豆腐皮常温贮藏过程中菌相变化及优势菌致腐性分析

Changes in the Bacterial Phases of Soybean Curd Sheets and Analysis of Spoilage Ability of Dominant Bacteria Stored at Normal Temperature

本文以中国传统豆类食品豆腐皮为研究对象,采用传统分离鉴定和高通量测序技术共同分析豆腐皮的腐败菌,并将优势菌回接至无菌豆腐皮,通过定期测定样品贮藏过程中菌落总数、挥发性盐基氮(TVB-N)、pH、感官评分和质构特性来确定优势菌对品质的影响,通过扫描电子显微镜、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和傅里叶红外光谱(Fourier Transform Infrared Spectroscopy,FTIR)来确定优势菌对蛋白的影响。结果表明,豆腐皮的菌相在贮藏过程中发生了明显变化,贮藏2 d的优势腐败菌主要为不动杆菌(Acinetobacter)、葡萄球菌(Staphylococcus)和库特氏菌(Kurthia)。对上述腐败菌,选择3株有代表性的进行回接,发现一株产蛋白酶的松鼠葡萄球菌和混菌的致腐能力最强,可以导致TVB-N、黏性显著增加,硬度、弹性和咀嚼性显著下降(P<0.05)。扫描电子显微镜显示接种松鼠葡萄球菌和混菌的样品表面蛋白结构扭曲,有许多孔洞;SDSPAGE结果表明大豆蛋白的7S亚基出现不同程度的褪色或消失,25 kDa以下区域明显加深且出现一个新的条带;傅里叶红外光谱显示样品的β-折叠降低,β-转角增加。因此,松鼠葡萄球菌被鉴定为豆腐皮常温贮藏的特定腐败菌,为后续靶向控制豆腐皮中腐败菌的生长提供参考。

The bacterial phase changes and the effect of dominant spoilage bacteria on quality were studied with traditional identification method and high-throughput sequence technique using Chinese traditional food soybean curd sheets as materials.Sterile soybean curd sheets were inoculated with three dominant bacteria and their combination.The total viable counts,total volatile base nitrogen(TVB-N),pH,sensory score,and texture characteristics of samples during normal temperature storage were measured periodically for spoilage potential.The scanning electron microscopy,sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE),and Fourier transform infrared spectroscopy(FTIR)were used to determine the effect of spoilage bacteria on the protein.The results indicated the bacterial phases of soybean curd sheets changed obviously during storage,and Acinetobacter,Staphylococcus as well as Kurthia became the dominant bacteria after 2 days of storage.The selected representative spoilage bacteria were re-inoculated to the samples,one protease-producing Staphylococcus sciuri and the mixed bacteria had the strongest spoilage potential,because the TVB-N and viscosity increased greatly,while the hardness,springiness,and chewiness decreased significantly(P<0.05).Scanning electron microscopy revealed that the surface protein structure of the samples was distorted and many holes appeared.SDS-PAGE result showed that the band of soybean protein 7S subunit faded or disappeared,however,the bands below 25 kDa were deepened obviously and a new band appeared.FTIR showed that the soybean proteinβ-sheet structure decreased,while theβ-turn structure increased.In conclusion,Staphylococcus sciur is identified as specific spoilage bacteria of soybean curd sheets during normal temperature storage,it provides a valuable reference for the spoilage bacteria control and prolong the product shelf-life.