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.     

Evaluation of direct plating methods to enumerate Alicyclobacillus in beverages

Ten agar media were evaluated for their suitability to support spore germination and colony development by six strains of Alicyclobacillus acidoterrestris, three strains of Alicyclobacillus acidocaldarius, and one strain of Alicyclobacillus cycloheptanicus. The influence of plating method (pour versus spread), incubation temperature (43 degrees C and 50 degrees C), and incubation time (up to 10 days) on colony development were determined. K agar, Alicyclobacillus medium (ALI agar), and Bacillus acidoterrestris thermophilic (BAT) agar recovered the highest numbers of spores. Orange serum agar and Hiraishi glucose yeast extract agar were the least suitable. Overall, surface plating was superior to pour plating and, with the exception of one strain of A. acidocaldarius which grew better at 50 degrees C, incubation of K agar, ALI agar, and BAT agar plates at 43 degrees C or 50 degrees C resulted in recovery of equivalent numbers of spores. Essentially all viable spores were detected on media incubated for 3 days at 43 degrees C. The ability of one strain of each Alicyclobacillus species to grow in ten non-carbonated commercially manufactured beverages at 30 degrees C and 43 degrees C was markedly affected by the composition of the beverages. Results show that surface plating samples on BAT agar, followed by incubating plates at 43 degrees C for 3 days provide the most suitable conditions to enumerate ten strains of three species of Alicyclobacillus most commonly responsible for spoilage of beverages.     

Survival and Recovery of Thermally Stressed Yeast in Orange Juice

Injury and survival of thermally stressed yeast in orange juice were assessed by plating on plate count agar, acidified potato dextrose agar, and orange serum agar. Thermally stressed yeast were found to have reduced plate counts on both orange serum agar and acidified potato dextrose agar in comparison to plate count agar. Severely injured populations were not able to repair injury in orange juice held at 25°C but survived storage at 25, 6, and -18°C.     

Aciduric and Heat Resistant Microorganisms in Apple Juice and Cider Processing Operations

Site visits to 15 apple juice or cider facilities were conducted in New York State. Washed apples, pulp, press juice, final product, and other samples, when appropriate, were sampled aseptically. Samples were plated on acidified potato dextrose agar, before and after heating at 70°C for 1-2 hr. For unheated samples, molds predominated in fruit, yeasts and molds in pulp, and yeasts in press and final juice samples. Average counts were 2.8 × 10⁴, 7.3 × 10⁴, 1.7 × 10⁵, and 1.4 × 10⁵/g, respectively. Heat resistant yeasts, molds, and bacteria were isolated frequently, generally at a level of less than 1 per log.     

Comparison of Media and Influence of Petri Plate Position (Inverted or Upright) for Determining Fungal Populations in Bulk-Stored, Dry, Seed-Based Foods

Total populations of fungi and the presence of aflatoxigenic aspergilli were determined in 109 bulk-stored foods predominantly from seed origin offered for sale by retail grocers. Six media were evaluated for their suitability to support development of fungal colonies. The influence of the position of the Petri dish (inverted or upright) on colony formation during incubation was determined. Overall, dichloran-rose bengal-chloramphenicol agar, oxytetracycline-glucose-yeast extract agar, antibiotic-supplemented plate count agar and rose bengal-chlortetracycline agar were superior to acidified potato dextrose agar for recovering yeasts and molds. Considering any given medium, the position of the plate did not significantly (P ≤ 0.05) influence colony development. Aflatoxigenic strains of Aspergillus flavus or A. parasiticus were detected in 3.7% of the samples.     

Isolation and characterization of thermo-acidophilic endospore-forming bacteria from the concentrated apple juice-processing environment

Forty-five thermo-acidophilic, spore-forming bacteria were isolated from a concentrated apple juice-processing environment. All of them were Gram-positive, rod shaped, and strictly aerobic that most likely belong to the genus of Alicyclobacillus. A fast identification method-16S rDNA PCR-RFLP was used to identify them. The results indicated that at the similarity level of 87%, apple juice isolates strains of 1-4 and 1-2-4 clustered with the reference strain of A. acidoterrstris DSM 3922T, and 4-2-1, S-22 and 5-1 with A. cycloheptanicus DSM 4006T, respectively. The other tested strains were different from all the reference strains in this study and may be new species of Alicyclobacillus genus or the other. In order to confirm this conclusion, we selected 7 16S rDNA PCR-RFLP identified strains and 5 type strains of Alicyclobacillus genus, carried on 51 kinds of phenotypic characteristics and analysis the data by unweighted pair group method with arithmetic mean (UPGMA). The results showed that the similarity degree between every two strains was lower than 80%. It also suggested that they may be different from each other and the unidentified strains may be new species. In addition, spoilage effects of them on 12 Brix apple juice were also studied. The result suggested that all 19 tested bacterial strains caused apple juice to become turbid, form a precipitate and off odor at varying rates when incubated at 37 degrees C up to 12 days. It suggested that these bacteria are associated with the spoilage of apple juice during storage.     

Yoghurt whey medium for food-borne yeasts

A selective medium based on yoghurt whey was developed for the enumeration of yeasts in foods. Yoghurt whey agar (YWA) was prepared by mixing one part of a sterile agar solution (5%) with two parts of a sterile whey filtrate obtained from autoclaved (121°C, 20min) plain set yoghurt. the performance of YWA in the enumeration of yeasts was compared with that of plate count agar plus antibiotics (APCA) and acidified potato dextrose agar (PDA-A) by examining food samples and broth cultures of known yeast isolates. APCA ranked first, confirming the superiority of antibiotic amended media over the acidified ones. the overall performance of YWA was comparable to that of PDA-A.     

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.     

酵母甘露聚糖处理对李果实褐腐病及贮藏特性的影响

研究酵母甘露聚糖对李褐腐菌(Monilinia fructicola)菌丝生长的影响及对李果实褐腐病的抑制作用。离体抑菌试验表明5、25、50、100g/L的甘露聚糖对李褐腐菌菌丝生长均有抑制作用,且质量浓度越高抑制效果越好。抑菌试验表明,甘露聚糖浸泡处理能有效抑制处理前/处理后损伤接菌的李果实褐腐病病斑扩展。酵母甘露聚糖处理还有效地降低了贮藏期间李果实的质量损失率和呼吸强度,推迟果实转色,从而维持李果实良好的贮藏品质。     

Heat resistance of Alicyclobacillus acidocaldarius in water, various buffers, and orange juice

The effect of the pH or the composition of the heating medium and of the sporulation temperature on the heat resistance of spores of a thermoacidophilic spore-forming microorganism isolated from a dairy beverage containing orange fruit concentrate was investigated. The species was identified as Alicyclobacillus acidocaldarius. The spores showed the same heat resistance in citrate-phosphate buffers of pH 4 and 7, in distilled water, and in orange juice at any of the temperatures tested (D120 degrees C = 0.1 min and z = 7 degrees C). A raise in 20 degrees C in the sporulation temperature (from 45 to 65 degrees C) increased the heat resistance eightfold (from D110 degrees C = 0.48 min when sporulated at 45 degrees C to 3.9 min when sporulated at 65 degrees C). The z-values remained constant for all sporulation temperatures. The spores of this strain of A. acidocaldarius were very heat resistant and could easily survive any heat treatment currently applied to pasteurize fruit juices.     

培养条件和果汁种类对脂环酸芽孢杆菌生长的影响

脂环酸芽孢杆菌是造成巴氏灭菌果汁腐败的重要细菌之一。鉴于目前脂环酸芽孢杆菌的培养方法尚未统一且污染特征不明确,本研究考察了pH、7种培养基和9种果汁对其生长的影响,同时采用固相微萃取(SPME)和气相色谱(GC)-质谱(MS)技术检测了该菌的主要代谢产物愈创木酚和2,6-二溴苯酚在果汁中的释放情况。结果表明,脂环酸芽孢杆菌在pH4.0时生长情况最好,pH2.5时生长较差,pH6.0~7.0时最差。AAM培养基和BAT培养基较其他5种培养基(YSG培养基、K氏培养基、SK培养基、PDB培养基和营养肉汤培养基)更适合用来培养脂环酸芽孢杆菌。脂环酸芽孢杆菌接种到9种果汁中后生长差异明显,在葡萄汁和橙汁中生长情况最好,细菌数量分别为1.5×104 CFU/mL和8.5×103 CFU/mL;在菠萝汁和西柚汁中生长缓慢,细菌数量分别为4.4×102 CFU/mL和1.9×102 CFU/mL。此外,9种果汁接种脂环酸芽孢杆菌后均未检测出2,6-二溴苯酚;但是,除葡萄汁外的8种果汁均检测出愈创木酚,浓度在8.6~23.9 μg/L之间。     

Factors affecting production of extracellular carbohydrate complexes by Escherichia coli O157:H7

Production of extracellular carbohydrate complexes (ECC) by foodborne pathogens on raw fruits and vegetables may result in protection against removal or inactivation by sanitizers. The influence of environmental conditions on cell growth, the total amount of ECC produced, and the amount of ECC produced on a per cell basis by Escherichia coli O157:H7 strains ATCC 43895 (wild type) and 43895-exopolysaccharides (EPS) (natural mutant, extensive EPS producer) was studied. To determine the effects of pH on the production of ECC on a per cell basis, E. coli O157:H7 was grown aerobically at 12 and 22 degrees C on tryptic soy agar (TSA) acidified to pH 7.0, 6.5, 6.0, 5.5, 5.0, 4.5, and 4.0. Lettuce, alfalfa sprout, cantaloupe, tomato, and apple juice agars (pH 4.46-6.50) were also evaluated for their support of the ECC production. Conditions generally favorable for growth of E. coli O157:H7 were a rich nutrient medium (TSA) vs. heated lettuce juice agar (HLJA) or minimal salts medium (MSM), 22 degrees C compared to 12 degrees C, and an aerobic atmosphere compared to modified atmosphere (1% O(2), 10% CO(2), and 89% N(2)). Conditions favorable for production of ECC on a per cell basis were HLJA, 12 degrees C, and an aerobic atmosphere. This suggests that modified atmosphere packaging of lettuce may not only decrease the growth of E. coli O157:H7 but also its propensity to form biofilm. There was a negative relationship between cell growth and production of ECC on a per cell basis, and environmental conditions that affected the total amount of ECC produced based on initial population reflected a combination of environmental conditions influencing both cell growth and ECC production on a per cell basis. A relative growth index factor (RGIF) was calculated to better understand ECC production as affected by various environmental conditions simultaneously. The production of ECC on a per cell basis by strain 43895-EPS showed a negative linear relationship with pH of TSA at both 12 and 22 degrees C. This strain generally produced a greater amount of ECC on fresh juice agar than on TSA at the same pH, but production of ECC on alfalfa sprout juice agar (FJA, pH 6.45) at 22 degrees C was significantly less than on TSA (pH 6.50). This indicates that nutrient limitation is not based only on nutrient availability. There may be other factors that repress the production of ECC on FJA, and the effects of those factors may be temperature dependent. Further studies will be required to better understand the relationship between nutrient availability and other factors on the production of ECC by E. coli O157:H7 on raw produce.     

Discrimination of Alicyclobacillus strains using nitrocellulose membrane filter and attenuated total reflectance 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. It would be helpful if a rapid method to detect and discriminate Alicyclobacillus strains was available. A simple and rapid sample preparation method using nitrocellulose membrane filter (NMF) and a single reflection horizontal attenuated total reflection (HATR) accessory with Fourier transform infrared (FT-IR) was developed here. Fourier transform infrared (FT-IR) spectroscopy was used and tested on 8 Alicyclobacillus strains (KF, WAC, NWN-13501, NWN-12697, NWN-12654, NWN-10682, 1016, 1101). A linear discriminant analysis (LDA) was established to discriminate Alicyclobacillus strains. The sample preparation method could successfully separated strains into different groups by principal component analysis (PCA). High identification accuracy (95%) was achieved with the LDA model. PRACTICAL APPLICATION: The method developed in the paper can be used to discriminate different Alicyclobacillus strains from each other making it possible to easily determine whether the strain of Alicyclobacillus present is associated with juice spoilage.     

椰毒假单胞菌酵米面亚种选择性分离培养基的比较研究

目的比较4种椰毒假单胞酵米面亚种选择性分离培养基(马铃薯葡萄糖琼脂,PDA;改良马铃薯葡萄糖琼脂,mPDA;椰毒假单胞菌酵米面亚种分离琼脂,PCFA;银耳卵黄氯霉素琼脂,TYCA)的分离效果,为修订GB/T4789.29—2003《食品卫生微生物学检验椰毒假单胞菌酵米面亚种检验》提供技术支持。方法接种椰毒假单胞菌酵米面亚种于4种选择性分离培养基,观察各培养基上目标菌菌落形态,计算生长率;接种10种常见致病菌和环境中常见菌于4种选择性分离培养基,进行生长特异性试验;通过直接涂布和增菌划线的加标试验,验证这4种选择性分离培养基对粪便、食品和环境土壤等不同标本/样品中椰毒假单胞菌酵米面亚种的分离检出情况。结果 mPDA和PCFA能够分别抑制8种和6种致病菌的生长,且椰毒假单胞菌酵米面亚种的生长率都高于75%;在mPDA和PCFA上,目标菌与多数杂菌形态有明显区别,而在PDA和TYCA上,形态相近不易区分;在粪便、土壤和食品等不同标本/样品的直接涂布和增菌划线分离的加标试验中,mPDA和PCFA上的检出率均超过80%,明显高于PDA和TYCA。结论 mPDA和PCFA分离效果比原标准的PDA明显提高,建议在标准修订中增加此两种选择性分离培养基。     

Alicyclobacillus in orange juice: occurrence and heat resistance of spores.

Spore suspensions of a pure culture of Alicyclobacillus acidoterrestris DSM 2498 were submitted to different heat treatments (60 degrees C for 60 min, 60 degrees C for 30 min, 70 degrees C for 20 min, 80 degrees C for 5 min, 80 degrees C for 10 min, 80 degrees C for 30 min, and boiling for 5 min) to determine the best activation conditions in orange juice. The best treatment for spore activation was shown to be 70 degrees C/20 min. Seventy-five samples of concentrated orange juice from 11 different suppliers were examined for the presence of thermophilic acid-tolerant spore formers by the most probable number technique using Bacillus acidocaldarius medium (BAM broth) and incubation at 44 degrees C for 5 days after a prior spore activation. After incubation, isolation was carried out using BAM agar medium incubating at 44 degrees C for 5 days. Typical colonies were submitted to a microscopic examination, evaluation for the presence of spores, and various biochemical tests. Of the orange juice samples examined, 14.7% were found to be positive for Alicyclobacillus. The thermal death time open tube method was used to determine the heat resistance of the spores of strains confirmed as being Alicyclobacillus. The D-values determined were in the range from 60.8 to 94.5 min at 85 degrees C, 10.0 to 20.6 min at 90 degrees C, and 2.5 to 8.7 min at 95 degrees C. The z-values were between 7.2 degrees C and 11.3 degrees C. The results demonstrated the occurrence of Alicyclobacillus in orange juice and the high heat resistance of the spores that could survive the heat treatments normally applied in the processing of orange juice.     

COMPARATIVE ANALYSIS OF TRYPAN BLUE AGAR AND CONGO RED AGAR FOR THE ENUMERATION OF YEAST AND MOLD USING THE HGMF SYSTEM1

Enumeration of yeast and molds from 39 food samples, including 6 dairy products, 2 meat products, 5 cereal products, 9 fruits, 4 vegetables, 6 beverages, 4 spices, and 3 condiments on Trypan blue (at 1: 10,000 dilution) in potato dextrose agar (Trypan blue agar) was compared with Congo red (at 25:1,000,000 dilution) in potato dextrose agar (Congo red agar) using the hydrophobic grid-membrane filter (HGMF) technique of the ISO-GRID system. Yeast and mold grew well on both Trypan blue agar and Congo red agar, producing blue and red colonies, respectively, in the ISO-GRID system, when examined in incan-descent light. However, not all yeast and mold colonies grown on Congo red agar fluoresced under ultraviolet (UV) light (a recommended observation procedure). Thus, results indicated that Trypan blue agar observed in incandescent light is more convenient to use for enumerating yeast and mold in food samples.     

Media for detecting and enumerating yeasts and moulds.

Dilution plating techniques are designed to determine populations of viable fungal propagules per unit weight or volume of food. Direct plating techniques, on the other hand, are designed to assess the internal mycoflora of individual pieces of foods, e.g., seeds or dried fruits, and results are expressed as a percentage of infected pieces. Both techniques are used by industry and regulatory agencies to monitor levels of fungal contamination at various stages of food handling, storing, processing and marketing. Peptone (0.1%) water is commonly used as a diluent for samples to be homogenized or blended. Buffered diluents containing up to 30% glucose or 60% sucrose are recommended for enumerating xerophiles. No one medium is satisfactory for detection or enumeration of yeasts and moulds in all foods. Dichloran rose bengal chloramphenicol agar, oxytetracycline glucose yeast extract agar and rose bengal chloramphenicol agar are superior to acidified potato dextrose agar for enumeration of yeasts and moulds. Dichloran 18% glycerol agar performs well for enumerating moderately xerophilic yeasts and moulds. Fastidious xerophiles require media containing high concentrations of sugars and/or sodium chloride. Media have been formulated to detect potentially aflatoxigenic aspergilli and mycotoxigenic strains of penicillia and fusaria, but increased selectivity and specificity of media for detecting mycotoxigenic moulds are needed. Heat-resistant mould ascospores often require heat treatment prior to plating in order to activate the germination process. The spread-plate technique is strongly preferred over the pour-plate technique for enumerating yeasts and moulds. The recommended incubation temperature is 25 degrees C, but incubation time between plating and counting colonies ranges from 5 days for determination of general populations of mycoflora to 4 weeks or more for fastidious xerophiles. There is a need for new and improved media for selectively isolating various groups, genera, species and/or strains of fungi capable of growing only under specific environmental conditions, e.g., low aw or, in the case of sublethally injured cells, under conditions which facilitate resuscitation. Improved media are needed which accurately detect moulds producing specific mycotoxins in a wide range of food types.     

3 种脂环酸芽孢杆菌分离培养基的性能评价

目的：比较3 个厂家的K培养基、酵母粉淀粉葡萄糖（yeast extract starch glucose medium,YSG）培养基和耐酸耐热芽孢菌（Alicyclobacillus）培养基（Bacillus acidoterrestris medium,BAT）对脂环酸芽孢杆菌的检测效果。方法：选用标准菌株、分离株及实际样品对3 个厂家（广东环凯微生物科技有限公司（简称HK,下同）、厂家Ⅰ和厂家Ⅱ）的K培养基、YSG培养基和BAT培养基进行生长菌落数及分离效果的评估。结果：酸土脂环酸芽孢杆菌标准株和脂环酸芽孢杆菌分离株4-2在K、YSG和BAT 3 种培养基上生长的菌落数（3 个厂家培养基上的菌落数平均数）均无显著性差异（P＞0.05）;酸热脂环酸芽孢杆菌标准株和脂环酸芽孢杆菌分离株5-3在K培养基上不生长,在YSG培养基和BAT培养基上生长良好,其菌落平均数统计学上无显著性差异（P＞0.05）;对实际样品中酸土脂环酸芽孢杆菌的分离,3 个厂家的同种培养基分离率和符合率相当;对实际样品中酸热脂环酸芽孢杆菌的分离,在YSG和BAT培养基上,HK和厂家Ⅱ的分离率和符合率相当,优于厂家Ⅰ。结论：K培养基不适合所有脂环芽孢杆菌,但分离酸土脂环酸芽孢杆菌效果很好,YSG培养基和BAT培养基适合所有脂环酸芽孢杆菌。HK和厂家Ⅱ的培养基优于厂家Ⅰ。     

Fate of Escherichia coli O157:H7 and Listeria monocytogenes in strawberry juice and acidified media at different pH values and temperatures

Survival and growth of Escherichia coli O157:H7 and Listeria monocytogenes in strawberry juice and acidified media at different pH levels (pH 3.4 to 6.8) and temperatures were studied. Sterile strawberry juice (pH 3.6) and acidified trypticase soy broth (TSB) media (pH 3.4 to 6.8) were inoculated with approximately 6.7 log CFU/ml E. coli O157:H7 or 7.3 log CFU/ ml L. monocytogenes, incubated for 3 days at 4 and 37 degrees C. Bacterial levels were determined after 2 h, 1 day, and 3 days using surface plating nonselectively on tryptic soy agar and selectively on sorbitol MacConkey agar for E. coli O157:H7 or modified Oxford agar for L. monocytogenes. A spectrophotometer (660 nm) was also used to study growth inhibition of L. monocytogenes in different TSB and strawberry juice media (pH 3.4 to 7.3). E. coli O157:H7 survived well at pH values of 3.4 to 6.8 at 4 degrees C, but the number of injured cells increased as pH decreased and incubation time increased. At 37 degrees C, E. coli O157:H7 was inactivated at pH of < or = 3.6 but could grow at pH 4.7. L. monocytogenes was quickly injured at pH of < or = 4.7 within 2 h of storage at 4 degrees C and then was slightly and gradually inactivated as storage time increased. L. monocytogenes survived well at pH 6.8 at 4 degrees C and grew well at 37 degrees C. Growth of L. monocytogenes at 37 degrees C was inhibited in TSB by 1% citric acid and 0.5% malic acids at pH 3.4 or by 50% strawberry juice at pH 4.7. Bacterial injury and inactivation appeared to be induced by the acids in strawberry juice. The acids, pH value, temperature, and time were important factors for bacterial survival, inactivation, and growth in the media tested.     

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.