虾加工副产物抗菌型水解液制备条件优化及抑菌作用

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

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。

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.