超高压对仙人掌有孢汉逊酵母的损伤机理

为探究超高压对仙人掌有孢汉逊酵母的损伤机理,该研究利用流式细胞仪、圆二色谱、扫描电镜与透射电镜等多种方法测定超高压处理(100、200、300、400、500 MPa)对菌体细胞膜、形态结构、胞内物质的影响。试验结果表明:选择300 MPa和300 s作为处理条件,超高压处理后仙人掌有孢汉逊酵母的死亡数为4.36 lg(CFU/mL),拥有较好的灭活效果。处理后细胞膜被破坏,70.50%的菌体可被碘化丙啶染色,核酸、蛋白、离子大量外泄,紫外260与280 nm吸光度分别达1.132及0.374,电导率升至26μS/cm,说明大部分菌体细胞膜通透性提高。同时胞内Na⁺/K⁺-ATP酶、Ca²⁺/Mg²⁺-ATP酶和总ATP酶的活力降低,抑制率分别达到31.13%,16.01%和20.06%,核酸、蛋白结构均发生改变,表明超高压可以影响胞内物质。扫描电镜及透射电镜图像显示,细胞膜穿孔,形态结构改变。以上结果表明,超高压处理对仙人掌有孢汉逊酵母菌体的损伤主要在于破坏细胞膜,并使得胞内物质失活。研究结果为超高压对非酿酒酵母的损伤机理提供参考。

Damage mechanism of Hanseniaspora opuntiae by high hydrostatic pressure

Non-Saccharomyces cerevisiae often appears on the surface of berries in the early natural fermentation of fruit wine,particularly on the most common varieties of Kloeckera apiculata and Hanseniaspora uvarum.However,the growth,reproduction,and metabolism of the non-Saccharomyces cerevisiae can easily produce ethanol and other undesired components,thereby causing the quality changes in most fruit and vegetable products,generally regarded the phenomenon as a spoilage strain.Thermal treatment is a common way to kill spoilage strains in fruit and vegetable juice,fresh fruit and non-alcoholic beverages.This approach can effectively inhibit the growth of spoilage strains.However,the loss of heat-sensitive active substances is accompanied during the treatment,leading inevitably to the decline in sensory quality of food.Alternatively,non-thermal sterilization technology,represented by high hydrostatic pressure(HHP),has widely been applied in food processing,which can effectively overcome the disadvantages of thermal treatment.Most previous studies were relatively comprehensive focuses on the bacteria in the sterilization mechanism of HHP on food microorganisms,but it is still lacking on the non-Saccharomyces cerevisiae.Furthermore,the growth and reproduction of non-Saccharomyces cerevisiae are the main factors resulting in the microbial spoilage of berry products.Therefore,it is necessary to effectively inhibit the physiological activity of non-Saccharomyces cerevisiae,thereby improving the product quality,while reducing the loss of flavor value.One kind of non-Saccharomyces cerevisiae,the Hanseniaspora opuntiae can be separated from natural berries.Therefore,it is feasible to investigate the damage mechanism of Hanseniaspora opuntiae with different sterilization treatments.In this study,Hanseniaspora opuntiae was isolated from naturally putrefied Patriot blueberry(Semen trigonellae)juice,and then treated with HHP to explore the damage mechanism,where the pressure of 300 MPa and the duration of 300 s were selected as the experimental condition.A flow cytometry,circular dichroism,scanning electron microscopy(SEM),and transmission electron microscopy(TEM)were adopted to determine the effects of HHP on the cell membrane,cell morphology and intracellular substances.The experimental results showed that the death count of HHP was 4.36 lg(CFU/mL),indicating an excellent inactivation effect.After treatment with HHP,the cell membrane was destroyed,and 70.50%of Hanseniaspora opuntiae was stained by PI.Nucleic acid,protein and ions were leaked out,where the OD260nm and OD280nm reached 1.132 and 0.374,respectively,and the electrical conductivity rose to 26μS/cm,indicating that the membrane permeability of most cells increased.Meanwhile,the activities of Na⁺/K⁺-ATPase,Ca²⁺/Mg²⁺-ATPase,and total ATPase decreased,where the inhibition rates reached 31.13%,16.01%,and 20.06%,respectively.The results of AO staining and circular dichroism spectrum demonstrated that the structure of nucleic acid and protein were changed,proving that the HHP can affect intracellular substances.In the images of SEM and TEM,there were perforations on the cell membrane and outflow of cytoplasm in large quantities.The results indicated that the HHP damage to Hanseniaspora opuntiae was due mainly to the destruction of the cell membrane,which caused the intracellular substances to lose their activities in an HHP treatment.