Use of Weibull frequency distribution model to describe the inactivation of Alicyclobacillus acidoterrestris by high pressure at different temperatures

The survival curves of Alicyclobacillus acidoterrestris by high hydrostatic pressure were obtained at two pressures (350 and 450 MPa) and three temperature levels (35, 45 and 50 °C) in BAM broth. Tailing (upward concavity) was observed in all survival curves. Weibull model was fitted to these data and goodness of fit of this model was investigated. Regression coefficients (R²), root mean square (RMSE) values and residual plot strongly suggested that Weibull model produced good fit to the data. A better fit was observed for the data at lower pressure (350 MPa). Shape factors of the Weibull model (n values) for 350 MPa at 35, 45 and 50 °C were significantly different from each other (P < 0.05). Two linear emprical equations were obtained for scale factors (b values) at the temperature values studied for 350 and 450 MPa. Such pressure–temperature inactivation models form the engineering basis for design, evaluation and optimization of high hydrostatic pressure processes as a new preservation technique.     

Aerobic treatment of black ripe olive processing streams to reduce biological contamination

A new aeration treatment of black ripe olive streams prior to their vacuum evaporation was studied. It was found that the aeration for two days of these solutions reduced the chemical oxygen demand (COD) of the distillate phase (>80%) generated during their vacuum evaporation; although the temperature (14–38 °C) and the air flow rate (10–50 L air/h/L) had a great influence on this reduction. Moreover, the COD in the distillate was mainly associated with the presence of acetic acid (~400 mg/L) and ethanol (~500 mg/L), the later substance being eliminated by strong aeration. Additionally, the growth of native aerobic microorganisms, mainly Acetobacter, increased the pH of the wastewater to 7–8 units, thus avoiding evaporation of the acetic acid salt formed. These results make the use of the distillate for plant irrigation viable and its discharge into municipal depuration plants possible, given the low COD (<500 mg/L) reached with this new method.Industrial relevanceThe worldwide production of table olives is annually increasing but the management of the solutions generated during olive processing is limiting this expansion, in particular due to their high salt content. Factories are nowadays concentrating these solutions by using vacuum evaporation but the contamination of the distillate is too high. The present work has been carried out based on industry demand for low cost and easy handle technology able to reduce COD in distillates. The results of the present study showed that the content in ethanol and acetic acid of distillates can be reduced to a large extend by simple aerating of the solutions prior to the vacuum evaporation step, and this open the possibility of their reuse for plant irrigation.     

Combined treatment of high pressure and heat on killing spores of Alicyclobacillus acidoterrestris in apple juice concentrate

Alicyclobacillus acidoterrestris, a thermoacidophilic and spore-forming bacterium, has been isolated from spoiled acidic juices and is considered to be one of the important target microorganisms in quality control of acidic canned foods. Combined high pressure and heat treatment showed an effectiveness to control A. acidoterrestris spores. However, the effectiveness of the combined treatment may change upon the apple juice concentration. Therefore, the objective of this study was to evaluate different levels of apple juice concentrate for reduction of Alicyclobacillus spores by high pressure and heat. Spores of A. acidoterrestris were inoculated into three different concentrations of apple juice (17.5, 35, and 70 degrees Brix), and subjected to three high-pressure treatments (207, 414, and 621 MPa) at four different temperatures (22, 45, 71, and 90 degrees C). High-pressure treatment (207, 414, and 621 MPa) at 22degrees C did not reduce the level of spores regardless of the apple juice concentration (P > 0.05). In diluted apple juice (17.5 degrees Brix), the combined treatment of high pressure and heat resulted in spore reductions of about 2 log at 45 degrees C, and more than 5 log at higher temperatures (71 and 90 degrees C) in a high-pressure and temperature-dependent manner. For apple juice with a higher concentration (30 degrees Brix), high-pressure treatment showed no effect at 45 degrees C but resulted in about 2 and 4 log reduction at 71 and 90 degrees C, respectively. However, for apple juice concentrate (70 degrees Brix), treatment with heat or high pressure alone, or their combinations showed no inactivation against spores of A. acidoterrestris. It is likely that differences in the water availability explain the greater resistance of spores to high-pressure inactivation in the juice concentrates than in diluted juices. Our results demonstrate that the effect of high pressure combined with heat against spores of A. acidoterrestris was highly dependent on the apple juice concentration.     

The effect of intermittent shaking, headspace and temperature on the growth of Alicyclobacillus acidoterrestris in stored apple juice

The presence of Alicyclobacillus acidoterrestris in stored juices can be difficult to detect. In this study the effects of storage temperature, headspace and agitation of juice containers was investigated. The results indicate that the amount of headspace has a significant effect on growth of vegetative cells and spores of A. acidoterrestris at 35 °C. Intermittent shaking before sampling increased growth and therefore probable detection rates at 30 °C, Agitating containers and sampling from several areas within containers is therefore recommended for determining whether A. acidoterrestris is present or absent from stored juice, especially in large containers.     

The use of chlorine dioxide to control Alicyclobacillus acidoterrestris spores in aqueous suspension and on apples

Alicyclobacillus acidoterrestris, a thermoacidophilic, spore-forming bacterium, has been identified as a spoilage organism in commercial, pasteurized fruit juices. This study was undertaken to evaluate chlorine dioxide for reducing numbers of A. acidoterrestris spores on laboratory media and on apples. A. acidoterrestris spores in aqueous suspension and on apple surfaces of four different cultivars were treated with several concentrations of chlorine dioxide. Spores in aqueous suspension treated with 40 ppm for 5 min were reduced by more than 4 log. Treatment with 80 ppm for 1 min and 120 ppm for 30 s resulted in about 1.8 log and 4.8 log reductions of spore viability, respectively, and treatment at 80 and 120 ppm for 5 min reduced spore viability to undetectable levels (<0.7 log CFU/ml). When applied to the surfaces of four different apple cultivars ('Red Delicious', 'Golden Delicious', 'Gala', and 'Fuji'), 40 ppm free chlorine dioxide reduced A. acidoterrestris spore numbers by 1.5, 3.2, 4.5, >4.8 log after 1-, 2-, 3-, and 4-min treatments, respectively. Spore numbers were reduced by >4.8 log with 120 ppm free chlorine dioxide after only 1-min treatment. However, there was no significant difference between apple cultivars (P>0.05) on spore reduction. These results show the great effectiveness of chlorine dioxide in controlling A. acidoterrestris spores both in aqueous suspension and on apple surfaces. There was no synergistic effect on spore reduction when chlorine dioxide treatment of aqueous suspension was followed by heat.     

Effects of microwave and ultrasonic wave treatment on inactivation of Alicyclobacillus

The objective of this study was to determine the effects of microwave (MW), ultrasonic wave (UW) and their combination treatments for the same length of time (10 or 30 min) on the inactivation of vegetative cells of three different strains of Alicyclobacillus, (Alicyclobacillus acidiphilus DSM 14558^T, Alicyclobacillus acidoterrestris DSM 3922^T and Alicyclobacillus cycloheptanicus DSM 4006^T) in a laboratory medium. The results showed that A. acidoterrestris DSM 3922^T seemed to be the most susceptible strain. It was also found that when MW or UW was employed separately, UW was more effective and the effectiveness of MW and UW was improved as the power and exposure time increased. By comparison, UW treatment followed by MW was significantly better than the treatment of MW followed by UW. Furthermore, combined MW and UW treatment had a markedly better effect than separate MW processing, but had a less effect than UW alone, except that the inactivation of A. cycloheptanicus DSM 4006^T was significantly increased (P < 0.05) by the 30‐min treatment of UW (400 W for 20 min) + MW (900 W for 10 min).     

Germination and inactivation of Bacillus coagulans and Alicyclobacillus acidoterrestris spores by high hydrostatic pressure treatment in buffer and tomato sauce

Acidothermophilic bacteria like Alicyclobacillus acidoterrestris and Bacillus coagulans can cause spoilage of heat-processed acidic foods because they form spores with very high heat resistance and can grow at low pH. The objective of this work was to study the germination and inactivation of A. acidoterrestris and B. coagulans spores by high hydrostatic pressure (HP) treatment at temperatures up to 60 °C and both at low and neutral pH. In a first experiment, spores suspended in buffers at pH 4.0, 5.0 and 7.0 were processed for 10 min at different pressures (100-800 MPa) at 40 °C. None of these treatments caused any significant inactivation, except perhaps at 800 MPa in pH 4.0 buffer where close to 1 log inactivation of B. coagulans was observed. Spore germination up to about 2 log was observed for both bacteria but occurred mainly in a low pressure window (100-300 MPa) for A. acidoterrestris and only in a high pressure window (600-800 MPa) for B. coagulans. In addition, low pH suppressed germination in A. acidoterrestris, but stimulated it in B. coagulans. In a second series of experiments, spores were treated in tomato sauce of pH 4.2 and 5.0 at 100 - 800 MPa at 25, 40 and 60 °C for 10 min. At 40 °C, results for B. coagulans were similar as in buffer. For A. acidoterrestris, germination levels in tomato sauce were generally higher than in buffer, and showed little difference at low and high pressure. Remarkably, the pH dependence of A. acidoterrestris spore germination was reversed in tomato sauce, with more germination at the lowest pH. Furthermore, HP treatments in the pH 4.2 sauce caused between 1 and 1.5 log inactivation of A. acidoterrestris. Germination of spores in the high pressure window was strongly temperature dependent, whereas germination of A. acidoterrestris in the low pressure window showed little temperature dependence. When HP treatment was conducted at 60 °C, most of the germinated spores were also inactivated. For the pH 4.2 tomato sauce, this resulted in up to 3.5 and 2.0 log inactivation for A. acidoterrestris and B. coagulans respectively. We conclude that HP treatment can induce germination and inactivation of spores from thermoacidophilic bacteria in acidic foods, and may thus be useful to reduce spoilage of such foods caused by these bacteria.     

微波提取苹果汁中脂环酸芽孢杆菌DNA的PCR检测方法研究

根据Genbank公布的酸土脂环酸芽孢杆菌(Alicyclobacillus acidoterrestris DSM 3922T)鲨烯环化酶序列自行设计一对引物,利用微波技术直接从果汁样品中提取目标菌DNA,对引物特异性、微波法提取DNA的扩增效果及检测灵敏度进行探讨。结果表明：微波功率1000W、处理时间30s、经5000r/min离心2min即可获得酸土脂环酸芽孢杆菌基因组DNA,所得模板质量符合聚合酶链式反应检测要求,目的条带清晰,检测时间仅为2h,检测限为200CFU/mL,有望真正应用于苹果汁生产的在线检测。     

Growth inhibition and inactivation of Alicyclobacillus acidoterrestris endospores in apple juice by hyperbaric storage at ambient temperature

Control of endospores of Alicyclobacillus acidoterrestris in pasteurized apple juice using hyperbaric storage at 18 to 23 °C was compared to storage at atmospheric pressure and 18 to 23 °C, as well as refrigeration at ~4 °C for up to 30 days. The juice samples were inoculated with approximately 1 × 10⁵ CFU/mL spores. The juice spoiled quickly at atmospheric pressure and ambient temperature, while under refrigeration spore levels remained unchanged for 30 days. Hyperbaric storage of inoculated apple juice at 25, 50 and 100 MPa at 18 to 23 °C resulted in spore inactivation at more rapid rates as pressure magnitudes increased, reaching levels below the detection limit of 10 CFU/mL at 50 and 100 MPa. In highly acid foods such as apple juice, hyperbaric storage at pressures ≤100 MPa and ambient temperature was effective in inactivating spores of A. acidoterrestris for periods up to 30 days.These results indicate hyperbaric storage at ambient temperature as a clearly more efficient preservation procedure to control the development of A. acidoterrestris endospores, compared to ambient temperature and refrigerated storage, in highly acidic foods as apple juice.     

The impact of high power ultrasound for controlling spoilage by Alicyclobacillus acidoterrestris: A population and a single spore assessment

Consumer demand for healthier, microbiologically safe and stable, higher quality and minimally processed foods has increased in the last decades, promoting the application of non-thermal process technologies. Ultrasound processing is gaining attention because of its potential for improving quality and safety while retaining product flavor, texture, color and nutrient composition. Recently, Alicyclobacillus acidoterrestris has been recognized by manufacturers and processors in the fruit industry as a new type of thermoacidophilic, endospore-forming spoilage bacterium for commercialized fruit juices that can withstand the high temperatures applied in pasteurization process and spoil them producing taint compounds. Based on the above, the present study was undertaken to investigate the separate and combined effect of ultrasound treatment operating at 26 kHz, 90 μm, 200 W for 5, 10, 15 min and heat stress (at 80 °C for 10 min) on the recovery of A. acidoterrestris spores in K broth adjusted to pH = 4.5 with 25% (w/v) citric acid at temperatures between 35 and 45 °C at population and individual spore level. At the population level, statistically significant differences in average lag time of A. acidoterrestris spores from different treatments at 35 and 45 °C have been found. After the applied ultrasound (at 100% Amplitude for 10 min) and heat shock (at 80 °C for 10 min), the average lag time of A. acidoterrestris spores at 35 °C (7.24 ± 0.34 h) and 45 °C (6.38 ± 0.18 h) has been shown to be longer compared to the control at 35 °C (5.68 ± 0.36 h) and at 45 °C (2.87 ± 0.19 h), while for this combination, the maximum specific growth rate was not significantly different from the control samples. Additionally, the findings of this study have shown that λ among individual spores was greatly variable when they were treated by ultrasound (at 100% Amplitude for 10 min) and/or thermal treatment (at 80 °C for 10 min). The application of ultrasound and thermal treatment resulted in a more pronounced effect on median lag phase duration (25.74 h) than the heat shock (11.63 h) and affected both the position and the spread of the λ distributions of A. acidoterrestris spores. This work would help to better assess and optimize the proposed combined treatments, since ultrasound and thermal treatments could work in a synergistic way on delaying the lag time of the tested bacterial spores.     

Slow-release chlorine dioxide gas treatment as a means to reduce Salmonella contamination on spices

Salmonella enterica is a major food safety issue for the spice industry. Slow-releasing chlorine dioxide (ClO₂) gas from self-contained sachets, which can be employed in a small-scale operation, was evaluated for its effectiveness in reducing Salmonella contamination on black pepper, cumin, and sesame seed. Three levels of chlorine dioxide gas (100, 200, or 500 mg ClO₂/kg spice) were applied to Salmonella-inoculated spices. Spices were sampled immediately after treatment, and at 1, 10, and 30-days post-treatment. The combined effect of ClO₂ gas treatment and storage time on Salmonella numbers on spices was evaluated. Salmonella reductions on black pepper, cumin, and sesame seed samples ranged from 0.81 to 2.74 log CFU/g after ClO₂ treatment. Storage time also had a significant impact on Salmonella numbers, as numbers decreased by up to 2.50 log CFU/g over 30 days of storage. Self-contained ClO₂ releasing sachets could provide small food processors a post-lethality treatment alternative for spices.     

Efficacy of disinfectants in killing spores of Alicyclobacillus acidoterrestris and performance of media for supporting colony development by survivors

Alicyclobacillus has recently emerged as a spoilage microorganism of concern in a wide range of pasteurized fruit products. The focus of this study was to determine the efficacy of chemical disinfectants in killing Alicyclobacillus acidoterrestris spores. Direct plating media were evaluated for their suitability to support germination and outgrowth of spores surviving exposure to these disinfectants. Significant (P < or = 0.05) reductions of about 2.2, 0.4, and 0.1 logs in the number of viable A. acidoterrestris spores in a five-strain mixture were achieved when spores were suspended in 200 ppm chlorine, 500 ppm acidified sodium chlorite, or 0.2% H2O2 solutions, respectively, for 10 min at 23 degrees C. When treated with either 1,000 ppm chlorine or 4% H2O2, the number of spores was reduced by more than 5 logs. Treatment with 8% trisodium phosphate or 80 ppm Tsunami did not significantly reduce numbers of viable spores. Spores of individual strains of A. acidoterrestris varied little in resistance to the same chemical treatment. K agar (pH 3.7) was judged best for recovering chemically treated spores, compared to orange serum agar (pH 5.0) and potato dextrose agar (pH 3.5). Experiments were done to determine the effectiveness of chemical treatments in killing a mixed-strain inoculum of A. acidoterrestris spores on the surface of apples. Treatment with 500 ppm chlorine or 1,200 ppm acidified sodium chlorite for 1 min significantly (P < or = 0.05) reduced the number of viable spores, but reductions were less than 1 log. Hydrogen peroxide (2%) was ineffective in killing spores remaining on the apple skin after treatment.     

Determination of guaiacol produced by Alicyclobacillus acidoterrestris in apple juice by using HPLC and spectrophotometric methods, and mathematical modeling of guaiacol production

In this study, easy detection of Alicyclobacillus acidoterrestris was performed by determination of guaiacol in apple juice. Guaiacol produced by A. acidoterrestris was determined by using HPLC, UV-Vis spectrophotometer, and Minolta spectrophotometer. Statistical analysis showed that the methods used for measuring the guaiacol concentrations were not significantly different (p > 0.05). Guaiacol formation in apple juice spiked with different levels of A. acidoterrestris spores was also analyzed using Gompertz, Logistic, and Richards models. In all cases, a good agreement between experimental data and fitted values was obtained. Using the modified Gompertz model, the derived biological parameters were calculated. Guaiacol formation rates (μ) and final guaiacol concentrations (A) were very similar in all cases, regardless of the initial A. acidoterrestris spore counts. However, lag phase durations (λ) were found to be dependent on the initial bacterial counts, and increased from 28.4 to 37.6 h, when initial inoculation level decreased from ∼10³ to ∼10¹ cfu/mL.     

Lactic acid and trisodium phosphate treatment of lamb breast to reduce bacterial contamination

Lactic acid and trisodium phosphate (TSP) were evaluated for the ability to reduce Escherichia coli and aerobic plate counts (APCs) on lamb breasts that were inoculated with a lamb fecal paste. A 90-s water rinse was applied followed by either a 9-s (55 degrees C) 2% lactic acid spray, a 60-s (55 degrees C) 12% TSP dip, or a combined treatment of both lactic acid and TSP treatments. Lactic acid reduced E. coli and APCs by 1.6 log10/cm2, and TSP caused a 1.8-log10/cm2 reduction in E. coli and a 0.7-log10/cm2 reduction in APCs. Combined reductions by the lactic acid spray followed by the TSP dip were 1.8 and 1.5 log10/cm2 for E. coli and APCs, respectively. Lactic acid and trisodium phosphate, used alone or in combination, were effective in reducing numbers of E. coli and could be useful as pathogen intervention steps in lamb slaughter processing.     

Effect of single and combined UV-C and ultra-high pressure homogenisation treatments on inactivation of Alicyclobacillus acidoterrestris spores in apple juice

Alicyclobacillus acidoterrestris is a spore-forming bacterium that can survive thermal pasteurization and acidic conditions. It produces changes in the odour and flavour of fruit juices leading to economical loses. A. acidoterrestris CECT 7094 spores were inoculated in clarified and cloudy apple juices (Golden delicious var.) in the range of 5–6 log₁₀ spores/mL and submitted to different short-wave ultraviolet light (UV-C) doses (7.2–28.7 J/mL) and ultra-high pressure homogenisation (UHPH) treatments (100–300 MPa), including their combination. A. acidoterrestris could be inactivated in clarified apple juice at a level of 4.8 log₁₀ CFU/mL by a 300 MPa-UHPH treatment when the inlet temperature was 80 °C. UV-C treatments showed to be more efficient achieving a lethality of 5.5 log₁₀ CFU/mL with a dose of 21.5 J/mL at 20 °C. In cloudy apple juice (2357 NTU) UV-C treatments were less efficient with a maximum lethality of 4.07 CFU/mL after a dose of 28.7 J/mL. A previous application of UHPH contributed with UV-C to obtain higher reductions of A. acidoterrestris spores at the doses of 14.3 and 21.5 J/mL compared with UV-C single treatments. On the other hand, this previous treatment also changed the properties of particles in the matrix which apparently reduced the effectiveness of UV-C at 28.7 J/mL.     

Detection of guaiacol produced by Alicyclobacillus acidoterrestris in apple juice by sensory and chromatographic analyses, and comparison with spore and vegetative cell populations

Spoilage of fruit juice by Alicyclobacillus acidoterrestris is characterized by a distinct medicinal or antiseptic off odor attributed to guaiacol, a metabolic by product of the bacterium. Detection of low populations of A. acidoterrestris that would precede sensory detection of guaiacol would enable juice processors to select appropriate processing and storage conditions that would minimize or eliminate spoilage. The objective of this study was to determine the recognition threshold of guaiacol in apple juice by sensory analysis and the population of A. acidoterrestris and incubation time at 21 and 37 degrees C necessary for chemical detection of guaiacol. Commercially sterilized apple juice (pH 3.54 +/- 0.04, 11.3 +/- 0.3 degrees Brix) was inoculated with a five-strain mixture of A. acidoterrestris spores (2.98 log10 CFU/ml) and stored at 21 or 37 degrees C for up to 61 days. Using an experienced sensory panel and the forced-choice ascending concentration method of limits, the best estimate threshold (BET) for recognition of guaiacol added to uninoculated apple juice was 2.23 ppb. Numbers of A. acidoterrestris spores and cells in inoculated juice remained constant during the 61-day storage period; however, the panel detected (P < or = 0.01) guaiacol in juice stored at 37 degrees C within 8 days. At three of four sampling times ranging from 13 to 61 days at which the sensory panel detected (P < or = 0.001) guaiacol, concentrations of 8.1 to 11.4 ppb were detected by chromatographic analysis. The panel detected (P < or = 0.1 to P < or = 0.01) guaiacol in five samples stored at 21 to 37 degrees C for 8 to 61 days in which the compound was not detected by chromatographic analyses. It appears that guaiacol content in apple juice inoculated with A. acidoterrestris is not always correlated with numbers of cells, and the limit of sensitivity of chromatographic quantitation of the compound is higher than the BET.