商品植酸酶及饲粮中植酸酶活性测定的影响因素及变异度

Influence Factors and Variation in Determination of Phytase Activity in Commercial Phytase and Diet Phytase

本试验旨在探讨商品植酸酶及饲粮中植酸酶活性测定的影响因素及变异度。试验分为4个部分。第1部分:考察国标方法中稀释缓冲液类型及溶液显色后离心速度对植酸酶活性测定值的影响。采用2×3两因素完全随机设计,稀释液分别为缓冲液1(乙酸/乙酸钠缓冲液)和缓冲液2(乙酸/乙酸钠+曲拉通X-100+牛血清白蛋白),显色静置10 min后离心速度分别设为3 000、5 000和8 000 r/min。第2部分:考察植酸酶的稀释倍数对植酸酶活性测定值的影响。商品植酸酶的稀释倍数分别设为150、200、250和300倍;饲粮中植酸酶的稀释倍数分别设为5、10和15倍。第3部分:考察饲料原料对植酸酶活性测定的干扰。采用单样本比较设计,在每千克玉米、豆粕、小麦麸、菜籽粕、棉籽粕中添加4 g植酸酶。第4部分:采用单因素完全随机设计,测定14 d内商品植酸酶及饲粮中植酸酶活性的变异。结果表明:1)使用缓冲液1时,饲粮中植酸酶活性的测定值在3 000、5 000和8 000 r/min间无显著差异(P>0.05);而使用缓冲液2时,饲粮中植酸酶活性的测定值随离心速度的增加而显著升高(P<0.05)。2)随着稀释倍数的增加,商品植酸酶活性的测定值先升高后下降(P<0.05),而饲粮中植酸酶活性测定值呈线性下降(P<0.05)。3)在饲料原料中添加植酸酶后,玉米中植酸酶总活性的实测值显著低于计算值(P<0.05),豆粕、小麦麸、菜籽粕和棉籽粕中植酸酶总活性的实测值显著高于计算值(P<0.05)。4)商品植酸酶活性测定值日内变异系数为3.13%,日间变异系数为6.11%,总变异系数为6.68%。饲粮中植酸酶活性测定值的日内变异系数为1.56%,日间变异系数为3.96%,总变异系数为4.19%。由此可见,乙酸/乙酸钠更适合作为植酸酶活性测定中的稀释液,饲粮中植酸酶活性的测定值受离心速度及稀释倍数的影响,而商品植酸酶的活性测定值仅受稀释倍数的影响。饲料原料明显地干扰了植酸酶活性的测定。饲粮中植酸酶活性测定值的变异程度低于商品植酸酶。

The objective of this experiment was to study the influence factors and variation in determination of phytase activity in commercial phytase and diet phytase. Four trials were included in this experiment. Trial 1 was to investigate the influence of the dilution buffer category and the centrifugal speed for the reaction solution after the color reaction on the determined value of phytase activity with the national standard method. Dilution solutions of buffer 1(acetic acid/sodium acetate buffer)or buffer 2(acetic acid/sodium acetate+triton X-100+BSA),and centrifugal speeds for the reaction solution of 3 000,5 000 or 8 000 r/min for the reaction solution were used in a 2×3 factorial arrangement in a completely randominze design. Trial 2 was to investigate the influence of dilution rate of phytase on the determined value of activity. The dilution rates were 4 treatments of150,200,250 or 300 times for commercial phytase and 3 treatments of 5,10 or 15 times for diet phytase. Trial 3 was to examine the interference of feed ingredients on the determined activity of phytase. A one-sample comparison design was adopted to each of corn,soybean meal,wheat bran,rapeseed meal,or cottonseed meal with the phytase added as 4 g per kilogram of feed ingredient. Trial 4 was to investigate the variation of phytase activity determined in commercial phytase and diet phytase in 14 days by a single-factor completely random design. The results showed as follows:1)the determined phytase activities in diet were not different at centrifugal speed of 3 000,5 000 or 8 000 r/min when the buffer 1 was used for the diluent solution(P>0.05). But the determined phytase activities of diets were increased by the centrifugal speed when the buffer 2 was used for the diluent solution(P<0.05). 2)With increment of the dilution rate,the determined activities of commercial phytase firstly increased and then decreased(P<0.05),however,the determined phytase activity in diets decreased linearly(P<0.05). 3)In feed ingredients added phytase,the determined value of total phytase activity in corn was significantly lower than the calculated value(P<0.05). But greater determined than calculated total phytase activities were observed in soybean meal,wheat bran,rapeseed meal and cottonseed meal(P<0.05).4)The intra-day,inter-day and total coefficient of variation(CV)of the determined phytase activity in the commercial phytase were 3.22%,6.29% and 6.88%. The intra-day,inter-day and total CV of the determined phytase activity in the diet supplemented with phytase were 1.55%,3.83% and 4.06%. In conclusion,acetic acid/sodium acetate buffer is more suitable to use as a diluent for determination of phytase activity. The determined value of phytase activity in diet is affected by the centrifugation speed and dilution rate,but the determined value of commercial phytase activity is only affected by the dilution rate. Feed ingredients significantly interfere with the determination of phytase activity. The variation in the determined phytase activity is lower in the diet than that in commercial phytase.