【作者】 赵峰；

【导师】 钱静亚；

【作者基本信息】 江苏大学 ， 食品工程（专业学位）， 2023， 硕士

【摘要】 酱油在生产过程中,容易受到有害微生物的污染。膜醭毕赤酵母菌(Pichia membranaefaciens)是一种耐盐和耐热的成膜酵母,会降解酱油中的有机物,分解营养物质,导致产品质量的下降。加热灭菌是当前酱油杀菌的主要方式,但热杀菌的高温,会使酱油颜色变深,产生焦味,致使氨基酸等营养物质的流失,同时会形成致癌物质氨基甲酸乙酯。因此,寻找新的酱油杀菌的方式对于酱油工业的发展具有积极意义。目前,超声波在新兴的食品保鲜技术中脱颖而出,它能很好地保持食品的色、香、味,最大限度地减少营养成分的损失。超声和中低温加热方式(热超声)的结合,不仅可以提高单独超声的杀菌效果,而且可以减轻热杀菌对食品感官和理化性质的影响。而山梨酸钾是主要的抑菌剂,在酱油中的添加可以延长其保质期。本论文研究了热超声联合山梨酸钾对酱油中膜醭毕赤酵母菌进行杀菌的杀菌效果、杀菌机理,并进一步研究了杀菌处理对酱油品质、风味及贮藏品质的影响,为此技术应用于酱油的工业化生产提供了一定的理论和科学依据。论文主要研究结果如下:(1)超声功率、间歇比和频率分别为300 W,10/3 s,20+40 k Hz,温度为60℃,山梨酸钾浓度为0.1 mg/m L时,热超声(TS)联合山梨酸钾(PS)处理20 min对酱油中膜醭毕赤酵母菌的杀菌效果最好,达到5.34 log的下降。分别在50、55、60、65℃下采用热联合山梨酸钾(T+PS)以及分别在25、40、50、55、60℃下采用热超声联合山梨酸钾(TS+PS)对膜醭毕赤酵母进行杀菌处理0～20 min,对杀菌动力学曲线进行拟合,发现Dose-Response模型相较于First Order和Weibull模型可以更好地描述T+PS和TS+PS对酱油中膜醭毕赤酵母的杀菌情况,拟合度最好。(2)SEM和TEM的成像结果显示TS+PS处理后膜醭毕赤酵母的细胞表面出现皱缩,细胞结构发生崩塌;细胞膜的严重破损导致胞内物质的外流,并同时出现不均匀分布的细胞质和电子密度。TS+PS处理20 min后,膜醭毕赤酵母胞内核酸泄漏OD值从0.013±0.002上升到0.196±0.002,蛋白泄漏量从0.30±0.07 ug/m L上升到24.09±0.47 ug/m L,TTC-脱氢酶活性下降57.53%,索氏平均粒径由11939.17 nm降低至6221.68 nm,细胞膜通透性显著增大且膜电位严重去极化,膜成分中的脂质和蛋白质含量降低。TS+PS处理后热休克蛋白Hsp82和Hsp70基因表达量下降,说明细胞受到严重的破坏,不能维持自身的稳态。(3)TS+PS处理对酱油的p H和总酸的影响较小;氨基酸态氮,还原糖在TS+PS处理后分别降低了16.4%和2.4%,而T90处理后,分别降低了20.0%和4.0%。TS+PS处理提高了酱油的亮度。蛋白酶活性(酸性、中性、碱性)在TS+PS处理后下降,但T90处理后的蛋白酶活性最低。T90和TS+PS处理分别使美拉德中间产物和美拉德终产物的增加最高。TS+PS处理后酱油粘度增加了11.57%,而T90处理后粘度减小了13.46%。酱油中挥发性风味物质的数量在TS+PS处理前后变化较小,但风味物质含量出现轻微下降。T90和TS+PS处理分别使挥发性风味物质含量下降了28.22%和14.12%,相比于高温热处理,TS+PS处理能够减少酱油中挥发性风味物质的损失,较好地保持酱油的风味。同时,根据挥发性风味物质的OAV值,鉴定出了29种主要香气活性物质。酱油在杀菌处理后,分别在4℃、25℃和37℃下进行贮藏。随着贮藏时间的不断延长,酱油的p H值不断减小,氨基酸态氮和还原糖含量不断下降,总酸含量不断增加,温度越高,变化幅度越明显。酱油的L~*,a~*,b~*值受贮藏温度的影响显著,温度越高,下降越快。但TS+PS处理的酱油的贮藏品质总体优于高温热处理的。更多还原

【Abstract】 Soy sauce is easy to be contaminated by harmful microorganisms during the production process.Pichia membranaefaciens,a salt and heat resistant film-forming yeast,degrades organic matter in soy sauce and decomposes nutrients,leading to a decrease in product quality.Thermal treatment is the main way to sterilize soy sauce currently,but the high temperature will cause the soy sauce to darken in color,produce a burnt taste,lose amino acids and other nutrients,and form carcinogenic substances such as ethyl carbamate.Therefore,it is important to look for new ways to sterilize soy sauce for the development of the soy sauce industry.Currently,ultrasound is proposed as one of the emerging food preservation technologies.Ultrasound can well maintain the color,aroma and taste of food and minimize the loss of nutrients.The combination of ultrasound and heat(thermoultrasound)can not only improve the inactivation efficacy of ultrasound alone,but also reduce the impact of high temperature on food sensory and physicochemical properties.Potassium sorbate is an antibacterial agent that can extend the shelf life of soy sauce.The inactivation effect and mechanism of thermoultrasound combined with potassium sorbate on P.membranaefaciens in soy sauce were investigated and the quality and storage quality of soy sauce after treatment were also evaluated in this paper.,which provided theoretical and scientific basis for the application of this technology in the soy sauce industry.The main findings of the paper are as follows:(1)When P.membranaefaciens was treated by thermoultrasound(TS)combined with potassium sorbate(PS)for 20 min at 300 W,10/3 s,20+40 k Hz,60°C and 0.1 mg/m L of potassium sorbate,the largest reduction of 5.34 log was achieved.The P.membranaefaciens was treated by heat combined with potassium sorbate(T+PS)at 50,55,60 and 65°C and thermoultrasound combined with potassium sorbate(TS+PS)at 25,40,50,55 and 60°C,respectively,for 0～20 min.The inactivation kinetic curves were fitted by models,and Dose-Response model fitted the curves better than First Order and Weibull models,indicating Dose-Response model could be used to describe the inactivation effect of T+PS and TS+PS on P.membranaefaciens in soy sauce.(2)SEM and TEM observation results showed that the cell surface of P.membranaefaciens wrinkled,cell structure collapsed,cell membrane damaged intracellular contents outflowed and cytoplasm and electron density unevenly distributed after TS+PS treatment.When P.membranaefaciens was treated by TS+PS for 20 min,the OD value of DNA leakage increased from 0.013±0.002 to 0.196±0.002,protein leakage increased from 0.30±0.07 ug/m L to24.09±0.47 ug/m L.TTC-dehydrogenase activity decreased by 57.53%,the mean particle size of Soxhlet decreased from 11939.17 nm to 6221.68 nm,the cell membrane permeability increased significantly,the membrane potential was severely depolarized and the lipid and the protein content of the membrane components decreased.The expression of heat shock protein Hsp82 and Hsp70 genes was down regulated after TS+PS treatment,indicating that the cells were severely damaged and could not maintain their homeostasis.(3)TS+PS treatment had little effect on p H and total acid of soy sauce.Amino acid nitrogen,reducing sugar decreased by 16.4%and 2.4%after TS+PS treatment,while decreased by 20.0%and 4.0%after T90 treatment.The brightness of soy sauce was improved by TS+PS treatment.Protease activity(acidic,neutral and alkaline)decreased after TS+PS treatment,but the lowest protease activity was observed after T90 treatment.T90 and TS+PS treatments resulted in the highest increments in the mid-products and end-products of the Maillard reaction,respectively.The viscosity of the soy sauce increased by 11.57%after TS+PS treatment,while decreased by13.46%after T90 treatment.The types of volatile flavor compounds in the soy sauce changed little before and after TS+PS treatment,but the content of flavor compounds slightly decreased,with the decreases by 28.22%and 14.12%after T90 and TS+PS treatments,respectively.Compared with heat treatment at high temperature,TS+PS treatment could reduce the loss of volatile flavor compounds and maintain the flavor of soy sauce.Meanwhile,a total of 29 major aroma active compounds were identified based on the OAV values of volatile flavor compounds.After treatment,soy sauce was stored at 4℃,25℃and 37℃respectively.With the increasing of storage time,the p H value,amino acid nitrogen and reducing sugar content decreased,and the total acid content of soy sauce increased,and the higher the temperature,the more change.The L~*,a~*and b~*values of soy sauce were significantly affected by the storage temperature,and the higher the temperature,the faster the decrease.However,the storage quality of soy sauce after TS+PS treatment was generally better than that of heat treatment at high temperature.更多还原