Application Of High Pressure Treatment To Improve The Preservation Of Cooked Ham

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 treatmentThe 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 hamIn 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 hamIn 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 analysisThe 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 hamIn 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℃).