INACTIVATION KINETICS AND REDUCTION OF BACILLUS COAGULANS SPORE BY THE COMBINATION OF HIGH PRESSURE AND MODERATE HEAT

The combination effect of high pressure (400, 500 and 600 MPa) and moderate heat (70 and 80C) on the inactivation kinetics and reduction of Bacillus coagulans spore in phosphate buffer and ultra-high temperature (UHT) whole milk was investigated. The pressure come-up time and corresponding logarithmic reduction of spore inactivation were considered during pressure-thermal treatment. B. coagulans spore had a much higher resistance to pressure in UHT whole milk than in phosphate buffer. Survival data were modeled using the linear, Weibull and log-logistic models to obtain relevant kinetic parameters. The tailing phenomenon occurred in all survival curves, indicating the linear model was not adequate for describing these curves. The mean square error and regression coefficient suggested that the log-logistic model produced best fits to all survival curves, followed by the Weibull model.

PRACTICAL APPLICATIONS

It becomes increasingly apparent that high-pressure treatment combined with moderate heat treatment for low acid and acid products is often required for effective bacterial spores' inactivation. Consequently, the prediction model of microbial survival curves is essential. Bacillus coagulans is a slightly pressure-resistant and relatively heat-resistant spoilage bacterium of considerable concern during the processing of acid foods. Spore inactivation effect during the pressure come-up time is sometimes considerable and should not beignored. The use of mathematical models to predict inactivation for spores could help the food industry further to develop optimum process conditions.     

高压二氧化碳杀菌机理研究进展

高压二氧化碳（HPCD）技术作为一种新型非热杀菌技术,越来越受到研究者的关注,但目前对其杀菌机理尚未明确。本文分析了目前国内外有关HPCD杀菌机理的研究进展,并分析了HPCD对微生物细胞内几个作用靶点包括细胞壁、细胞膜、细胞质、蛋白质、酶和核质的影响,以期为进一步研究和应用提供理论依据。     

响应曲面法建立乳酸菌高压二氧化碳杀菌模型

采用三因素三水平的Box-Behnken设计,利用响应曲面法(RSM)对影响高压二氧化碳(HPCD)杀菌效果的主要因素(温度、压力和时间)进行研究,以杀灭乳酸菌的对数值为响应函数,建立了猪肉糜中乳酸菌的HPCD杀菌模型.对模型进行变异分析、决定系数分析、t值检验、共线性分析和显著性因子对杀菌效果影响的分析结果表明,采用二阶效应模式构建的模型显著性好、准确度高;显著因子时间、压力的二次方、温度-压力交互项、压力、温度对模型的重要性依次递增且其独立性可靠;温度、压力和时间均能增强HPCD对乳酸菌的杀菌效果.     

A comparative study of inactivation of peach polyphenol oxidase and carrot polyphenol oxidase induced by high‐pressure carbon dioxide

The inactivation of polyphenol oxidase (PPO) in peach juice and PPO in carrot juice was investigated by high‐pressure carbon dioxide (HPCD), and their inactivation kinetics was analysed and compared. The temperature was 35–55 °C, the pressure was 5–15 MPa under HPCD condition. Results showed that HPCD enhanced the inactivation effect of the temperature on the two PPOs. The inactivation kinetics of peach PPO was well fitted to a first‐order kinetic model, of carrot PPO to a fraction‐conversion model as a function of temperatures or pressures. Susceptibility of the rate constant k of peach PPO was not altered and of carrot PPO was lessened to the temperature, but the susceptibility of the rate constant k of peach PPO and carrot PPO to the pressure was not changed when the pressure was >8 or 12 MPa, indicating the presence of a threshold pressure.     

Inactivation of natural microorganisms in litchi juice by high-pressure carbon dioxide combined with mild heat and nisin

The individual and combined effects of high-pressure carbon dioxide (HPCD), mild heat (MH) and nisin (200 ppm) on the inactivation of natural microorganisms, including aerobic bacteria (AB), yeasts and molds (Y&;M), in litchi juice were evaluated. The samples were treated at a pressure of 10 MPa and temperatures of 32, 42 or 52 °C for 5, 10, 15, 20, 25 or 30 min. Temperature played a prominent role in the inactivation of both AB and Y&;M when combined with HPCD, particularly for AB at 52 °C and Y&;M at temperatures ≥42 °C. Nisin increased the susceptibility of AB to the combined treatment of HPCD and MH (HPCD + MH). A reduction of 4.19 log cycles was achieved by HPCD + MH at 52 °C for 15 min, and complete inactivation of AB was obtained by combination of HPCD, MH and nisin (HPCD + MH + nisin). No significant effect of nisin was found on the inactivation of Y&;M.     

Synergetic effects of high-pressure carbon dioxide and nisin on the inactivation of Escherichia coli and Staphylococcus aureus

Synergetic effects of high-pressure carbon dioxide (HPCD) and nisin on Escherichia coli and Staphylococcus aureus were evaluated. Changes in morphology, interior structure, and membrane permeability were analyzed by scanning and transmission electron microscopy, and flow cytometry. Synergetic effects were found, especially in S. aureus. HPCD alone or with nisin led to morphological and intracellular alterations in both bacteria, but nisin alone led to these damages only in S. aureus. A positive correlation between membrane damage and inactivation was found, but ratios of inactivation were higher, probably because of viable but non-culturable state. Mechanisms were proposed for synergism: for E. coli, outer membrane was damaged first by HPCD, and then HPCD and nisin jointly acted on and destroyed the cytoplasmic membrane, leading to further intracellular damage by HPCD; for S. aureus, HPCD and nisin acted on the cytoplasmic membrane together leading to cell death.Industrial RelevanceEscherichia coli and Staphylococcus aureus are two common microorganisms, which exist widely in the environment and easily contaminate food such as vegetables and dairy products, respectively. Considering heat treatment may destroy some heat-sensitive quality of the products, this study evaluated synergetic effects of high-pressure carbon dioxide (HPCD) combined with the bacteriocin nisin. The investigations provided evidence for potentially combined application of HPCD and nisin to help keep food safe in the industry.     

高压二氧化碳在肉品杀菌保鲜中的应用研究进展

肉及肉制品营养价值高,是人类优质蛋白质的重要来源。但是肉品在加工和流通等过程中容易受到各种来源的污染,导致肉质腐败,甚至诱发食源性疾病。高压二氧化碳（High pressure carbon dioxide, HPCD）是一种新型非热加工技术,可在相对温和的压力和温度条件下达到显著的杀菌效果。本文总结了HPCD的杀菌机制,综述了近年来国内外HPCD技术在肉品杀菌保鲜中的应用研究进展,并阐述了单一HPCD技术及其与其它方法联合处理对肉品中微生物的杀灭效果、肉品理化品质、质构特性和微生物稳定性的影响,针对当前存在的问题,对HPCD技术的发展前景进行了展望。     

Inactivation of microorganisms naturally present in raw bovine milk by high‐pressure carbon dioxide

This study assessed the inactivation of microorganisms naturally present in raw bovine milk by high‐pressure carbon dioxide (HPCD) at 10–30 MPa and 20–50 °C for 20–70 min. The log reduction of microorganisms increased as raw bovine milk was exposed to higher pressures and temperatures and longer treatment times. The maximum reduction of aerobic bacteria (AB) was 4.96‐log at 25 MPa and 50 °C for 70 min. At lower temperatures and treatment times, a complete inactivation of yeasts and moulds (Y&M) and coliform bacteria (CB) was obtained at 25 MPa. Changes in microorganisms naturally present in raw bovine milk during storage were also assessed. There were 1.83‐log survival of AB, 0.65‐log survival of Y&M and a complete inactivation of CB in raw bovine milk when subjected to HPCD at 25 MPa and 40 °C for 50 min. Moreover, the AB, Y&M and the CB in raw bovine milk exhibited insignificant alterations during storage at 4 °C for 15 days, indicating a potential capability of HPCD to extend the shelf life of milk.     

Effect of dissolved carbon dioxide on thermal inactivation of microorganisms in milk

Postpasteurization addition of CO2 inhibits growth of certain microorganisms in dairy products, but few workers have investigated the effect of CO2 on the thermal inactivation of microorganisms during pasteurization. Concentrations of CO2 ranging from 44 to 58 mM added to raw whole milk significantly (P < 0.05) reduced the number of surviving standard plate count (SPC) organisms in milk heated over the range of 67 to 93 degrees C. A decrease in thermal survival rates (D-values) for Pseudomonas fluorescens R1-232 and Bacillus cereus ATCC 14579 spores in milk was positively correlated with CO2 concentrations (1 to 36 mM). D(50 degrees C)-values for P. fluorescens significantly decreased (P < 0.05) in a linear fashion from 14.4 to 7.2 min. D(89 degrees C)-values for B. cereus spores were significantly (P < 0.05) decreased from 5.56 min in control milk to 5.29 min in milk containing 33 mM CO2. The Weibull function was used as a model to describe the thermal inactivation of P. fluorescens, B. cereus spores, and SPC organisms in raw milk. Nonlinear parameters for the Weibull function were estimated, and survival data fitted to this model had higher R2 values than when fitted to the linear model, further providing support that the thermal inactivation of bacteria does not always follow first-order reaction rate kinetics. These results suggest that CO2 could be used as a processing aid to enhance microbial inactivation during pasteurization.     

预测微生物学在高压二氧化碳杀菌动力学的应用进展

高压二氧化碳(HPCD)技术是目前越来越受关注的非热杀菌技术之一。目前有关HPCD杀菌预测模型的研究报道较少。利用食品预测微生物学方法和理论,通过研究各个因素对HPCD杀菌过程的影响,建立预测模型和回归方程,可以更好的理解HPCD杀菌动力学及机理,提高其杀菌效果,确保食品安全。本文就食品预测微生物学在HPCD杀菌动力学研究中的应用进行综述分析,并提出未来可能的研究方向。     

High pressure carbon dioxide inactivation of microorganisms in foods: the past, the present and the future

Thermal pasteurization is a well known and old technique for reducing the microbial count of foods. Traditional thermal processing, however, can destroy heat-sensitive nutrients and food product qualities such as flavor, color and texture. For more than 2 decades now, the use of high-pressure carbon dioxide (HPCD) has been proposed as an alternative cold pasteurization technique for foods. This method presents some fundamental advantages related to the mild conditions employed, particularly because it allows processing at much lower temperature than the ones used in thermal pasteurization. In spite of intensified research efforts the last couple of years, the HPCD preservation technique has not yet been implemented on a large scale by the food industry until now. This review presents a survey of published knowledge concerning the HPCD technique for microbial inactivation, and addresses issues of the technology such as the mechanism of carbon dioxide bactericidal action, the potential for inactivating vegetative cells and bacterial spores, and the regulatory hurdles which need to be overcome. In addition, the review also reflects on the opportunities and especially the current drawbacks of the HPCD technique for the food industry.     

Enzyme Inactivation in Food Processing using High Pressure Carbon Dioxide Technology

High pressure carbon dioxide (HPCD) is an effective non-thermal processing technique for inactivating deleterious enzymes in liquid and solid food systems. This processing method avoids high temperatures and exerts a minimal impact on the nutritional and sensory properties of foods, but extends shelf life by inhibiting or killing microorganisms and enzymes. Indigenous enzymes in food such as polyphenol oxidase (PPO), pectin methylesterase (PME), and lypoxygenase (LOX) may cause undesirable chemical changes in food attributes, showing the loss in color, texture, and flavor. For more than two decades, HPCD has proved its effectiveness in inactivating these enzymes. The HPCD-induced inactivation of some microbial enzymes responsible for microbial metabolism is also included. This review presents a survey of the published knowledge regarding the use of HPCD for the inactivation of these enzymes, and analyzes the factors controlling the efficiency of HPCD and speculates on the underlying mechanism that leads to enzyme inactivation.     

Membrane permeabilization and cellular death of Escherichia coli, Listeria monocytogenes and Saccharomyces cerevisiae as induced by high pressure carbon dioxide treatment

In this study, the relationship between (irreversible) membrane permeabilization and loss of viability in Escherichia coli, Listeria monocytogenes and Saccharomyces cerevisiae cells subjected to high pressure carbon dioxide (HPCD) treatment at different process conditions including temperature (35–45 °C), pressure (10.5–21.0 MPa) and treatment time (0–60 min) was examined. Loss of membrane integrity was measured as increased uptake of the fluorescent dye propidium iodide (PI) with spectrofluorometry, while cell inactivation was determined by viable cell count. Uptake of PI by all three strains indicated that membrane damage is involved in the mechanism of HPCD inactivation of vegetative cells. The extent of membrane permeabilization and cellular death increased with the severity of the HPCD treatment. The resistance of the three tested organisms to HPCD treatment changed as a function of treatment time, leading to significant tailing in the survival curves, and was dependent on pressure and temperature. The results in this study also indicated a HPCD-induced damage on nucleic acids during cell inactivation. Transmission electron microscopy showed that HPCD treatment had a profound effect on the intracellular organization of the micro-organisms and influenced the permeability of the bacterial cells by introducing pores in the cell wall.     

The contribution of high pressure carbon dioxide in the inactivation kinetics and structural alteration of myrosinase

High pressure carbon dioxide (HPCD) has been verified to be an efficient way of inactivating enzyme activity. This work investigates the influence of temperature (T), pressure (P), exposure times (t) on the activity of commercial myrosinase (MYR) submitted to HPCD. Results showed that only 1.00% of MYR activity retained at 22 MPa and 65 °C for 5 min. Moreover, the first‐order reaction kinetic data of MYR inactivation as influenced by pressure of HPCD were analysed. With the pressure rising from 8 to 22 MPa at 55 °C, the inactivation rate constant (k) increased from 0.015 to 0.024 min^?1, while the decimal reduction time (D) decreased from 157.2 to 96.1 min. Additionally, a series of exploratory experiments were conducted to investigate the contribution of the HPCD parameters (T, P and CO₂ dissolution), with analysing circular dichroism spectroscopy and tryptophan fluorescence spectra, illustrate that CO₂ dissolution plays a dominant role in MYR inactivation and structural alteration.     

高压CO2杀菌技术对猪肉糜中枯草芽孢的作用

通过对影响高压CO2杀菌技术(HPCD)的主要因素温度、压力、保压时间进行分析,利用正交试验,以杀灭猪肉糜中枯草芽孢的致死率为指标,对猪肉糜中枯草芽孢的杀菌条件进行优化,并优选出辅助试剂来协同HPCD进行灭菌,在此基础上,推测HPCD致死枯草芽孢的机理.结果表明:HPCD能有效杀灭枯草芽孢,3个因素对杀菌效果的影响程度为压力>保压时间>温度,最优参数组合为30MPa、40℃、保压60min,致死率可以达到3.39.添加辅助试剂的最佳杀菌方式为:既添加芽孢的萌发剂又添加天然抑菌剂,致死率可达5.16.单独添加萌发剂的效果要好于单独添加天然抑菌剂的,故猜测HPCD致死芽孢的机理为先促使芽孢萌发而后将其营养体杀死.     

高压二氧化碳技术速冻香菇工艺

采用高压二氧化碳技术(high pressure carbon dioxide,HPCD)对香菇进行速冻,以期解决国内食用菌速冻技术存在的能耗大、成本高的问题。在原料热烫钝酶的基础上,通过单因素试验和正交试验,并结合速冻产品感官评价对工艺条件进行优化。结果表明:HPCD速冻香菇最佳工艺参数为处理釜初温6℃、处理釜设定压力7MPa、卸压时间4min,且卸压时间为影响速冻产品品质的最显著因素。     

Temperature and treatment time influence high hydrostatic pressure inactivation of feline calicivirus, a norovirus surrogate

Interest in high hydrostatic pressure processing as a nonthermal pasteurization process for foods continues to increase. Feline calicivirus (FCV), a propagable virus that is genetically related to the nonpropagable human noroviruses, was used for detailed evaluation of the high pressure processing parameters necessary for virus inactivation. Pressure inactivation curves of FCV strain KCD in Dulbecco's modified Eagle medium with 10% fetal bovine serum were obtained at 200 and 250 MPa as a function of time at room temperature. Pressure inactivation curves at 200 and 250 MPa also were determined as a function of temperature ranging from --10 to 50 degrees C at treatment times of 4 and 2 min, respectively. Tailing was observed for inactivation as a function of treatment time, indicating that the linear model was not adequate for describing these curves. The two nonlinear models, the log logistic and Weibull functions, consistently produced better fit to inactivation curves than did the linear model. The mean square errors were 0.381 for the log logistic model, 0.425 for the Weibull model, and 1.546 for the linear model. For inactivation as a function of temperature, FCV was most resistant to pressure at 20 degrees C. Temperatures above and below 20 degrees C significantly increased pressure inactivation of FCV. A 4-min treatment of 200 MPa at --10 and 50 degrees C reduced the titer of FCV by 5.0 and 4.0 log units, respectively; whereas at 20 degrees C the same treatment only reduced the titer by 0.3 log units. These novel results point to the potential for using temperatures above and particularly below room temperature to lower the pressure needed to cause the desired level of virus inactivation.     

Effects on microbial inactivation and quality attributes in frozen lychee juice treated by supercritical carbon dioxide

In this paper, we described the use of high-pressure carbon dioxide (HPCD) for the inactivation of natural microbes in lychee juice and evaluated its effects on lychee juice quality, compared to a conventional high-temperature, short-time (HTST) method. The HPCD treatments were carried out using a HPCD unit (8 MPa, 36 °C, 2 min), and the HTST was performed at 90 °C for 60 s. The results showed that five log reduction for yeasts and molds and total aerobic microorganisms occurred at 8 MPa for 2 min. And effects of the treatments on pH and concentrations of microbes, organic acids, titratable acidity (TA), total soluble solid (TSS), sugars, polyphenols, color, and free amino acids were also investigated. HPCD could efficiently maintain the concentration of polyphenols and original color at 8 MPa, 36 °C for 2 min. Insignificant differences in colors were observed between unprocessed and HPCD juices, while significant differences were observed between unprocessed and HTST juices. Furthermore, HTST decreased the total free amino acids, whereas HPCD caused a significant increase (increased by 45.92% at 8 MPa) (p < 0.05). The increase in total amino acids induced by HPCD treatment is beneficial for nutritional value of commercial ready-to-drink lychee juice. In general, HPCD treatment had less influence on the measured quality parameters of lychee juice than HTST treatment. Therefore, HPCD treatment could be a useful alternative to traditional heat treatment.     

Use of the Weibull model for lactococcal bacteriophage inactivation by high hydrostatic pressure

Four lactococcal bacteriophages (phiLl6-2, phiLl35-6, phiLd66-36 and phiLd67-42) in M17 broth were pressurized at 300 and 350 MPa at room temperature and their survival curves were determined at various time intervals. Tailing (monotonic upward concavity) was observed in all survival curves. The resulting non-linear semi-logarithmic survival curves were described by the Weibull model and goodness of fit of this model was investigated. Regression coefficients (R2), root mean square error (RMSE), residual and correlation plots strongly suggested that Weibull model produced a better fit to the data than the traditional linear model. Hazard plots suggested that the Weibull model was fully appropriate for the data being analyzed. These results have confirmed that the Weibull model, which is mostly utilized to describe the inactivation of bacterial cells or spores by heat and pressure, could be successfully used in describing the lactococcal bacteriophage inactivation by high hydrostatic pressure.