However, there are large differences in the fat content of different tissues of soft fish. For example, the fat content of muscle tissue is low, with an average of 1%-2% of wet weight; while the fat content of digestive gland tissue is very high, with an average of 1% of wet weight. 30%–50%. Therefore, when extracting these types of tissue fat, different extraction protocols need to be set for different tissues, which will bring more time costs and systematic errors to the experiment. In particular, when extracting fat from digestive gland tissue, the time will be relatively long and the fat will be severely oxidized. For this reason, when measuring fatty acids in soft fish tissue, it is necessary to consider the complete extraction and oxidation of tissue fat at the same time to obtain accurate fatty acid mass fraction data.
发明内容Contents of the invention
本发明的主要目的是提供一种柔鱼类不同组织脂肪酸质量分数的测定方法，解决柔鱼类不同组织脂肪萃取时间差异而导致的组织脂肪萃取不完全以及组织脂肪萃取过程中被氧化等问题，准确测定并得出柔鱼类肌肉、消化腺、性腺不同组织的脂肪酸质量分数。The main purpose of the present invention is to provide a method for determining the mass fraction of fatty acids in different tissues of squid, so as to solve the problems of incomplete tissue fat extraction and oxidation of tissue fat during extraction caused by the difference in fat extraction time of different tissues of squid, and to accurately determine and obtain the mass fraction of fatty acids in different tissues of squid muscle, digestive gland and gonad.
为实现上述目的，本发明采用如下技术方案：In order to achieve the above objects, the present invention adopts the following technical solutions:
本发明提供一种柔鱼类不同组织脂肪酸质量分数的测定方法，基于组织脂肪索氏萃取法进行柔鱼类肌肉、消化腺、性腺组织的粗脂肪萃取和脂肪酸测定，得到不同组织脂肪酸质量分数，包括以下步骤：The present invention provides a method for measuring the fatty acid mass fractions of different tissues of soft fish. Based on the tissue fat Soxhlet extraction method, crude fat extraction and fatty acid measurement of muscles, digestive glands, and gonad tissues of soft fish are performed to obtain the fatty acid mass fractions of different tissues. Includes the following steps:
步骤1、采集柔鱼类胴体肌肉组织、消化腺组织和性腺组织的鲜样各5.00g；Step 1, collecting 5.00 g of fresh samples of muscle tissue, digestive gland tissue and gonad tissue from carcasses of squid;
步骤2、肌肉组织、消化腺组织和性腺组织的鲜样零下80℃冷冻保存24–36小时，分别置入冷冻干燥机冷冻干燥至恒重；Step 2. Fresh samples of muscle tissue, digestive gland tissue and gonad tissue are frozen and stored at minus 80°C for 24-36 hours, and placed in a freeze dryer to freeze-dry to constant weight;
步骤3、称量肌肉组织、消化腺组织和性腺组织的干燥样品重量，研磨粉碎，每个组织称取0.3–0.5g干燥的粉碎样品，精确到±0.1mg；Step 3. Weigh the dry samples of muscle tissue, digestive gland tissue and gonad tissue, grind and grind them. Weigh 0.3–0.5g of dried and ground samples for each tissue, accurate to ±0.1mg;
步骤4、使用组织脂肪索氏萃取法对上述肌肉组织、消化腺组织和性腺组织样品中脂肪进行两个阶段萃取：Step 4. Use the tissue fat Soxhlet extraction method to perform a two-stage extraction of fat from the above muscle tissue, digestive gland tissue and gonad tissue samples:
第一个阶段：分别萃取肌肉、消化腺和性腺样品组织的部分脂肪含量，萃取时间均为30分钟，萃取得到的部分脂肪物质经旋转蒸发、氮吹至恒重，记录脂肪含量，精确到±0.1mg；随后组织脂肪直接用于脂肪酸甲酯化，置入气相色谱-质谱联用仪的脂肪酸测样瓶，测定每个组织的脂肪酸浓度，单位为mg/mL；The first stage: extract part of the fat content of muscle, digestive gland and gonad sample tissues respectively. The extraction time is 30 minutes. The extracted part of the fat material is rotary evaporated and blown with nitrogen to constant weight. The fat content is recorded to the nearest ± 0.1 mg; then the tissue fat is directly used for fatty acid methyl esterification, placed into the fatty acid sample bottle of the gas chromatograph-mass spectrometer, and the fatty acid concentration of each tissue is measured in mg/mL;
第二个阶段：对已经萃取部分脂肪含量后的组织样品继续使用组织脂肪索氏萃取法进行组织脂肪完全脱脂，完全脱脂后的组织样品置入干燥烘箱干燥至恒重，称量此时的组织样品干重，根据肌肉、消化腺和性腺样品组织脱脂前和脱脂后的干重，得出每个组织的脂肪含量，精确到±0.1mg；The second stage: continue to use the tissue fat Soxhlet extraction method to completely delipidate tissue fat from the tissue sample that has extracted part of the fat content. The completely defatted tissue sample is placed in a drying oven to dry to constant weight, and the tissue at this time is weighed. Sample dry weight, based on the dry weight of muscle, digestive gland and gonad sample tissues before and after degreasing, the fat content of each tissue is obtained, accurate to ±0.1mg;
步骤5、根据上述步骤中肌肉、消化腺、性腺组织的脂肪含量(以下称作“总脂肪”)，以及用于脂肪酸测定的部分脂肪含量(以下称作“脂肪酸脂肪”)，得出脂肪酸脂肪占总脂肪的百分比，记作“脂肪酸脂肪占比”，单位％；Step 5. According to the fat content of muscles, digestive glands, and gonadal tissues in the above steps (hereinafter referred to as "total fat"), and the partial fat content used for fatty acid determination (hereinafter referred to as "fatty acid fat"), the fatty acid fat is obtained The percentage of total fat is recorded as "fatty acid fat ratio" in %;
步骤6、根据脂肪萃取用的肌肉、消化腺、性腺组织样品干燥重量以及脂肪酸脂肪占比，得出每个组织脂肪酸脂肪所对应的样品干燥重量，记作“脂肪酸脂肪样品干重”，单位mg；Step 6. Based on the dry weight of the muscle, digestive gland, and gonad tissue samples used for fat extraction and the proportion of fatty acids and fats, obtain the dry weight of the samples corresponding to the fatty acids and fats of each tissue, which is recorded as "fatty acid fat sample dry weight" in mg. ;
步骤7、根据测定的肌肉、消化腺、性腺组织的脂肪酸浓度(mg/mL)，得出每个组织的脂肪酸质量分数，公式为：Step 7. Based on the measured fatty acid concentration (mg/mL) of muscle, digestive gland, and gonad tissue, obtain the fatty acid mass fraction of each tissue. The formula is:
式中，Fa为组织的脂肪酸质量分数，单位为mg/mg；c为GC-MS测定的脂肪酸浓度，单位为mg/mL；hv为组织脂肪酸甲酯化时加入的正己烷体积，单位为mL；sw为脂肪酸脂肪所对应的样品干燥重量，单位为mg；k为肌肉、消化腺、或性腺组织；i为GC-MS测定的每个单体脂肪酸。Where Fa is the mass fraction of fatty acids in the tissue, in mg/mg; c is the fatty acid concentration determined by GC-MS, in mg/mL; hv is the volume of n-hexane added during the methyl esterification of tissue fatty acids, in mL; sw is the dry weight of the sample corresponding to the fatty acid fat, in mg; k is muscle, digestive gland, or gonad tissue; i is each monomer fatty acid determined by GC-MS.
作为优选，所述柔鱼类选为阿根廷滑柔鱼。Preferably, the soft fish is Argentine soft fish.
本发明还提供上述柔鱼类组织脂肪酸含量的检测方法在海洋生态学和繁殖生物生态学领域脂肪酸生物标志物测定与分析中的应用。The present invention also provides the application of the above method for detecting fatty acid content in soft fish tissue in the determination and analysis of fatty acid biomarkers in the fields of marine ecology and reproductive ecology.
与现有技术相比，本发明的有益效果在于：本发明基于组织脂肪索氏抽提处理的基础上获得精确的组织脂肪含量，并准确测定和得出组织的脂肪酸质量分数，最大限度地降低由于不同组织脂肪含量差异及萃取时间不同而产生的实验误差，解决柔鱼类不同组织脂肪萃取时间差异而导致的组织脂肪萃取不完全以及组织脂肪萃取过程中被氧化等问题，准确测定并得出柔鱼类肌肉、消化腺、性腺不同组织的脂肪酸质量分数，定量测定组织的单位质量脂肪酸含量，通过对5尾阿根廷滑柔鱼的胴体、消化腺、性腺等组织进行脂肪萃取并得出脂肪酸质量分数，结果显示胴体组织的C20:4n6、C20:5n3和C22:6n3质量分数分别为1.05±0.09mg/g、9.83±2.35mg/g和41.46±6.07mg/g；性腺组织的C20:4n6、C20:5n3和C22:6n3质量分数分别为2.20±0.31mg/g、22.10±3.74mg/g和65.70±11.45mg/g；消化腺的C20:4n6、C20:5n3和C22:6n3质量分数分别为2.30±1.19mg/g、15.62±5.16mg/g和37.64±7.72mg/g；可应用于海洋生态学、繁殖生物生态学等领域中开展脂肪酸生物标志物的测定与分析。Compared with the prior art, the invention has the following beneficial effects: the invention obtains accurate tissue fat content based on the Soxhlet extraction of tissue fat, accurately determines and obtains the fatty acid mass fraction of the tissue, minimizes the experimental error caused by the difference in fat content of different tissues and the difference in extraction time, solves the problems of incomplete tissue fat extraction caused by the difference in fat extraction time of different tissues of squid and oxidation of tissue fat during extraction, accurately determines and obtains the fatty acid mass fraction of different tissues of squid, including muscle, digestive gland and gonad, quantitatively determines the fatty acid content per unit mass of the tissue, extracts fat from the carcass, digestive gland, gonad and other tissues of 5 Argentine smooth squid, and obtains the fatty acid mass fraction, and the results show that the C20:4n6, C The mass fractions of C20:5n3 and C22:6n3 were 1.05±0.09 mg/g, 9.83±2.35 mg/g and 41.46±6.07 mg/g, respectively; the mass fractions of C20:4n6, C20:5n3 and C22:6n3 in gonadal tissue were 2.20±0.31 mg/g, 22.10±3.74 mg/g and 65.70±11.45 mg/g, respectively; the mass fractions of C20:4n6, C20:5n3 and C22:6n3 in digestive gland were 2.30±1.19 mg/g, 15.62±5.16 mg/g and 37.64±7.72 mg/g, respectively; it can be applied to the determination and analysis of fatty acid biomarkers in the fields of marine ecology, reproductive biological ecology, etc.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1是实施例中柔鱼类组织脂肪酸质量分数的测定流程图。Figure 1 is a flow chart for the determination of the fatty acid mass fraction of soft fish tissue in the embodiment.
图2是实施例中阿根廷滑柔鱼的胴体、消化腺、性腺组织C20:4n6、C20:5n3、C22:6n3的质量分数。Figure 2 shows the mass fractions of C20:4n6, C20:5n3, and C22:6n3 in the carcass, digestive glands, and gonad tissues of Argentinus elegans in the example.
具体实施方式Detailed ways
为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例的附图，对本发明实施例的技术方案进行清楚、完整地描述。显然，所描述的实施例是本发明的一部分实施例，而不是全部的实施例。基于所描述的本发明的实施例，本领域普通技术人员在无需创造性劳动的前提下所获得的所有其它实施例，都属于本发明保护的范围。In order to make the purpose, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings of the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. Based on the described embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.
实施例1Example 1
如图1所示，本实施例提供一种柔鱼类不同组织脂肪酸质量分数的测定方法，基于组织脂肪索氏萃取法进行柔鱼类肌肉、消化腺、性腺组织的粗脂肪萃取和脂肪酸测定，得出组织脂肪酸质量分数，步骤以下：As shown in Figure 1, this embodiment provides a method for measuring the mass fraction of fatty acids in different tissues of soft fish. Based on the tissue fat Soxhlet extraction method, crude fat extraction and fatty acid determination of muscle, digestive gland, and gonad tissue of soft fish are performed. To obtain the tissue fatty acid mass fraction, the steps are as follows:
1、采集柔鱼类胴体肌肉组织、消化腺组织和性腺组织的鲜样各5.00g；1. Collect 5.00g each of fresh samples of soft fish carcass muscle tissue, digestive gland tissue and gonad tissue;
2、肌肉组织、消化腺组织和性腺组织鲜样零下80℃冷冻保存24–36小时后，分别置入冷冻干燥机，冷冻干燥至恒重；2. Fresh samples of muscle tissue, digestive gland tissue and gonad tissue are frozen and stored at minus 80°C for 24-36 hours, then placed in a freeze dryer and freeze-dried to constant weight;
3、称量肌肉组织、消化腺组织和性腺组织的干燥样品重量，研磨粉碎。每个组织称取0.3g–0.5g干燥的粉碎样品，精确到±0.1mg；3. Weigh the dry samples of muscle tissue, digestive gland tissue and gonad tissue, and grind them into powder. Weigh 0.3g-0.5g of dry powdered sample of each tissue, accurate to ±0.1mg;
4、使用组织脂肪索氏萃取法，进行肌肉、消化腺和性腺样品组织脂肪的两个阶段萃取；4. Use tissue fat Soxhlet extraction method to perform two-stage extraction of tissue fat from muscle, digestive gland and gonad samples;
第一个阶段：分别萃取肌肉、消化腺和性腺样品组织的部分脂肪含量，萃取时间均为30分钟。萃取得到的部分脂肪物质经旋转蒸发、氮吹至恒重，记录脂肪含量，精确到±0.1mg。随后，组织脂肪直接用于脂肪酸甲酯化，置入气相色谱-质谱联用仪专用的脂肪酸测样瓶，测定每个组织的脂肪酸浓度，单位为mg/mL；The first stage: extract part of the fat content of muscle, digestive gland and gonad sample tissues respectively, and the extraction time is 30 minutes. The extracted part of the fat material is rotary evaporated and nitrogen blown to constant weight, and the fat content is recorded with an accuracy of ±0.1mg. Subsequently, the tissue fat is directly used for fatty acid methyl esterification and placed in a dedicated fatty acid sample bottle for gas chromatography-mass spectrometry to measure the fatty acid concentration of each tissue in mg/mL;
第二个阶段：对已经萃取部分脂肪含量后的组织样品，继续使用组织脂肪索氏萃取法，实现组织脂肪的完全脱脂。完全脱脂后的组织样品置入干燥烘箱，干燥至恒重，称量此时的组织样品干重。根据肌肉、消化腺和性腺等样品组织脱脂前和脱脂后的干重，得出每个组织的脂肪含量，精确到±0.1mg。The second stage: continue to use the tissue fat Soxhlet extraction method for tissue samples that have already extracted part of their fat content to achieve complete degreasing of tissue fat. The completely defatted tissue sample is placed in a drying oven, dried to constant weight, and the dry weight of the tissue sample at this time is measured. Based on the dry weight of sample tissues such as muscle, digestive glands and gonads before and after defatting, the fat content of each tissue was obtained, accurate to ±0.1 mg.
5、根据肌肉、消化腺、性腺组织的脂肪含量(以下称作“总脂肪”)，以及用于脂肪酸测定的部分脂肪含量(以下称作“脂肪酸脂肪”)得出脂肪酸脂肪占总脂肪的百分比，记作“脂肪酸脂肪占比”，单位％；5. According to the fat content of muscles, digestive glands, and gonadal tissues (hereinafter referred to as "total fat"), and the partial fat content used for fatty acid determination (hereinafter referred to as "fatty acid fat"), the percentage of fatty acid fat in total fat is obtained , recorded as "fatty acid fat ratio", unit %;
6、根据脂肪萃取用的肌肉、消化腺、性腺组织样品干燥重量以及脂肪酸脂肪占比，得出每个组织脂肪酸脂肪所对应的样品干燥重量，记作“脂肪酸脂肪样品干重”，单位mg；6. Based on the dry weight of the muscle, digestive gland, and gonad tissue samples used for fat extraction and the proportion of fatty acid fat, the dry weight of the sample corresponding to the fatty acid fat of each tissue is obtained, which is recorded as "fatty acid fat sample dry weight" in mg;
7、根据测定的肌肉、消化腺、性腺组织的脂肪酸浓度(mg/mL)，得出每个组织的脂肪酸质量分数，得出公式为：7. According to the measured fatty acid concentration (mg/mL) of muscle, digestive gland, and gonad tissue, the fatty acid mass fraction of each tissue is obtained. The formula is:
式中，Fa为组织的脂肪酸质量分数，单位为mg/mg；c为GC-MS测定的脂肪酸浓度，单位为mg/mL；hv为组织脂肪酸甲酯化时加入的正己烷体积，单位为mL；sw为脂肪酸脂肪所对应的样品干燥重量，单位为mg；k为肌肉、消化腺、或者性腺组织；i为GC-MS测定的每个单体脂肪酸。In the formula, Fa is the fatty acid mass fraction of the tissue, in mg/mg; c is the fatty acid concentration measured by GC-MS, in mg/mL; h v is the volume of n-hexane added during methyl esterification of tissue fatty acids, in units of mL; s w is the dry weight of the sample corresponding to the fatty acid fat, in mg; k is muscle, digestive gland, or gonad tissue; i is each monomer fatty acid measured by GC-MS.
根据实施例1中柔鱼类不同组织脂肪酸质量分数的测定方法，对5尾阿根廷滑柔鱼的胴体、消化腺、性腺组织进行脂肪酸质量分数测定，得出的C20:4n6、C20:5n3和C22:6n3的质量分数结果如图2所示，胴体组织的C20:4n6、C20:5n3和C22:6n3的质量分数分别为1.05±0.09mg/g、9.83±2.35mg/g和41.46±6.07mg/g；性腺组织的C20:4n6、C20:5n3和C22:6n3的质量分数分别为2.20±0.31mg/g、22.10±3.74mg/g和65.70±11.45mg/g；消化腺的C20:4n6、C20:5n3和C22:6n3的质量分数分别为2.30±1.19mg/g、15.62±5.16mg/g和37.64±7.72mg/g。According to the method for measuring the fatty acid mass fractions of different tissues of soft fish in Example 1, the fatty acid mass fractions of the carcasses, digestive glands, and gonad tissues of 5 Argentine soft fish were measured, and the obtained C20:4n6, C20:5n3 and C22 The mass fraction results of :6n3 are shown in Figure 2. The mass fractions of C20:4n6, C20:5n3 and C22:6n3 in the carcass tissue are 1.05±0.09mg/g, 9.83±2.35mg/g and 41.46±6.07mg/ respectively. g; the mass fractions of C20:4n6, C20:5n3 and C22:6n3 in gonad tissue are 2.20±0.31mg/g, 22.10±3.74mg/g and 65.70±11.45mg/g respectively; the mass fractions of C20:4n6 and C20 in digestive glands The mass fractions of :5n3 and C22:6n3 are 2.30±1.19mg/g, 15.62±5.16mg/g and 37.64±7.72mg/g respectively.
以上所述，仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应所述以权利要求的保护范围为准。The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013104025A1 (en) * | 2012-01-11 | 2013-07-18 | Adelaide Research & Innovation Pty Ltd | Stabilising and analysing fatty acids in a biological sample stored on solid media |
| CN107356688A (en) * | 2017-07-07 | 2017-11-17 | 上海海洋大学 | A kind of method for distinguishing multiple geographical population jumbo flying squids using muscle essential fatty acid composition |
| CN109387585A (en) * | 2018-10-23 | 2019-02-26 | 江汉大学 | The method of content of fatty acid in gas-chromatography, mass spectrometric hyphenated technique detection nematode |
| CN112255328A (en) * | 2020-09-21 | 2021-01-22 | 河南师范大学 | Optimized method for measuring fatty acids in different tissues of fish body |
| RU2758932C1 (en) * | 2020-07-17 | 2021-11-03 | Федеральное государственное бюджетное учреждение науки Федеральный исследовательский центр комплексного изучения Арктики имени академика Н.П. Лаверова Уральского отделения Российской академии наук (ФГБУН ФИЦКИА УрО РАН) | Method for measuring mass concentration of methyl esters of fatty acids in biological media by gas-liquid chromatography method |
-
2022
- 2022-08-09 CN CN202210948819.5A patent/CN115308078B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013104025A1 (en) * | 2012-01-11 | 2013-07-18 | Adelaide Research & Innovation Pty Ltd | Stabilising and analysing fatty acids in a biological sample stored on solid media |
| CN107356688A (en) * | 2017-07-07 | 2017-11-17 | 上海海洋大学 | A kind of method for distinguishing multiple geographical population jumbo flying squids using muscle essential fatty acid composition |
| CN109387585A (en) * | 2018-10-23 | 2019-02-26 | 江汉大学 | The method of content of fatty acid in gas-chromatography, mass spectrometric hyphenated technique detection nematode |
| RU2758932C1 (en) * | 2020-07-17 | 2021-11-03 | Федеральное государственное бюджетное учреждение науки Федеральный исследовательский центр комплексного изучения Арктики имени академика Н.П. Лаверова Уральского отделения Российской академии наук (ФГБУН ФИЦКИА УрО РАН) | Method for measuring mass concentration of methyl esters of fatty acids in biological media by gas-liquid chromatography method |
| CN112255328A (en) * | 2020-09-21 | 2021-01-22 | 河南师范大学 | Optimized method for measuring fatty acids in different tissues of fish body |
Non-Patent Citations (3)
| Title |
|---|
| Biochemical composition in relation to the energetics of growth and sexual maturation in the ommastrephid squid Illex argentinus;Clarke A 等;Philosophical Transactions of the Royal Society of London Series B: Biological Sciences;19941231;第344卷(第1308期);201-212 * |
| 阿根廷滑柔鱼雄性个体的能量积累特点及与环境因子关系;刘伟 等;水生生物学报;20240229;第48卷(第2期);342-349 * |
| 雌性阿根廷滑柔鱼性腺发育阶段的脂肪酸组成及其变化;林东明;孙程婕;宣思鹏;韩飞;陈新军;;上海海洋大学学报;20190515(第03期);93-102 * |
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