饲用酶制剂生物学价值评价技术研究
摘要
随着饲用酶制剂在饲料工业中的广泛应用,对酶制剂的生物学价值评价成为目前企业及养殖户关注的焦点。目前,对饲用酶制的评价主要依据酶活测定和进行动物体内消化试验来判断。酶活测定是在特定的条件下对特定酶的活性进行测定,是最理想状态下的酶的应用效果。但酶制剂的应用环境是动物体,在这一复杂的系统中酶活受到多种因素的影响。酶活测定的结果在动物体内环境下产生的效果如何,尚有疑问。动物试验法可以较准确地判断饲用酶制剂的生物学价值,但易受环境、动物种类及动物的生理状态、生理阶段及动物所采食的饲料种类等多种因素的影响。因此,很有必要建立一种快速、准确、可靠的评价体系对饲用酶制剂的使用效果进行评价。本研究采用对酶制剂使用效果比较敏感的仔猪作为试验动物,对它的胃和小肠的具体消化环境进行实际测定,结合相关文献作为参考。根据测定结果对其体内消化环境进行体外模拟,对主要影响体外评价方法的条件进行组合筛选,确定体外评价体系的最佳模拟参数,并以植酸酶、木聚糖酶的体内法对建立的体外评价体系进行验证,用建立的体外评价体系对甘露聚糖酶生物学价值进行评价。具体研究内容如下:
试验一:仔猪的主要消化参数测定。选择健康的15kg杜长大三元杂交仔猪6头,饲喂符合NRC标准的玉米-豆粕型日粮,日喂三次(8:00、12:00、20:00),自由饮水。防疫、防病等饲养管理按常规方法进行。饲养15天后,试猪于早上饲喂后1h屠宰,立即打开腹腔,结扎幽门瓣、回盲瓣,立即测定胃部消化温度和胃及小肠各段的pH值,取胃、十二指肠、空肠、回肠食糜,迅速放入液氮中冷冻保存,所有速冻样品立即放入-70℃中保存。样品经过解冻、预处理后测定酶活。结果如下:胃温度37.5～38.4℃;pH值:1.85～3.48;胃蛋白酶浓度:192.53～255.67 U/ml。空肠食糜中酶浓度,淀粉酶:714.08～893.75 U/g;脂肪酶:720.09～1156.69 U/g;胰蛋白酶:522.96～653.08 U/g;糜蛋白酶:432.10～595.81 U/g,空肠中段pH值6.77～7.91。
试验二:体外评价体系的建立。根据实际测定结果,结合相关参考文献对体外条件进行模拟和优化选择,试验采用L9(34)两个正交设计对仔猪胃、小肠的主要消化参数进行模拟。胃部温度37℃、38℃、39℃;pH值1.5、2.5、3.5;胃蛋白酶浓度:150 U/ml、220 U/ml、290 U/ml;消化时间:2h、3h、4h。以干物质及粗蛋白的体外消化率作为筛选指标。结果表明胃部体外消化的最佳参数为:39℃、pH1.5、220 U/ml、4h,此时干物质及粗蛋白消化率最高。在胃部消化的基础上对小肠段主要是空肠的pH值、酶浓度、缓冲液体积、食糜在小肠段的停留时间进行模拟。pH值:6.0、7.0、8.0;食糜中酶浓度25 mg/g、35 mg/g、45mg/g;缓冲液体积:300ml、400ml、500ml;食糜在小肠段的停留时间3h、6h、12h。以干物质和粗蛋白的体外消化率作为筛选指标。结果表明小肠最佳消化参数为pH6.0、45 mg/g、12h、300ml,此时干物质及粗蛋白消化率最高。据此建立一套体外评价体系。
试验三:用植酸酶对建立的体外评价体系进行验证。通过体内法和体外评价体系研究添加植酸酶对仔猪生产性能及营养物质消化率的影响,对体内法和体外评价体系所得结果进行相关性分析,结果表明:干物质、粗蛋白、能量、钙、磷的消化率体内法和体外法有较强的相关性(P<0.05),其相关系数分别为:0.88,0.59,0.65,0.90,0.95。证明此方法可以用来评价不同剂量植酸酶的生物学价值。添加植酸酶对钙和磷的体内外消化率有极显著的影响,且体内外相关性强(P<0.05),因此初步确立钙和磷作为植酸酶生物学价值体外评价的主要指标。可根据体外评价体系所测钙和磷的消化率对植酸酶的实际生产效果进行可靠的预测,以此来评价植酸酶的应用效果。
试验四:用木聚糖酶进一步验证该体外评价体系对其它酶制剂评价的有效性。采用体内法和体外评价体系研究添加木聚糖酶对仔猪生产性能及营养物质消化率的影响,对体内法和体外评价体系所得结果进行相关性分析,结果表明:干物质、粗蛋白、能量、粗纤维、粗脂肪的体内外消化率有较强的相关性(P<0.01),其相关系数分别为:0.87、0.83、0.94、0.95、0.89。证明此方法可以用来评价不同酶制剂的生物学价值。添加木聚糖酶对能量、粗纤维的消化率有极显著的影响(P<0.01),且体内外相关性极强(P<0.01),初步确定能量和粗纤维消化率作为木聚糖酶生物学价值体外评价的主要指标。因此可根据体外评价体系所测能量和粗纤维的消化率对木聚糖酶的实际生产效果进行初步的预测,以此来评价木聚糖酶的应用效果。
试验五:用所建立的体外评价体系对甘露聚糖酶进行体外评价。结果表明:添加甘露聚糖酶可提高玉米-豆粕型日粮干物质、粗蛋白和能量的体外消化率。当甘露糖酶添加水平为600U/kg日粮时的干物质、粗蛋白、能量的体外消化率最高。这与其它文献资料所得结论相符。证明该方法的实用性。
With the widely application of feed enzyme in feed industry, the evaluation of feed enzyme effect is becoming the focus of enterprises and farmers. At present, there are two ways to evaluate the effect of enzyme, One is enzyme activities determination, the other is to determine digestibility in vivo.The method of activities determination have done under strict experimental condition with sepicial enzymes,but the digested conditions in animals are very complex system which have effected the evaluation of enzyme activities by variety of factors. Animal experiment is a good approach to evaluate the biological value of feed enzyme .But the experiment is easily to be affected by environment, animal species, physiological stage,and the feeds the animals intake ,etc.So it's necessary to establish a rapid, accurate and reliable method for enzyme evaluation. In this experiment, we choose the piglets as experimental animals because of sensibility to enzyme.We determine the actual digestive environment of piglet’s stomach and small intestine, combined with relative references , on the basis of determination, we imitate the digestion environment in vitro to find out the conditions that affect the evaluation of this in vitro method,and then, we choose the best conditions of this in vitro method, and we use phytase, xylanase as a model in vivo method to verificate the above established methods and use the method to evaluate the biological value of mannanase. All of the experiments were as follows:
Experiment 1.Determine the main digestive parameters of piglet.Six healthy piglet its bodyweight is 15kg were provide with NRC corn-soybean meal. During the trial period, all animals were fed with three times a day and drunk water ad libltum.The management keep accordence with normal piglets. After 15 days, the piglets were slaughtered when they were feeded in 1h and rapidly splited the abdominal cavity, ligated pyloric valve and ileocecal valve. The sample took from the stomach and small intestine were immediately determined some parameters including pH and tempreture. The samples were pretreatment in liquid nitrogen and stored at -70℃,digestive enzyme activities of chyme in duodenum, jejunum and ileum will be measured after treatment.The results as follows:the tempreture of the stomach: 37.5～38.4℃,pH value:1.85～3.48,pepsin activity:192.53～255.67 U/ml ; the enzymes activities of small intestine(U/g chyme): amylase:714.08～893.75;lipase:720.09～1156.69;trypsin:522.96～653.08;chymotrypsin:432.10～595.81.pH:6.77-7.91.
Experiment 2. Establishing a digestion method in vitro to imitate digestion in vivo and its optimization parameters.Two orthogonal design were conducted to imitate the important factors of the stomach and small intestine.Tempreture of the stomach: 37℃、38℃、39℃;pH:1.5、2.5、3.5;pepsin level:150 U/ml、220 U/ml、290 U/ml;time:2h、3h、4h.The screening indexes include digestibility of dry matter and crude protein. After stomach digestion, the best parameters of the stomach is 39℃,pH:1.5, pepsin concentration: 220 U/ml, time:4h. Base on the optimization condition, DM and CP have the highest digestibility. After stomach digestion, imitate the optimization parameters of the small intestine, pH value:6.0、7.0、8.0; the retention time of chyme: 3h、6h、12h; buffer :300ml 400ml 500ml.enzyme activity 25mg/g、35 mg/g、45mg/g chyme.Considered DM and CP digestibility as determined items.The results suggest:the best digestion parameters of the intestine is pH6.0,enzyme :45mg/g chime,retention time: 12h, buffer: 300ml.Therefore, the DM and CP have the higher digestibility than the others in the small intestine.
Experiment 3: Veirfy the in vitro method by studying the effect of additive phytase on piglet productivity and digestibilities of nutrient in vitro and in vivo. The result shows:the digestibility of DM, CP, DE, Ca ,P are high correlation coefficient between in vitro and in vivo methods(P<0.05). the R2 were 0.88,0.59,0.65,0.90,0.95. It was concluded that the methods we found was efficient and reliable. It can be used to evaluate feed enzyme biological value. Additive phytase to low- phosphorus diet has significant effect on the digestibility of Ca and P and has high correlation coefficient between in vitro and in vivo methods(P<0.05).So the digestibility of Ca and P in vitro could be used to predict the biological evaluation of phytase.
Experiment 4: In order to verify the methods efficiency and reliability in diferent enzymes, the diet applied with xylanase got the further research.The result shows: The digestibility of DM CP DE CF EE are high correlations between in vitro and in vivo methods(P<0.01),the correlation coefficient is respectively of 0.87、0.83、0.94、0.95、0.89.It showed that the methods that had been established in experiments were efficiency and reliability and can be used to evaluate biological value of different enzymes . Additive xylanase to low-energy diet has significant effect on the digestibility of DE and CF and has high correlation coefficient between in vitro and in vivo methods(P<0.01).So the digestibility of DE and CF in vitro could be used to predict the biological evaluation of xylanase.
Experiment 5:Evaluation the biological value of mannanase by using the in vitro method . The results show: mannanase had increased the digestibility of DM, CP and DE of corn-soybean diet in vitro .The group which was applied with 600U mannanase had the highest digestibility in DM、CP and DE in experiment.The results are agree with many other references and verify the method are practical.
试验一:仔猪的主要消化参数测定。选择健康的15kg杜长大三元杂交仔猪6头,饲喂符合NRC标准的玉米-豆粕型日粮,日喂三次(8:00、12:00、20:00),自由饮水。防疫、防病等饲养管理按常规方法进行。饲养15天后,试猪于早上饲喂后1h屠宰,立即打开腹腔,结扎幽门瓣、回盲瓣,立即测定胃部消化温度和胃及小肠各段的pH值,取胃、十二指肠、空肠、回肠食糜,迅速放入液氮中冷冻保存,所有速冻样品立即放入-70℃中保存。样品经过解冻、预处理后测定酶活。结果如下:胃温度37.5～38.4℃;pH值:1.85～3.48;胃蛋白酶浓度:192.53～255.67 U/ml。空肠食糜中酶浓度,淀粉酶:714.08～893.75 U/g;脂肪酶:720.09～1156.69 U/g;胰蛋白酶:522.96～653.08 U/g;糜蛋白酶:432.10～595.81 U/g,空肠中段pH值6.77～7.91。
试验二:体外评价体系的建立。根据实际测定结果,结合相关参考文献对体外条件进行模拟和优化选择,试验采用L9(34)两个正交设计对仔猪胃、小肠的主要消化参数进行模拟。胃部温度37℃、38℃、39℃;pH值1.5、2.5、3.5;胃蛋白酶浓度:150 U/ml、220 U/ml、290 U/ml;消化时间:2h、3h、4h。以干物质及粗蛋白的体外消化率作为筛选指标。结果表明胃部体外消化的最佳参数为:39℃、pH1.5、220 U/ml、4h,此时干物质及粗蛋白消化率最高。在胃部消化的基础上对小肠段主要是空肠的pH值、酶浓度、缓冲液体积、食糜在小肠段的停留时间进行模拟。pH值:6.0、7.0、8.0;食糜中酶浓度25 mg/g、35 mg/g、45mg/g;缓冲液体积:300ml、400ml、500ml;食糜在小肠段的停留时间3h、6h、12h。以干物质和粗蛋白的体外消化率作为筛选指标。结果表明小肠最佳消化参数为pH6.0、45 mg/g、12h、300ml,此时干物质及粗蛋白消化率最高。据此建立一套体外评价体系。
试验三:用植酸酶对建立的体外评价体系进行验证。通过体内法和体外评价体系研究添加植酸酶对仔猪生产性能及营养物质消化率的影响,对体内法和体外评价体系所得结果进行相关性分析,结果表明:干物质、粗蛋白、能量、钙、磷的消化率体内法和体外法有较强的相关性(P<0.05),其相关系数分别为:0.88,0.59,0.65,0.90,0.95。证明此方法可以用来评价不同剂量植酸酶的生物学价值。添加植酸酶对钙和磷的体内外消化率有极显著的影响,且体内外相关性强(P<0.05),因此初步确立钙和磷作为植酸酶生物学价值体外评价的主要指标。可根据体外评价体系所测钙和磷的消化率对植酸酶的实际生产效果进行可靠的预测,以此来评价植酸酶的应用效果。
试验四:用木聚糖酶进一步验证该体外评价体系对其它酶制剂评价的有效性。采用体内法和体外评价体系研究添加木聚糖酶对仔猪生产性能及营养物质消化率的影响,对体内法和体外评价体系所得结果进行相关性分析,结果表明:干物质、粗蛋白、能量、粗纤维、粗脂肪的体内外消化率有较强的相关性(P<0.01),其相关系数分别为:0.87、0.83、0.94、0.95、0.89。证明此方法可以用来评价不同酶制剂的生物学价值。添加木聚糖酶对能量、粗纤维的消化率有极显著的影响(P<0.01),且体内外相关性极强(P<0.01),初步确定能量和粗纤维消化率作为木聚糖酶生物学价值体外评价的主要指标。因此可根据体外评价体系所测能量和粗纤维的消化率对木聚糖酶的实际生产效果进行初步的预测,以此来评价木聚糖酶的应用效果。
试验五:用所建立的体外评价体系对甘露聚糖酶进行体外评价。结果表明:添加甘露聚糖酶可提高玉米-豆粕型日粮干物质、粗蛋白和能量的体外消化率。当甘露糖酶添加水平为600U/kg日粮时的干物质、粗蛋白、能量的体外消化率最高。这与其它文献资料所得结论相符。证明该方法的实用性。
With the widely application of feed enzyme in feed industry, the evaluation of feed enzyme effect is becoming the focus of enterprises and farmers. At present, there are two ways to evaluate the effect of enzyme, One is enzyme activities determination, the other is to determine digestibility in vivo.The method of activities determination have done under strict experimental condition with sepicial enzymes,but the digested conditions in animals are very complex system which have effected the evaluation of enzyme activities by variety of factors. Animal experiment is a good approach to evaluate the biological value of feed enzyme .But the experiment is easily to be affected by environment, animal species, physiological stage,and the feeds the animals intake ,etc.So it's necessary to establish a rapid, accurate and reliable method for enzyme evaluation. In this experiment, we choose the piglets as experimental animals because of sensibility to enzyme.We determine the actual digestive environment of piglet’s stomach and small intestine, combined with relative references , on the basis of determination, we imitate the digestion environment in vitro to find out the conditions that affect the evaluation of this in vitro method,and then, we choose the best conditions of this in vitro method, and we use phytase, xylanase as a model in vivo method to verificate the above established methods and use the method to evaluate the biological value of mannanase. All of the experiments were as follows:
Experiment 1.Determine the main digestive parameters of piglet.Six healthy piglet its bodyweight is 15kg were provide with NRC corn-soybean meal. During the trial period, all animals were fed with three times a day and drunk water ad libltum.The management keep accordence with normal piglets. After 15 days, the piglets were slaughtered when they were feeded in 1h and rapidly splited the abdominal cavity, ligated pyloric valve and ileocecal valve. The sample took from the stomach and small intestine were immediately determined some parameters including pH and tempreture. The samples were pretreatment in liquid nitrogen and stored at -70℃,digestive enzyme activities of chyme in duodenum, jejunum and ileum will be measured after treatment.The results as follows:the tempreture of the stomach: 37.5～38.4℃,pH value:1.85～3.48,pepsin activity:192.53～255.67 U/ml ; the enzymes activities of small intestine(U/g chyme): amylase:714.08～893.75;lipase:720.09～1156.69;trypsin:522.96～653.08;chymotrypsin:432.10～595.81.pH:6.77-7.91.
Experiment 2. Establishing a digestion method in vitro to imitate digestion in vivo and its optimization parameters.Two orthogonal design were conducted to imitate the important factors of the stomach and small intestine.Tempreture of the stomach: 37℃、38℃、39℃;pH:1.5、2.5、3.5;pepsin level:150 U/ml、220 U/ml、290 U/ml;time:2h、3h、4h.The screening indexes include digestibility of dry matter and crude protein. After stomach digestion, the best parameters of the stomach is 39℃,pH:1.5, pepsin concentration: 220 U/ml, time:4h. Base on the optimization condition, DM and CP have the highest digestibility. After stomach digestion, imitate the optimization parameters of the small intestine, pH value:6.0、7.0、8.0; the retention time of chyme: 3h、6h、12h; buffer :300ml 400ml 500ml.enzyme activity 25mg/g、35 mg/g、45mg/g chyme.Considered DM and CP digestibility as determined items.The results suggest:the best digestion parameters of the intestine is pH6.0,enzyme :45mg/g chime,retention time: 12h, buffer: 300ml.Therefore, the DM and CP have the higher digestibility than the others in the small intestine.
Experiment 3: Veirfy the in vitro method by studying the effect of additive phytase on piglet productivity and digestibilities of nutrient in vitro and in vivo. The result shows:the digestibility of DM, CP, DE, Ca ,P are high correlation coefficient between in vitro and in vivo methods(P<0.05). the R2 were 0.88,0.59,0.65,0.90,0.95. It was concluded that the methods we found was efficient and reliable. It can be used to evaluate feed enzyme biological value. Additive phytase to low- phosphorus diet has significant effect on the digestibility of Ca and P and has high correlation coefficient between in vitro and in vivo methods(P<0.05).So the digestibility of Ca and P in vitro could be used to predict the biological evaluation of phytase.
Experiment 4: In order to verify the methods efficiency and reliability in diferent enzymes, the diet applied with xylanase got the further research.The result shows: The digestibility of DM CP DE CF EE are high correlations between in vitro and in vivo methods(P<0.01),the correlation coefficient is respectively of 0.87、0.83、0.94、0.95、0.89.It showed that the methods that had been established in experiments were efficiency and reliability and can be used to evaluate biological value of different enzymes . Additive xylanase to low-energy diet has significant effect on the digestibility of DE and CF and has high correlation coefficient between in vitro and in vivo methods(P<0.01).So the digestibility of DE and CF in vitro could be used to predict the biological evaluation of xylanase.
Experiment 5:Evaluation the biological value of mannanase by using the in vitro method . The results show: mannanase had increased the digestibility of DM, CP and DE of corn-soybean diet in vitro .The group which was applied with 600U mannanase had the highest digestibility in DM、CP and DE in experiment.The results are agree with many other references and verify the method are practical.
引文
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