应用超高压技术延长低温火腿的货架期
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摘要
低温肉制品,是指采用较低的温度(68～85℃)进行热加工,在严格的低温车间生产,在低温冷链下进行运输、销售的一类熟肉制品。此类肉制品最大程度保持了肉品的营养价值及天然风味,加工技术先进、市场广阔,代表了肉品发展的主导方向。然而,由于生产中热加工温度较低,产品杀菌不彻底,以及后续加工过程中存在的二次污染隐患等原因,当前我国低温肉制品货架期普遍偏短。严重制约了我国低温肉制品市场的发展。食品超高压灭菌技术主要是依据压力的致死、致伤作用,在不改变(或较小改变)食品现有感官、质地及风味的前提下实现对微生物的杀灭、抑制,延长食品货架期。超高压能够有效改善低温肉制品存在的货架期短问题,目前该技术在国外发达国家已应用于低温肉制品产业,很多超高压食品面市,而我国超高压低温肉制品产业刚刚进入起步阶段,行业内对超高压加工技术相对还比较陌生,相关研究亟待开展。本论文以低温熏煮猪肉切片火腿为研究对象,通过对超高压处理后低温火腿的贮藏特性、微生物种群结构以及超高压抑菌机制的研究,确立了低温火腿的超高压加工工艺,以期为解决我国低温肉制品货架期短的问题,推进超高压肉品产业化进程提供数据参考和理论指导。具体研究内容和结论如下：
1.超高压处理后低温火腿贮藏特性研究
参考国外研究成果,选择了400 MPa和600 MPa的压力条件,室温条件下(12℃)对低温火腿进行10 min超高压处理,在4℃贮藏条件下定期评定样品的微生物、理化和感官品质变化,确定超高压对低温肉制品腐败菌的抑制效应,评定其对肉制品品质指标的影响效果。以期在延长产品货架期的同时有效保证产品的食用品质和营养价值。结果表明：400 MPa和600 MPa的超高压处理均能够有效抑制低温火腿中的腐败微生物,使乳酸菌数处于相当长的低水平期,很大程度上缓解了由乳酸菌繁殖造成的低温火腿产气、发黏、涨袋等腐败现象,对肉制品中常见污染的肠杆菌和耐冷菌显示了很强的抑制效果。初始污染菌数相对较低时,600MPa/12℃/10 min的超高压处理,4℃贮藏条件下能够将产品货架期延长至10周,对低温火腿的脂肪氧化、颜色、质构特性等理化指标和感官指标影响较小,不会或较小破坏低温火腿脂肪水解、蛋白质水解等与营养价值相关的各项指标。
2.超高压对低温火腿微生物种群结构及动态变化的影响研究
参考相关研究,采用400 MPa和600 MPa的压力在室温(22℃)条件下对低温火腿进行10 min超高压处理,于4℃贮藏0(高压前),1(高压后),30,90 d,通过直接提取样品微生物种群的基因组DNA和总RNA,分别进行PCR-和RT-PCR-DGCE (DGGE:变性梯度凝胶电泳)指纹图谱分析,揭示超高压对低温火腿中主要腐败微生物的抑制效应,进一步深入了解超高压处理后存活微生物的消长规律,为超高压技术在低温肉制品杀菌中的应用提供更全面的数据支撑。结果表明：低温火腿中污染的微生物种群主要由乳杆菌属、乳球菌属、明串珠菌属、魏斯菌属和链球菌属五个属中的细菌组成；RT-PCR-DGGE技术能够有效揭示超高压处理后各微生物种群的存活状况；400 MPa和600 MPa的超高压处理能够有效抑制低温火腿中的清酒乳杆菌(Lactobacillus sakei).弯曲乳杆菌(Lactobacillus curvatus)等优势腐败微生物的生长繁殖；600 MPa的压力处理后,绝大多数的微生物种群未检出生长,低温火腿微生物种群结构变得非常单一,仅有绿色魏斯菌(Weissella viridescens)和肠膜明串珠菌(Leuconostoc mesenteroides)存活；超高压处理在降低微生物多样性的同时进一步提高了低温火腿的食用安全性。
3.超高压耐受菌的分类鉴定及耐压特性研究
运用变性梯度凝胶电泳技术对分离自600 MPa超高压处理组样品中的84株超高压耐受菌进行分类,并通过16S rDNA序列分析技术以及细菌生理生化试验进行鉴定；以分离到的超高压耐受菌为供试对象,比较研究了超高压处理后耐压菌在平板计数琼脂、MRS琼脂以及二者组合制备的薄层平板培养琼脂上的生长繁殖状况；在此基础上,以乳酸链球菌素和超高压作为栅栏因子,研究了二者协同处理对超高压耐受菌存活率的影响效果,为评价超高压耐受菌的耐压特性,寻找有效的控制措施进行了初步尝试。结果表明：84株待分类的超高压耐受菌在DGGE图谱中仅表现出两个位置不同的迁移条带,经鉴定后这两株超高压耐受菌为绿色魏斯菌(Weissella viridescens)和肠膜明串珠菌(Leuconostoc mesenteroides);采用薄层平板培养方法作为评价手段,揭示出超高压与乳酸链球菌素结合处理对这两株超高压耐受菌具有显著的协同抑制效果,200μg/mL的乳酸链球菌素添加量结合500MPa/5min/20℃的超高压处理条件,能够彻底抑制初始菌数为109 CFU/mL的绿色魏斯菌的生长繁殖。乳酸链球菌素的添加能够有效降低工业化生产中高压力的使用。
4.超高压与热处理抑菌机制的比较性研究
运用扫描/透射电镜观察和流式细胞多参数分析技术比较研究了超高压处理和热处理后绿色魏斯菌生理生化状态的变化,揭示出两种不同的抑菌手段对绿色魏斯菌细胞膜完整性和酯酶活性的破坏情况,为更好的理解超高压抑菌机制提供理论补充。结果表明：超高压处理对绿色魏斯菌的菌体形态未发现显著影响,600 MPa处理时细胞胞质成分出现暗色区域及部分气泡性结构；在压力由100 MPa升高至600 MPa的过程中,细胞膜完整性受到破坏的绿色魏斯菌数量显著增加,然而整个过程中绿色魏斯菌的酯酶活性却未见显著变化。热处理能够引起绿色魏斯菌菌体形态发生严重变形,在温度由60℃升高到100℃的过程中,菌体形变明显加剧,细胞内成分发生变性凝集,出现大面积白色区域。随热处理温度的升高,绿色魏斯菌细胞膜完整性及酯酶活性均显著丧失。超高压处理与热处理相同,二者都能够破坏绿色魏斯菌的细胞膜完整性,通过引起胞体蛋白质发生变性影响其生物化学反应,最终达到抑制微生物的目的。
5.低温火腿超高压杀菌工艺研究
参考国内外研究成果,选择压力、时间、温度以及乳酸链球菌素添加量四个因素作为考察因子,以低温火腿为基质,评定了四因子对超高压杀灭绿色魏斯菌和肠膜明串珠菌的影响效果；在此基础上,利用Box-Behnken设计法,以绿色魏斯菌死亡数量级为响应值,对影响超高压杀灭效果的压力、时间和乳酸链球菌素添加量三个主要因子进行了最佳水平的优化,同时利用模型的响应面及其等高线对影响超高压杀菌的关键因子及其交互作用进行探讨,建立了低温肉制品超高压灭菌的二次多项数学模型,确立了低温火腿超高压杀菌工艺。结果表明：所考察的四个因子对超高压杀菌效果均有不同程度的影响,所建立的二次多项模型经检验证明是合理可靠的,优化出了能够杀灭4个数量级绿色魏斯菌的工艺参数：压力417.57 MPa,保压时间9.89 min和乳酸链球菌素添加量1.98 mg/kg,并根据实际可操作性将参数修正为压力420 MPa,保压时间10 min和乳酸链球菌素添加量2 mg/kg,处理温度10℃。
Cooked meat products maintain more nutrition and flavour properties, with advanced procedure and wide markets, which orient the future meat industry. Due to low-temperature heating process and recontamination phenomena during slicing and packaging procedures, cooked meat products are likely to be contaminated after processing. The shorter shelf-life problem of these products limited the development of meat industry in China.
High hydrostatic pressure (HHP) processing offers an alternative preservation method for processed meat and meat products. When compared to thermal treatments, HHP causes little or no effects on nutritional or quality parameters, but micro-organisms can be inactivated at higher pressures. Now, HHP is being used commercially in a number of countries in Japan, Europe and the USA, a few pressurised food products become commercially available and it seems probable that more will be developed in the near future. However, in our country, high pressure technology still in the initial stage, a great many work need to do to follow and offer new developments. In the present work, the storage characteristic, such as microbiological, quality and sensory parameters, of HHP treated sliced vacuum-packaged cooked ham was investigated. Microbial diversity and dynamic changes during refrigerated storage after high pressure processing was also revealed. Moreover, the effect of high pressure and thermal treatments on Weissella viridescens was evaluated by flow cytometric analysis. Finally, a second order quadratic equation of HHP inactivation was built and pressure-sterilization technics of cooked ham was developed. We would like to provide a theory basis and techniques guidance for quality and safety assurance of cooked meat products during processing, storage and distribution. The WHOLE work includes five parts, the specific contents and results are as follows:
1. The storage characteristic of cooked ham after high pressure treatment
The objective of this part is to evaluate the microbiological, quality parameters and sensory properties of sliced vacuum-packaged cooked ham after high pressure treatment and determine whether HHP processing is a valid preservation method to reduce the growth of spoilage microorganisms without modification of its quality properties. Slices of cooked ham were submitted to high pressure treatments at 400 MPa or 600 MPa for 10 min at 12℃and then stored at 4℃. Counts of aerobic mesophiles, lactic acid bacteria, enterobacteria and psychrotrophs were determined. In addition, TBA, colour, FFA, FAA, TPA as well as sensory analysis were performed at the same time on the treated and non-treated (NT) slices. It was found that HHP caused a highly degree of microbiological inactivation, which increased with pressure level. High pressure treatment inactivated lactic acid bacteria at a rather low level (< 104 CFU/g) for a long period, Enterobacteriaceae were detected below the detection limit during the entire storage (102 CFU/g), and the initial competition abilities of Psychrotrophs and Enterobacteriaceae were lost after HHP. The firmness of pressurized hams was decreased a little, no other changes were found in quality and sensory parameters after HHP and during refrigerated storage. The application of HHP on meat products after final packaging is an efficient method for delaying the growth of spoilage microorganisms in sliced cooked ham. High pressure treated at 600 MPa for 10 min at 12℃will extend the shelf life of cooked ham to 10 weeks, with little or no modification of quality parameters.
2. Microbial diversity and dynamic changes of pressurized cooked ham
In this part, culture-dependent and culture-independent approaches were used to reveal the microbial diversity and dynamic changes occurring in sliced vacuum-packaged cooked ham after high pressure processing (400 MPa or 600 MPa for 10 min at 22℃) during refrigerated storage over 90 days. Direct extraction of genome DNA and total RNA from meat samples, followed by PCR-denaturing gradient gel electrophoresis (DGGE) and RT-PCR-DGGE on 16S rDNA V3 region, was performed to define the structure of the bacterial populations and active species in pressurized cooked ham. Results showed that HHP affected differently the various species detected. The predominant spoilage organisms of cooked ham, such as Lactobacillus sakei and Lactobacillus curvatus, were found to be very sensitive to pressure as they were unable to be detected in HHP samples at any time during refrigerated storage. Weissella viridescens and Leuconostoc mesenteroides survived HHP at 600 MPa for 10 min at 22℃and were responsible for the final spoilage. An RNA-based DGGE approach clearly has potential for the analysis of active species that have survived in pressurized cooked ham. High pressure processing at 400 or 600 MPa for 10 min at room temperature (22℃) has a powerful inhibitory effect on the major spoilage bacteria of sliced vacuum-packed cooked ham. High pressure treatment may lead to reduced microbial diversity and improve the products'safety.
3. Classification, identification and characterization of representative bacteria in pressurized cooked ham
In this part, a total of 84 strains isolated from MRS medium which treated at 600 MPa for 10 min at 22℃were identified by molecular methods by means of the PCR-DGGE. Strains with the same DGGE profiles were grouped and representatives of each group were amplified and sequenced. Identified representative strains then used for evaluating the culture effectiveness by PCA, MRS and TAL plates methods. Finally, the cooperate effect between high pressure and nisin content in inactivating pressure-resistant bacteria of cooked meat products was investigated. Results showed that, there are only two comigrations exhibited accounted for 84 unique bands, they represented two different species, Weissella viridescens and Leuconostoc mesenteroides. Both of the two strains grow better on TAL plates than the other two culture media. With the growing addition of nisin, numbers of the two strains were falling rapidly. When nisin content was up to 200μg/mL, these inactivate effect went to maximum. Significant reduction in bacteria numbers was found at high pressure treated at 400 MPa and 300 MPa for W. viridescens and L. mesenteroides, respectively. However, when high pressure treated with 200μg/mL nisin content, the pressure level fallen to 100 MPa both for these two bacteria. High pressure treated at 500 MPa for 5 min at 20℃, with 200μg/mL nisin content, inactivated W. viridescens counts by 9 log10 CFU/mL. We have provided evidence that there surely exist combined effect between high pressure and nisin when used for delaying bacteria grows. Nisin addition can effectively lower the pressure level used during industrial decontamination processes.
4. Comparison of inactivation pathways of high pressure and thermal inactivated Weissella viridescens by flow cytometry analysis
The effect of high pressure and thermal treatment on pressure-resistant Weissella viridescens was evaluated by flow cytometric analysis in conjunction to standard cultivation techniques. Carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) and propidium iodide (PI) were used to monitor bacterial esterase activity and membrane integrity, respectively. Single staining with these two probes rapidly and noticeably reflected the behavior of bacterial cells during pressure and heat exposure. However, the flow cytometry results tended to overestimate the viability compared to plate counts both in thermal and pressure treated samples. Exposure of W. viridescens cells to high pressure at increasing pressure level from 100 to 600 MPa resulted in a gradual shift of cells from Gate LR (cF+PI-) to Gate UR (cF+PI+), whereas practically no cells were found in Gate UL (cF-PI+). Cells exposed to 600 MPa still showed an extraordinary high level cF-accumulation capacity, but cultivation results demonstrated that the cell counts was already below the detection limit (< 1 log10 CFU/mL). The particular metabolic activity (cF-accumulation capacity) of W. viridescens cells did not correlate with their ability to reproduce and form visible colonies on agar. During heat treatment from 60℃to 100℃, the membrane integrity of W. viridescens was gradually damaged prior to esterase activity. At temperature treated at 80℃, all cells were lost their membrane integrity and esterase activity. No different fluorescence behaviors of thermal and high pressure treatment were found. These insights, which are not explicitly assessable by conventional cultivation techniques, will be important in understanding HHP inactivation during industrial decontamination processes.
5. Study of the high pressure inactivation conditions in the industrial production of cooked ham
In this part, pressure, pressure holding time, temperature and nisin content on HHP inactivation of Weissella viridescens and Leuconostoc mesenteroides were investigated by single factor method. Response surface methodology (RSM) was then employed and a second order quadratic equation for high hydrostatic pressure (HHP) inactivation was built. The adequacy of the model equation for predicting the optimum response values was verified effectively by the validation data. By analyzing the response surface plots and their corresponding contour plots as well as solving the quadratic equation, the optimum process parameters for inactivation of Weissella viridescens of four log cycles were obtained as: pressure 420 MPa, pressure holding time 10 min and nisin content 2 mg/kg (processing medium temperature under 20℃).
1.超高压处理后低温火腿贮藏特性研究
参考国外研究成果,选择了400 MPa和600 MPa的压力条件,室温条件下(12℃)对低温火腿进行10 min超高压处理,在4℃贮藏条件下定期评定样品的微生物、理化和感官品质变化,确定超高压对低温肉制品腐败菌的抑制效应,评定其对肉制品品质指标的影响效果。以期在延长产品货架期的同时有效保证产品的食用品质和营养价值。结果表明：400 MPa和600 MPa的超高压处理均能够有效抑制低温火腿中的腐败微生物,使乳酸菌数处于相当长的低水平期,很大程度上缓解了由乳酸菌繁殖造成的低温火腿产气、发黏、涨袋等腐败现象,对肉制品中常见污染的肠杆菌和耐冷菌显示了很强的抑制效果。初始污染菌数相对较低时,600MPa/12℃/10 min的超高压处理,4℃贮藏条件下能够将产品货架期延长至10周,对低温火腿的脂肪氧化、颜色、质构特性等理化指标和感官指标影响较小,不会或较小破坏低温火腿脂肪水解、蛋白质水解等与营养价值相关的各项指标。
2.超高压对低温火腿微生物种群结构及动态变化的影响研究
参考相关研究,采用400 MPa和600 MPa的压力在室温(22℃)条件下对低温火腿进行10 min超高压处理,于4℃贮藏0(高压前),1(高压后),30,90 d,通过直接提取样品微生物种群的基因组DNA和总RNA,分别进行PCR-和RT-PCR-DGCE (DGGE:变性梯度凝胶电泳)指纹图谱分析,揭示超高压对低温火腿中主要腐败微生物的抑制效应,进一步深入了解超高压处理后存活微生物的消长规律,为超高压技术在低温肉制品杀菌中的应用提供更全面的数据支撑。结果表明：低温火腿中污染的微生物种群主要由乳杆菌属、乳球菌属、明串珠菌属、魏斯菌属和链球菌属五个属中的细菌组成；RT-PCR-DGGE技术能够有效揭示超高压处理后各微生物种群的存活状况；400 MPa和600 MPa的超高压处理能够有效抑制低温火腿中的清酒乳杆菌(Lactobacillus sakei).弯曲乳杆菌(Lactobacillus curvatus)等优势腐败微生物的生长繁殖；600 MPa的压力处理后,绝大多数的微生物种群未检出生长,低温火腿微生物种群结构变得非常单一,仅有绿色魏斯菌(Weissella viridescens)和肠膜明串珠菌(Leuconostoc mesenteroides)存活；超高压处理在降低微生物多样性的同时进一步提高了低温火腿的食用安全性。
3.超高压耐受菌的分类鉴定及耐压特性研究
运用变性梯度凝胶电泳技术对分离自600 MPa超高压处理组样品中的84株超高压耐受菌进行分类,并通过16S rDNA序列分析技术以及细菌生理生化试验进行鉴定；以分离到的超高压耐受菌为供试对象,比较研究了超高压处理后耐压菌在平板计数琼脂、MRS琼脂以及二者组合制备的薄层平板培养琼脂上的生长繁殖状况；在此基础上,以乳酸链球菌素和超高压作为栅栏因子,研究了二者协同处理对超高压耐受菌存活率的影响效果,为评价超高压耐受菌的耐压特性,寻找有效的控制措施进行了初步尝试。结果表明：84株待分类的超高压耐受菌在DGGE图谱中仅表现出两个位置不同的迁移条带,经鉴定后这两株超高压耐受菌为绿色魏斯菌(Weissella viridescens)和肠膜明串珠菌(Leuconostoc mesenteroides);采用薄层平板培养方法作为评价手段,揭示出超高压与乳酸链球菌素结合处理对这两株超高压耐受菌具有显著的协同抑制效果,200μg/mL的乳酸链球菌素添加量结合500MPa/5min/20℃的超高压处理条件,能够彻底抑制初始菌数为109 CFU/mL的绿色魏斯菌的生长繁殖。乳酸链球菌素的添加能够有效降低工业化生产中高压力的使用。
4.超高压与热处理抑菌机制的比较性研究
运用扫描/透射电镜观察和流式细胞多参数分析技术比较研究了超高压处理和热处理后绿色魏斯菌生理生化状态的变化,揭示出两种不同的抑菌手段对绿色魏斯菌细胞膜完整性和酯酶活性的破坏情况,为更好的理解超高压抑菌机制提供理论补充。结果表明：超高压处理对绿色魏斯菌的菌体形态未发现显著影响,600 MPa处理时细胞胞质成分出现暗色区域及部分气泡性结构；在压力由100 MPa升高至600 MPa的过程中,细胞膜完整性受到破坏的绿色魏斯菌数量显著增加,然而整个过程中绿色魏斯菌的酯酶活性却未见显著变化。热处理能够引起绿色魏斯菌菌体形态发生严重变形,在温度由60℃升高到100℃的过程中,菌体形变明显加剧,细胞内成分发生变性凝集,出现大面积白色区域。随热处理温度的升高,绿色魏斯菌细胞膜完整性及酯酶活性均显著丧失。超高压处理与热处理相同,二者都能够破坏绿色魏斯菌的细胞膜完整性,通过引起胞体蛋白质发生变性影响其生物化学反应,最终达到抑制微生物的目的。
5.低温火腿超高压杀菌工艺研究
参考国内外研究成果,选择压力、时间、温度以及乳酸链球菌素添加量四个因素作为考察因子,以低温火腿为基质,评定了四因子对超高压杀灭绿色魏斯菌和肠膜明串珠菌的影响效果；在此基础上,利用Box-Behnken设计法,以绿色魏斯菌死亡数量级为响应值,对影响超高压杀灭效果的压力、时间和乳酸链球菌素添加量三个主要因子进行了最佳水平的优化,同时利用模型的响应面及其等高线对影响超高压杀菌的关键因子及其交互作用进行探讨,建立了低温肉制品超高压灭菌的二次多项数学模型,确立了低温火腿超高压杀菌工艺。结果表明：所考察的四个因子对超高压杀菌效果均有不同程度的影响,所建立的二次多项模型经检验证明是合理可靠的,优化出了能够杀灭4个数量级绿色魏斯菌的工艺参数：压力417.57 MPa,保压时间9.89 min和乳酸链球菌素添加量1.98 mg/kg,并根据实际可操作性将参数修正为压力420 MPa,保压时间10 min和乳酸链球菌素添加量2 mg/kg,处理温度10℃。
Cooked meat products maintain more nutrition and flavour properties, with advanced procedure and wide markets, which orient the future meat industry. Due to low-temperature heating process and recontamination phenomena during slicing and packaging procedures, cooked meat products are likely to be contaminated after processing. The shorter shelf-life problem of these products limited the development of meat industry in China.
High hydrostatic pressure (HHP) processing offers an alternative preservation method for processed meat and meat products. When compared to thermal treatments, HHP causes little or no effects on nutritional or quality parameters, but micro-organisms can be inactivated at higher pressures. Now, HHP is being used commercially in a number of countries in Japan, Europe and the USA, a few pressurised food products become commercially available and it seems probable that more will be developed in the near future. However, in our country, high pressure technology still in the initial stage, a great many work need to do to follow and offer new developments. In the present work, the storage characteristic, such as microbiological, quality and sensory parameters, of HHP treated sliced vacuum-packaged cooked ham was investigated. Microbial diversity and dynamic changes during refrigerated storage after high pressure processing was also revealed. Moreover, the effect of high pressure and thermal treatments on Weissella viridescens was evaluated by flow cytometric analysis. Finally, a second order quadratic equation of HHP inactivation was built and pressure-sterilization technics of cooked ham was developed. We would like to provide a theory basis and techniques guidance for quality and safety assurance of cooked meat products during processing, storage and distribution. The WHOLE work includes five parts, the specific contents and results are as follows:
1. The storage characteristic of cooked ham after high pressure treatment
The objective of this part is to evaluate the microbiological, quality parameters and sensory properties of sliced vacuum-packaged cooked ham after high pressure treatment and determine whether HHP processing is a valid preservation method to reduce the growth of spoilage microorganisms without modification of its quality properties. Slices of cooked ham were submitted to high pressure treatments at 400 MPa or 600 MPa for 10 min at 12℃and then stored at 4℃. Counts of aerobic mesophiles, lactic acid bacteria, enterobacteria and psychrotrophs were determined. In addition, TBA, colour, FFA, FAA, TPA as well as sensory analysis were performed at the same time on the treated and non-treated (NT) slices. It was found that HHP caused a highly degree of microbiological inactivation, which increased with pressure level. High pressure treatment inactivated lactic acid bacteria at a rather low level (< 104 CFU/g) for a long period, Enterobacteriaceae were detected below the detection limit during the entire storage (102 CFU/g), and the initial competition abilities of Psychrotrophs and Enterobacteriaceae were lost after HHP. The firmness of pressurized hams was decreased a little, no other changes were found in quality and sensory parameters after HHP and during refrigerated storage. The application of HHP on meat products after final packaging is an efficient method for delaying the growth of spoilage microorganisms in sliced cooked ham. High pressure treated at 600 MPa for 10 min at 12℃will extend the shelf life of cooked ham to 10 weeks, with little or no modification of quality parameters.
2. Microbial diversity and dynamic changes of pressurized cooked ham
In this part, culture-dependent and culture-independent approaches were used to reveal the microbial diversity and dynamic changes occurring in sliced vacuum-packaged cooked ham after high pressure processing (400 MPa or 600 MPa for 10 min at 22℃) during refrigerated storage over 90 days. Direct extraction of genome DNA and total RNA from meat samples, followed by PCR-denaturing gradient gel electrophoresis (DGGE) and RT-PCR-DGGE on 16S rDNA V3 region, was performed to define the structure of the bacterial populations and active species in pressurized cooked ham. Results showed that HHP affected differently the various species detected. The predominant spoilage organisms of cooked ham, such as Lactobacillus sakei and Lactobacillus curvatus, were found to be very sensitive to pressure as they were unable to be detected in HHP samples at any time during refrigerated storage. Weissella viridescens and Leuconostoc mesenteroides survived HHP at 600 MPa for 10 min at 22℃and were responsible for the final spoilage. An RNA-based DGGE approach clearly has potential for the analysis of active species that have survived in pressurized cooked ham. High pressure processing at 400 or 600 MPa for 10 min at room temperature (22℃) has a powerful inhibitory effect on the major spoilage bacteria of sliced vacuum-packed cooked ham. High pressure treatment may lead to reduced microbial diversity and improve the products'safety.
3. Classification, identification and characterization of representative bacteria in pressurized cooked ham
In this part, a total of 84 strains isolated from MRS medium which treated at 600 MPa for 10 min at 22℃were identified by molecular methods by means of the PCR-DGGE. Strains with the same DGGE profiles were grouped and representatives of each group were amplified and sequenced. Identified representative strains then used for evaluating the culture effectiveness by PCA, MRS and TAL plates methods. Finally, the cooperate effect between high pressure and nisin content in inactivating pressure-resistant bacteria of cooked meat products was investigated. Results showed that, there are only two comigrations exhibited accounted for 84 unique bands, they represented two different species, Weissella viridescens and Leuconostoc mesenteroides. Both of the two strains grow better on TAL plates than the other two culture media. With the growing addition of nisin, numbers of the two strains were falling rapidly. When nisin content was up to 200μg/mL, these inactivate effect went to maximum. Significant reduction in bacteria numbers was found at high pressure treated at 400 MPa and 300 MPa for W. viridescens and L. mesenteroides, respectively. However, when high pressure treated with 200μg/mL nisin content, the pressure level fallen to 100 MPa both for these two bacteria. High pressure treated at 500 MPa for 5 min at 20℃, with 200μg/mL nisin content, inactivated W. viridescens counts by 9 log10 CFU/mL. We have provided evidence that there surely exist combined effect between high pressure and nisin when used for delaying bacteria grows. Nisin addition can effectively lower the pressure level used during industrial decontamination processes.
4. Comparison of inactivation pathways of high pressure and thermal inactivated Weissella viridescens by flow cytometry analysis
The effect of high pressure and thermal treatment on pressure-resistant Weissella viridescens was evaluated by flow cytometric analysis in conjunction to standard cultivation techniques. Carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) and propidium iodide (PI) were used to monitor bacterial esterase activity and membrane integrity, respectively. Single staining with these two probes rapidly and noticeably reflected the behavior of bacterial cells during pressure and heat exposure. However, the flow cytometry results tended to overestimate the viability compared to plate counts both in thermal and pressure treated samples. Exposure of W. viridescens cells to high pressure at increasing pressure level from 100 to 600 MPa resulted in a gradual shift of cells from Gate LR (cF+PI-) to Gate UR (cF+PI+), whereas practically no cells were found in Gate UL (cF-PI+). Cells exposed to 600 MPa still showed an extraordinary high level cF-accumulation capacity, but cultivation results demonstrated that the cell counts was already below the detection limit (< 1 log10 CFU/mL). The particular metabolic activity (cF-accumulation capacity) of W. viridescens cells did not correlate with their ability to reproduce and form visible colonies on agar. During heat treatment from 60℃to 100℃, the membrane integrity of W. viridescens was gradually damaged prior to esterase activity. At temperature treated at 80℃, all cells were lost their membrane integrity and esterase activity. No different fluorescence behaviors of thermal and high pressure treatment were found. These insights, which are not explicitly assessable by conventional cultivation techniques, will be important in understanding HHP inactivation during industrial decontamination processes.
5. Study of the high pressure inactivation conditions in the industrial production of cooked ham
In this part, pressure, pressure holding time, temperature and nisin content on HHP inactivation of Weissella viridescens and Leuconostoc mesenteroides were investigated by single factor method. Response surface methodology (RSM) was then employed and a second order quadratic equation for high hydrostatic pressure (HHP) inactivation was built. The adequacy of the model equation for predicting the optimum response values was verified effectively by the validation data. By analyzing the response surface plots and their corresponding contour plots as well as solving the quadratic equation, the optimum process parameters for inactivation of Weissella viridescens of four log cycles were obtained as: pressure 420 MPa, pressure holding time 10 min and nisin content 2 mg/kg (processing medium temperature under 20℃).
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