Heat resistance of five spoilage microorganisms in a carbonated broth.

Despite their acidic pH, carbonated beverages can be contaminated by spoilage microorganisms. Thermal treatments, before and/or after carbonation, are usually applied to prevent the growth of these microorganisms. However, the impact of CO2 on the heat resistance of spoilage microorganisms has never been studied. A better understanding of the combined impact of CO2 and pH on the heat resistance of spoilage microorganisms commonly found in carbonated beverages might allow to optimize thermal treatment. Five microorganisms were selected for this study: Alicyclobacillus acidoterrestris (spores), Aspergillus niger (spores), Byssochlamys fulva (spores), Saccharomyces cerevisiae (vegetative cells), and Zygosaccharomyces parabailii (vegetative cells). A method was developed to assess the impact of heat treatments in carbonated media on microbial resistance. The heat resistances of the five studied species are coherent with the literature, when data were available. However, neither the dissolved CO2 concentration (from 0 to 7 g/L), nor the pH (from 2.8 to 4.1) have an impact on the heat resistance of the selected microorganisms, except for As. niger, for which the presence of dissolved CO2 reduced the heat resistance. This study improved our knowledge about the heat resistance of some spoilage microorganisms in presence of CO2.

Quality attributes optimization of orange juice subjected to multi-frequency thermosonication: Alicyclobacillus acidoterrestris spore inactivation and applied spectroscopy ROS characterization.

This is the first time to investigate the synergistic inactivation effect and mechanism of multifrequency ultrasound (MTUS) on A. acidoterrestris (AAT) vegetative cells and spores, nutrients and enzymes of orange juice. The optimized results of MTUS (using Box Behnken design- surface responsemethodology) and further comparison with different mode of ultrasound (mono-and multi-frequency) revealed that 20/40 kHz, 24 min and 64 °C were the best optimum results. The AAT spores and vegetative cells were inactivated by 2 and 4 logs, respectively, without deteriorating orange juice contents. In addition, AAT inactivation indicated an inversely proportional relationship with ROS production. FT-IR and UV-Vis spectroscopy characterization confirmed the existence of ROS in treated orange juice and LF-NMR analysis confirmed the inactivation of AAT spores. The findings illustrated the successfully used dual-frequency ultrasound technology for fruit beverages, promoting beneficial changes in physical properties without any significant effects on the quality of ascorbic acid.

New insights into thermo-acidophilic properties of Alicyclobacillus acidoterrestris after acid adaptation.

Alicyclobacillus acidoterrestris has unique thermo-acidophilic properties and is the main cause of fruit juice deterioration. Given the acidic environment and thermal treatment during juice processing, the effects of acid adaptation (pH 3.5, 3.2, and 3.0) on the resistance of A. acidoterrestris to heat (65 °C, 5 min) and acid (pH = 2.2, 1 h) stresses were investigated for the first time. The results showed that acid adaptation induced cross-protection against heat stress of A. acidoterrestris and acid tolerance response, and the extent of induced tolerance was increased with the decrease of adaptive pH values. Acid adaptation treatments did not disrupt the membrane potential stability and intracellular pH homeostasis, but reduced intracellular ATP concentration, increased cyclic fatty acids content, and changed the acquired Fourier transform infrared spectra. Transcription levels of stress-inducible (dnaK, grpE, clpP, ctsR) genes and genes related to spore formation (spo0A, ctoX) were up-regulated after acid adaptation, and spore formation was observed by scanning electron microscopy. This study revealed that the intracellular microenvironment homeostasis, expression of chaperones and proteases, and spore formation played a coordinated role in acid stress adaptive responses, with implications for applications in fruit juice processing.

Heat resistance and genomics of spoilage Alicyclobacillus spp. Isolated from fruit juice and fruit-based beverages.

Alicyclobacillus acidoterrestris is a spore-forming bacterium of importance to the fruit juice industry due to its remarkable heat resistance and production of guaiacol taint. Whole genome sequencing analysis reveals species demarcation corresponds to the two major genotypic groups to which A. acidoterrestris isolates belong. Heat resistance was significantly different between genotypic groups 1 and 2 with D90 values of 15.5 and 9.3 min, respectively (p < 0.01). Comparison of squalene-hopene cyclase (shc) encoding sequences reveals non-synonymous changes and the alteration of glutamine residues. Glutamine absence may link to the stability reinforcement of the enzyme structure against thermal denaturation. Genomic islands harbouring heavy metal resistance genes are found in the majority of genotypic group 1 genomes (63%) but occurs in only one genome (5%) of genotypic group 2. Distribution of the genomic islands in the genotypic groups 1 and 2 is also consistent with phylogenetic trees and ANI and dDDH values. Subsequently, we propose genotypic group 1 as a new species closely related to A. acidoterrestris that possesses enhanced heat resistance.

Effects of heating and recovery media pH on the heat resistance of Alicyclobacillus acidoterrestris Ad 746 spores.

AIMS: Alicyclobacillus acidoterrestris is a sporulating, acidophilic bacterial species which spoils acidic beverages such as fruit juices. This work aims to quantify the heat resistance of A. acidoterrestris spores and their recovery potential as a function of heating and recovery media pH. METHODS AND RESULTS: The heat treatments were carried out with the strain of A. acidoterrestris Ad 746 in Bacillus acidoterrestris thermophilic medium. The pH of the heating medium from pH 7 to pH 2 nonsignificantly reduced the heat resistance. However, the pH levels of the recovery media strongly affected the apparent heat resistance of this strain. The maximum heat resistance was found when the pH was 4·70 and decreased when the pH decreased to pH 2·8, close to the minimum growth pH and when the recovery medium pH increased to pH 5·3. CONCLUSION: The heating medium pH has a slight effect on the spore heat resistances of this acidophilic species. However, the pH of the recovery media strongly affected the apparent heat resistance of this strain. SIGNIFICANCE AND IMPACT OF THE STUDY: The obtained parameters quantifying the heat resistance of A. acidoterrestris spores are tools to optimize the heat treatments and to control its development.

Analysis of differential expression proteins reveals the key pathway in response to heat stress in Alicyclobacillus acidoterrestris DSM 3922T.

For the purpose of investigating the heat resistance mechanism of Alicyclobacillus acidoterrestris, label-free quantification was used to reveal some cellular changes in A. acidoterrestris during heat stress. Totally, 545 differential expression proteins were respectively identified at heat stress of 65 °C for 5 min, of which 258 proteins were up-regulated and 287 proteins were down-regulated. These significantly changed proteins were mapped to 100 pathways and some of them were mostly related to protection or repair of macromolecules such as proteins and DNA, cell wall formation, which indicated that these proteins might play crucial roles in response to heat stress. The KEGG pathway analysis combined with protein functional analysis and further validation at mRNA level suggested that A. acidoterrestris sensed the temperature rise in environment through alterations in the secondary structure of DNA and RNA molecules. The biosynthesis of antibiotics pathway and the ribosomes might be involved in signal transduction in heat stress and further trigger a large number of proteins playing a critical role in the regulation of heat stress in A. acidoterrestris. The study firstly demonstrated the global physiological response to heat stress and the results provided a better understanding of thermal adaption mechanism of A. acidoterrestris.

Physico-chemical approach to adhesion of Alicyclobacillus cells and spores to model solid materials.

Acidothermophilic bacteria of the genus Alicyclobacillus are frequent contaminants of fruit-based products. This study is the first attempt to characterize the physico-chemical surface properties of two Alicyclobacillus sp. and quantify their adhesion disposition to model materials [diethylaminoethyl (DEAE), carboxyl- and octyl-modified magnetic beads] representing materials with different surface properties used in the food industry. An insight into the mechanism of adhesion was gained through comparison of experimental adhesion intensities with predictions of a colloidal interaction model (XDLVO). Experimental data (contact angles, zeta potentials, size) on interacting surfaces (cells and materials) were used as inputs into the XDLVO model. The results revealed that the most significant adhesion occurred at pH 3. Adhesion of both vegetative cells and spores of two Alicyclobacillus sp. to all materials studied was the most pronounced under acidic conditions, and adhesion was influenced mostly by electrostatic attractions. The most intensive adhesion of vegetative cells and spores at pH 3 was observed for DEAE followed by hydrophobic octyl and hydrophilic carboxyl surfaces. Overall, the lowest rate of adhesion between cells and model materials was observed at an alkaline pH. Consequently, prevention of adhesion should be based on the use of alkaline sanitizers and/or alkaline rinse water.

Dipicolinic Acid Release and the Germination of Alicyclobacillus acidoterrestris Spores under Nutrient Germinants.

The presence of Alicyclobacillus, a thermoacidophilic and spore-forming bacterium, in acidic fruit juices poses a serious problem for the processing industry. A typical sign of spoilage in contaminated juices is a characteristic phenolic off-flavour associated with the production of guaiacol. Spores are formed in response to starvation and in a natural environment re-access the nutrients, e.g.: L-alanine and AGFK - a mixture of asparagine, glucose, fructose and potassium, triggers germination. The aim of this study was to estimate the impact of L-alanine and AGFK on the germination of the spores of two Alicyclobacillus acidoterrestris strains and to evaluate the relationship of the germination rate with dipicolinic acid (DPA) release. The spores were suspended in apple juice or in buffers at pH 4 and pH 7, followed by the addition of L-alanine and AGFK. Suspensions were or were not subjected, to a temperature of 80°C/10 min and incubated for various periods of time at 45°C. Optical density (OD660) was used to estimate the number of germinated spores. The amount of DPA released was determined using HPLC. The results indicate that the degree of germination of A. acidoterrestris spores depended on the strain and time of incubation and the nutritious compounds used. The data obtained show that the amount of DPA released correlated to the number of A. acidoterrestris spores germinated.

Evaluation of hydrophobicity and quantitative analysis of biofilm formation by Alicyclobacillus sp.

Alicyclobacillus sp. are acidothermophilic bacteria frequently contaminating fruit based products (juices and juice concentrates). These sporulating bacteria are able to survive at elevated temperatures and highly acidic environments which causes difficulties in their removal from industrial environments. Although numerous literature data examine Alicyclobacillus sp. presence in fruit based products and methods of their elimination, there is still a limited knowledge on ability of these bacteria to adhere to abiotic surfaces. Therefore, the objective of this study was to determine Alicyclobacillus sp. cells' hydrophobicity and capability of biofilm formation on a glass surface. The degree of cells hydrophobicity, according to Microbial Adhesion to Hydrocarbon (MATH) and Salt Aggregation Test (SAT), was investigated for eleven environmental isolates from natural Polish habitats, identified as Alicyclobacillus sp., and a Alicyclobacillus acidoterrestris DSM 3922 reference strain. The dynamics of biofilm formation within 3-day incubation on a glass surface was evaluated and quantified by a plate count method both, for cultures with and without agitation. All of the bacterial strains tested expressed ability to colonize a glass surface and four environmental isolates were classified as fast-adherent strains. The mature biofilm structures were predominantly formed after 48 hours of incubation. Dynamic culturing conditions were observed to accelerate the biofilm formation. The majority of strains expressed a moderate hydrophobicity level both, in SAT (41.7%) and MATH-PBS (75.0%), as well as MATH-PUM (91.7%) tests. However, no correlation between hydrophobicity and cell adherence to a glass slide surface was observed.

Fate of Alicyclobacillus spp. in enrichment broth and in juice concentrates.

In this study, the fate of Alicyclobacillus acidoterrestris spores in different types of juice concentrates stored under different conditions was investigated. In addition, the impact of dilution procedures during the enrichment step for the detection of Alicyclobacillus in lemon juice concentrates was studied. Pear, red grape, mango, tangerine, carrot and lemon juice concentrates (50-69.4°Brix, pH1.7-4.3) were inoculated with A. acidoterrestris spores (10(3) spore/mL) and stored at 4 °C and 20 °C, after which the spores were counted at 0, 2, 5, 9, 17, 21, 28, 36, 43, and 50 days. No significant differences in the number of Alicyclobacillus spores were observed at storage temperatures of 4 °C and 20 °C (p>0.05). The results also indicated that the number of spores of A. acidoterrestris remained stable in all types of juice concentrates during the storage period, except in lemon juice concentrate. In lemon juice concentrate, a decline in A. acidoterrestris spore populations of 0.3-0.8 log CFU/mL was observed within 5-10 days of storage. The decline in A. acidoterrestris spore populations was more pronounced in cloudy lemon juice concentrate, which contained higher concentrations of flavonoids (mainly eriocitrin and hesperidin) than clarified lemon juice concentrate. It was also found that dilution of lemon juice concentrate samples in the proportion of 1:19 allowed the germination of A. acidoterrestris spores and the growth of populations of up to 10(7) CFU/mL. In contrast, the proportion (1:9) recommended in internationally recognized methods led to a reduction in the population of this microorganism that would yield false negative results. Data presented in this study demonstrated that Alicyclobacillus spores remain stable in most juice concentrates during storage, but that natural antimicrobial compounds present in some of them may decrease spore counts and inhibit their recovery by detection procedures.

Germination and Inactivation of Alicyclobacillus acidoterrestris Spores Induced by Moderate Hydrostatic Pressure.

Given the importance of spoilage caused by Alicyclobacillus acidoterrestris for the fruit juice industry, the objective of this work was to study the germination and inactivation of A. acidoterrestris spores induced by moderate hydrostatic pressure. Hydrostatic pressure treatment can induce the germination and inactivation of A. acidoterrestris spores. At low pH, spore germination of up to 3.59-3.75 log and inactivation of 1.85-2.04 log was observed in a low pressure window (200-300 MPa) applied at 50 degrees C for 20 min. Neutral pH suppressed inactivation, the number of spores inactivated at pH 7.0 was only 0.24-1.06 log. The pressurization temperature significantly affected spore germination and inactivation. The degree of germination in apple juice after pressurization for 30 min with 200 MPa at 20 degrees C was 2.04 log, with only 0.61 log of spores being inactivated, while at 70 degrees C spore germination was 5.94 log and inactivation 4.72 log. This temperature strongly stimulated germination and inactivation under higher (500 MPa) than lower (200 MPa) pressure. When the oscillatory mode was used, the degree of germination and inactivation was slightly higher than at continuous mode. The degree of germination and inactivation was inversely proportional to the soluble solids content and was lowest in concentrated apple juice.

Spore inactivation and DPA release in Alicyclobacillus acidoterrestris under different stress conditions.

This paper reports on the inactivation of spores of 5 strains of Alicyclobacillus acidoterrestris under different stress conditions (acidic and alkaline pH, high temperature, addition of lysozyme, hydrogen peroxide and p-coumaric acid). The research was divided into two different steps; first, each stress was studied alone, thus pointing out a partial uncoupling between spore inactivation and DPA release, as H2O2 reduced spore level below the detection but it did not cause the release of DPA. A partial correlation was found only for acidic and alkaline pH. 2nd step was focused on the combination of pH, temperature and H2O2 through a factorial design; experiments were performed on both fresh and 4 month-old spores and pinpointed a different trend for DPA release as a function of spore age.

External pH is a cue for the behavioral switch that determines surface motility and biofilm formation of Alicyclobacillus acidoterrestris.

Bacteria use different strategies to survive unfavorable environmental conditions. Alicyclobacillus acidoterrestris is a bacterium capable of surviving extremely harsh conditions, for instance, during industrial food processing. A. acidoterrestris is a spore-forming, thermoacidophilic, nonpathogenic bacterium that commonly contaminates commercial pasteurized fruit juices and is, therefore, considered a major microbiological contaminant in the juice industry. The purpose of this study was to elucidate whether A. acidoterrestris is capable of multicellular behavior by testing its ability of biofilm formation and surface motility. A. acidoterrestris was found to be proficient in migration over a surface that is apparently powered by flagella. It was further shown that lowering the external pH leads to inhibition in surface motility of these bacteria. Concomitantly, the reduction in the external pH triggered biofilm formation of A. acidoterrestris cells. Thus, although no significant biofilm was formed at pH 4.5, robust cell adhesion and confluent biofilm formation was seen below the pH 3.6. These findings indicate that the reduction of external pH is an environmental cue for the behavioral switch that inhibits surface motility and triggers biofilm formation of A. acidoterrestris. Gaining insight into the multicellular behavior that facilitates A. acidoterrestris survival in food contact surfaces may contribute to the development of novel antimicrobial means to prevent cross-contamination caused by this bacterium.

Effect of sporulation medium on wet-heat resistance and structure of Alicyclobacillus acidoterrestris DSM 3922-type strain spores and modeling of the inactivation kinetics in apple juice.

Alicyclobacillus acidoterrestris is a spoilage bacterium in fruit juices leading to high economic losses. The present study evaluated the effect of sporulation medium on the thermal inactivation kinetics of A. acidoterrestris DSM 3922 spores in apple juice (pH3.82±0.01; 11.3±0.1 °Brix). Bacillus acidocaldarius agar (BAA), Bacillus acidoterrestris agar (BATA), malt extract agar (MEA), potato dextrose agar (PDA) and B. acidoterrestris broth (BATB) were used for sporulation. Inactivation kinetic parameters at 85, 87.5 and 90°C were obtained using the log-linear model. The decimal reduction times at 85°C (D85°C) were 41.7, 57.6, 76.8, 76.8 and 67.2min; D87.5°C-values were 22.4, 26.7, 32.9, 31.5, and 32.9min; and D90°C-values were 11.6, 9.9, 14.7, 11.9 and 14.1min for spores produced on PDA, MEA, BATA, BAA and BATB, respectively. The estimated z-values were 9.05, 6.60, 6.96, 6.15, and 7.46, respectively. The present study suggests that the sporulation medium affects the wet-heat resistance of A. acidoterrestris DSM 3922 spores. Also, the dipicolinic acid content (DPA) was found highest in heat resistant spores formed on mineral containing media. After wet-heat treatment, loss of internal volume due to the release of DPA from spore core was observed by scanning electron microscopy. Since, there is no standardized media for the sporulation of A. acidoterrestris, the results obtained from this study might be useful to determine and compare the thermal resistance characteristics of A. acidoterrestris spores in fruit juices.

Alicyclobacillus tengchongensis sp. nov., a thermo-acidophilic bacterium isolated from hot spring soil.

A thermo-acidophilic bacterium, designated strain ACK006(T), was isolated from the soil of a hot spring at Tengchong in China. Cells were Gram-staining-positive, motile, catalase-positive and oxidase-negative, spore-forming rods. The isolate grew aerobically at 30-50°C (optimum at 45°C), pH 2.0-6.0 (optimum pH 3.2) and 0-5.0% (w/v) NaCl (optimum 1% NaCl). Phylogenetic analyses based on 16S rRNA gene sequences indicated that strain ACK006(T) belongs to the genus Alicyclobacillus with the sequence similarity of 92.3, 92.4, 92.5, and 92.8% to Alicyclobacillus cycloheptanicus SCH(T), Alicyclobacillus ferrooxydans TC-34(T), Alicyclobacillus contaminans 3-A191(T) and Alicyclobacillus disulfidooxidans SD-11(T), respectively. Similarity to other species of the genus Alicyclobacillus was 90.3-92.8% and similarity to species of the genus Tumebacillus was 85.9-87.8%. The genomic DNA G+C content was 53.7 mol%. The predominant menaquinone was MK-7. Major fatty acids were ω-cycloheptane C18:0, iso-C17:0 and anteiso-C17:0. The cell-wall peptidoglycan was the A1γ type; containing meso-diaminopimelic acid as the diagnostic diamino acid. On the basis of polyphasic analysis from this study, strain ACK006(T) represents a novel species of the genus Alicyclobacillus for which the name Alicyclobacillus tengchongensis sp. nov. is proposed. The type strain is ACK006(T) (=KCTC 33022(T) =DSM 25924(T)).

Growth of Alicyclobacillus acidoterrestris in the Hypoxic Environment of Bottled Fruit Juice.

Alicyclobacillus acidoterrestris, an obligate aerobe and one of the most harmful bacteria in acidic beverages, requires oxygen for growth. However, the relationship between oxygen availability and its growth has not yet been quantified. We examined the correlation between A. acidoterrestris growth and oxygen availability to determine whether A. acidoterrestris can be controlled by restricting oxygen. Airtight containers were filled with YSG broth and apple juice at various oxygen concentrations. Positive correlation (R(2)=0.9329) was observed between A. acidoterrestris growth and oxygen availability in YSG broth, and a lower but nonetheless slight correlation (R(2)=0.5604) was observed for apple juice. These results indicate that decreased oxygen availability in a container could restrict growth. As results, the addition of reducing compounds along with airtight conditions may help prevent the deterioration of beverages caused by the proliferation of A. acidoterrestris.

Inactivation of Alicyclobacillus acidoterrestris spores in aqueous suspension and on apples by neutral electrolyzed water.

Alicyclobacillus acidoterrestris can be difficult to control in fruit juices as their spores survive juice pasteurization temperatures and may subsequently germinate and grow. Contaminated fruits can be regarded as a major source of spoilage caused by A. acidoterrestris in fruit juices. The objective of this study was to evaluate the efficacy of neutral electrolyzed water (NEW) in reducing the number of A. acidoterrestris spores in aqueous suspension and on surface-inoculated apples. Its effectiveness was compared with that of sodium hypochlorite (SH) solutions at free chlorine concentrations of 50 and 200mg/L. Viable spore counts in test suspensions were significantly (P<0.05) reduced after exposure to NEW (200mg/L free chlorine) for 1 min. However, NEW (50mg/L free chlorine) and SH solutions were unable to significantly (P>0.05) reduce the number of viable spore during the same exposure period. More than 5 log reduction in spore counts was achieved by NEW solution containing 200mg/L free chlorine after 5 min of exposure. Exposure to NEW solutions for 3 min yielded more than 4 log reductions in the number of viable spores on apple surfaces. At the same concentrations of free chlorine, NEW was three to more than ten-fold effective than SH in reducing viability of A. acidoterrestris spores in aqueous suspension and on apple surfaces. This finding suggests that NEW can be considered as an effective disinfectant for the control of A. acidoterrestris on fruits.

An immunomagnetic separation-real-time PCR system for the detection of Alicyclobacillus acidoterrestris in fruit products.

Alicyclobacillus acidoterrestris is the most important spoilage species within the Alicyclobacillus genus and has become a major issue in the pasteurized fruit juice industry. The aim of this study was to develop a method combining immunomagnetic separation (IMS) with real-time PCR system (IMS-PCR) for rapid and specific detection of A. acidoterrestris in fruit products. A real-time PCR with the TaqMan system was designed to target the 16S rDNA genes with specific primer and probe set. The specificity of the assay was confirmed using 9 A. acidoterrestris strains and 21 non-A. acidoterrestris strains. The results indicated that no combination of the designed primers and probe was found in any Alicyclobacillus genus except A. acidoterrestris. The detection limit of the established IMS-PCR was less than 10CFU/mL and the testing process was accomplished in 2-3h. For the three types of samples (sterile water, apple juice and kiwi juice), the correlation coefficient of standard curves was greater than 0.991, and the calculated PCR efficiencies were from 108% to 109%. As compared with the standard culture method performed concurrently on the same set of samples, the sensitivity, specificity and accuracy of IMS-PCR for 196 naturally contaminated fruit products were 90.0%, 98.3% and 97.5%, respectively. The results exhibited that the proposed IMS-PCR method was effective for the rapid detection of A. acidoterrestris in fruit products.

Control of Alicyclobacillus acidoterrestris in fruit juices by a newly discovered bacteriocin.

Alicyclobacillus acidoterrestris is one of the most spoilage-causing bacteria in fruit juices. Control of A. acidoterrestris in fruit juices by bificin C6165 (Pei et al. in J Appl Microbiol 114(5):1273-1284, 2013), a bacteriocin produced by Bifidobacterium animalis subsp. animalis CICC 6165, was described in this study. Activity spectrum of bificin C6165 was investigated and sixteen strains of A. acidoterrestris were sensitive to bificin C6165 in diluted Apple Juices. In the commercial fruit juices, vegetative cells of A. acidoterrestris were inactivated by bificin C6165 at 40 µg/ml. The inhibitory effect of bificin C6165 was better at lower pH (pH 3.5) and at a higher temperature of 45 °C. Furthermore, electron microscopy examination of the vegetative cells treated with bacteriocin revealed substantial cell damage and bacterial lysis. The result suggested that primary mode of action of bificin C6165 was most probably due to pore formation. Although no significantly activity of bificin C6165 was observed against the endospores of A. acidoterrestris in commercial apple juice, the addition of bacteriocin contributed to the reduction of the thermal resistance of A. acidoterrestris spores. Additionally, encapsulation of bificin C6165 with Ca-alginate gel was investigated. Encapsulation of bificin C6165 provided a promising method to control A. acidoterrestris in food juice industry.

1,6-Diphenyl-1,3,5-hexatrine as a reporter of inner spore membrane fluidity in Bacillus subtilis and Alicyclobacillus acidoterrestris.

A method to determine the inner membrane fluidity of spore populations was developed using the hydrophobic probe 1,6-diphenyl-1,3,5-hexatriene (DPH). DPH incorporated into the inner spore membrane of Bacillus subtilis ATCC 6633 and Alicyclobacillus acidoterrestris N1100, during static and dynamic fluidity experiments. The membrane fluidity of A. acidoterrestris spores stripped of the outer spore coats was the same as that of native spores. In addition, laser scanning confocal microscopy confirmed the presence of DPH in the spore membrane. This is the first report of spores containing fluid regions of their inner membranes.