半胱胺和海南霉素对山羊肌内共轭亚油酸含量与脂肪酸组成的影响及其机理研究
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摘要
针对人们日益增长的肉品品质需求，本实验选择波杂一代山羊为研究对象，研究日粮添加半胱胺(cs)和海南霉素(HM)对山羊骨骼肌肌内脂肪、共轭亚油酸(CLA)含量以及脂肪酸组成的影响，并对其可能的作用机制进行初步探讨。
1．确定并优化肌肉样品中脂肪酸甲酯的气相色谱测定条件
选取2克山羊肌肉样品，匀浆后，用Folch法抽提肌肉样品中的脂质(氯仿／甲醇V／V 2∶1溶液)。取其中40mg脂质用2ml正已烷溶解，采用0.5M甲醇钠进行甲酯化反应，生成脂肪酸甲酯。脂肪酸甲酯在Agilent6890N气相色谱仪上使用强极性的SP-2560脂肪酸甲酯测定专用毛细管柱(100m×0.25mm×0.20μm，Supelco Inc，Bellefonte，PA)进行分离，通过氢火焰离子化检测器(FID)进行测定。为了提高毛细管柱的分离效果，采用程序升温条件，初始温度为150℃，保持5分钟以后以2℃／分钟的速率升至175℃，保持15分钟后以7℃／分钟的速率升至200℃，保持20分钟以5℃／的分钟速率升至220℃，保持25分钟。最后，通过与标准品比较，对山羊肌肉样品中各种脂肪酸成份进行定性与定量分析。
2．半胱胺(CS)和海南霉素(HM)对山羊骨骼肌肌内脂肪与CLA含量的影响
选取健康、体重一致(15±1kg)的3月龄育肥山羊90头，随机分成三组：组Ⅰ饲喂基础日粮，组Ⅱ基础日粮中添加900 mg·kg-1 CS，组Ⅲ添加5mg·kg-1 HM。60天后每组随机选10头宰杀，取背最长肌和半腱肌，采用索氏抽提法测定肌内脂肪含量，采用气相色谱分析法测定肌肉中CLA以及其它具有重要生理功能的脂肪酸的含量。结果显示：对照组背最长肌肌内脂肪含量比半腱肌高62.3％(P＜0.05)，每克肌肉中CLA含量比半腱肌高41.9％(P＜0.05)；半胱胺和海南霉素对山羊肌内脂肪含量无显著影响，但半胱胺能够有效提高羊肉中CLA含量，半胱胺处理组半腱肌肌内脂肪中CLA含量与对照组相比增加26.5％(P＜0.05)；日粮添加海南霉素使背最长肌肌内脂肪中CLA含量提高10.1％，但作用不显著。
3．半胱胺(CS)和海南霉素(HM)对山羊骨骼肌肌内脂肪酸影响的机制
反刍动物体内富含具有特殊生理功能的共轭亚油酸(CLA)，其主要通过组织中的硬脂酰辅酶A脱氢酶(SCD)对瘤胃生成的反式油酸(TVA)作用生成。半胱胺(CS)能耗竭机体内的生长抑素(SS)，促进生长激素、IGF-1、胰岛素、甲状腺素、B-内啡肽之类与生长相关的激素分泌；生长激素又能够影响动物体内SCD的表达。因而CS可能通过作用SCD影响反刍动物体内CLA含量。本试验以上述动物的半腱肌和肝脏为样品，对其中SCDmRNA的表达进行了测定。结果显示，日粮中添加CS对山羊半腱肌和肝脏中SCDmRNA的表达有提高趋势。结果提示，CS提高山羊半腱肌中CLA以及脂肪含量可能部分是通过CS促进SCDmRNA的表达实现的。海南霉素(HM)与CS比较，对山羊半腱肌和肝脏中SCDmRNA的表达作用都不明显。
In this study, Boer×Suining White crossbred goats were used to investigate the effects of dietary Cysteamine (CS) and Hainan-mycin (HM) supplementation on the content of intramuscular fat, conjugated linoleic acid (CLA) and fatty acids composition in goats. The possible mechanism was also studied.
1. Identification and optimization of the conditions for the determination of fatty acids of intramuscular fat (IMF) by the gas chromatograph (GC)
The intramuscular fat of 2g muscle sample in goat was extracted with chloroform/methanol (2:1, vol/vol) according to Folch et al. 40mg IMF was re-dissolved in 2ml hexane, then 0.5M sodium methylate was added to produce fatty acids ethyl ester (FAME). The FAME were separated by a SP2560 capillary column (100m×0.25mm×0.20μm, Supelco Inc, Bellefonte, PA) installed in Agilent 6890N GC, determined by a hydrogen flame ionisation detector (FID). In order to improve separation effect of fatty acids, temperature programme was used: initial oven temperature was 150℃, held for 5 min, subsequently increased to 175℃at a rate of2℃min~(-1), held for 15 min, then to 200℃at 7℃min~(-1), held for 20 min, in the end to 220℃at 5℃min~(-1) and held for 25 minutes. Finally, the fatty acid composition of IMF in goats was analysed qualitatively and quantitatively by comparation with Sigma and Matreya FAME standard.
2. The effects of Cysteamine (CS) and Hainan-mycin (HM) on the content of intramuscular fat and CLA in goats
90 healthy goats (15±1kg, 3 months of age ) were randomly assigned to three groups: groupⅠwas fed with basal diet, groupⅡandⅢwas provided with basal diet supplemented with 900 mg·kg-1 Cysteamine and 5mg·kg-1 Hainan-mycin, respectively. 10 goats from each group were randomly slaughtered to collect longissimus dorsi (LD) and semitendinosus (SD) muscle samples after 60d. The Intramuscular fat (IMF) content was determined by Soxhlet method, and the conjugated linoleic acid (CLA) and other important fatty acids content was analysed with gas chromatography (GC). The IMF content in LD was 62.3% higher compared with that in SD (P＜0.05), and CLA content per g of muscle mass was higher in LD than SD by 41.9% (P＜0.05). There was no significant effect by CS and HM supplementation on the intramuscular fat content in both LD and SD, however, CLA content in IMF of SD was increased by 26.5% with CS supplementation (P＜0.05). Dietary HM increased the CLA content by 10.1% in IMF of LD but no significance (P＞0.05).
3. The mechanism of Cysteamine (CS) and Hainan-mycin (HM) on intramuscular CLA in goats
Natural conjugated linoleic acid (CLA) mainly exists in ruminant products, which has special physiological functions. CLA deposited in tissues is formed by trans-oleic acid (TVA) from rumen catalyzed by the stearyl-dehydrogenase (SCD). CS can deplete the endogenous SS and promote secretion of the hormone related to growth, eg: growth hormone, IGF-1, insulin, thyroxine,β-endorphin et al. Growth hormone can affect SCD mRNA expression in animal. There is a hypothesis that CS might affect CLA content of ruminant animals by SCD. To verify the above hypothesis, the semitendinosus muscles and livers of the above experimental animals were sampled to investigate the expression of SCD mRNA. The results showed that dietary supplementation of CS improve the expression of SCD mRNA of goat liver and muscle, but not significantly. The results suggest that the reason that CS increased the CLA content in semitendinosus muscles and the IMF content in goats might result from SCDmRNA expression level partly by CS supplementation. The effect of HM on the expression of SCD mRNA in liver and muscle of goat was not significant compared with CS supplementation.
1．确定并优化肌肉样品中脂肪酸甲酯的气相色谱测定条件
选取2克山羊肌肉样品，匀浆后，用Folch法抽提肌肉样品中的脂质(氯仿／甲醇V／V 2∶1溶液)。取其中40mg脂质用2ml正已烷溶解，采用0.5M甲醇钠进行甲酯化反应，生成脂肪酸甲酯。脂肪酸甲酯在Agilent6890N气相色谱仪上使用强极性的SP-2560脂肪酸甲酯测定专用毛细管柱(100m×0.25mm×0.20μm，Supelco Inc，Bellefonte，PA)进行分离，通过氢火焰离子化检测器(FID)进行测定。为了提高毛细管柱的分离效果，采用程序升温条件，初始温度为150℃，保持5分钟以后以2℃／分钟的速率升至175℃，保持15分钟后以7℃／分钟的速率升至200℃，保持20分钟以5℃／的分钟速率升至220℃，保持25分钟。最后，通过与标准品比较，对山羊肌肉样品中各种脂肪酸成份进行定性与定量分析。
2．半胱胺(CS)和海南霉素(HM)对山羊骨骼肌肌内脂肪与CLA含量的影响
选取健康、体重一致(15±1kg)的3月龄育肥山羊90头，随机分成三组：组Ⅰ饲喂基础日粮，组Ⅱ基础日粮中添加900 mg·kg-1 CS，组Ⅲ添加5mg·kg-1 HM。60天后每组随机选10头宰杀，取背最长肌和半腱肌，采用索氏抽提法测定肌内脂肪含量，采用气相色谱分析法测定肌肉中CLA以及其它具有重要生理功能的脂肪酸的含量。结果显示：对照组背最长肌肌内脂肪含量比半腱肌高62.3％(P＜0.05)，每克肌肉中CLA含量比半腱肌高41.9％(P＜0.05)；半胱胺和海南霉素对山羊肌内脂肪含量无显著影响，但半胱胺能够有效提高羊肉中CLA含量，半胱胺处理组半腱肌肌内脂肪中CLA含量与对照组相比增加26.5％(P＜0.05)；日粮添加海南霉素使背最长肌肌内脂肪中CLA含量提高10.1％，但作用不显著。
3．半胱胺(CS)和海南霉素(HM)对山羊骨骼肌肌内脂肪酸影响的机制
反刍动物体内富含具有特殊生理功能的共轭亚油酸(CLA)，其主要通过组织中的硬脂酰辅酶A脱氢酶(SCD)对瘤胃生成的反式油酸(TVA)作用生成。半胱胺(CS)能耗竭机体内的生长抑素(SS)，促进生长激素、IGF-1、胰岛素、甲状腺素、B-内啡肽之类与生长相关的激素分泌；生长激素又能够影响动物体内SCD的表达。因而CS可能通过作用SCD影响反刍动物体内CLA含量。本试验以上述动物的半腱肌和肝脏为样品，对其中SCDmRNA的表达进行了测定。结果显示，日粮中添加CS对山羊半腱肌和肝脏中SCDmRNA的表达有提高趋势。结果提示，CS提高山羊半腱肌中CLA以及脂肪含量可能部分是通过CS促进SCDmRNA的表达实现的。海南霉素(HM)与CS比较，对山羊半腱肌和肝脏中SCDmRNA的表达作用都不明显。
In this study, Boer×Suining White crossbred goats were used to investigate the effects of dietary Cysteamine (CS) and Hainan-mycin (HM) supplementation on the content of intramuscular fat, conjugated linoleic acid (CLA) and fatty acids composition in goats. The possible mechanism was also studied.
1. Identification and optimization of the conditions for the determination of fatty acids of intramuscular fat (IMF) by the gas chromatograph (GC)
The intramuscular fat of 2g muscle sample in goat was extracted with chloroform/methanol (2:1, vol/vol) according to Folch et al. 40mg IMF was re-dissolved in 2ml hexane, then 0.5M sodium methylate was added to produce fatty acids ethyl ester (FAME). The FAME were separated by a SP2560 capillary column (100m×0.25mm×0.20μm, Supelco Inc, Bellefonte, PA) installed in Agilent 6890N GC, determined by a hydrogen flame ionisation detector (FID). In order to improve separation effect of fatty acids, temperature programme was used: initial oven temperature was 150℃, held for 5 min, subsequently increased to 175℃at a rate of2℃min~(-1), held for 15 min, then to 200℃at 7℃min~(-1), held for 20 min, in the end to 220℃at 5℃min~(-1) and held for 25 minutes. Finally, the fatty acid composition of IMF in goats was analysed qualitatively and quantitatively by comparation with Sigma and Matreya FAME standard.
2. The effects of Cysteamine (CS) and Hainan-mycin (HM) on the content of intramuscular fat and CLA in goats
90 healthy goats (15±1kg, 3 months of age ) were randomly assigned to three groups: groupⅠwas fed with basal diet, groupⅡandⅢwas provided with basal diet supplemented with 900 mg·kg-1 Cysteamine and 5mg·kg-1 Hainan-mycin, respectively. 10 goats from each group were randomly slaughtered to collect longissimus dorsi (LD) and semitendinosus (SD) muscle samples after 60d. The Intramuscular fat (IMF) content was determined by Soxhlet method, and the conjugated linoleic acid (CLA) and other important fatty acids content was analysed with gas chromatography (GC). The IMF content in LD was 62.3% higher compared with that in SD (P＜0.05), and CLA content per g of muscle mass was higher in LD than SD by 41.9% (P＜0.05). There was no significant effect by CS and HM supplementation on the intramuscular fat content in both LD and SD, however, CLA content in IMF of SD was increased by 26.5% with CS supplementation (P＜0.05). Dietary HM increased the CLA content by 10.1% in IMF of LD but no significance (P＞0.05).
3. The mechanism of Cysteamine (CS) and Hainan-mycin (HM) on intramuscular CLA in goats
Natural conjugated linoleic acid (CLA) mainly exists in ruminant products, which has special physiological functions. CLA deposited in tissues is formed by trans-oleic acid (TVA) from rumen catalyzed by the stearyl-dehydrogenase (SCD). CS can deplete the endogenous SS and promote secretion of the hormone related to growth, eg: growth hormone, IGF-1, insulin, thyroxine,β-endorphin et al. Growth hormone can affect SCD mRNA expression in animal. There is a hypothesis that CS might affect CLA content of ruminant animals by SCD. To verify the above hypothesis, the semitendinosus muscles and livers of the above experimental animals were sampled to investigate the expression of SCD mRNA. The results showed that dietary supplementation of CS improve the expression of SCD mRNA of goat liver and muscle, but not significantly. The results suggest that the reason that CS increased the CLA content in semitendinosus muscles and the IMF content in goats might result from SCDmRNA expression level partly by CS supplementation. The effect of HM on the expression of SCD mRNA in liver and muscle of goat was not significant compared with CS supplementation.
引文
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