Radiation-heat synergism for inactivation of Alicyclobacillus acidoterrestris spores in citrus juice

The objective of this study was to investigate the influence of electron-beam and gamma-ray irradiation and temperature (85 to 95 degrees C) on Alicyclobacillus acidoterrestris GD3B strain (NCIMB 13137) spores by calculating and comparing the decimal reduction dose or time (D-values). The survival rate of A. acidoterrestris spores decreased exponentially with irradiation doses of an electron beam or gamma ray. D-values determined for electron-beam and gamma-ray irradiated spores on filter paper ranged from 1.02 to 1.10 kGy. On the other hand, the thermal sterilization effect showed a single exponential decrease within 1.5-log decreases in cell numbers (D85 degrees C = 70.5 min, D90 degrees C = 16.1 min, and D95 degrees C = 5.19 min and z-value [change in temperature required to change the D-value] was 8.83 degrees C), and prolonged heating produced an increase of 10 to 13 times that of the thermal resistance. However, within all time ranges studied (5 to 360 min), a linear decrease in the D-value was observed with an increase in the temperature. A combination of two different methods, irradiation before heating, was appropriate for reducing the duration of the heat treatment required to achieve the inactivation of conidia. Moreover, a necessary radiation dosage for complete inactivation of A. acidoterrestris spores that contaminated dextrin was examined. Dextrin is often used in the juice industry as an augmentor, and it is known to be sometimes contaminated by these spores. The D-values of the spores in dextrin for electron-beam and gamma-ray irradiations were 1.72 and 1.79 kGy, respectively. The doses required for elimination of the spores could be lowered by using irradiation in combination with heat sterilization. When dextrin powder contaminated with 10(4) CFU/g of A. acidoterrestris was preirradiated at 1.0 kGy of electron beam, the citrus juice containing dextrin at a concentration of 10% (wt/vol) was completely sterilized by heating for 20 min at 95 degrees C.     

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.     

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.     

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.     

Antibacterial activity and mechanism of cinnamic acid and chlorogenic acid against Alicyclobacillus acidoterrestris vegetative cells in apple juice

The aim of this study was to investigate the antibacterial activity and mechanism of cinnamic acid and chlorogenic acid against Alicyclobacillus acidoterrestris. Minimum inhibitory concentrations of cinnamic acid and chlorogenic acid were 0.375 and 2.0 mg mL⁻¹, and the minimum bactericidal concentrations were 0.50 and 4.0 mg mL⁻¹, respectively. The apple juice ingredients had little influence on the inactivation of A. acidoterrestris. After treatment with cinnamic acid and chlorogenic acid, the morphology of A. acidoterrestris cells were severely destroyed; the leakage of nucleic acids and proteins increased significantly. SDS-PAGE investigation of bacterial proteins proved that the loss of soluble proteins was obvious as well. These results demonstrated that cinnamic acid and chlorogenic acid exerted their antibacterial activity mainly by an action mode of membrane disruption. This study provides an alternative method for the control of A. acidoterrestris-related spoilage in the fruit juice/beverage industry.     

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.     

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.     

Use of nisin for inhibition of Alicyclobacillus acidoterrestris in acidic drinks

The inhibitory effects of nisin on the growth of the thermoacidophilic spoilage bacteriumAlicyclobacillus acidoterrestris were investigated for the purpose of preventing flat-sour-type spoilage in acidic drinks. Minimum inhibitory concentration values of nisin against the spores were from less than 0·78 to 12·5 IU ml⁻¹and from 25 to 100 IU ml⁻¹on mYPGA plates at pH 3·4 and 4·2, respectively. The levels of nisin inhibition against the vegetative cells were, however, higher than those of the spores. In determining the effects of nisin on the thermal resistance of A. acidoterrestris spores, the addition of nisin contributed to the reduction of the thermal resistance of A. acidoterrestris spores in acidic drinks. Furthermore, the outgrowth of A. acidoterrestris spores was inhibited by the addition of 25–50 IU ml⁻¹nisin in both orange and fruit-mixed drinks, but was not inhibited by the higher level (600 IU ml⁻¹) addition in a clear-apple drink. From these findings, we conclude that it would be useful to add nisin for preventing the spoilage caused by A. acidoterrestris in all but clear-apple acidic drinks.     

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.     

Rapid discrimination of Alicyclobacillus strains in apple juice by Fourier transform infrared spectroscopy

Alicyclobacillus spp. are thermoacidophilic, spore-forming bacteria. Some of which cause spoilage in pasteurized and heat-treated apple juice products through the production of guaiacol. Fourier transform infrared (FT-IR) spectroscopy was used to discriminate between eight Alicyclobacillus strains (WAC, 81-2, Oly#21, 51-1, KF, 1016, 1101, and A-Gala A4) in apple juice. FT-IR vibrational combination bands reflected compositional differences in the cell membranes of Alicyclobacillus strains in the "fingerprint region" at wavenumbers between 1500 and 800 cm(-1). Distinctive segregation among spectral sample clusters of different Alicyclobacillus strains was observed using principal component analysis (PCA). Two closely related strains (1016 and 1101) of Alicyclobacillus acidoterrestris could be distinguished, suggesting that this method can be highly selective. Results of soft independent modeling of class analogy (SIMCA) demonstrated that guaiacol-producing and non-guaiacol producing Alicyclobacillus strains could be differentiated up to 89% of the time. This technique may provide a tool for fruit juice producers to detect Alicyclobacillus rapidly and to monitor and control guaiacol formation.     

苹果梨汁的常温护色方法

为抑制苹果梨汁褐变,在果实榨汁前,以护色剂对苹果梨汁抑制褐变率和色差值的影响为指标,在5组单因素试验的基础上,通过5因素2水平正交试验,运用正交试验的极差分析,确定了柠檬酸、Na_2SO_3、L-半胱氨酸3种对褐变抑制率及色差值影响较大的护色剂,之后通过软件进行试验设计及响应面法分析。试验结果表明,最佳的苹果梨汁护色剂配方为:柠檬酸添加量为0.67 g/100 mL,Na_2SO_3添加量为0.01 g/100 mL,L-半胱氨酸添加量为0.01 g/100 mL,在此配方条件下预测苹果梨汁褐变抑制率为58.1%,色差值为25.8 NBS。实际值为苹果梨汁平均褐变抑制率58.6%、平均色差值25.3 NBS,预测值与试验值吻合程度分别为99.14%和98.06%。     

Alicyclobacillus acidoterrestris in fruit juices and its control by nisin

Summary The acid-tolerant and heat-resistant bacterium Alicyclobacillus acidoterrestris is a spoilage problem in pasteurized and heat-treated fruit juices. In this study it was shown to grow in orange juice, grapefruit juice and apple juice to produce detectable taint at levels of about 10⁴–10⁵ c.f.u. ml⁻¹. Decimal reduction times were determined at 80 °, 90 ° and 95 °C in each juice and confirmed the heat-resistant nature of the spores under normal juice pasteurization conditions. They also confirmed that heat sensitivity increased with decreasing pH but that this effect was less pronounced at higher temperatures. The organism was, however, sensitive to the bacteriocin food preservative nisin. The presence of nisin during heating decreased the D value by up to 40% and the MIC for nisin against spores at 25 °C was only 5 International Units (IU) ml⁻¹. The results indicate that use of nisin is a potentially useful way of controlling this organism in fruit juices and fruit juice-containing products.     

Thermal inactivation of Alicyclobacillus acidoterrestris spores under different temperature, soluble solids and pH conditions for the design of fruit processes

Alicyclobacillus acidoterrestris, a thermoacidophilic, non-pathogenic and spore-forming bacterium has been detected in several spoiled commercial pasteurised fruit juices. A. acidoterrestris spores, besides being resistant to the pasteurisation treatment conditions normally applied to acidic fruit products, can germinate and grow causing spoilage. Therefore, this microorganism was suggested as the target to be used in the design of adequate pasteurisation processes. The objectives of this work were to investigate the influence of temperature (T: 85-97 degrees C), total soluble solids (SS: 5-60 degrees Brix or % by weight) and pH (2.5-6.0) on D-values (decimal reduction time) of Alicyclobacillus acidoterrestris (type strain, NCIMB 13137) spores, and to fit a model using response surface methodology. A central composite face-centred experimental design was used, and the response, D-value determined in malt extract broth, ranged between 0.498+/-0.045 and 94.9+/-6.7 min. Within the factor ranges studied, temperature was the parameter that most affected the D-value. Following this was the SS and, lastly, the pH value. A linear decrease in D-value was observed with decreasing SS and pH, and a non-linear decrease in D-value was noticed with increasing temperature. A second order polynomial was successfully fitted to the data (R2 = 0.98). In general, D-values measured in real fruit systems, such as orange, apple and grape juices, blackcurrant concentrates, cupua?u (exotic fruit) extract and orange juice drink, were higher than those predicted by the malt extract broth model. This result emphasises the importance of experimental validation of any model-derived process.     

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.     

Characterization of a wild strain of Alicyclobacillus acidoterrestris: heat resistance and implications for tomato juice

This article reports the characterization of a wild strain of Alicyclobacillus acidoterrestris and describes the implications of the heat resistance of this microorganism in tomato juice. The strain (labeled as A. acidoterrestrisγ4) showed pH and temperature ranges for growth typical of the species (3.0 to 6.0 for the pH and 35 to 60 °C for the temperature); heat resistance in tomato juice was as follows: D(T) values of 40.65, 9.47, and 1.5 min (at 85, 90, and 95 °C, respectively) and z-value of 7 °C. A treatment at 70 °C for 15 min was found to be optimal for spore activation, whereas Malt Extract Agar, acidified to pH 4.5, showed good results for spore recovery. Concerning the implications of heat resistance of A. acidoterrestris on tomato juice, high temperatures required for spore inactivation determined a general decrease of the antioxidant activity (increase of the redox potential and reduction of the chain-breaking activity), but not the formation of brown compounds (namely, hydroxymethylfurfural), thus suggesting an effect on the secondary antioxidants (carotenoids and ascorbic acid) rather than on lycopene. PRACTICAL APPLICATION: Alicyclobacillus acidoterrestris is an emerging spore-forming microorganism, capable of causing spoilage in tomato juice. Due to their high thermal resistance, spores could be used as targets for the optimization of heat processing; this article reports on the assessment of thermal resistance of a wild strain of A. acidoterrestris, then focusing on the effect of the thermal treatment necessary to inactivate spores on the quality of tomato juice.     

酸土环脂芽孢杆菌检测与控制技术研究进展

酸土环脂芽孢杆菌是造成果汁变质的一种主要腐败菌。在生产实践中快速、准确的检测手段必不可少,同时对该菌进行控制是提高产品质量、减少污染的重要环节,特别是对其耐热芽孢的抑制。通过对酸土环脂芽孢杆菌生长、代谢特性的分析,重点阐述了该菌污染的检测技术和菌种的分离、鉴定方法;同时从果汁生产环节入手探讨了控制该菌的各种方法,并指出今后应该进行的研究方向。     

Control of Alicyclobacillus acidoterrestris in fruit juices by enterocin AS-48

Alicyclobacillus acidoterrestris is a spoilage-causing bacterium in fruit juices. Control of this bacterium by enterocin AS-48 from Enterococcus faecalis A-48-32 is described. Enterocin AS-48 was active against one A. acidocaldarius and three strains of A. acidoterrestris tested. In natural orange and apple juices incubated at 37 degrees C, vegetative cells of A. acidoterrestris DSMZ 2,498 were inactivated by enterocin AS-48 (2.5 microg/ml) and no growth was observed in 14 days. In commercial fruit juices added of AS-48 (2.5 microg/ml) and inoculated with vegetative cells or with endospores of strain DSMZ 2,498, no viable cells were detected during 90 days of incubation at temperatures of 37 degrees C, 15 degrees C or 4 degrees C, except for apple, peach and grapefruit juices inoculated with vegetative cells and incubated at 37 degrees C which were protected efficiently for up to 60 days. Remarkably, in all commercial fruit juices tested, no viable cells were detected as early as 15 min after incubation with the bacteriocin. Endospores incubated for a very short time (1 min) with increasing bacteriocin concentrations were inactivated by 2.5 microg/ml AS-48. Electron microscopy examination of vegetative cells and endospores treated with enterocin AS-48 revealed substantial cell damage and bacterial lysis as well as disorganization of endospore structure.