乳中主要共轭亚油酸异构体的分析
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摘要
共轭亚油酸(conjugated linoleic acid, CLA)是一类含共轭双键亚油酸的位置(6-8,7-9,9-11,11-13,13-15)和几何(cis,cis; cis,trans; trans,cis; trans,trans)异构体的总称,是反刍动物乳、肉中存在的天然脂肪酸。CLA具有多种有益的生理活性。研究证实了不同的CLA有不同的生理活性,已经公认的如9c,11t-CLA具有抗癌活性,抗粥样动脉硬化,免疫调节的功能；10t,12c-CLA可抑制脂肪的合成,具有减肥的作用。因而建立可靠的分析方法确定食品中共轭亚油酸异构体组成,分析CLA异构体的种类,对于研究CLA结构与生理活性的关系以及共扼亚油酸在食品中的应用,都有着十分重要的意义。乳脂肪是丰富的天然CLA的来源,全乳平均1g脂肪含CLA约有10 mg,牛奶中CLA的主要异构体是生理活性很强的9c,11t-CLA,约占90%。为分析不同乳中CLA的准确含量和类型,了解各种因素对乳及乳制品中CLA含量的影响,建立快速准确的分析方法是非常必要的。
本研究采用银离子高效液相色谱法(Ag+-HPLC)法和胶束电动色谱法(MEKC)对几种奶样中主要的CLA异构体的含量进行了分析测定,研究中对乳脂的提取,样品的处理方法以及仪器分析条件进行了优化,较好地提高了分析水平,主要结果如下：
1.对不同的乳脂提取方法进行比较。结果表明,在提取试剂为正己烷：异丙醇=1：1(v/v)时,采用超声波辅助提取乳脂,提取乳脂的效果最佳。
2.比较不同的甲酯化方法对对乳脂中CLA的酯化效果的影响,确定NaOCH3-CH3OH进行酯化的效果较好,甲酯化较完全,并以响应面法对其进行优化,其最优条件为NaOCH3-CH3OH添加量为8.56 mL,反应温度为24.4℃,反应时间为12.8 min。
3.采用一根银离子柱的Ag+-HPLC对CLA异构体进行分离,可实现对两种生理活性较强的CLA异构体9c,11t-CLA和10t,12c-CLA的初步分离。在7.812～500μg/mL范围内,HPLC分析的峰面积与9c,11t-CLA的进样量呈现良好的线性关系,可用于对样品中的9c,11t-CLA进行定量分析。
4.优化了MEKC的分离条件：分离电压25 kV,柱温20℃,进样时间10 s。在优化的条件下(分离电压25 kV,柱温20℃,进样时间10 s),MEKC可达到对9c,11t-CLA与10t,12c-CLA标品的良好分离。以MEKC法对鲜奶、巴氏奶、超高温瞬时灭菌奶(UHT)中CLA含量及组成进行分析,CLA总含量(9c,11t-CLA与10t,12c-CLA含量的总和)：鲜奶为9.281 mg/g乳脂、巴氏奶为9.129 mg/g乳脂、UHT为8.631 mg/g乳脂,各奶样间9c,11t-CLA与10t,12c-CLA比例无显著差异；牛奶、水牛奶、羊奶中CLA总含量(9c,11t-CLA与10t,12c-CLA含量的总和)分别为9.220 mg/g乳脂、2.972 mg/g乳脂、6.054 mg/g乳脂,各奶样间9c,11t-CLA与10t,12c-CLA比例差异显著。
5.采用Ag+-HPLC对不同处理和条件的奶样进行分析,发现热处理(巴氏杀菌、超高温瞬时灭菌)对奶中9c,11t-CLA的含量均有影响,超高温瞬时灭菌可降低CLA的含量,影响最大；保存时间也可影响奶中的9c,11t-CLA含量,随保存时间增加,各奶样中的9c,11t-CLA含量均下降；下降幅度从大到小依次为乳脂,巴氏杀菌奶,鲜奶,UHT奶；不同季节也可影响奶样中CLA含量,夏季奶中CLA含量明显高于其他季节的奶；对不同公司生产的品牌牛奶的CLA含量进行了考察,发现不同品牌牛奶CLA含量存在显著差异；检测结果也表明不同动物奶样中的9c,11t-CLA的含量也有显著差异。
Conjugated linoleic acid (CLA) is a technical term for a mixture of positional (6-8,7-9,9-11,11-13,13-15) and geometric (cis,cis; cis,trans; trans,cis; trans,trans) isomers of linoleic acid that contain conjugated double bonds. The major source of naturel CLA is milk and meat from ruminant. Milk fat is a very common and good source for natural CLA. On average, per gram of milk fat contains 10mg CLA, most of which is biological activity isomer 9c,11t-CLA. CLA have many positive biological activities, and the different isomers show different bioactivities. For instance, based on testing in animal models,9c,11t-CLA showed the function of anti-carcinogenesis, anti-atherosclerosis and immunomodulation.10t,12c-CLA could reduce the body fat, and it also is a potent inhibitor of milk-fat synthesis. Considering the use of CLA for medicinal and nutraceutical purposes, a safe isomer-selective process is required. Therefore, it is necessary to establish the reliable methods for analysis of CLA isomer.
In the present study, conditions of milk fat extraction, sample pretreatment and HPLC and MEKC analysis were optimized. The major CLA isomers from milk samples were separated and tested by silver-ion high performance liquid chromatography (Ag+-HPLC) and micellar electrokinetic chromatography (MEKC). The main results were as follows:
1. Different solvent combinations were evaluated for the extraction of milk fat. In comparison with others, hexane/isopropanol with the assistance of ultrasound displayed better extraction ability.
2. Methylation of CLA with NaOCH3-CH3OH in mild conditions showed favorable effect. Box-Behnken design was applied to find the optimum methylation conditions. The results showed that the optimum conditions for the methylation of CLA were as follows:8.56 mL of 0.5 M sodium methoxide-methanol, reaction temperature 24.4℃, reaction time 12.8 min.
3. Ag+-HPLC has been shown to be effective in the resolution of the major CLA isomers 9c,11t-CLA and 10t,12c-CLA, which were known as their beneficial biological activities. In the concentration of standard isomers from 7.812μg/mL to 500μg/mL, there was a good linear relationship between the peak area and injection concentration.
4. The milk treated with heat, including pasteurization and ultra-high temperature instantaneous sterilization (UHT), could reduce the content of CLA significantly. The varations of CLA content in milk was investigated during storage by using Ag+-HPLC. With the increase in preservation time, there was sharp decline in CLA levels in all the samples. During shelf life,9c,11t-CLA content in row milk decreased by 9%,9c,11t-CLA in pasteurized milk decreased by 16%, and in UHT milk decreased by 8%. Season was another important influencing factor. The result showed that the CLA content in summer milk was significantly higher than other seasons. Additionally, there was a significantly difference in the 9c, 11t-CLA content among the cow milk, goat milk and buffalo milk (P<0.001). The CLA content of milk from different companies showed statistically difference (P< 0.001).
5. Under the optimum conditions,80 mM borate (pH 9.0),54 mM sodium dodecyl sulphate,4%(w/v)β-cyclodextrin,8M urea,4%(v/v) ethanol,25 kV and 20℃, the major CLA isomers 9c,11t-CLA and 10t,12c-CLA in milk could be separated by MEKC effectively within 15 min. The result indicated that there was a significant difference in the total CLA content among raw milks, pasteurized milk, UHT milk (P<0.001). However, the content of 10t,12c-CLA from all samples was almost similar (about 3%). The result also showed there was a significant difference in the total CLA content and 10t,12c-CLA content among the cow milk, goat milk and buffalo milk (P<0.05). The total content of CLA in these samples was 9.220mg/g milk fat in the cow milk,2.972 mg/g milk fat in the buffalo milk and 6.054 mg/g milk fat in the goat milk.
本研究采用银离子高效液相色谱法(Ag+-HPLC)法和胶束电动色谱法(MEKC)对几种奶样中主要的CLA异构体的含量进行了分析测定,研究中对乳脂的提取,样品的处理方法以及仪器分析条件进行了优化,较好地提高了分析水平,主要结果如下：
1.对不同的乳脂提取方法进行比较。结果表明,在提取试剂为正己烷：异丙醇=1：1(v/v)时,采用超声波辅助提取乳脂,提取乳脂的效果最佳。
2.比较不同的甲酯化方法对对乳脂中CLA的酯化效果的影响,确定NaOCH3-CH3OH进行酯化的效果较好,甲酯化较完全,并以响应面法对其进行优化,其最优条件为NaOCH3-CH3OH添加量为8.56 mL,反应温度为24.4℃,反应时间为12.8 min。
3.采用一根银离子柱的Ag+-HPLC对CLA异构体进行分离,可实现对两种生理活性较强的CLA异构体9c,11t-CLA和10t,12c-CLA的初步分离。在7.812～500μg/mL范围内,HPLC分析的峰面积与9c,11t-CLA的进样量呈现良好的线性关系,可用于对样品中的9c,11t-CLA进行定量分析。
4.优化了MEKC的分离条件：分离电压25 kV,柱温20℃,进样时间10 s。在优化的条件下(分离电压25 kV,柱温20℃,进样时间10 s),MEKC可达到对9c,11t-CLA与10t,12c-CLA标品的良好分离。以MEKC法对鲜奶、巴氏奶、超高温瞬时灭菌奶(UHT)中CLA含量及组成进行分析,CLA总含量(9c,11t-CLA与10t,12c-CLA含量的总和)：鲜奶为9.281 mg/g乳脂、巴氏奶为9.129 mg/g乳脂、UHT为8.631 mg/g乳脂,各奶样间9c,11t-CLA与10t,12c-CLA比例无显著差异；牛奶、水牛奶、羊奶中CLA总含量(9c,11t-CLA与10t,12c-CLA含量的总和)分别为9.220 mg/g乳脂、2.972 mg/g乳脂、6.054 mg/g乳脂,各奶样间9c,11t-CLA与10t,12c-CLA比例差异显著。
5.采用Ag+-HPLC对不同处理和条件的奶样进行分析,发现热处理(巴氏杀菌、超高温瞬时灭菌)对奶中9c,11t-CLA的含量均有影响,超高温瞬时灭菌可降低CLA的含量,影响最大；保存时间也可影响奶中的9c,11t-CLA含量,随保存时间增加,各奶样中的9c,11t-CLA含量均下降；下降幅度从大到小依次为乳脂,巴氏杀菌奶,鲜奶,UHT奶；不同季节也可影响奶样中CLA含量,夏季奶中CLA含量明显高于其他季节的奶；对不同公司生产的品牌牛奶的CLA含量进行了考察,发现不同品牌牛奶CLA含量存在显著差异；检测结果也表明不同动物奶样中的9c,11t-CLA的含量也有显著差异。
Conjugated linoleic acid (CLA) is a technical term for a mixture of positional (6-8,7-9,9-11,11-13,13-15) and geometric (cis,cis; cis,trans; trans,cis; trans,trans) isomers of linoleic acid that contain conjugated double bonds. The major source of naturel CLA is milk and meat from ruminant. Milk fat is a very common and good source for natural CLA. On average, per gram of milk fat contains 10mg CLA, most of which is biological activity isomer 9c,11t-CLA. CLA have many positive biological activities, and the different isomers show different bioactivities. For instance, based on testing in animal models,9c,11t-CLA showed the function of anti-carcinogenesis, anti-atherosclerosis and immunomodulation.10t,12c-CLA could reduce the body fat, and it also is a potent inhibitor of milk-fat synthesis. Considering the use of CLA for medicinal and nutraceutical purposes, a safe isomer-selective process is required. Therefore, it is necessary to establish the reliable methods for analysis of CLA isomer.
In the present study, conditions of milk fat extraction, sample pretreatment and HPLC and MEKC analysis were optimized. The major CLA isomers from milk samples were separated and tested by silver-ion high performance liquid chromatography (Ag+-HPLC) and micellar electrokinetic chromatography (MEKC). The main results were as follows:
1. Different solvent combinations were evaluated for the extraction of milk fat. In comparison with others, hexane/isopropanol with the assistance of ultrasound displayed better extraction ability.
2. Methylation of CLA with NaOCH3-CH3OH in mild conditions showed favorable effect. Box-Behnken design was applied to find the optimum methylation conditions. The results showed that the optimum conditions for the methylation of CLA were as follows:8.56 mL of 0.5 M sodium methoxide-methanol, reaction temperature 24.4℃, reaction time 12.8 min.
3. Ag+-HPLC has been shown to be effective in the resolution of the major CLA isomers 9c,11t-CLA and 10t,12c-CLA, which were known as their beneficial biological activities. In the concentration of standard isomers from 7.812μg/mL to 500μg/mL, there was a good linear relationship between the peak area and injection concentration.
4. The milk treated with heat, including pasteurization and ultra-high temperature instantaneous sterilization (UHT), could reduce the content of CLA significantly. The varations of CLA content in milk was investigated during storage by using Ag+-HPLC. With the increase in preservation time, there was sharp decline in CLA levels in all the samples. During shelf life,9c,11t-CLA content in row milk decreased by 9%,9c,11t-CLA in pasteurized milk decreased by 16%, and in UHT milk decreased by 8%. Season was another important influencing factor. The result showed that the CLA content in summer milk was significantly higher than other seasons. Additionally, there was a significantly difference in the 9c, 11t-CLA content among the cow milk, goat milk and buffalo milk (P<0.001). The CLA content of milk from different companies showed statistically difference (P< 0.001).
5. Under the optimum conditions,80 mM borate (pH 9.0),54 mM sodium dodecyl sulphate,4%(w/v)β-cyclodextrin,8M urea,4%(v/v) ethanol,25 kV and 20℃, the major CLA isomers 9c,11t-CLA and 10t,12c-CLA in milk could be separated by MEKC effectively within 15 min. The result indicated that there was a significant difference in the total CLA content among raw milks, pasteurized milk, UHT milk (P<0.001). However, the content of 10t,12c-CLA from all samples was almost similar (about 3%). The result also showed there was a significant difference in the total CLA content and 10t,12c-CLA content among the cow milk, goat milk and buffalo milk (P<0.05). The total content of CLA in these samples was 9.220mg/g milk fat in the cow milk,2.972 mg/g milk fat in the buffalo milk and 6.054 mg/g milk fat in the goat milk.
引文
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