低盐泡菜作为我国传统特色发酵食品，脆嫩酸爽,具有开胃益生的功效，深受消费者喜爱。在实际生产中，考虑到其独特风味和益生特性在高温下极易损失，该类产品通常不经热杀菌而选择低温贮运，但高酸度和高水分活度的特点极易引起氧化型产膜酵母大量繁殖并引发泡菜生花腐败，因此采用冷杀菌技术实现对低盐泡菜杀菌保鲜具有重要的现实意义。低温等离子体技术具有成本低、易于操作、安全无残留的特点，在鲜切果蔬、肉类及其制品、水产品及其制品等多类生鲜产品的保质、保鲜及货架期延长方面展现出明显优势，目前在泡菜冷杀菌方面刚刚起步。本研究采用低温等离子体冷杀菌（dielectric barrier discharge, DBD）对低盐泡菜进行处理，探究了DBD对泡菜生花腐败的抑制及贮藏品质的影响，分离鉴定致生花腐败的主要酵母菌，探究不同激发因素及不同基质环境下DBD的杀菌动力学特性，并进一步探究DBD对酵母细胞的损伤作用，为低盐泡菜DBD保鲜及品质调控提供研究基础，具体研究内容如下：

1.低温等离子体冷杀菌对低盐泡菜生花腐败抑制及贮藏品质的影响

采用DBD对低盐泡菜处理并进行25 °C贮藏期实验，通过微生物、理化指标、色差、质构及挥发性风味物质分析，探究DBD对低盐泡菜生花腐败的抑制及贮藏品质的影响。结果表明：DBD电压70 kv、频率50 Hz下，处理180 s循环4次条件下，可将菌落总数和酵母菌数分别减少3.55 lg CFU/g和4.21 lg CFU/g，完全杀灭大肠菌群并保留一定乳酸菌，抑制产膜酵母生长，有效控制生花腐败。在品质指标方面，DBD可抑制pH上升，泡菜脆度、亮度、黄度及发酵酸度显著高于巴氏杀菌（P<0.05）；虽处理后亚硝酸盐含量有所上升，但峰值远低于国标限量，且随发酵进行含量逐渐降低；贮藏期间可抑制二甲基硫化物增加导致的异味产生。以pH和产膜酵母数衡量，常温下至少可将货架期延长66.7 %。综上所述：DBD处理可在保证低盐泡菜品质特性的前提下，有效控制贮藏期间的生花腐败，延长货架期。

2.低盐泡菜中毕赤酵母的低温等离子体冷杀菌动力学研究

分离鉴定致生花腐败的主要酵母菌，采用动力学模型探究不同激发因素及基质环境下DBD的杀菌动力学特性。结果表明：致生花腐败的主要酵母菌为曼氏毕赤酵母（Pichia manshurica）。修正的 Gompertz模型适于不同激发因素下DBD杀菌过程的拟合，增大电压可显著提高致死速率并显著降低细胞存活率。DBD电压80 kv，频率50 Hz，处理时间7.5 min条件下可将毕赤酵母降低4.84 lg CFU/mL。Weibull模型适于不同基质环境下DBD杀菌效果的拟合。2～8 %盐度下DBD对残存酵母细胞的致死效果显著增强，7.5 min内将6 lg CFU/mL的毕赤酵母全部灭活；0.2～0.8 %的乳酸协同DBD可减小杀菌的初始阻力，在90 s内将酵母菌数降低3.5 lg CFU/mL；降低pH至3～5，不仅减小了杀菌的初始阻力，还显著削弱残存细胞对DBD的抗性，在120 s内将6 lg CFU/mL的毕赤酵母全部灭活。综上所述： 盐、乳酸、低pH协同高DBD电压对于减小初始杀菌阻力，降低残存细胞抗性具有积极意义。

3.低温等离子体对低盐泡菜中毕赤酵母的细胞损伤研究

以分离所得毕赤酵母为研究对象，通过不同DBD处理时间组毕赤酵母生长曲线、产膜速率的测定，SEM和TEM观察，ROS含量、MDA含量、SOD与CAT酶活性及核酸、蛋白泄露的测定，探究DBD对毕赤酵母的损伤作用。结果表明： DBD电压80 kv，频率50 Hz条件下，处理时间超过4.5min后对数生长期明显滞后，生长速率和成膜速率显著降低。随处理时间增加，一方面细胞表面皱缩甚至严重形变、破裂，内容物部分聚集，部分大量流失，出芽生殖受到抑制；另一方面抗氧化酶活显著降低，细胞ROS水平显著升高，造成严重的脂质过氧化，处理7.5 min MDA含量为2.25μM，达对照组的5倍。此外，核酸、蛋白泄露量逐渐增加， 7.5min处理核酸、蛋白泄露量为对照组的1.9倍和2.7倍，反映了毕赤酵母细胞膜通透性的增强。综上所述：DBD处理显著降低毕赤酵母的生长及生膜速率，在处理时间的累积效应下，细胞形态结构上的物理性损伤、细胞氧化损伤及膜通透性的增大导致了细胞死亡。

As a traditional characteristic fermented food in China, low-salt pickle is deeply loved by consumers because of their crispy, sour and refreshing mouthfeel, as well as their appetizing and probiotic functions. During the industrial production and processing, pickle products are usually stored and transported at low temperature without heat sterilization, considering that its unique flavor and probiotic properties are easily lost at high temperatures. However, the product has high acidity and water activity, which promotes the mass reproduction of oxidative pellicle-forming yeast, and eventually leads to the pellicle-spoilage. Therefore, it is of great significance to find a cold sterilization technology for the preservation of low-salt pickle. Due to the advantages of low cost, convenient operation, high safety, and no pollutants remain, cold plasma technology has been studied in the sterilization, preservation and shelf-life extension of fresh-cut fruits and vegetables, meat, aquatic products, etc, but there were few studies on the sterilization of pickle. In this research, dielectric barrier discharge (DBD) technology was used to treat the low-salt pickle to explore its effect on the pellicle-spoilage and storage quality. Through the isolation and identification, it was found that the main pellicle-forming yeast was Pichia manshurica. After that, the sterilization kinetics of DBD under different excitation factors and different microbial environments were explored. In addition, the damaging effect was further explored. This study provides a research basis for DBD preservation and quality control of low-salt pickle. The specific research contents are as follows:

1．Effect of cold plasma treatment on the inhibition of pellicle-spoilage and the storage quality of low-salt pickle.

To explore the effect of DBD treatment on the inhibition of pellicle-spoilage and the storage quality of low-salt pickle, the microorganisms, physical and chemical indicators, color difference, hardness and volatile flavor compounds during storage at 25°C were investigated. The results showed that DBD treatment (70 kv, 50 Hz, 180 s×4) could reduce the numbers of total bacterial count and yeast by 3.55 lg CFU/g and 4.21 lg CFU/g respectively, and eliminate all coliforms while allowing the retention of lactic acid bacteria. Moreover, DBD treatment inhibited the growth of pellicle-forming yeast which could effectively control the biofilm-spoilage of low-salt pickle during storage. In terms of quality indicators, DBD treatment inhibited the increase of pH obviously, compared with the pasteurized group, the hardness, brightness, yellowness and total acid content of the low-salt pickle in the DBD treated group were increased significantly (P<0.05). At the same time, the peak value of nitrite content in the DBD treated group was much lower than the national standard limit, and its content decreased gradually during the storage. Additionally, DBD treatment had little effect on the main volatile flavor compounds within a short time, and could inhibit the odor caused by the increase of dimethyl sulfide during storage. Measured by pH and the number of pellicle-forming yeast, the shelf life at room temperature could be extended by 66.7 %. Therefore, DBD treatment could control the biofilm-spoilage of low-salt pickle during storage while ensuring the quality characteristics, thereby effectively extending the shelf life.

1. The sterilization kinetics of Pichia manshurica in low-salt pickle by low temperature plasma treatment

After isolation and identification of the main yeast that lead to pellicle-spoilage, the bactericidal kinetics of DBD under different excitation factors and different substrate environment were explored. The results showed that the main yeast causing pellicle-spoilage was Pichia manshurica. The Modified Gompertz model was most suitable for the fitting of curves under different excitation factors. Increasing the voltage increased the lethal rate and reduced the cell residues significantly. Under the condition of DBD voltage of 80 kv, frequency of 50 Hz, and treatment time of 7.5 min, Pichia manshurica can be reduced by 4.84 lg CFU/mL. The Weibull model was most suitable for the fitting of curves under different substrate environment.The lethal effect of DBD on the remaining yeast was significantly improved by increasing the salt content of the bacterial suspension (2～8%), and the Pichia manshurica could be completely inactivated within 7.5 minutes. The combination of lactic acid (0.2～0.8%) and DBD reduced the initial resistance of sterilization, and reduced the number of the yeast by 3.5 lg CFU/mL within 90 s. The low pH environment (3～5) not only reduced the initial resistance to sterilization, but also significantly weakened the resistance of the remaining cells to DBD. Pichia manshurica could be completely inactivated within 120 s. Therefore: NaCl, lactic acid and low pH combined with high voltage of DBD have positive significance for reducing the initial sterilization resistance and the residual cell resistance.

3．The damaging effect of Pichia manshurica in low-salt pickle by cold plasma treatment.

Taking the isolated Pichia manshurica as the experimental strain, growth curves, pellicle-forming rate, SEM, TEM, the content of ROS and MDA, the activities of SOD and CAT, and the leakage of nucleic acid and protein were determined to explore the damaging effect of DBD on Pichia manshurica. The results showed that, DBD treatment (80 kv, 50 Hz) delayed the logarithmic growth phase significantly when the treatment time exceeds 4.5min, and the growth rate and pellicle-forming rate of yeast were decreased. With the increase of DBD treatment time, on the one hand, the surface of yeast cells shrank or even seriously deformed and ruptured. Part of the cytoplasmic material was accumulated, the other part was lost in large quantities, and the budding reproduction was inhibited, on the other hand , the activity of antioxidant enzymes decreased significantly, and the content of ROS was significantly increased, resulting in severe lipid peroxidation. The content of the MDA was 2.25 μM after treated for 7.5 min, which was 5 times that of the control group. In addition, the leakage of nucleic acid and protein were 1.9 times and 2.7 times that of the control group respectively, indicating that DBD treatment led to the cell membrane permeabilization. Therefore, the DBD treatment significantly reduced the growth and pellicle-forming rate of Pichia manshurica. With the cumulative effect of sterilization time, the severe physical damage of the cell morphology, the aggravated oxidative damage and the membrane permeabilization caused the death of the Pichia manshurica.