基于偶氮酪蛋白法研究饲料蛋白来源对饲料中蛋白酶活性测定的影响

doi:10.16431/j.cnki.1671-7236.2025.11.012

摘要/Abstract

摘要： 【目的】试验旨在优化偶氮酪蛋白法来测定蛋白酶活性，并探究不同蛋白质原料对饲粮中蛋白酶活性测定的影响。【方法】采用2×3两因素完全随机试验设计，研究pH对底物偶氮酪蛋白、酶解产物偶氮肽特征峰分布的影响，以确定偶氮肽的检测波长。采用单因素完全随机试验设计，分别研究不同浓度三氯乙酸(TCA)对偶氮酪蛋白的沉淀作用以及不同酶解反应时间对酶解反应速度的影响，并测定优化后的偶氮酪蛋白法的线性和精密度。采用2×3两因素完全随机试验设计，研究饲粮处理方法(10%、20%提取)和蛋白源(豆粕、花生粕、玉米蛋白粉)对蛋白酶酶促反应动力学的影响。【结果】偶氮酪蛋白底物和酶解产物的特征峰分布接近，当pH变为碱性时，偶氮酪蛋白底物和酶解产物特征峰由330～340 nm转移至415～425 nm。当TCA浓度为220.3和440.5 mg/mL时空白对照的吸光度值最低，440.5 mg/mL对偶氮酪蛋白沉淀效果最好。蛋白酶酶解速度随反应时间延长而降低，10和20 min时酶解速度与初始速度无显著差异(P＞0.05)，最适酶解反应时间为20 min。偶氮酪蛋白法在0.0016～0.1000 mg/mL蛋白酶测定中具有良好的线性关系(R²=0.993)，批内变异系数为1.59%～3.30%，批间变异系数为6.26%～13.12%。与空白对照组相比，10%饲粮提取液显著降低了蛋白酶酶促动力学最大反应速度(V_max，P＜0.05)，但对米氏常数(K_m)无显著影响(P＞0.05)；20%饲粮提取液显著降低了V_max(P＜0.05)，并显著提高了K_m(P＜0.05)；10%水平不同蛋白源对V_max和K_m均无显著影响(P＞0.05)。【结论】偶氮酪蛋白法测定蛋白酶活性具有灵敏度高、稳定性好的特点，可用于饲粮中蛋白酶活性测定。不同浓度饲粮提取液对蛋白酶酶促反应的抑制作用不同，但与饲粮蛋白质来源相关性较小。

关键词: 蛋白酶活性; 偶氮酪蛋白; 饲粮蛋白; 酶促动力学

Abstract: 【Objective】 This study aimed to optimize the azocasein method to determine protease activity and investigate the effect of protein sources on in-feed protease activity measurement. 【Method】 Using a 2×3 completely randomized factorial design, the effect of pH on the distribution of featured peaks for substrate azocasein and hydrolysate azopeptide was investigated to obtain the detection wavelength for azopeptide. The precipitation effects of different concentrations of trichloroacetic acid (TCA) and the hydrolyzing velocity at different time points were studied using a single-factor completely randomized design, and the linearity and precision of the azocasein method were analyzed. The effects of feed pretreatment method (10% and 20% extract) and protein sources (soybean meal, peanut meal, and corn gluten meal) on the protease hydrolyzing kinetics were explored using a 2×3 completely randomized factorial design. 【Result】 The azocasein hydrolysate had similar featured peak distribution as the azocasein substrate, which transited from 330-340 to 415-425 nm when pH turned to alkaline. The blank control had the least absorbance when TCA was included at 220.3 and 440.5 mg/mL, and 440.5 mg/mL TCA exhibited the best precipitation effect on azocasein. The hydrolyzing velocity diminished progressively along with the extension of time, but hydrolyzing velocity at 10 and 20 min did not significantly differ from the initial rate (P＞0.05), establishing 20 min as the optimal reaction duration. A good linearity (R²=0.993) was confirmed at 0.0016-0.1000 mg/mL proteas, the within-batch CV was 1.59%-3.30%, and the total batch CV was 6.26%-13.12%. Compared with blank control group, 10% feed extract significantly reduced V_max (P＜0.05) of the protease hydrolyzing reaction without affecting the K_m (P＞0.05), While 20% feed extract significantly reduced V_max (P＜0.05) and increased K_m (P＜0.05), respectively. Different protein resources (10% level) did not affect V_max and K_m (P＞0.05). 【Conclusion】 The optimized azocasein method had high sensitivity and stability, which made it suitable for the measurement of protease activity in feed. Inhibitory effect on the protease kinetics varied when different amounts of feed extract were included, but it had a relatively small correlation with feed protein resources.

Key words: protease activity; azocasein; feed protein; enzymatic kinetics

中图分类号:

S816.4

矫松君, 刘学涛, 李铁钊, 赵峰, 王钰明, 侯嘉, 解竞静. 基于偶氮酪蛋白法研究饲料蛋白来源对饲料中蛋白酶活性测定的影响[J]. 中国畜牧兽医, 2025, 52(11): 5147-5156.

JIAO Songjun, LIU Xuetao, LI Tiezhao, ZHAO Feng, WANG Yuming, HOU Jia, XIE Jingjing. Exploring the Influences of Feed Protein Resources on the Measurement of Protease Activity Using Azocasein Method[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(11): 5147-5156.

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