鱼蛋白胨的制备与质量分析

Preparation and quality analysis of fish peptone

目的制备鱼蛋白胨,并进行质量分析。方法将鱼糜加工下脚料原料经绞碎、石油醚脱脂,得到鱼糜浆,用0.1%木瓜蛋白酶酶解,离心收集上清(即鱼水解蛋白胨溶液),经300 Da纳滤膜脱盐和浓缩,喷雾干燥后,获得淡黄色粉末,即鱼蛋白胨样品FZU01。对鱼蛋白胨FZU01和鱼粉蛋白的主要理化指标(澄明度、亚硝酸盐和碱性沉淀、酸碱度、含氮量、氨基氮、干燥失重、炽灼残渣)进行检测;在固定酵母提取物、Na Cl浓度、初始p H和接种量的基础上,分别用两种蛋白胨配制LB培养基,接种大肠埃希菌,检测A600值;采用分子筛高效液相色谱法(size-exclusion highperformance liquid chromatography,SEC-HPLC)检测两种蛋白胨的相对分子质量。分别将50、150、300、600、900、1 200和1 500 mg/L组胺以及0.1、0.5、1.0、5.0、10.0、20.0、30.0、40.0和50.0 mg/L重金属离子(Hg2+、Cd2+、As3+、Cr6+和Pb2+)加至大肠埃希菌中,37℃培养12 h,检测A600值。结果两种蛋白胨理化指标检测结果均符合《中国生物制品主要原辅材料质控标准》规定,酸碱度、含氮量、氨基氮、干燥失重和炽灼残渣之间差异无统计学意义(P>0.05),鱼蛋白胨FZU01相对分子质量主要分布在1 000以内,占92.5%,鱼粉蛋白胨相对分子质量主要分布在10 000,小于1 000占56.1%;与鱼粉蛋白胨组相比,鱼蛋白胨FZU01组生长速率明显增加(P<0.05)。随着组胺浓度的增加,大肠埃希菌的生长抑制率逐渐增加,与组胺浓度呈正相关,IC50为257.3 mg/L,当组胺浓度大于600 mg/L时,完全抑制大肠埃希菌的生长;大肠埃希菌的生长抑制率与重金属离子浓度呈正相关,Hg^(2+)、Cd^(2+)、As^(3+)、Cr^(6+)和Pb^(2+)对大肠埃希菌的IC50分别为1.35、4.67、7.37、11.04和20.19 mg/L。结论应增加蛋白质相对分子质量、组胺和重金属作为蛋白胨产品重要质量参数,控制一定的浓度限量范围,才能保证蛋白胨产品的稳定性和安全性。

Objective To prepare fish peptone and analyze its quality. Methods The leftovers during process of surimi were ground and degreased, and the obtained oar was digested with 0. 1% papain, of which the supernatant（fish peptone digest） was collected by centrifugation, de-salted with a 300 Da nanofiltration membrane, concentrated then dried by nebulization to obtain fish peptone sample FZU01 in a form of light yellow powder. Both the sample FZU01 and fish powder protein were tested for main physiochemical indexes, including clarity, nitrite and basic precipitations, acidity and alkalinity, nitrogen content, amino nitrogen, loss on drying and ignition residue. LB media were prepared with the two kinds of peptones respectively at the same yeast extract and sodium chloride concentrations, initial p H value and inoculum concentrations, and inoculated with E. coli, of which the A600 values were determined. The relative molecular masses of two peptones were determined by size-exclusion high-performance liquid chromatography（SEC-HPLC）. E. coli was added with 50, 150, 300, 600, 900, 1 200 and 1 500 mg / L histamine and 0. 1, 0. 5, 1. 0, 5. 0, 10. 0, 20. 0, 30. 0, 40. 0 and50. 0 mg / L heavy metal ions（Hg^（2＋）, Cd^（2＋）, As^（3＋）, Cr^（6＋） and Pb^（2＋））respectively, cultured at 37 ℃ for 12 h and determined for A600 value. Results All the physiochemical indexes of two peptones met the Standard for Quality Control of Main Raw and Auxiliary Materials for Biologics in China, of which the acidity and alkalinity, nitrogen content, amino nitrogen, loss on drying and ignition residue showed no significant difference（P〉0. 05）. The relative molecular masses of 92. 5% of FZU01 were less than 1 000. The relative molecular masses of a major of fish powder peptone were 10 000, while those of56. 1% were less than 1 000. The growth rate of E. coli in FZU01 group increased significantly as compared with that in fish powder peptone group（P〈0. 05）. However, the inhibiting rate to growth of E. coli was positively related to the histamine concentration, with an IC50 of 257. 3 mg / L. The histamine at a concentration of more than 600 mg / L inhibited the growth of E. coli completely. The inhibiting rate of growth of E. coli was positively related to the heavy metal ion concentration. The IC50 of Hg2＋, Cd2＋, As3＋, Cr6＋and Pb2＋to E. coli were 1. 35, 4. 67, 7. 37, 11. 04 and 20. 19 mg / L respectively. Conclusion Relative molecular mass, histamine and heavy metal ion may be used as the important parameters for quality control of peptone product, of which the concentrations should be control within a specified range to ensure the stability and safety of the product.