Identification and haplotype distribution of Alicyclobacillus spp. from different juices and beverages

Alicyclobacillus spp. is an important thermoacidophilic, spore-forming spoilage bacterium that is a major concern for beverage and juice industries. In order to develop effective control strategies and adequately address the prevalence of contamination sources, it is necessary to characterize Alicyclobacillus' ecology in fruit, juice and beverage production and processing environments.Alicyclobacillus spp. isolates were collected from juice, beverage, ingredients, and environmental samples over a period of ten years. A total of 141 isolates were characterized as Alicyclobacillus spp. by 16S rRNA analysis and the most frequently isolated species was found to be Alicyclobacillus acidoterrestris (45%), A. acidocaldarius subsp. acidocaldarius (30%), and A. acidocaldarius (11%).The majority of thermotolerant sporeformers isolated from apple juices and concentrates was found to be A. acidoterrestris (24 out of 36 total apple isolates); while A. acidoterrestris was most frequently associated with citrus, citrus concentrates, and their associated environments, isolated by University of Florida (UF) (15 out of total 28 UF citrus isolates). However, A. acidocaldarius and subsp. acidocaldarius were frequently isolated by Cornell University (CU) (29 out of 35 CU citrus isolates), from citrus juices made from concentrate. Four major haplotypes of Alicyclobacillus spp. were identified based on the 16S rRNA gene sequencing from the 141 isolates tested. The Allelic Types (ATs) matched the phylogenetic analysis grouping of the different Alicyclobacillus spp. based on the isolation source.Our results suggest a predisposition for certain ATs of Alicyclobacillus spp. depending on juice or ingredient isolation source.     

Alicyclobacillus acidoterrestris in pasteurized exotic Brazilian fruit juices: Isolation,genotypic characterization and heat resistance

In this study, the population of Alicyclobacillus spp. was estimated in pasteurized exotic Brazilian fruit juices using the most probable number (MPN) technique followed by biochemical tests. Pasteurized passion fruit (n = 57) and pineapple (n = 50) juices were taken directly from Brazilian manufacturers. While Alicyclobacillus spp. was isolated from passion fruit juice, the microorganism was not found in any pineapple juice samples. A higher incidence of Alicyclobacillus was observed in samples taken in June and July (dry months in Brazil) in comparison to the other months (March, April, May and August), and the highest Alicyclobacillus counts were recovered from these samples(>23 MNP/100 mL). Sixteen (n = 16) Alicyclobacillus strains were typed using the randomly amplified polymorphic DNA method (RAPD-PCR). RAPD-PCR revealed great genetic similarity between the passion fruit juice strains and Alicyclobacillus acidoterrestris DSM 2498. The heat resistance of three isolates was determined, and the mean D_95°  (1.7 min) and z (7.6 °C) values in the passion fruit juice were not significantly different (p > 0.05) from those obtained for the DSM 2498 strain (D_95°  = 1.5 min and z = 7.1 °C). This is the first report on the isolation of A. acidoterrestris from exotic fruit juices such as passion fruit juice. It is worth pointing out the importance of applying good agricultural practices in the field and applying controls for the fruit selection and washing steps, as well as controlling the time/temperature conditions for pasteurization so as to reduce the incidence and chances of A. acidoterrestris spoilage in these juices.     

Use of a multivariate approach to assess the incidence of Alicyclobacillus spp. in concentrate fruit juices marketed in Argentina: results of a 14-year survey

The purpose of this study was to determine the incidence of Alicyclobacillus spp. in fruit/vegetable juices (concentrated pulps and clarified and non-clarified juices) marketed in Argentina between 1996 and 2009.The presence of Alicyclobacillus was determined in a total of 8556 samples of fruit and vegetable juices (apple, pear, grape, peach, blend of juices, tangerine, pineapple, orange, mango, plum, guava, apricot, lemon, banana, kiwi, carrot, strawberry, grapefruit, and beetroot) collected in seven Argentinean provinces. Multiple factor analysis (MFA) was carried out on a data matrix that contained the percentage of positive samples, type of juice, raw material and production year.Except for kiwi and orange, Alicyclobacillus was found in juices from all the evaluated raw materials. The highest percentage of positive samples was found for beetroot, strawberry, banana, peach, mango, carrot and plum juices. The percentage of positive samples for these juices ranged from 100% to 24%.Furthermore, the application of multivariate techniques provided an insight on the relationship between the incidence of Alicyclobacillus and production variables. This approach enabled the identification of the most relevant variables that increased the percentage of positive samples among the juices, which could help in developing strategies to avoid the incidence of this bacterium.By means of hierarchical cluster analysis seven groups (clusters) of juices which showed different percentages of positive samples for Alicyclobacillus spp. were identified. This analysis showed that pineapple, peach, strawberry, mango and beetroot juices had higher rates of positivity for Alicyclobacillus than the rest of the evaluated juices. MFA analysis also showed that some clear relationships could be highlighted between the percentage of samples positive for Alicyclobacillus and five types of fruit juices (strawberry, beetroot, grapefruit, pineapple and mango). It was observed that a large proportion of juices produced in 2000, 2005 and 2008 were located in clusters with higher incidence of Alicyclobacillus spp., whereas a larger proportion of clarified concentrate juice and concentrate pulp samples showed higher probability of incidence of Alicyclobacillus in these products. Data presented in this study brings a contribution to the ecology of Alicyclobacillus in fruit/vegetable juices marketed in Argentina. This information would be useful to enhance the microbiological stability of fruit juices regarding the presence of Alicyclobacillus spp.     

Isolation,identification and typification of Alicyclobacillus acidoterrestris and Alicyclobacillus acidocaldarius strains from orchard soil and the fruit processing environment in South Africa

Alicyclobacillus acidoterrestris and Alicyclobacillus acidocaldarius are thermo-acidophilic, non-pathogenic, spore-forming bacteria that can survive the typical heat processing of fruit juices and concentrates. Bacterial endospores then germinate, grow and cause spoilage of acid food products. Species of Alicyclobacillus were isolated from orchard soil and a fruit concentrate production factory in South Africa. Preliminary identification of the isolates was based on morphological, biochemical and physiological properties. Identification at species level was done by PCR amplification using genus-specific primers and 16S ribosomal RNA (rRNA) gene sequencing. The majority of isolates belonged to the species A. acidoterrestris, but A. acidocaldarius was also isolated and identified. As far as we could determine, this is the first report of the isolation of A. acidoterrestris from wash water and soil outside a fruit processing plant, as well as the isolation of A. acidocaldarius from vinigar flies. The genotypic relatedness between strains of A. acidoterrestris and between strains of A. acidocaldarius was determined by RAPD-PCR. Sixteen isolates identified as A. acidoterrestris grouped into four clusters based on RAPD-PCR banding patterns, suggesting that they belong to at least four genotypic groups. Three isolateT:/PGN/ELSEVIER/YFMIC/web/00001155/s identified as A. acidocaldarius gave three unique banding patterns.     

Isolation and identification of Alicyclobacillus acidocaldarius by 16S rDNA from mango juice and concentrate

In this study we investigate the spoilage of ultra high temperature UHT mango juice as well as a carbonated fruit juice blend to identify organisms contributing to the spoilage. The mango concentrate, the final product, as well as the other ingredients used during manufacturing, were tested for the presence of Alicyclobacillus by polymerase chain reaction (PCR) and sequencing analyses. Microbiological examination of the mango pureé and spoiled fruit juices, using YSG agar [yeast extract 2 g, glucose 1 g, soluble starch 2 g, pH 3.7 (adjust with 2N H₂SO₄), H₂O 1000 mL, bacto agar 15 g] incubated at 55 °C, detected sporeforming, acid dependent and thermotolerant bacteria. The hyper variable region of the 16S rDNA was amplified. The nucleotide sequence of the PCR fragments was determined using the ABI Prism 310 automated DNA sequencer and the collected sequencing data were analysed and compared with the non‐redundant database using NCBI‐BLAST. Alicyclobacillus acidocaldarius were isolated and identified by 16S rDNA gene sequences analyses. The results indicated that the mango purèe, as well as the final product of mango juice and the fruit juice blend, were positive for Alicyclobacillus. The preventative measures of low pH, pasteurization of mango juice and the subsequent use of aseptic packaging were not regarded as sufficient to prevent the outgrowth of Alicyclobacillus spoilage organisms.     

Discrimination of <Emphasis Type="Italic">Alicyclobacillus</Emphasis> Strains by Lipase and Esterase Fingerprints

Alicyclobacillus spp. have recently become a major issue in the fruit juice industry due to its implication in the spoilage of pasteurized juices/beverages. In this study, lipase and esterase fingerprints of 37 Alicyclobacillus strains and 14 Bacillus strains in apple juice were collected using a five-substrate cocktail including four C8-esters and one C4-diester. Characterization of each strain could be finished in a single assay followed by high-performance liquid chromatography (HPLC) analysis for product separation and quantification. The obtained lipase and esterase fingerprints were highly species-dependent. Differences between the relative peak areas of each product for different species were significant. Coupled to chemometric techniques including principal component analysis (PCA) and stepwise linear discriminant analysis (SLDA), the lipase and esterase profiles led to 100 % of correct species identification for Alicyclobacillus and Bacillus strains. This approach is promising, reliable, and with good repeatability and could be used as an alternative tool to discriminate Alicyclobacillus spp. in fruit juices.     

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.     

Isolation of Alicyclobacillus and the influence of different growth parameters

Alicyclobacillus species are thermo-acidophilic, endospore-forming bacteria that are able to survive pasteurisation and have been implicated in a number of spoilage incidents involving acidic foods and beverages. The aim of this study was to compare three isolation methods used for the detection of Alicyclobacillus acidoterrestris and to investigate the influence of incubation temperature on the growth of A. acidoterrestris and A. acidocaldarius. Peach juice samples inoculated with A. acidoterrestris K47 were analysed using either the International Federation of Fruit Juice Producers (IFU) Method No. 12 (Method A), which involved spread plating onto Bacillus acidoterrestris (BAT) agar at pH 4.0; Method B, which involved pour plating using potato dextrose agar (PDA) at pH 3.7; or Method C, which made use of membrane filtration followed by incubation on K agar at pH 3.7. The performance of the three methods differed significantly, with the IFU Method No. 12 recovering the highest percentage of cells at 75.97%, followed by Method B at 66.79% and Method C at 3.43%. These findings strengthen the proposal of the IFU for the use of the IFU Method No. 12 as a standard international method for the detection of Alicyclobacillus. To investigate the effect on growth of different incubation temperatures A. acidoterrestris (three strains) and A. acidocaldarius (two strains) were incubated at either 45 °C or 25 °C. Growth at 25 °C was slower and maximum cell concentrations were lower (1 × 10⁵-10⁶ cfu/mL compared to 1 × 10⁷-10⁸ cfu/mL) than at 45 °C for A. acidoterrestris. A. acidocaldarius was unable to grow at 25 °C and cell concentrations decreased by 1-2 logs. Since a growth temperature of 25 °C could not inhibit growth of A. acidoterrestris, cooling to room temperature (20°-25 °C) is not an effective control measure for A. acidoterrestris inhibition.     

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.     

Mitigation of Alicyclobacillus spp. spores on food contact surfaces with aqueous chlorine dioxide and hypochlorite

The prevalence of Alicyclobacillus spp. and other spore-forming spoilage organisms in food handling and processing environments presents a sanitation challenge to manufacturers of products such as juices and beverages. The objectives of this study were to determine the efficacy of chlorine dioxide and sodium hypochlorite in killing Alicyclobacillus spores in situ and to evaluate the efficacy of various chlorine dioxide and hypochlorite sanitizing regimes on Alicyclobacillus spp. spores on stainless steel, wood, and rubber conveyor material. Five or two log CFU/ml spore concentrations were left in aqueous solution or inoculated onto stainless steel, rubber, or wood coupons and challenged with sanitizer for varied time intervals. After treatment, the coupons were placed in sterile sample bags, massaged with neutralizing buffer, and enumerated on Ali agar. Surfaces were also examined before and after treatment by scanning electron microscopy to confirm destruction or removal of the spores. For both five and two log CFU/ml spore concentrations, treatments of 50 and 100 ppm of chlorine dioxide and 1000 and 2000 ppm of hypochlorite, respectively, were the most effective. Of the range of chlorine dioxide concentrations and contact time regimes evaluated for all surfaces, the most effective concentration/time regime applied was 100 ppm for 10 min. Reductions ranged from 0 to 4.5 log CFU/coupon. Chlorine dioxide was least effective when applied to wood. Hypochlorite was not efficient at eliminating Alicyclobacillus spores from any of the food contact surfaces at any time and concentration combinations tested. Chlorine dioxide is an alternative treatment to kill spores of Alicyclobacillus spp. in the processing environment.     

The Effect of RF Treatment Combined with Nisin Against <Emphasis Type="Italic">Alicyclobacillus</Emphasis> Spores in Kiwi Fruit Juice

In the present study, the efficiency of the treatment of radio frequency (RF) combined with nisin against the spores of three strains of Alicyclobacillus from two species was examined with the response surface methods (RSM). The results showed that the combination of RF and nisin was effective on killing Alicyclobacillus acidoterrestris and Alicyclobacillus contaminans spores in kiwi fruit juice. The killing effect increased with the increase of temperature, holding time, and nisin concentration, but the increment was not significant at high levels of temperature and nisin concentration. The killing effect between A. acidoterrestris and A. contaminans strains was different in kiwi fruit juice. The strain with the highest killing effect was DSM 17975^T, followed by DSM 3923^T and C-ZJB-12-33. The reduction of spore number of DSM 17975^T at the central design points of RSM was about 2.4. Under experimental conditions, nisin showed hardly any effect on killing DSM 17975^T spores when combined with RF.     

Alicyclobacillus acidoterrestris: an increasing threat to the fruit juice industry?

The quality of food can be defined in various ways with processors and consumers considering flavour, odour and appearance as well as extended shelf life among the most important of its attributes. Spoilage of food by bacterial contamination may occur at any point during the processing, altering any one, or all, of these characteristics, rendering the product unusable. Alicyclobacillus spp. and Alicyclobacillus acidoterrestris, in particular, are emerging food spoilage organisms in the fruit juice and fruit juice products industry. Spores of the latter are able to survive heat treatments presently used in the industry and their elimination from products may be used as a measure of the effectiveness of any processing protocol to remove potential spoilage. This paper reviews the history, methods of detection, both traditional and rapid and the protocols that may be effective in controlling the growth of the organism and hence spoilage in the finished product.     

Characterisation of the volatile profile of orange juice contaminated with Alicyclobacillus acidoterrestris

A rapid and reliable analytical method, based on the characterisation of the volatile profile by dynamic headspace extraction followed by gas chromatography mass–spectrometry, was developed in order to early detect Alicyclobacillus acidoterrestris spoilage in orange juice. Gas chromatographic peak areas were submitted to multivariate statistical analysis (principal component and linear discriminant analysis) in order to visualise clusters within samples and to detect the volatile compounds able to differentiate contaminated from not-contaminated samples. Significant differences in the volatile profile of the analysed samples were found, assessing the reliability of the proposed method to detect the A. acidoterrestris contamination in orange juice.     

GC–MS quantification and sensory thresholds of guaiacol in orange juice and its correlation with Alicyclobacillus spp.

Guaiacol is a profoundly negative taint/off-flavour produced by the increasingly common microbial contamination of fruit juices with Alicyclobacillus spp. The objectives of this study included: determining sensory thresholds for guaiacol in orange juice (OJ), developing an analytical method whose detection limits were equivalent to sensory thresholds and determining levels of Alicyclobacillus spp. that would produce detectable levels of guaiacol. A 12 member trained panel was used to establish guaiacol detection and recognition thresholds. Guaiacol ortho and retro nasal detection thresholds in OJ were 0.70 and 0.53 μg/l respectively. Odour recognition threshold was 2.0 μg/l. A SPME GC–MS Selected Ion Monitoring (SIM) procedure was optimised to achieve analytical detection limits of 0.5 μg/l. Optimum guaiacol detection limit was achieved using only responses from m/z 109 and 124. Ion ratios (m/z 109/124) and linear retention index value matching were used to confirm the identification of guaiacol. Quantification was achieved using peak areas from standard guaiacol additions in orange juice between 0.5 and 100 μg/l. Alicyclobacillus growth of 2.2 log CFU/ml in OJ produced just detectable levels (0.7 μg/l) of guaiacol.     

Alicyclobacillus acidoterrestris spores in fruit products and design of pasteurization processes

Alicyclobacillus acidoterrestris is a thermoacidophilic, non-pathogenic and sporeforming bacterium which has been found in commercial pasteurized fruit juices in the past. Only few and recent studies were available in the literature, since only in 1984 Cerny et al. [Cerny, G., Hennlich, W., & Poralla, K. (1984). Fruchtsaftverderb durch bacillen: isolierung und charakterisierung des verderbserregers. Z. Lebensmitt. Unters. Forsch. 179, 224–227] reported a spoiled aseptically packaged apple juice with A. acidoterrestris and in 1987 Deinhard et al. [Deinhard, G., Blanz, P., Poralla, K., & Altan, E. (1987). Bacillus acidoterrestris sp. nov., a new thermotolerant acidophile isolated from different soils. Systematic and Applied Microbiology, 10, 47–53] named first this species. Detection and identification methods for A. acidoterrestris were reviewed and data regarding heat resistance of spores and growth in fruits were collected. Finally, a new methodology to design pasteurization processes for high acidic fruit products is presented.     

Development of Polyclonal Antibody‐Based Indirect Enzyme‐Linked Immunosorbent Assay for the Detection of Alicyclobacillus Strains in Apple Juice

Abstract: A sort of specific polyclonal anti‐Alicyclobacillus antibody was generated by immunizing New Zealand white rabbits, and a sensitive indirect enzyme‐linked immunosorbent assay (ELISA) was developed for Alicyclobacillus detection in apple juice. A set of experimental parameters such as concentration of antigen, dilutions of the antibody and goat anti‐rabbit IgG‐horseradish peroxidase conjugate, selection of the blocking reagent, incubation time, and temperature was optimized. The cross‐reactivity of the antibody was evaluated by ELISA and the result was consistent with Western blot analysis. The detection limit of the ELISA was about 10⁵ colony forming units (CFU)/mL in apple juice samples. Samples were detected by ELISA and conventional culture method, and the ELISA results gave a good agreement with the results obtained by plating on Alicyclobacillus acidoterrestris medium agar. ELISA takes a total detection time of 6 to 7 h, which is less than the time of conventional techniques requiring more than 24 to 48 h. These results indicated that the established ELISA was a potential useful analytical method for detection of Alicyclobacillus in apple juice.     

Behaviour of aroma volatiles during the evaporative concentration of some tropical fruit juices and pulps

The dynamics of aroma separation from some tropical fruit juices and pulps was studied using a thin film evaporator. In the case of mango (Mangifera indica Linn, Alphonso and Totapuri varieties) and guava (Psidium guajava Linn) pulps, 90% of each of the volatile fraction, esters, carbonyls and alcohols, was removed at 30–35% evaporation of the water in a single pass. In the case of pineapple (Ananas sativa Schult F) juice, 90% separation of esters and carbonyls required about 80% and 47% evaporation, respectively. However, the behaviour of alcohols in pineapple juice was found to be similar to that of mango and guava. Mango pulp from Alphonso and Totapuri varieties on 60% evaporation showed a loss in oxygenated terpenes of 80% and 63%, respectively, while guava pulp lost about 58% oxygenated terpenes on 60% concentration. The dynamics of the total aroma volatiles separation from the fruit juices and pulps showed that, in a single pass evaporation, 85–90% volatiles were removed at a juice evaporation degree of 60% water, while the same extent of aroma removal occurred at 32–35% total evaporation by multiple pass.     

Recovery of alicyclobacillus from inhibitory fruit juice concentrates

Growth of Alicyclobacillus in low-pH fruit juices may result in off-odors and off-flavors due to the production of compounds such as guaiacol (2-methoxy phenol). An important step in preventing Alicyclobacillus contamination of fruit juices is the screening of incoming ingredients. Many fruit juice concentrates contain compounds that inhibit Alicyclobacillus growth, but beverages produced from the concentrates may not contain sufficient amounts of the active component to prevent spoilage. Therefore, accurate screening of juice concentrates is essential to prevent false-negative test results and product spoilage. The objective of this study was to evaluate isolation methods for detection of Alicyclobacillus in inhibitory juice concentrates. Recovery of Alicyclobacillus spores from inoculated and naturally contaminated concentrates was compared by using pour plate, spread plate, and filtration methods. Pour plates consistently recovered the lowest number of spores from inoculated concentrates. Spread plating was the most effective method used to recover spores from inoculated apple and pomegranate juice concentrates, while filtration resulted in the highest recovery from cranberry concentrate. When tested on naturally contaminated concentrates, the pour plate method failed to detect Alicyclobacillus in many samples. Filtration was much more effective. The filtration method increased the likelihood of detecting Alicyclobacillus contamination of fruit juice concentrates containing inhibitory compounds.     

Heat resistance of Neosartorya fischeri in various juices

Heat resistance of Neosartorya fischeri was studied in three different juices (apple, pineapple and papaya). The optimum heat activation temperature and time for the ascospores of the N. fischeri (growth for 30 days at 30 °C) was 85 °C for 10 min. Of the three juices tested, apple juice exhibited maximum 1/k values at 80, 85 and 90 °C (208.3, 30.1 and 2.0 min, respectively). The 1/k values for papaya juice (129.9, 19.0 and 1.9 min) and pineapple juice (73.5, 13.2 and 1.5 min) decreased with acidity and °Brix/acidity (ratio) level. The Z^* values for apple, papaya and pineapple juices were 5, 5.5 and 5.9 °C, respectively. The sterilization F values (4-log reduction) for apple, pineapple and papaya juices were 56.3, 38.0 and 7.2 s, respectively. Considering the thermal treatments commercially applied to pineapple (96 °C/30 s) and apple juices (95 °C/30 s), it is concluded that such treatments will not guarantee that less than 1 ascospore in each set of 10³ packs survive. Only the treatment applied to papaya juice (100 °C/30 s) will be sufficient because the F value is less than 30 s.     

Formation of guaiacol from vanillin by<Emphasis Type="Italic"> Alicyclobacillus acidoterrestris</Emphasis> in apple juice: a model study

In this study, the formation of guaiacol from vanillin by Alicyclobacillus acidoterrestris in apple juice was investigated. Apple juices spiked with 10 and 100 mg/L of vanillin were inoculated with 1×10³ CFU/mL and 1×10⁵ CFU/mL spores of A. acidoterrestris and incubated at 25 and 46 °C. A. acidoterrestris started to form guaiacol from vanillin when the spore count exceeded the critical level of 1×10⁴ CFU/mL. Increasing the vanillin concentration also increased the concentration of guaiacol formed, especially at 46 °C. The growth of A. acidoterrestris was not favorable at 25 °C. Thus, the formation of guaiacol is strongly limited at room temperature.