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

Abstract

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

CLC Number:

S816.4

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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Exploring the Influences of Feed Protein Resources on the Measurement of Protease Activity Using Azocasein Method

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