Phylogenetic and spectroscopic analysis of Alicyclobacillus isolates by 16S rDNA sequencing and mid-infrared spectroscopy

Alicyclobacillus spp. are a group of thermophilic, acidophilic, spore-forming bacteria, some of which cause spoilage in commercially pasteurized fruit and vegetable juice products including apple, orange, tomato and carrot juice. In this study, we characterized seven isolates of Alicyclobacillus (14-2, KF, A-Gala 2-1, C-Fuji 6, Gala 9-2, 1016, 18-1) and a reference strain A. acidiphilus DSM 14558 by a 16S rDNA sequencing technique and Fourier transform infrared (FT-IR) spectral features. The degree of similarity between Alicyclobacillus isolates based upon phylogenetic analysis of sequence data and multivariate statistical analysis of FT-IR spectral data was determined. Results from both methods were consistent, suggesting that a combination of methods targeting both 16S rDNA and mid-infrared spectroscopic signatures may be a suitable and more accurate approach to either identify or discriminate Alicyclobacillus isolates from fruit and vegetable juice products.     

Prevalence, concentration, spoilage, and mitigation of Alicyclobacillus spp. in tropical and subtropical fruit juice concentrates

The presence of Alicyclobacillus in fruit juices and concentrates poses a serious problem for the juice industry. This study was undertaken to determine the (i) prevalence, concentration, and species of Alicyclobacillus in tropical and subtropical concentrates; (ii) efficacy of aqueous chlorine dioxide in reducing Alicyclobacillus spp. spores on tropical and subtropical fruit surfaces; and (iii) fate of and off-flavor production by Alicyclobacillus acidoterrestris in mango and pineapple juices. One hundred and eighty tropical and subtropical juice concentrates were screened for the presence and concentration of Alicyclobacillus spp. If found, the species of Alicyclobacillus was determined by 16S rDNA sequencing and analysis with NCI BLAST. Of these samples, 6.1% were positive for Alicyclobacillus, and nine A. acidoterrestris strains and two Alicyclobacillus acidocaldarius strains were identified. A five-strain cocktail of Alicyclobacillus spp. was inoculated onto the surface of fruits (grapefruit, guava, limes, mangoes, oranges and pineapple), which were then washed with 0, 50, or 100 ppm aqueous chlorine dioxide. Significant reductions due to chlorine dioxide were only seen on citrus fruits. A five-strain cocktail of A. acidoterrestris was inoculated into mango and pineapple juices. Microbial populations were enumerated over a 16-day period. Aroma compounds in the juice were analyzed by GC-olfactometry (GC-O) and confirmed using GC-MS. GC-O of mango juice identified previously reported medicinal/antiseptic compounds. GC-O of pineapple juice revealed an unexpected “cheese” off-aroma associated with 2-methylbutyric acid and 3-methylbutyric acid.     

Alicyclobacillus spoilage and isolation--a review

Until recently, acidic products such as fruit juice and fruit based products were generally thought to be susceptible to spoilage by yeasts, mycelia fungi and lactic acid bacteria, as the low pH of these products acts as natural control measures against spoilage by most bacteria. Alicyclobacillus seem to be prevalent in fruit based products as they survive the acidic fruit juice environment, even when they are exposed to pasteurisation temperatures during production. In this review the historical background of the discovery of these bacteria is summarised. The bacterial characteristics and the reported spoilage incidences caused by members of this genus are discussed. As the isolation methods for these bacteria are controversial, this review includes a discussion of the various media that have been reported in the literature for the use in the isolation and enumeration of members of the genus Alicyclobacillus.     

Discrimination between Bacillus and Alicyclobacillus Isolates in Apple Juice by Fourier Transform Infrared Spectroscopy and Multivariate Analysis

Alicyclobacillus is a causative agent of spoilage in pasteurized and heat‐treated apple juice products. Differentiating between this genus and the closely related Bacillus is crucially important. In this study, Fourier transform infrared spectroscopy (FT‐IR) was used to identify and discriminate between 4 Alicyclobacillus strains and 4 Bacillus isolates inoculated individually into apple juice. Loading plots over the range of 1350 and 1700 cm^?1 reflected the most distinctive biochemical features of Bacillus and Alicyclobacillus. Multivariate statistical methods (for example, principal component analysis and soft independent modeling of class analogy) were used to analyze the spectral data. Distinctive separation of spectral samples was observed. This study demonstrates that FT‐IR spectroscopy in combination with multivariate analysis could serve as a rapid and effective tool for fruit juice industry to differentiate between Bacillus and Alicyclobacillus and to distinguish between species belonging to these 2 genera.     

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.     

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.     

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.     

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.     

Occurrence of Alicyclobacillus in the fruit processing environment--a review

Concentrated fruit products have a significant place in modern consumption markets and are valuable semi-prepared food components to the bakery, dairy, confectionary, canning, baby food, frozen food, distilling and beverage industries. There is continuous pressure on the beverage industry to improve the quality of concentrated fruit products in order for reconstituted fruit beverages to compete with beverages that are made from fresh fruits. In recent years, Alicyclobacillus spp. have become a major concern to the beverage industry worldwide as many high-acid, concentrated fruit products have been found to be contaminated with these spoilage microbes. The thermo-acidophilic nature of alicyclobacilli and highly resistant endospores allows for their survival during the production of concentrated fruit products. Under favourable conditions, endospores can germinate and multiply to numbers high enough to cause spoilage and product deterioration through the production of chemical taint compounds. It is imperative to understand the nature of Alicyclobacillus within the fruit concentrate processing environment so as to develop effective control strategies and to prevent spoilage in juice and beverage products that are reconstituted from fruit concentrates. This paper reviews the occurrence of alicyclobacilli in the fruit processing environment, control measures, as well as detection, identification and standardised test methods that are currently used for Alicyclobacillus in concentrated fruit products.     

Spoilage by Alicyclobacillus Bacteria in Juice and Beverage Products: Chemical,Physical, and Combined Control Methods

Alicyclobacillus is a genus of spoilage bacteria causing contamination of juices and other beverage products that cannot easily be contaminated by other microbes because of their high acid contents. During the last 4 decades since the first species of Alicyclobacillus was isolated in 1967, Alicyclobacillus has become a major concern to the global juice and beverage industries, and many promising methods have been developed and applied to control them. After introducing the history and general characteristics of Alicyclobacillus, as well as their heat resistance and spoilage, this review focuses on the control methods against Alicyclobacillus, including chemical and physical methods and combined methods. All these control methods show inhibitory or killing effects against Alicyclobacillus to some extent and, moreover, some of them have been put to use in the juice and beverage industries for decades and shown to be quite effective, although further developments can be achieved and new methods are constantly being established and investigated. Although it is difficult to compare the effects with one another among these methods because of the different experimental conditions in different reports, some of them, such as the treatments of nisin and high hydrostatic pressure, are well studied and proved to have extensive application prospect. The inhibitory factors, test strains and media, and especially the detailed experimental conditions and main results of these control methods are summarized here. The limitations of some methods mainly relating to the changes of products’ sensory qualities are also presented.     

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.     

Diversity of the Predominant Spoilage Bacteria in Water-Boiled Salted Duck during Storage

Abstract: The spoilage microbiota in water-boiled salted duck during storage at 4 °C was determined using culture-dependent and independent methods. Analysis of the denaturing gradient gel electrophoresis (DGGE) patterns of PCR amplicons targeting the V3 region of the 16S rDNA and sequencing of the bands allowed profiling of the microbiota present in the duck. Community DNA extracts were prepared directly from water-boiled salted duck and from culturable bacterial fractions harvested from both MRS and PCA media. The spoilage bacteria mainly consisted of Staphylococcus saprophyticus, Macrococcus caseolyticus, Weissella, Halomonas sp. or Cobetia sp., and Exiguobacterium sp. based on sequencing and homology search of the DGGE bands. It appeared that both the bacterial counts and diversity increased during storage time. By plating method, bacterial counts in MRS agar increased from 10⁴ to 10⁸ CFU/g from day 1 to 10, while total bacterial counts in PCA agar reached 10⁹ CFU/g after 10 d. Total of 14 strains isolated from PCA and MRS agar were identified as M. caseolyticus (2), S. saprophyticus (7), S. sciuri (1), W. paramesenteroides (2), and W. confusa (2) by 16S rDNA sequencing. The identification of the spoilage-related microbiota is helpful to better understand the bacteria ecology in water-boiled salted duck and may lead to the discovery of appropriate preservation strategies.     

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.     

Development of Molecular Typing Methods for Bacillus spp. and Paenibacillus spp. Isolated from Fluid Milk Products

Bacillus spp. and related sporeformers are important food spoilage organisms. While use of molecular subtyping methods has provided important information on the ecology and transmission of foodborne pathogens, the lack of rapid, reliable, and affordable subtyping methods for Bacillus spp. has limited our ability to understand and control their transmission throughout the food chain. We used a previously described collection of Bacillus spp. and Paenibacillus spp. isolated from dairy products to develop a DNA sequencing‐based subtyping approach for these spoilage microorganisms. After optimization of polymerase chain reaction (PCR) parameters, primers targeting the rpoB housekeeping gene allowed for successful amplification in all isolates. rpoB sequencing allowed differentiation of 29 subtypes (that is, sequence types) among the 57 isolates characterized. Phylogenetic analyses of rpoB sequences revealed distinct monophyletic lineages that correlated with bacterial genera (Bacillus and Paenibacillus) as well as with species or species‐like assemblages within each genus. rpoB sequencing provided improved subtype discrimination over 16S rDNA sequencing; therefore, rpoB sequencing allows for both sensitive subtype discrimination as well as for species and genus identification. Analysis of subtypes isolated over time in dairy products revealed the presence of both persistent and transient bacterial subtypes, indicating that application of these methods can improve our understanding of the ecology of these spoilage organisms and can help in identification of bacterial niches that may contribute to the persistence of these spoilage organisms in food systems.     

Isolation and identification of spoilage microorganisms using food-based media combined with rDNA sequencing: Ranch dressing as a model food

Investigating microbial spoilage of food is hampered by the lack of suitable growth media and protocols to characterize the causative agents. Microbial spoilage of salad dressing is sporadic and relatively unpredictable, thus processors struggle to develop strategies to minimize or prevent spoilage of this product. The objectives of this study were to (i) induce and characterize spoilage events in ranch-style dressing as a model food, and (ii) isolate and identify the causative microorganisms using traditional and food-based media, coupled with rDNA sequence analysis. Ranch dressing (pH 4.4) was prepared and stored at 25 °C for 14 d and microbial populations were recovered on MRS agar and ranch dressing agar (RDA), a newly formulated food-based medium. When isolates suspected as the spoilage agents were inoculated into ranch dressing and held at 25 °C for 9–10 d, three unique spoilage events were characterized. Using rDNA sequence comparisons, spoilage organisms were identified as Lactobacillus brevis, Pediococcus acidilactici, and Torulaspora delbrueckii. P. acidilactici produced flat-sour spoilage, whereas Lb. brevis resulted in product acidification and moderate gas production. The RDA medium allowed for optimum recovery of the excessive gas-producing spoilage yeast, T. delbrueckii. The isolation and identification strategy utilized in this work should assist in the characterization of spoilage organisms in other food systems.     

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.     

Enhancing disease resistance in harvested mango (Mangifera indica L. cv. ‘Matisu’) fruit by salicylic acid

To learn how salicylic acid (SA) may affect disease resistance in mango, mango fruit (Mangifera indica L. cv. ‘Matisu’) were treated with 1 mmol L^?1 SA solution under vacuum infiltration for 2 min at a low pressure (?80 kPa) and for an additional 10 min at air pressure. The fruit were inoculated with anthracnose (Colletotichum gloeosporioides Penz.) spore suspension (1 × 10⁴ CFU mL^?1) and incubated at 13 °C, 85–95% RH. Disease incidence and lesion diameter in/on the SA‐treated fruit were 37.5% and 20.9% lower than that in/on control fruit on the 4th day of incubation. The study further showed that activities of defensive enzymes in the fruit were significantly enhanced by SA treatment. The activity of phenylalanine ammonia‐lyase (PAL) and β‐1,3‐glucanase in the SA‐treated fruit was over 6‐ or 0.9‐fold higher than that in control fruit on the 4th day after the fruit being treated with SA, respectively. Level of hydrogen peroxide (H₂O₂) or superoxide radicals (O₂^?) generation rate in SA‐treated fruit was 22.3% or 79.4% higher than that in controls on the 8th day after the fruit being treated with SA, respectively. These results suggested that PAL and β‐1,3‐glucanase, as well as H₂O₂ or O₂^?, may be involved in the enhancement of disease resistance in mango fruit. Copyright © 2006 Society of Chemical Industry     

我国首例因Asaia sp.污染而导致含果汁及果肉饮料变质的案例分析

某含椰果粒椰汁饮料发生变质,外观正常,经检测发现细菌严重超标。污染菌通过16S rDNA序列比对鉴定为Asaia sp.,这是该属微生物第一次在我国检出。该菌能耐受pH值3.0~3.5的酸性环境,能耐受75℃、30min或80℃、10min热处理,耐受0.8g/kg浓度的山梨酸钾。尤其值得重视的是:该菌在36℃~37℃平板计数琼脂培养基上不生长,所以根据GB 47892-2010食品安全国家标准食品微生物检验菌落总数测定规定的方法无法检出该污染菌。因其耐酸、耐热、耐防腐剂和多种抗生素、产生物膜能力较强的特性,Asaia sp.细菌可能对水果、果汁等产品的安全性造成不可忽视的影响。     

Modulation of thermal resistance of ascospores of Neosartorya fischeri by acidulants and preservatives in mango and grape juice

Minimal thermal processing is desirable for near natural organoleptic and nutritional qualities of fruit based products. In the present investigation, the effect of heat (85°C) in combination with acidulants or common preservatives on inactivation of ascospores of Neosartorya fischeri, a heat resistant mould isolated from grapes, has been studied in mango and grape juice. The ascospores were found to survive for >300 min of heating at 70, 75 and 80°C in these fruit juices and complete inactivation required 120 min of heating at 85°C. The synergistic effect of heat and organic acids or preservatives in fruit juices was noticed. The thermal death rate (1/k_85°C) values did not vary much in the presence of lactic (20), malic (20) and citric (19) acids, but tartaric acid showed least inactivation effect (1/k_85°C=54 min) in mango juice. The 1/k_85°Cvalues for ascospores of N. fischeri in mango juice containing 0·1% of potassium sorbate or sodium benzoate or combination of both at 0·05% were found to be 44, 35 and 29 min respectively. These values were respectively, 32, 13 and 14 min in grape juice. Nearly 50 and 67% of the heating time was reduced by the use of potassium sorbate and sodium benzoate (0·05% each) in mango and grape juice to inactivate 3 log number of ascospores of N. fischeri. These results may be useful in thermal processing of fruit juices.     

Ascospore Heat Resistance and Control Measures for Talaromyces flaws Isolated From Fruit Juice Concentrate

Talarotnyces flavus were isolated from fruit juice concentrates in two unrelated spoilage incidences of packaged reconstituted fruit juice. The population of survivors in heated fruit concentrate was 0.6 colony forming units (CFU) per gram. The ellipsoidal ascospores diameters were 4.5 ± 6 μm. Potassium sorbate and sodium benzoate prevented outgrowth of T. flavus, the concentration required was lower when the pH of the growth medium was 3.5 instead of 5.4. Conidia were sensitive to 70% ethanol, but ascospores were resistant. Ascospores required heat activation before maximum outgrowth occurred, the time for activation was less with higher activation temperature. Thermal death rate curves were nonlogarithmic but approached logarithmic death rate at higher temperatures. A calculated thermal destruction rate, using the exponential formula, for 1 log₁₀ was 2–7 min at 90°C depending upon the strain; and the approximate z-value was 10.3°C.