虾加工副产物抗菌型水解液制备条件优化及抑菌作用

谷萝; 宋茹

doi:10.13386/j.issn1002-0306.2023050066

虾加工副产物抗菌型水解液制备条件优化及抑菌作用

谷萝,
宋茹

Optimization of Hydrolysis Conditions and Antibacterial Activity of Hydrolysate from Shrimp Processing By-products

GU Luo,
SONG Ru

摘要

摘要: 目的：为了探究水产品加工副产物用于抗菌型水解液制备，并研究其抑菌作用，本文以南美白对虾优势腐败菌（Specific spoilage organisms of Penaeus vannamei，PV-SSOs）为实验用菌，采用虾加工副产物制备抗菌型水解液（Antibacterial hydrolysate from shrimp processing by-products，SPPH），考察对PV-SSOs的抑菌作用。方法：以中华管鞭虾加工副产物为原料，分别采用5种蛋白酶水解，测定水解液对PV-SSOs的抑菌作用。以抑菌效果最强酶为实验用酶，研究加酶量、酶解时间、酶解温度和料液比对PV-SSOs的抑菌效果影响，然后采用响应面法优化SPPH酶解条件，高效液相色谱法分析SPPH中肽段分子量分布，采用膜渗漏法测定SPPH对PV-SSOs的细胞膜渗透性影响，进一步通过扫描电子显微镜观察SPPH作用后PV-SSOs菌体微观结构变化。结果：选用胃蛋白酶为水解用酶，在酶解反应pH2.0和料液比（1:2，w/v）条件下，经响应面Box-Behnke三因素三水平试验确定抑制PV-SSOs的SPPH最佳制备条件为：胃蛋白酶添加量700 U/g，酶解2.3 h，酶解温度33 ℃。经测定SPPH对PV-SSOs的抑菌直径达到24.10±0.43 mm。SPPH中分子量小于3000 Da肽组分的相对百分含量接近70%。PV-SSOs经SPPH作用2~12 h菌体细胞膜渗透性均显著高于菌对照组（P<0.05），扫描电镜下PV-SSOs经SPPH处理12 h可见部分菌体发生扭曲、皱缩，细胞膜表面形成凹陷、孔洞及内容物渗出。结论：中华管鞭虾加工副产物可用于制备抗菌型水解液，SPPH通过膜损伤方式抑制PV-SSOs，本研究为SPPH进一步用于南美白对虾保鲜研究提供了理论依据。

Abstract: Objective: In order to investigate the use of by-products from aquatic product processing in the preparation of antibacterial hydrolysates and their antibacterial effects, in this study, the specific spoilage organisms of Penaeus vannamei (PV-SSOs) were used as the experimental bacteria and the antibacterial hydrolysates from shrimp processing by-products (SPPH) were prepared, as well as the antibacterial effects of SPPH on PV-SSOs were determined. Methods: Five different proteases were used to hydrolyze the processing by-products of Solenocera crassicornis, and the antibacterial effects of these hydrolysates generated on PV-SSOs were compared. An appropriate protease was selected to prepare SPPH. The effects of hydrolysis conditions including enzyme addition content, hydrolysis time, hydrolysis temperature and solid-liquid ratio (w/v) on the inhibition of PV-SSOs were determined. Then, the response surface methodology was applied to optimize the hydrolysis conditions for SPPH preparation. Furthermore, the molecular weight distribution of peptidic fractions in SPPH was analyzed using high-performance liquid chromatography (HPLC). In addition, the effect of SPPH on the cell membrane permeability of PV-SSOs was measured using membrane leakage method, and the microstructure changes of PV-SSOs after SPPH treatment were further observed through scanning electron microscopy (SEM). Results: Pepsin was selected as the appropriate protease. Under the conditions of pH2.0 and solid-liquid ratio 1:2 (w/v) in the hydrolysis reaction, the optimal conditions for antibacterial hydrolysates preparation that inhibited PV-SSOs were obtained through the Box-Behnke experiment using response surface methodology at three levels and three factors as follows: 700 U/g of pepsin addition, 2.3 h of hydrolysis time, and 33 ℃ of hydrolysis temperature. The diameter of the inhibitory zone of SPPH on PV-SSOs was 24.10±0.43 mm. The result of HPLC showed that the relative percentage of peptidic fractions in SPPH with molecular weight less than 3000 Da was close to 70%. After treated with SPPH for 2 h to 12 h, the cell membrane permeability of PV-SSOs was significantly increased as compared to the control of PV-SSOs (P<0.05). Under SEM observation, some bacteria of PV-SSOs were twisted and shrunk, forming depressions, pores, and exudation of contents on the surface of the cell membrane after SPPH treatment for 12 hours. Conclusions: The processing by-products of Solenocera crassicornis can be applied to prepare antibacterial hydrolysates. SPPH inhibits PV-SSOs through membrane damage mode. All these findings will provide a theoretical basis for further development of SPPH for the preservation of Penaeus vannamei.