School of Food Science, Henan Institute of Science and Technology, Xinxiang, Henan 453003, China.
School of Food Science, Henan Institute of Science and Technology, Xinxiang, Henan 453003, China.
Food Res Int. 2019 Jan;115:580-588. doi: 10.1016/j.foodres.2018.09.033. Epub 2018 Sep 12.
Inhibition of spore germination or sterilization after induction of spore germination would effectively control low pH food spoilage caused by Alicyclobacillus acidoterrestris spores. However, the characteristics and mechanisms of A. acidoterrestris spore germination in low ambient pH remains poorly understood. In this study, the germination rate of A. acidoterrestris spores at different ambient pH conditions was determined, and subsequently the proteomic profiles of A. acidoterrestris in spore germination were analysed by label-free quantification, in which the specific metabolic pathways involved were identified and key functional proteins were screened and validated using RT-qPCR (real time quantitative PCR). The suitable ambient pH value for the spore germination of A. acidoterrestris ranged from 3.0 to 5.0 with the optimum pH of 4.0. According to the LC-ESI-MS/MS (liquid chromatography electrospray ionization tandem mass spectrometry) analysis, 98 proteins of geminated spores of A. acidoterrestris incubated for 2 h at pH 3.0 were changed significantly in comparison to non-germinated spores, the expression of 20 proteins were up-regulated and that of 78 proteins down-regulated respectively. Those differential expressed proteins were mainly involved in cell wall hydrolysis, cell morphological changes, protein synthesis and folding, perception of external stimuli and signal transduction etc., and we observed that germination receptor D (GerD), cell wall hydrolase, transpeptidase, peptidase S1 and two-component regulatory system phoR were significantly up-regulated, but hydrolase NlpC/P60, peptidoglycan glycosyltransferase, spore coat proteins CotX, CotJB and the Lrp/AsnC (leucine-responsive regulatory protein/asparagine synthase C products) protein were significantly down-regulated in the experiment, which implied the important roles of identified proteins during the spore germination. Furthermore, the pathway analysis showed the possible involvement of differentially expressed proteins in the β-lactam resistance, ribosome, biosynthesis of secondary metabolites, pyruvate metabolism, two-component system and other metabolic pathways, which indicated that synthesis and hydrolysis of cell wall, intracellular substance synthesis, energy generation and signal transduction were likely associated with the initiation of spore germination and restoration of vegetative growth. In conclusion, the quantitative proteomic landscape of A. acidoterrestris spores could provide the theoretic and experimental evidences for the hazard control of A. acidoterrestris spores in the thermal pasteurization process of acidic beverages industry.
抑制孢子萌发或在诱导孢子萌发后进行杀菌，可有效控制低 pH 值食品腐败，而 Alicyclobacillus acidoterrestris 孢子是造成这种腐败的主要原因。然而，A. acidoterrestris 孢子在低环境 pH 值下萌发的特性和机制仍知之甚少。本研究通过无标记定量法测定了 A. acidoterrestris 孢子在不同环境 pH 值条件下的萌发率，分析了 A. acidoterrestris 在孢子萌发过程中的蛋白质组图谱，鉴定了涉及的特定代谢途径，并使用 RT-qPCR（实时定量 PCR）筛选和验证了关键功能蛋白。A. acidoterrestris 孢子萌发的适宜环境 pH 值范围为 3.0 至 5.0，最适 pH 值为 4.0。根据 LC-ESI-MS/MS（液相色谱-电喷雾串联质谱）分析，与未萌发的孢子相比，在 pH 3.0 下孵育 2 小时的 A. acidoterrestris 萌发孢子的 98 种蛋白质发生了显著变化，20 种蛋白质表达上调，78 种蛋白质表达下调。这些差异表达的蛋白质主要涉及细胞壁水解、细胞形态变化、蛋白质合成和折叠、对外界刺激的感知和信号转导等，我们观察到萌发受体 D（GerD）、细胞壁水解酶、转肽酶、肽酶 S1 和双组分调节系统 phoR 显著上调，但水解酶 NlpC/P60、肽聚糖糖基转移酶、孢子壳蛋白 CotX、CotJB 和 Lrp/AsnC（亮氨酸反应调节蛋白/天冬酰胺合成酶 C 产物）蛋白显著下调，这表明鉴定出的蛋白质在孢子萌发过程中具有重要作用。此外，途径分析表明差异表达蛋白可能参与了β-内酰胺抗性、核糖体、次生代谢物生物合成、丙酮酸代谢、双组分系统和其他代谢途径，这表明细胞壁的合成和水解、细胞内物质的合成、能量产生和信号转导可能与孢子萌发的启动和营养生长的恢复有关。综上所述，A. acidoterrestris 孢子的定量蛋白质组图谱为酸性饮料工业热巴氏杀菌过程中 A. acidoterrestris 孢子危害控制提供了理论和实验依据。
