CN113008822A - 反刍动物瘤胃液中丙酮酸肌酸降解率的测定方法 - Google Patents
反刍动物瘤胃液中丙酮酸肌酸降解率的测定方法 Download PDFInfo
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Abstract
本发明公开了一种反刍动物瘤胃液中丙酮酸肌酸降解率的测定方法,其包括以下步骤:预热发酵瓶至39℃,取瘘管牛晨饲前的瘤胃液,配制前置溶液,配制瘤胃缓冲液,配制微生物混合培养液,配置丙酮酸测定液,配制肌酸检测液,分别测定微生物混合培养液在0和24h两个时间点实验组和对照组的丙酮酸和肌酸的浓度。分别配置丙酮酸测定液和肌酸检测液从而分别检测出反刍动物瘤胃液中丙酮酸和肌酸含量,极大的提高了丙酮酸肌酸降解率测定的准确性。
Description
技术领域
本发明属于畜牧专业领域,尤其涉及一种反刍动物瘤胃液中丙酮酸肌酸降解率的测定方法。
背景技术
丙酮酸肌酸(creatine pyruvate,CrPyr)是一种新型功能营养素,其主要效应成分为机体内正常的代谢中间产物—丙酮酸和肌酸,其分子中肌酸和丙酮酸的比例为60:40,分子式是C7H13N3O5,不会结合任何矿物质且具有较高的溶解性,其溶解后容易分解为丙酮酸和肌酸两种物质。
非专利文献“丙酮酸、丙酮酸肌酸和肌酸对肉鸡肌肉蛋白/酶表达谱的影响”(陈娟等,畜牧与兽医发表时间:2012-12-31,期刊)研究表明,饲喂CrPyr对肉鸡肌肉能量代谢的调控作用效果明显高于其单独的有效成分肌酸和丙酮酸。此外,非专利文献“丙酮酸肌酸对肉鸡脂肪代谢和蛋白质代谢的影响及其机制研究”(陈娟,南京农业大学发表时间:2011-04-01,博士)发现饲粮中添加CrPyr提高了肉鸡肌肉中肌酸激酶的活性,增加磷酸肌酸的含量;同时降低磷酸化酶b激酶的活性,通过降低糖原的分解进而增加肌肉的糖原储备。
前人的研究只是针对单胃动物,对于反刍动物能否降解消化吸收CrPyr还未见相关报道。目前国内尚未公认的有关瘤胃内CrPyr降解率的测定方法,测定瘤胃中CrPyr的含量可以直接反应瘤胃内糖代谢、氮代谢及氨态氮生成情况,间接表明微生物蛋白与瘤胃微生物区系之间的关系,对阐明CrPyr在反刍动物当中的使用提供一种可靠的依据,因此具有重要的实用价值。
发明内容
本发明提供了一种反刍动物瘤胃液中丙酮酸肌酸降解率的测定方法,以解决现有的动物饲养技术方案中涉及的管理环节不够全面,无法有效且准确计算动物瘤胃的对丙酮酸肌酸降解率的问题,从而为丙酮酸肌酸在反刍动物营养上的应用提供一种理论依据,为提升养殖效率奠定基础。
本发明的技术方案是这样实现的:
一种反刍动物瘤胃液中丙酮酸肌酸降解率的测定方法,其特征在于包括以下步骤:
(1)、将发酵瓶置于39℃培养箱预热至39℃,发酵瓶包括实验组和对照组,实验组中添加0.8%的丙酮酸肌酸的常规底物,对照组仅添加常规底物;
(2)、取瘘管牛晨饲前的瘤胃液,瘤胃液经四层纱布过滤后置于预先预热39℃的发酵瓶中进行发酵操作;
(3)、配制前置溶液:
微量元素溶液(A):称取13.2g CaCl2·2H2O+10.0g MnCl2·4H2O+1.0g CoCl2·6H2O+8.0g FeCl3·6H2O,加蒸馏水溶解,定容至1000mL;
缓冲液(B):称取4.0g NH4HCO3+35g NaHCO3,加蒸馏水溶解,定容至1000mL;
常量元素溶液(C):称取9.45g Na2HPO4·12H2O(或者5.7g Na2HPO4·无水)+6.2gKH2PO4·无水+0.6g MgSO4·7H2O,加蒸馏水溶解,定容至1000mL;
刃天青溶液(D):称取100mg刃天青溶解于1000mL蒸馏水即可;
还原液(E):称取4.0mL NaOH(1mol/L)+625mg Na2S·9H2O+625mg半胱氨酸盐酸盐+95mL蒸馏水;
(4)、配制瘤胃缓冲液:依次添加以下剂量份的溶液,520.2mL蒸馏水+208.1mL缓冲液(B)+208.1mL常量元素溶液(C)+0.1mL微量元素溶液(A)+1.0mL刃天青溶液(D)+62.4mL还原液(E),通入CO2,直至溶液由淡蓝色转变为无色;
(5)、配制微生物混合培养液:将瘤胃液与瘤胃缓冲液按1:2的体积比混合,搅拌均匀即可;
(6)、配置丙酮酸测定液:
(a)配制8%的三氯乙酸:取8g的三氯乙酸溶解在100mL的水中;
(b)配制0.1%的2,4-二硝基苯肼液:取100mg 2,4-二硝基苯肼溶于2mol/L的HCl中,100mL容量瓶定容;
(c)配制1.5mol/L NaOH:取60g NaOH溶于水中,1000mL容量瓶定容;
(d)丙酮酸标准液的配制:称取丙酮酸钠7.5mg于烧杯中,用8%三氯乙酸溶解,100mL容量瓶定容;
(7)、配制肌酸检测液:
利用高效液相色谱法对肌酸含量进行检测;
(a)配制5%高氯酸:10mL 70%-72%高氯酸,130mL双蒸水;
(b)配制0.8mol/L K2CO3:取5.52g K2CO3溶于水中,用50mL容量瓶定容;
(c)配制流动相A:色谱级乙腈;
(d)配制流动相B(磷酸盐缓冲液):29.4mmol/L KH2PO4缓冲液,其中缓冲液内含1.15mmol/L TBAHS,调节pH为5.10。配2L需加7.997gKH2PO4;0.781g TBAHS。配好的流动相经0.45μm滤膜过滤,并超声波去除溶液中的气泡;
(e)配制标准品溶液:称取0.025g肌酸标准品用配好的磷酸盐缓冲液定容到50mL容量瓶中,制成标品母液,初始浓度为500μg/mL,然后通过再次添加磷酸盐缓冲液的方式逐级稀释到以下浓度梯度:250μg/mL标品,
100μg/mL标品,50μg/mL标品,25μg/mL标品,10μg/mL标品;
(8)、用自动分液器将混合培养液分别加入发酵瓶中,将对照组和添加组各取6个发酵瓶放入冰盒中,记为培养发酵0h,第一次对发酵液中丙酮酸和肌酸含量的测定;另外12个发酵瓶迅速放入39℃人工瘤胃培养箱中,培养发酵至24h,迅速放入冰盒当中终止发酵,第二次对发酵液中丙酮酸和肌酸含量的测定。发酵液用四层尼龙网布过滤,方便用于检测发酵液中丙酮酸和肌酸的含量;
在本发明的这种反刍动物瘤胃液中丙酮酸肌酸降解率的测定方法中,步骤(1)中所述常规底物添加步骤为:准确称取0.200g(DM)饲料风干样,将样品用长柄勺送至发酵瓶底部,将发酵瓶活塞的前1/3部位均匀涂抹适量凡士林,装好后将发酵瓶置于39℃培养箱预热。
在本发明的这种反刍动物瘤胃液中丙酮酸肌酸降解率的测定方法中,发酵液中丙酮酸和肌酸含量的测定方法如下:
①丙酮酸含量的测定:取1.0mL发酵上清液于10.0mL试管中,加2mL 8%三氯乙酸,加1.0mL 0.1%2,4-二硝基苯肼液,摇匀,再加5.0mL的1.5mol/L NaOH溶液,摇匀显色,使用紫外分光光度计,在520nm波长下进行比色;其吸光度带入标准曲线中就可得出发酵液中丙酮酸的浓度;
②肌酸含量的测定:取2.0mL发酵上清液于5.0mL试管中,加2mL预冷的5%高氯酸溶液,浸提15min,并于在4℃下以10000r/min离心10min,移取上清液加入900μL 0.8mol/L的K2CO3,浸提10min,再于4℃以10000r/min离心15min,吸取上清液,经0.45μm滤膜过滤后用高效液相色谱仪进行分析。根据步骤(7)(e)所述标准品溶液,用高效液相色谱仪分析得到标准曲线,用来求得发酵液中肌酸浓度。
在本发明的这种反刍动物瘤胃液中丙酮酸肌酸降解率的测定方法中,所述用于色谱分析的高效液相色谱仪的测定参数为:色谱柱:Agilent ZorbaXSB-C18 5μm(4.6mm×250mm);流速:1.0mL/min;柱温:25℃;进样量:20μL;紫外检测波长:210nm;运行时间:22min。
在本发明的这种反刍动物瘤胃液中丙酮酸肌酸降解率的测定方法中,所述丙酮酸肌酸降解率的计算公式如下:
C0为发酵0h,实验组中发酵液中丙酮酸、肌酸的浓度;
C0对为发酵0h,对照组中发酵液中丙酮酸、肌酸的浓度;
C24为发酵24h,实验组中发酵液中丙酮酸、肌酸的浓度;
C24对为发酵24h,对照组中发酵液中丙酮酸、肌酸的浓度。
在本发明的这种反刍动物瘤胃液中丙酮酸肌酸降解率的测定方法中,逐级稀释方法为:
3mL 500μg/mL标品+3mL磷酸盐缓冲液—250μg/mL标品
2mL 250μg/mL标品+3mL磷酸盐缓冲液—100μg/mL标品
3mL 100μg/mL标品+3mL磷酸盐缓冲液—50μg/mL标品
3mL 50μg/mL标品+3mL磷酸盐缓冲液—25μg/mL标品
2mL 25μg/mL标品+3mL磷酸盐缓冲液—10μg/mL标品
实施本发明的这种反刍动物瘤胃液中丙酮酸肌酸降解率的测定方法,具有以下有益效果:
1、分别配置丙酮酸测定液和肌酸检测液从而分别识别动物瘤胃液中丙酮酸和肌酸,极大的提高了丙酮酸肌酸降解率检测准确性。
2、在不同的发酵时间点分别进行检测,并通过四层纱布进行过滤瘤胃液,肌酸检测过程中还有进一步浸提离心操作,使发酵液中肌酸含量能够更准确测定,从而提取出更深层次、更精密的数据。
3、该技术方案中事先配置好各种检测液,在最终检测的过程中只需要采用简单的紫外分光光度计和高效液相色谱仪进行测定,整体设备成本大幅度降低,便于在养殖业中进行普及推广。
附图说明
图1为本发明的这种反刍动物瘤胃液中丙酮酸肌酸降解率的测定方法的流程示意图。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将结合附图对本发明实施例中的技术方案进行清楚、完整地描述。
如附图1所示,本发明的这种反刍动物瘤胃液中丙酮酸肌酸降解率的测定方法,其包括以下步骤:
(1)、将发酵瓶置于39℃培养箱预热至39℃,发酵瓶包括实验组和对照组,实验组中添加0.8%的丙酮酸肌酸的常规底物,对照组仅添加常规底物;常规底物为:准确称取0.200g(DM)饲料风干样,将样品用长柄勺送至发酵瓶底部,将发酵瓶活塞的前1/3部位均匀涂抹适量凡士林,装好后将发酵瓶置于39℃培养箱预热。总共设置24个发酵瓶,12个常规底物作为对照组,12个添加0.8%的丙酮酸肌酸。
(2)、取瘘管牛晨饲前的瘤胃液,瘤胃液经四层纱布过滤后置于预先预热39℃的发酵瓶中,然后放入培养箱中进行发酵操作,
(3)、配制前置溶液:
微量元素溶液(A):称取13.2g CaCl2·2H2O+10.0g MnCl2·4H2O+1.0g CoCl2·6H2O+8.0g FeCl3·6H2O,加蒸馏水溶解,定容至1000mL;
缓冲液(B):称取4.0g NH4HCO3+35g NaHCO3,加蒸馏水溶解,定容至1000mL;
常量元素溶液(C):称取9.45g Na2HPO4·12H2O(或者5.7g Na2HPO4·无水)+6.2gKH2PO4·无水+0.6g MgSO4·7H2O,加蒸馏水溶解,定容至1000mL;
刃天青溶液(D):称取100mg刃天青溶解于1000mL蒸馏水即可;
还原液(E):称取4.0mL NaOH(1mol/L)+625mg Na2S·9H2O+625mg半胱氨酸盐酸盐+95mL蒸馏水;
(4)、配制瘤胃缓冲液:依次添加以下剂量份的溶液,520.2mL蒸馏水+208.1mL缓冲液(B)+208.1mL常量元素溶液(C)+0.1mL微量元素溶液(A)+1.0mL刃天青溶液(D)+62.4mL还原液(E),通入CO2,直至溶液由淡蓝色转变为无色;
(5)、配制微生物混合培养液:将瘤胃液与瘤胃缓冲液按1:2的体积比混合,搅拌均匀即可;
(6)、配置丙酮酸测定液:
(a)配制8%的三氯乙酸:取8g的三氯乙酸溶解在100mL的水中;
(b)配制0.1%的2,4-二硝基苯肼液:取100mg 2,4-二硝基苯肼溶于2mol/L的HCl中,100mL容量瓶定容;
(c)配制1.5mol/L NaOH:取60g NaOH溶于水中,1000mL容量瓶定容;
(d)丙酮酸标准液的配制:称取丙酮酸钠7.5mg于烧杯中,用8%三氯乙酸溶解,100mL容量瓶定容;
测定液的配制曲线如下表:
(7)、配制肌酸检测液:
利用高效液相色谱法对肌酸含量进行检测;
(a)配制5%高氯酸:10mL 70%-72%高氯酸,130mL双蒸水;
(b)配制0.8mol/L K2CO3:取5.53g K2CO3定容到50mL容量瓶中;
(c)配制流动相A:色谱级乙腈;
(d)配制流动相B(磷酸盐缓冲液):29.4mmol/L KH2PO4缓冲液,其中缓冲液内含1.15mmol/L TBAHS,调节pH为5.10。配2L需加7.997g KH2PO4;0.781g TBAHS。配好的流动相经0.45μm滤膜过滤,并超声波去除溶液中的气泡;
(e)配制标准品溶液:称取0.025g肌酸标准品用配好的磷酸盐缓冲液定容到50mL容量瓶中,制成标品母液,初始浓度为500μg/mL,然后通过再次添加磷酸盐缓冲液的方式逐级稀释到以下浓度梯度:250μg/mL标品,100μg/mL标品,50μg/mL标品,25μg/mL标品,10μg/mL标品;
(8)、用自动分液器将混合培养液分别加入发酵瓶中,将部分发酵瓶放入冰盒中,记为培养发酵0h,第一次对发酵液中丙酮酸和肌酸含量的测定;另一部分发酵瓶迅速放入39℃人工瘤胃培养箱中,培养发酵至24h,迅速放入冰盒当中终止发酵,第二次对发酵液中丙酮酸和肌酸含量的测定。发酵液用四层尼龙网布过滤,方便用于检测发酵液中丙酮酸和肌酸的含量;
较佳的,发酵液中丙酮酸和肌酸含量的测定方法如下:
①丙酮酸含量的测定:取1.0mL发酵上清液于10.0mL试管中,加2mL 8%三氯乙酸,加1.0mL 0.1%2,4-二硝基苯肼液,摇匀,再加5.0mL的1.5mol/L NaOH溶液,摇匀显色,使用紫外分光光度计,在520nm波长下进行比色;其吸光度带入标准曲线中就可得出发酵液中丙酮酸的浓度;
②肌酸含量的测定:取2.0mL发酵上清液于5.0mL试管中,加2mL预冷的5%高氯酸溶液,浸提15min,并于在4℃下以10000r/min离心10min,移取上清液加入900μL 0.8mol/L的K2CO3,浸提10min,再于4℃以10000r/min离心15min,吸取上清液,经0.45μm滤膜过滤后用高效液相色谱仪进行分析。根据步骤(7)(e)所述标准品溶液,用高效液相色谱仪分析得到标准曲线,用来求得发酵液中肌酸浓度。
较佳的,色谱柱:Agilent ZorbaXSB-C18 5μm(4.6mm×250mm);流速:1.0mL/min;柱温:25℃;进样量:20μL;紫外检测波长:210nm;运行时间:22min。
本实施例中的丙酮酸肌酸降解率的计算公式如下:
C0为发酵0h,实验组中发酵液中丙酮酸、肌酸的浓度;
C0对为发酵0h,对照组中发酵液中丙酮酸、肌酸的浓度;
C24为发酵24h,实验组中发酵液中丙酮酸、肌酸的浓度;
C24对为发酵24h,对照组中发酵液中丙酮酸、肌酸的浓度。
较佳的,逐级稀释方法为:
3mL 500μg/mL标品+3mL磷酸盐缓冲液—250μg/mL标品
2mL 250μg/mL标品+3mL磷酸盐缓冲液—100μg/mL标品
3mL 100μg/mL标品+3mL磷酸盐缓冲液—50μg/mL标品
3mL 50μg/mL标品+3mL磷酸盐缓冲液—25μg/mL标品
2mL 25μg/mL标品+3mL磷酸盐缓冲液—10μg/mL标品
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改,等同替换和改进等,均应包含在本发明的保护范围之内。
Claims (6)
1.一种反刍动物瘤胃液中丙酮酸肌酸降解率的测定方法,其特征在于包括以下步骤:
(1)、将发酵瓶置于39℃培养箱预热至39℃,发酵瓶包括实验组和对照组,实验组常规底物中添加0.8%的丙酮酸肌酸,对照组仅添加常规底物;
(2)、取瘘管牛晨饲前的瘤胃液,瘤胃液经四层纱布过滤后置于预先预热39℃的发酵瓶中进行发酵操作;
(3)、配制前置溶液:
微量元素溶液(A):称取13.2g CaCl2·2H2O+10.0g MnCl2·4H2O+1.0g CoCl2·6H2O+8.0g FeCl3·6H2O,加蒸馏水溶解,定容至1000mL;
缓冲液(B):称取4.0g NH4HCO3+35g NaHCO3,加蒸馏水溶解,定容至1000mL;
常量元素溶液(C):称取9.45g Na2HPO4·12H2O(或者5.7g Na2HPO4·无水)+6.2gKH2PO4·无水+0.6g MgSO4·7H2O,加蒸馏水溶解,定容至1000mL;
刃天青溶液(D):称取100mg刃天青溶解于1000mL蒸馏水即可;
还原液(E):称取4.0mL NaOH(1mol/L)+625mg Na2S·9H2O+625mg半胱氨酸盐酸盐+95mL蒸馏水;
(4)、配制瘤胃缓冲液:依次添加以下剂量份的溶液,520.2mL蒸馏水+208.1mL缓冲液(B)+208.1mL常量元素溶液(C)+0.1mL微量元素溶液(A)+1.0mL刃天青溶液(D)+62.4mL还原液(E),通入CO2,直至溶液由淡蓝色转变为无色;
(5)、配制微生物混合培养液:将瘤胃液与瘤胃缓冲液按1:2的体积比混合,搅拌均匀即可;
(6)、配置丙酮酸测定液:
(a)配制8%的三氯乙酸:取8g的三氯乙酸溶解在100mL的水中;
(b)配制0.1%的2,4-二硝基苯肼液:取100mg 2,4-二硝基苯肼溶于2mol/L的HCl中,100mL容量瓶定容;
(c)配制1.5mol/L NaOH:取60g NaOH溶于水中,1000mL容量瓶定容;
(d)丙酮酸标准液的配制:称取丙酮酸钠7.5mg于烧杯中,用8%三氯乙酸溶解,100mL容量瓶定容;
(7)、配制肌酸检测液:
利用高效液相色谱法对肌酸含量进行检测;
(a)配制5%高氯酸:10mL 70%-72%高氯酸,130mL双蒸水;
(b)配制0.8mol/L K2CO3:取5.53g K2CO3定容到50mL容量瓶中;
(c)配制流动相A:色谱级乙腈;
(d)配制流动相B(磷酸盐缓冲液):29.4mmol/L KH2PO4缓冲液,其中缓冲液内含1.15mmol/L四丁基硫酸氢铵(TBAHS),调节pH为5.10;配2L需加7.997g KH2PO4;0.781gTBAHS;配好的流动相经0.45μm滤膜过滤,并超声波去除溶液中的气泡;
(e)配制标准品溶液:称取0.025g肌酸标准品用配好的磷酸盐缓冲液定容到50mL容量瓶中,制成标品母液,初始浓度为500μg/mL,然后通过再次添加磷酸盐缓冲液的方式逐级稀释到以下浓度梯度:250μg/mL标品,100μg/mL标品,50μg/mL标品,25μg/mL标品,10μg/mL标品;
(8)、用自动分液器将混合培养液分别加入发酵瓶中,将部分发酵瓶放入冰盒中,记为培养发酵0h,第一次对发酵液中丙酮酸和肌酸含量的测定;另一部分发酵瓶迅速放入39℃人工瘤胃培养箱中,培养发酵至24h,迅速放入冰盒当中终止发酵,第二次对发酵液中丙酮酸和肌酸含量的测定;发酵液用四层尼龙网布过滤,方便用于检测发酵液中丙酮酸和肌酸的含量。
2.根据权利要求1所述的反刍动物瘤胃液中丙酮酸肌酸降解率的测定方法,其特征在于,步骤(1)中所述常规底物添加步骤为:准确称取0.200g(DM)饲料风干样,将样品用长柄勺送至发酵瓶底部,将发酵瓶活塞的前1/3部位均匀涂抹适量凡士林,装好后将发酵瓶置于39℃培养箱预热。
3.根据权利要求1所述的反刍动物瘤胃液中丙酮酸肌酸降解率的测定方法,其特征在于,发酵液中丙酮酸和肌酸含量的测定方法如下:
①丙酮酸含量的测定:取1.0mL发酵上清液于10.0mL试管中,加2mL 8%三氯乙酸,加1.0mL 0.1%2,4-二硝基苯肼液,摇匀,再加5.0mL的1.5mol/L NaOH溶液,摇匀显色,使用紫外分光光度计,在520nm波长下进行比色;
其吸光度带入标准曲线中就可得出发酵液中丙酮酸的浓度;
②肌酸含量的测定:取2.0mL发酵上清液于5.0mL试管中,加2mL预冷的5%高氯酸溶液,浸提15min,并于在4℃下以10000r/min离心10min,移取上清液加入900μL 0.8mol/L的K2CO3,浸提10min,再于4℃以10000r/min离心15min,吸取上清液,经0.45μm滤膜过滤后用高效液相色谱仪进行分析;根据步骤(7)(e)所述标准品溶液,用高效液相色谱仪分析得到标准曲线,用来求得发酵液中肌酸浓度。
4.根据权利要求3所述的反刍动物瘤胃液中丙酮酸肌酸降解率的测定方法,其特征在于,所述用于色谱分析的高效液相色谱仪的测定参数为:色谱柱:Agilent ZorbaXSB-C18 5μm(4.6mm×250mm);流速:1.0mL/min;柱温:25℃;进样量:20μL;紫外检测波长:210nm;运行时间:22min。
6.根据权利要求1所述的反刍动物瘤胃液中丙酮酸肌酸降解率的测定方法,其特征在于,逐级稀释方法为:
3mL 500μg/mL标品+3mL磷酸盐缓冲液—250μg/mL标品
2mL 250μg/mL标品+3mL磷酸盐缓冲液—100μg/mL标品
3mL 100μg/mL标品+3mL磷酸盐缓冲液—50μg/mL标品
3mL 50μg/mL标品+3mL磷酸盐缓冲液—25μg/mL标品
2mL 25μg/mL标品+3mL磷酸盐缓冲液—10μg/mL标品。
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