Inactivation of Saccharomyces cerevisiae and Bacillus cereus by pulsed electric fields technology

The effect of pulsed electric field (PEF) treatment, applied in a continuous system, on Saccharomyces cerevisiae and Bacillus cereus cells and spores was investigated. S. cerevisiae inoculated into sterilised apple juice and B. cereus cells and spores inoculated into sterilised 0.15% NaCl were treated with electric field strengths of 10–28 kV/cm using an 8.3 pulse number and with pulse numbers of 4.2–10.4 at 20 kV/cm, respectively. The inactivation of S. cerevisiae depended on the electric field intensity and number of pulses. The yeast inactivation increased when the applied electric field intensity and pulse number were increased. Approximately four log cycles reduction was achieved in apple juice using 10.4 pulses at 20 kV/cm. B. cereus cells were less sensitive to PEF treatment. The reduction in microbial count of B. cereus cells was hardly more than one log cycle using 10.4 pulses at 20 kV/cm. The applied PEF treatment was ineffective on Bacillus cereus spores.     

Inactivation of tomato juice peroxidase by high-intensity pulsed electric fields as affected by process conditions

A response surface was used to establish the high-intensity pulsed electric fields (HIPEF) conditions in processing tomato juice to obtain the greatest peroxidase (POD) inactivation. Keeping constant the electric field strength at 35 kV/cm and the temperature below 35 °C, the treatments were set at pulse frequency from 50 to 250 Hz, pulse width from 1 to 7 μs and treatment time from 1000 to 2000 μs, using monopolar or bipolar mode. The effect of these parameters on POD inactivation was evaluated through a second order model that adequately fitted the experimental data (p = 0.0001), with a determination coefficient (R²) of 0.85. HIPEF treatment resulted to be more effective in bipolar than monopolar mode to reduce POD activity and the longer the treatment time, the greater the reduction on the enzyme activity. A pulse frequency of 200 Hz was enough to reach a minimum value of residual POD activity. The significant interaction term pulse frequency and treatment time was included in the model, showing that different combinations of both variables can lead to the same level of residual POD activity. The effect of pulse width was enhanced by using a bipolar mode, being feasible to maximize POD inactivation selecting pulse width higher than 5.5 μs in bipolar mode.     

Inactivation of Saccharomyces cerevisiae and Lactobacillus plantarum in orange juice using ultra high-pressure homogenisation

Yeasts and lactic acid bacteria are the usual contaminants in orange juice and responsible for decreasing the shelf life of the product. Ultra high-pressure homogenisation has been shown to be an alternative to the traditional thermal pasteurisation of pumpable foods. The product was pumped through a homogeniser valve at 100 MPa, 150 MPa, 200 MPa, 250 MPa and 300 MPa using two synchronized overlapped intensifiers at a flow rate of approximately 270 mL/min. The inlet temperature was kept at 10 °C, pH at 4.1 and soluble solids at 10.0 °Bx. After processing, the product was immediately cooled to 4 °C and the microbiological count was determined. The study showed that Lactobacillus plantarum and Saccharomyces cerevisiae are sensible to ultra high-pressure homogenisation treatment. The results indicated that pressures higher than 250 MPa were able to completely destroy initial loads of 1.2 × 107 UFC/mL of L. plantarum and 2.9 × 105 UFC/mL of S. cerevisiae in orange juice, making this technology a promising way to nonthermally process orange juices.Industrial relevanceThis paper deals with inactivation of microorganism contaminants of orange juice using dynamic ultra high process technology. It is of industrial interest and relevance to evaluate the use of this non-thermal emerging technology to process fluid foods that may result into better taste, optimum product functionality, safety and quality characteristics.     

高压脉冲电场对酿酒酵母杀菌效果影响和模型分析研究

研究了高压脉冲电场(pulsed electric field,PEF)对接种于胡萝卜汁中酿酒酵母(Saccharomyces cerevisiae,CGMCC2.604)的杀菌效果并进行了模型分析。结果表明,随电场强度和脉冲时间增加,PEF对酿酒酵母的杀灭效果增强,25kV/cm、2173μs时酿酒酵母降低了4.5个对数。Weibull模型和Log-Logistic模型均能很好的拟合PEF处理酿酒酵母的失活曲线,模型评价参数(Af,Bf,SS,RMSE和R2)分析表明,Log-Logistic模型比Weibull模型能更好地拟合PEF处理下酿酒酵母失活动力学变化。     

Inactivation of Saccharomyces cerevisiae with radio frequency electric fields

The application of radio frequency (RF) electric fields as a nonthermal alternative to thermal inactivation of microorganisms in liquids was investigated. A novel RF system producing frequencies in the range of 20 to 60 kHz was developed. Electric field strengths of 20 and 30 kV/cm were applied to suspensions of Saccharomyces cerevisiae in water over a temperature range of 35 to 55 degrees C. The flow rate was 1.2 liters/min. The S. cerevisiae population was reduced by 2.1 +/- 0.1 log units following exposure to a 30-kV/cm field at 40 degrees C. The results of the present study provide the first evidence that strong RF electric fields inactivate microorganisms at moderately low temperatures. Increasing the field strength, the number of treatments, and the temperature enhanced inactivation. Frequency had no effect on inactivation over the range of frequencies studied.     

Inactivation of orange juice peroxidase by high‐intensity pulsed electric fields as influenced by process parameters

The inactivation of orange juice peroxidase (POD) under high‐intensity pulsed electric fields (HIPEF) was studied. The effects of HIPEF parameters (electric field strength, treatment time, pulse polarity, frequency and pulse width) were evaluated and compared with conventional heat pasteurization. Samples were exposed to electric field strengths from 5 to 35 kV cm^?1 for up to 1500 µs using square wave pulses in mono‐ and bipolar mode. Effect of pulse frequency (50–450 Hz), pulse width (1–10 µs) and electric energy on POD inactivation by HIPEF were also studied. Temperature was always below 40 °C. POD was totally inactivated by HIPEF and the treatment was more effective than thermal processing in inactivating orange juice POD. The extent of POD inactivation depended on HIPEF processing parameters. Orange juice POD inhibition was greater when the electric field strength, the treatment time, the pulse frequency and the pulse width increased. Monopolar pulses were more effective than bipolar pulses. Orange juice POD activity decreased with electric energy density input. The Weibull distribution function adequately described orange juice POD inactivation as a function of the majority of HIPEF parameters. Moreover, reduction of POD activity related to the electric field strength could be well described by the Fermi model. Copyright © 2005 Society of Chemical Industry     

高压脉冲电场处理草莓汁中损伤亚致死大肠杆菌与酿酒酵母的检测

为探寻高压脉冲电场对果汁中微生物的杀灭情况,揭示亚损伤微生物的存在及其影响因素。以草莓汁为原料,研究了不同处理条件下,高压脉冲电场对草莓汁中大肠杆菌和酿酒酵母损伤亚致死细胞产生的影响。结果表明,草莓汁中大肠杆菌亚致死细胞数目受高压脉冲电场处理条件的影响较为显著,在一定处理条件下(25kV/cm、200μs、10℃)大肠杆菌亚致死对数值最高达到1.339,亚致死细胞数目较多;但酿酒酵母亚致死情况受处理条件的影响较小,且亚致死对数均低于0.4,亚致死细胞产生数目较大肠杆菌少。      

Inactivation of Salmonella enterica Ser. Enteritidis in tomato juice by combining of high-intensity pulsed electric fields with natural antimicrobials

ABSTRACT:  The effect of high-intensity pulsed electric field (HIPEF) treatment (35kV/cm, 4 μs pulse length in bipolar mode without exceeding 38 °C) as influenced by treatment time (200, 600, and 1000 μs) and pulse frequency (100, 150, and 200 Hz) for inactivating Salmonella enterica ser. Enteritidis inoculated in tomato juice was evaluated. Similarly, the effect of combining HIPEF treatment with citric acid (0.5%, 1.0%, 1.5%, and 2.0%[wt/vol]) or cinnamon bark oil (0.05%, 0.10%, 0.2%, and 0.3%[vol/vol]) as natural antimicrobials against S. Enteritidis in tomato juice was also studied. Higher treatment time and lower pulse frequency produced the greater microbial inactivation. Maximum inactivation of S. Enteritidis (4.184 log₁₀ units) in tomato juice by HIPEF was achieved when 1000 μs and 100 Hz of treatment time and pulse frequency, respectively, were applied. However, a greater microbial inactivation was found when S. Enteritidis was previously exposed to citric acid or cinnamon bark oil for 1 h in tomato juice. Synergistic effects were observed in HIPEF and natural antimicrobials. Nevertheless, combinations of HIPEF treatment with 2.0% of citric acid or 0.1% of cinnamon bark oil were needed for inactivating S. Enteritidis by more than 5.0 log₁₀ units (5.08 and 6.04 log₁₀ reductions, respectively). Therefore, combinations of HIPEF with organic acids or essential oils seem to be a promising method to achieve the pasteurization in these kinds of products.     

Inactivation of Salmonella Typhimurium in orange juice containing antimicrobial agents by pulsed electric field

Combinations of different hurdles, including moderately high temperatures (<60 degrees C), antimicrobial compounds, and pulsed electric field (PEF) treatment, to reduce Salmonella in pasteurized and freshly squeezed orange juices (with and without pulp) were explored. Populations of Salmonella Typhimurium were found to decrease with an increase in pulse number and treatment temperature. At a field strength of 90 kV/cm, a pulse number of 20, and a temperature of 45 degrees C, PEF treatment did not have a notable effect on cell viability or injury. At and above 46 degrees C, however, cell death and injury were greatly increased. Salmonella numbers were reduced by 5.9 log cycles in freshly squeezed orange juice (without pulp) treated at 90 kV/cm, 50 pulses, and 55 degrees C. When PEF treatment was carried out in the presence of nisin (100 U/ml of orange juice), lysozyme (2,400 U/ml), or a mixture of nisin (27.5 U/ml) and lysozyme (690 U/ml), cell viability loss was increased by an additional 0.04 to 2.75 log cycles. The combination of nisin and lysozyme had a more pronounced bactericidal effect than did either nisin or lysozyme alone. An additional Salmonella count reduction of at least 1.37 log cycles was achieved when the two antimicrobial agents were used in combination. No significant difference (P > 0.05) in cell death was attained by lowering the pH value; only cell injury increased. Inactivation by PEF was significantly more extensive (P < 0.05) in pasteurized orange juice than in freshly squeezed orange juice under the same treatment conditions. This increase might be due to the effect of the chemical composition of the juices.     

Effect of pulsed electric fields upon accumulation of magnesium in Saccharomyces cerevisiae

Cultures of Saccharomyces cerevisiae were treated with PEF to improve accumulation of magnesium in the biomass. Under optimized conditions, that is, on 15-min exposure of the 20-h grown culture to PEF of the 2,000 V and 20-μs pulse width, accumulation of magnesium in the yeast biomass reached maximum 3.98 mg/g dm. It constituted 40% of the total magnesium in the medium. That accumulation significantly correlated against magnesium concentration in the medium. Neither multiple exposure of the cultures to PEF nor intermittent supplementation of the cultures with magnesium increased the magnesium accumulation. The intermittent supplementation of the cultures with magnesium could even reduce the accumulation efficiency by 30%.     

Color and viscosity of watermelon juice treated by high-intensity pulsed electric fields or heat

The effects of high-intensity pulsed electric field (HIPEF) processing (35 kV/cm for 1727 μs applying 4-μs pulses at 188 Hz in bipolar mode) on color, viscosity and related enzymes in watermelon juice were evaluated during 56 days of storage and compared to thermal treatments (90 °C for 60 s or 30 s). HIPEF-treated juice maintained brighter red color than thermally treated juices along the storage time. In addition, the application of HIPEF as well as heat at 90 °C for 60 s led to juices with higher viscosity than those untreated for 56 days of storage. On the other hand, peroxidase (POD) was inactivated more efficiently after HIPEF processing than after applying heat treatments. However, the thermally processed juice at 90 °C for 60 s kept the lowest residual POD activity values beyond day 7 of storage. Differences in lipoxygenase (LOX) activity among treatments were not appreciated at day 0. However, storage time had a strong reducing influence on the enzyme activity of heat-treated samples. A substantial loss of pectin methylesterase (PME) activity (more than 50%) was observed in all the treated juices, whereas a slight reduction in polygalacturonase (PG) activity was only achieved after the HIPEF treatment. The use of HIPEF technology could be an alternative to thermal treatments and could contribute to better maintain valuable attributes of watermelon juice.Industrial RelevanceHIPEF processing is a feasible alternative to thermal treatments to obtain watermelon juice, achieving optimal inactivation of deleterious microorganisms and quality-related enzymes. HIPEF-treated watermelon juices exhibit better physical properties such as color or viscosity than thermally treated juices throughout storage. Thus, HIPEF technology can help processors to obtain juices that keep their fresh characteristics, thus being better accepted by consumers.     

Effects of pulsed electric fields on the survival behaviour of Saccharomyces cerevisiae suspended in single solutions of low concentration

This paper investigates the survival behaviour of Saccharomyces cerevisiae cells suspended in NaCl solutions with various low concentrations after exposure to pulsed electric field (PEF) treatments. The input and heat‐generation pulse power during each PEF treatment experiment were measured directly. In addition, detailed information on sublethally PEF‐injured S. cerevisiae cells was obtained by the use of image analysis. Results showed that both the field intensity and the total treatment time had significantly affected the survival behaviour of S. cerevisiae cells, whereas the concentration of NaCl solution had less effect. When the S. cerevisiae cells were suspended in the solution with conductivity of 300 μS cm^?1, a high ratio of surviving cells was observed. Image analysis indicated a large number of sublethally injured cells (SICs) after the PEF treatment with low field intensity. Results indicate that the survival behaviour of cells suspended in solutions of low concentration was influenced by PEF intensity.     

高压脉冲电场杀菌技术在橙汁中的应用

食品冷杀菌技术是一类不用热能杀死微生物,不影响食品营养、质构、色泽和风味的新兴杀菌技术。主要阐述了高压脉冲电场杀菌（PEF）的基本原理和优点,讨论了其在橙汁中的应用,并对比了PEF处理和热处理（90℃,15s）对橙汁微生物的杀灭效果及橙汁理化指标的变化。     

PEF作用下亚致死损伤酿酒酵母修复条件的初步研究

为进一步探究高压脉冲电场（PEF）对酿酒酵母的灭菌机理,初步研究了PEF处理下亚致死损伤酿酒酵母（Saccharomyces cerevisiae）的修复条件。采用酵母浸出粉、蛋白胨和葡萄糖缓冲溶液作为模拟酿酒酵母的培养修复环境,通过选择性培养基与非选择性培养基对PEF（20k V/cm,400μs,15℃）处理前后亚致死酵母细胞进行菌落计数,结果显示PEF作用下亚致死酿酒酵母于三种模拟体系下可在30⁷0min内完成修复,在酵母浸出粉复杂模拟体系中修复速度最快。通过在p H7.2的磷酸盐缓冲液（PBS）模拟体系和实际食品体系（鲜草莓汁）添加K+、Ca2+和Mg2+离子,结果表明在模拟和实际体系中添加金属阳离子对均会产生不同的修复效果。      

Radio frequency electric fields processing of orange juice

The non-thermal process of radio frequency electric fields (RFEF) has been shown to inactivate bacteria in apple juice at moderately low temperatures, but has yet to be extended to inactivate bacteria in orange juice. An 80 kW RFEF pasteurizer was used to process pulp-free orange juice at flow rates of 1.0 and 1.4 l/min. Escherichia coli K12 in orange juice was exposed to electric field strengths of 15 and 20 kV/cm at frequencies of 21, 30, and 40 kHz. Ascorbic acid (Vitamin C) content and color of the juice before and after treatment were analyzed. Electrical energy costs were calculated using the measured voltage and current. An energy balance was performed using the inlet and outlet temperatures. Processing at an outlet temperature of 65 °C reduced the population of E. coli by 3.3 log relative to the control. Increasing the treatment time and temperature and decreasing the frequency enhanced the level of inactivation. Varying the electric field strength over the range of conditions used had no effect on the inactivation. No loss in ascorbic acid or enzymatic browning was observed due to RFEF processing. The electrical energy determined using the voltage and current was 180 J/ml. This was in good agreement with the energy calculated using the temperature data. The electrical cost was $0.0026/l of orange juice. The results provided the first evidence that the RFEF process inactivates bacteria in orange juice at moderately low temperatures.

Industrial relevance

The RFEF process has been shown to inactivate E. coli in apple juice at moderately low temperatures, but has yet to be extended to inactivate bacteria in orange juice. An RFEF pilot plant pasteurizer was used to process orange juice at rates of up to 1.4 l/min. RFEF processing reduced the population of E. coli by 99.3% at 60 °C and a hold time of 3 s, whereas conventional heating at the same conditions had no effect on the E. coli. This work demonstrated that the non-thermal RFEF process can be extended to inactivate bacteria in orange juice.     

柚皮苷与脉冲电场对酿酒酵母的协同致死作用

研究了抑菌剂柚皮苷和脉冲电场(PEF)对酿酒酵母的协同致死效果。结果表明,柚皮苷和PEF单独作用时,均对酿酒酵母有一定的抑制或者致死作用,且当两者协同处理酿酒酵母后,其致死效果得到显著提升。酿酒酵母经0.7 g/L柚皮苷处理5 h后,在20 kV/cm场强下分别处理2400、3600、4800μs,酿酒酵母协同处理致死菌率分别是PEF单独处理时的2.29、1.95和1.16倍;是柚皮苷单独处理时的1.95、2.41和2.84倍。这说明抑菌剂柚皮苷与脉冲电场在对微生物杀菌方面具有协同增效作用。     

Inactivation of microorganisms using pulsed electric fields: the influence of process parameters on Escherichia coli,Listeria innocua,Leuconostoc mesenteroides and Saccharomyces cerevisiae

This study examines the killing effect of pulsed electric fields (PEF) on four organisms suspended in a model medium. Escherichia coli, Listeria innocua, Leuconostoc mesenteroides and Saccharomyces cerevisiae differ in size, shape and cell wall construction. The electric field strength, pulse duration and number of pulses were varied in the ranges of 25–35 kV/cm, 2–4 μs and 20–40 pulses, respectively. The results showed that S. cerevisiae was the most sensitive organism with a 6-log reduction, followed by E. coli with a 5.4-log reduction, when they were exposed to 30 kV/cm, and 20 pulses with 4 μs duration. The most resistant organisms were L. innocua and L. mesenteroides with only a 3-log reduction, however, by increasing the parameters to 35 kV/cm and 40 pulses with 4 μs pulse duration; marked viability reductions of 8 and 7 log, respectively, were observed. Heat, which is generated during the process, has limited killing effect on the cells, hence the observed reduction can be ascribed to the PEF treatment. Although transmission electron microscopy of PEF treated cells did not confirm membrane damage, observations suggest that PEF treatments have profound direct or indirect effects on the intracellular organisation of microorganisms.     

Optimizing critical high-intensity pulsed electric fields treatments for reducing pectolytic activity and viscosity changes in watermelon juice

A response surface methodology was used to determine the combined effect of high-intensity pulsed electric fields (HIPEF) variables such as frequency, pulse width and polarity on the inactivation of pectolytic enzymes involved in viscosity changes of juices. Pectin methylesterase (PME) and polygalacturonase (PG) activities as well as viscosity were determined in watermelon juices processed at pulse frequencies from 50 to 250 Hz and pulse widths ranging from 1.0 to 7.0 μs in monopolar or bipolar mode. Electric field strength and total treatment time were maintained constant in all treatments at 35 kV/cm and 1,000 μs. Second-order expressions were accurate enough to fit the experimental results. The great PME reduction contrasted with the low impact of HIPEF on the PG activity of watermelon juice within the range of assayed conditions. Minimal residual PME activity values (15%) were obtained by selecting pulse widths higher than 5.5 μs at 250 Hz in bipolar mode, whereas the lowest PG residual activities (60%) were achieved after applying 7.0-μs bipolar pulses at 250 Hz. Moreover, watermelon juice viscosity increased throughout the range of studied conditions. The highest viscosity observed in the juice after applying 7.0-μs bipolar pulses at 250 Hz was related to the lowest PME activities obtained in the product treated under those conditions. Hence, the HIPEF processing optimization through frequency, pulse width and polarity could contribute to assure enzymatic inactivation while keeping valuable attributes of juices.     

酿酒酵母菌含量对控制橙汁腐败的影响

以酿酒酵母为模式微生物,研究了GB 19297-2003中规定的酵母菌含量对橙汁中酿酒酵母生长延滞期的影响,以及在5、8、25、35℃下,低菌含量酿酒酵母在橙汁中的生长规律.研究表明,产品酵母数量≤20cfu/mL的橙汁,虽然符合GB 19297-2003要求,但是在25℃下仍能发生腐败,导致胀袋;允许酵母残留的橙汁产品,其温度必须控制在5℃以下.