Comparative assessment of antibacterial activity of Matricaria chamomilla L. extract, nisin and of its combination against Alicyclobacillus spp.

Alicyclobacillus spp. is a potential spoiling agent of acidic products and citrus drinks, leading to sensory alterations in contaminated products and consequent economic losses. Treatments such as pasteurization eliminate vegetative cells, but also create a favorable atmosphere for spore germination. To guarantee quality and safety, the application of natural substances as bioconservatives is a considerable and promising alternative for the food industry. This study evaluated the effect of hexane extract of Matricaria chamomilla L. (HE), Nisin (N) and their combination (HE + N). These compounds are present in some studies describing their antibacterial action, but no studies were found on the association of these compounds against the species Alicyclobacillus spp. This study aimed to analyze the antioxidant activity (AA) for the DPPH• (0,23 µmol Trolox/mg) and ABTS (27.93 µmol Trolox/mg), the Checkboard test revealed synergism between HE and N with a fractional inhibitory index (FIC) of 0.068., and to study the antibacterial and sporicidal effect. The antibacterial and sporicidal activity was satisfactory against Alicyclobacillus acidoterrestris with MIC and MBC of 1.95 µg/mL and MSC of 7.81 µg/mL in analyzes using HE + N. The application in orange juice proved to be effective, with an MBC of 0.007 µg/mL. The MIC results served as a parameter for other tests carried out in this study, such as flow cytometry and Scanning Electron Microscopy (SEM), and for the evaluation of sensory characteristics with Electronic Nose (E-nose).

Influence of physicochemical characteristics on the growth and guaiacol production of Alicyclobacillus acidoterrestris in fruit juices.

Alicyclobacillus acidoterrestris is a bacterium known for causing spoilage in the taste and odour of fruit juices due to its thermoacidophilic nature. Its spoilage is attributed to the formation of guaiacol, which requires the presence of suitable precursors in the juices that A. acidoterrestris can metabolize. Therefore, A. acidoterrestris could exhibit different behaviour depending on the physicochemical characteristics the juice. In this study, we aimed to evaluate the behaviour of five A. acidoterrestris strains in seven different fruit juices by monitoring total cell and spore populations and quantifying guaiacol production. Also, physicochemical and phenolic profile, focusing on antimicrobials and guaiacol precursors, were analysed to better understand differences. Results showed growth in orange, apple, and plum juices for all the tested strains, with total cell populations reaching approximately 7 log cfu/mL, except for plum juice. In persimmon juice, growth was only observed in 3 out of 5 strains, for both total cells and spores. In contrast, all strains were inhibited in peach, black grape, and strawberry juices, maintaining a consistent population around 4 log cfu/mL. A strong negative correlation was observed between bacterial population and compounds such as kaempferol (for strains R3, R111, and P1), cyanidin chloride (for strains R111 and P1), and p-coumaric acid (for strain 7094 T). Regarding guaiacol production, orange and persimmon juices exhibited the highest guaiacol levels, with strain P1 (362.3 ± 12.6 ng/mL) and strain EC1 (325.1 ± 1.4 ng/mL) as the top producers, respectively. Plum, black grape, and strawberry juices showed similar guaiacol concentrations (16.9 ± 2.8 to 105.0 ± 33.7 ng/mL). Vanillin was showed positive correlations with guaiacol production in almost all strains (7094 T, R3, R111, and P1), with correlation coefficients of 0.97, 0.99, 0.82, and 0.87, respectively. We have reported different behaviour of A. acidoterrestris strains depending on juice type. Despite growth inhibition observed in some juices, enough guaiacol quantities to spoil the juice can be produced. This highlights the necessity of exploring strategies to prevent guaiacol production, even under growth restriction.

Efficacy, kinetics, inactivation mechanism and application of cold plasma in inactivating Alicyclobacillus acidoterrestris spores.

As spores of Alicyclobacillus acidoterrestris can survive traditional pasteurization, this organism has been suggested as a target bacterium in the fruit juice industry. This study aimed to investigate the inactivation effect of cold plasma on A. acidoterrestris spores and the mechanism behind the inactivation. The inactivation effect was detected by the plate count method and described by kinetic models. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), the detection of dipicolinic acid (DPA) release and heat resistance detection, the detection and scavenging experiment of reactive species, and cryo-scanning electron microscopy were used to explore the mechanism of cold plasma inactivation of A. acidoterrestris. The results showed that cold plasma can effectively inactivate A. acidoterrestris spores in saline with a 3.0 ± 0.3 and 4.4 ± 0.8 log reduction in CFU/mL, for 9 and 18 min, respectively. The higher the voltage and the longer the treatment time, the stronger the overall inactivation effect. However, a lower gas flow rate may increase the probability of spore contact with reactive species, resulting in better inactivation results. The biphasic model fits the survival curves better than the Weibull model. SEM and TEM revealed that cold plasma treatment can cause varying degrees of damage to the morphology and structure of A. acidoterrestris spores, with at least 50 % sustaining severe morphological and structural damage. The DPA release and heat resistance detection showed that A. acidoterrestris spores did not germinate but died directly during the cold plasma treatment. 1O2 plays the most important role in the inactivation, while O3, H2O2 and NO3- may also be responsible for inactivation. Cold plasma treatment for 1 min reduced A. acidoterrestris spores in apple juice by 0.4 ± 0.0 log, comparable to a 12-min heat treatment at 95 °C. However, as the treatment time increased, the survival curve exhibited a significant tailing phenomenon, which was most likely caused by the various compounds in apple juice that can react with reactive species and exert a physical shielding effect on spores. Higher input power and higher gas flow rate resulted in more complete inactivation of A. acidoterrestris spores in apple juice. What's more, the high inactivation efficiency in saline indicates the cold plasma device provides a promising alternative for controlling A. acidoterrestris spores during apple washing. Overall, our study provides adequate data support and a theoretical basis for using cold plasma to inactivate A. acidoterrestris spores in the food industry.

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.

Alicyclobacillus spp. in fruit-based products: Isolation, identification, quantitative assessment (SPME/GC-MS) of spoilage compounds and spore's resistance to thermal shocks.

Alicyclobacillus spp. is the cause of great concern for the food industry due to their spores' resistance (thermal and chemical) and the spoilage potential of some species. Despite this, not all Alicyclobacillus strains can spoil fruit juices. Thus, this study aimed to identify Alicyclobacillus spp. strains isolated from fruit-based products produced in Argentina, Brazil, and Italy by DNA sequencing. All Alicyclobacillus isolates were tested for guaiacol production by the peroxidase method. Positive strains for guaiacol production were individually inoculated at concentration of 103 CFU/mL in 10 mL of orange (pH 3.90) and apple (pH 3.50) juices adjusted to 11°Brix, following incubation at 45 °C for at least 5 days to induce the production of the following spoilage compounds: Guaiacol, 2,6-dichlorophenol (2,6-DCP) and 2,6-dibromophenol (2,6-DBP). The techniques of micro-solid phase extraction by headspace (HS-SPME) and gas-chromatography with mass spectrometry (GC-MS) were used to identify and quantify the spoilage compounds. All GC-MS data was analyzed by principal component analysis (PCA). The effects of different thermal shock conditions on the recovery of Alicyclobacillus spores inoculated in orange and apple juice (11°Brix) were also tested. A total of 484 strains were isolated from 48 brands, and the species A. acidocaldarius and A. acidoterrestris were the most found among all samples analyzed. In some samples from Argentina, the species A. vulcanalis and A. mali were also identified. The incidence of these two main species of Alicyclobacillus in this study was mainly in products from pear (n = 108; 22.3 %), peach (n = 99; 20.5 %), apple (n = 86; 17.8 %), and tomato (n = 63; 13 %). The results indicated that from the total isolates from Argentina (n = 414), Brazil (n = 54) and Italy (n = 16) were able to produce guaiacol: 107 (25.8 %), 33 (61.1 %) and 13 (81.2 %) isolates from each country, respectively. The PCA score plot indicated that the Argentina and Brazil isolates correlate with higher production of guaiacol and 2,6-DCP/2,6-DBP, respectively. Heatmaps of cell survival after heat shock demonstrated that strains with different levels of guaiacol production present different resistances according to spoilage ability. None of the Alicyclobacillus isolates survived heat shocks at 120 °C for 3 min. This work provides insights into the incidence, spoilage potential, and thermal shock resistance of Alicyclobacillus strains isolated from fruit-based products.

Fabrication and characterization of antibacterial epsilon-poly-L-lysine anchored dicarboxyl cellulose beads.

Pathogens in the food and environment pose a great threat to human health. To solve this problem, we described a novel route to synthesize antibacterial epsilon-poly-L-lysine (EPL) anchored dicarboxyl cellulose beads. Cellulose beads were prepared via a sol-gel transition method and oxidized by sodium periodate and sodium chlorite to form carboxyl groups. EPL was anchored on the beads using carbodiimide mediated amidation. The structure and morphology of beads were characterized by FTIR, XPS, XRD, SEM, and TGA. After dissolution and regeneration, the crystalline form of cellulose is transformed from cellulose I to cellulose II. The thermal degradation temperature of the beads is 200∼300 °C.The samples displayed excellent antimicrobial activity against Staphylococcus aureus, Alicyclobacillus acidoterrestris and Escherichia coli within 12 h. The beads could be biodegraded in soil after 20 days. The biodegradable beads exhibited great potential in food and environmental applications.

Control of the growth of Alicyclobacillus acidoterrestris in industrialized orange juice using rosemary essential oil and nisin.

The use of rosemary essential oil (RO) and its combination with nisin (RO+N) in preventing the multiplication of Alicyclobacillus acidoterrestris in orange juice was evaluated. The minimum inhibitory and bactericidal concentrations (MIC and MBC) for RO were both 125 µg ml-1 while RO+N displayed a synergistic effect. The use of RO and RO+N at concentrations of 1, 4 and 8× MIC in orange juice for 96 h was evaluated in terms of their sporicidal effectiveness. With regard to the action against A. acidoterrestris spores, RO at 8× MIC was sporostatic, whereas RO+N at 1× MIC was sporicidal. Morphological changes in the structure of the micro-organism after treatment were also observed by microscopy. Furthermore, flow cytometric analysis showed that most cells were damaged or killed after treatment. In general, the antioxidant activity after addition of RO+N decreased with time. The results demonstrate that using the combination of RO and nisin can prevent the A. acidoterrestris growth in orange juice.

Development of the Saltatory Rolling Circle Amplification Assay for Rapid and Visual Detection of Alicyclobacillus acidoterrestris in Apple Juice.

A novel nucleic acid isothermal amplification method based on saltatory rolling circle amplification (SRCA) for rapid and visual detection of Alicyclobacillus acidoterrestris in apple juice was established. Fourteen A. acidoterrestris strains and 44 non-A. acidoterrestris strains were used to confirm the specificity. The sensitivity of SRCA was 4.5 × 101 CFU/mL by observing the white precipitate with the naked eye, while it was 4.5 × 100 CFU/mL by fluorescence visualization. The detection limit of SRCA in artificially inoculated apple juice was 7.1 × 101 and 7.1 × 100 CFU/mL via visualization of the white precipitate and fluorescence, respectively. Compared with the traditional PCR method, SRCA exhibited at least a 100-fold higher sensitivity and 100-fold lower detection limit. Seventy samples were investigated for A. acidoterrestris contamination, and the results showed 100% sensitivity, 97.01% specificity, and 97.14% accuracy compared with those by the conventional microbiological cultivation method. Overall, this method is a potentially useful tool for visual and rapid detection of A. acidoterrestris.

Effect of ultraviolet (UV-C) radiation on spores and biofilms of Alicyclobacillus spp. in industrialized orange juice.

Bacteria of the genus Alicyclobacillus pose serious quality problems for the juice processing industries that have sought effective alternatives for its control. The present study evaluated the effect of UV-C radiation on the reduction of spores and biofilm formation of Alicyclobacillus spp. on stainless steel and rubber surfaces using industrialized orange juice as a culture medium. Four reference Alicyclobacillus spp. species and different UV-C dosages were investigated. After exposed for 20 min (16.8 kJ/m2) to UV-C, the spores of Alicyclobacillus acidoterrestris, Alicyclobacillus herbarius, and Alicyclobacillus cycloheptanicus decreased drastically more of 4 log CFU/mL, with counts below the detection limit of the method (<1.7 log CFU/mL), while the Alicyclobacillus acidocaldarius spores were more sensitive to UV-C, once this spore reduction was observed within 15 min (12.6 kJ/m2). Morphological changes in the Alicyclobacillus acidoterrestris spores were observed by scanning electron microscopy. A reduction of biofilm formation was observed for all UV-C treatments, and the higher reductions (approximately 2 log CFU/mL) were found for the Alicyclobacillus acidocaldarius species after 30 min (26.2 kJ/m2), on the stainless steel and rubber surfaces. The results suggest that UV-C can be used to reduce the biofilm formation and could be a promising alternative for controlling Alicyclobacillus spp. spores in industrialized orange juice.

Label free quantitative analysis of Alicyclobacillus acidoterrestris spore germination subjected to low ambient pH.

Inhibition of spore germination or sterilization after induction of spore germination would effectively control low pH food spoilage caused by Alicyclobacillus acidoterrestris spores. However, the characteristics and mechanisms of A. acidoterrestris spore germination in low ambient pH remains poorly understood. In this study, the germination rate of A. acidoterrestris spores at different ambient pH conditions was determined, and subsequently the proteomic profiles of A. acidoterrestris in spore germination were analysed by label-free quantification, in which the specific metabolic pathways involved were identified and key functional proteins were screened and validated using RT-qPCR (real time quantitative PCR). The suitable ambient pH value for the spore germination of A. acidoterrestris ranged from 3.0 to 5.0 with the optimum pH of 4.0. According to the LC-ESI-MS/MS (liquid chromatography electrospray ionization tandem mass spectrometry) analysis, 98 proteins of geminated spores of A. acidoterrestris incubated for 2 h at pH 3.0 were changed significantly in comparison to non-germinated spores, the expression of 20 proteins were up-regulated and that of 78 proteins down-regulated respectively. Those differential expressed proteins were mainly involved in cell wall hydrolysis, cell morphological changes, protein synthesis and folding, perception of external stimuli and signal transduction etc., and we observed that germination receptor D (GerD), cell wall hydrolase, transpeptidase, peptidase S1 and two-component regulatory system phoR were significantly up-regulated, but hydrolase NlpC/P60, peptidoglycan glycosyltransferase, spore coat proteins CotX, CotJB and the Lrp/AsnC (leucine-responsive regulatory protein/asparagine synthase C products) protein were significantly down-regulated in the experiment, which implied the important roles of identified proteins during the spore germination. Furthermore, the pathway analysis showed the possible involvement of differentially expressed proteins in the ß-lactam resistance, ribosome, biosynthesis of secondary metabolites, pyruvate metabolism, two-component system and other metabolic pathways, which indicated that synthesis and hydrolysis of cell wall, intracellular substance synthesis, energy generation and signal transduction were likely associated with the initiation of spore germination and restoration of vegetative growth. In conclusion, the quantitative proteomic landscape of A. acidoterrestris spores could provide the theoretic and experimental evidences for the hazard control of A. acidoterrestris spores in the thermal pasteurization process of acidic beverages industry.

Development and validation of predictive models for the effect of storage temperature and pH on the growth boundaries and kinetics of Alicyclobacillus acidoterrestris ATCC 49025 in fruit drinks.

This study was undertaken to provide quantitative tools for predicting the behavior of the spoilage bacterium Alicyclobacillus acidoterrestris ATCC 49025 in fruit drinks. In the first part of the study, a growth/no growth interface model was developed, predicting the probability of growth as a function of temperature and pH. For this purpose, the growth ability of A. acidoterrestris was studied at different combinations of temperature (15-45 °C) and pH (2.02-5.05). The minimum pH and temperature where growth was observed was 2.52 (at 35 and 45 °C) and 25 °C (at pH ≥ 3.32), respectively. Then a logistic polynomial regression model was fitted to the binary data (0: no growth, 1: growth) and, based on the concordance index (98.8%) and the Hosmer-Lemeshow statistic (6.226, P = 0.622), a satisfactory goodness of fit was demonstrated. In the second part of the study, the effects of temperature (25-55 °C) and pH (3.03-5.53) on A. acidoterrestris growth rate were investigated and quantitatively described using the cardinal temperature model with inflection and the cardinal pH model, respectively. The estimated values for the cardinal parameters Tmin, Tmax, Topt and pHmin, pHmax, pHopt were 18.11, 55.68, 48.60 °C and 2.93, 5.90, 4.22, respectively. The developed models were validated against growth data of A. acidoterrestris obtained in eight commercial pasteurized fruit drinks. The validation results showed a good performance of both models. In all cases where the growth/no growth interface model predicted a probability lower than 0.5, A. acidoterrestris was, indeed, not able to grow in the tested fruit drinks; similarly, when the model predicted a probability above 0.9, growth was observed in all cases. A good agreement was also observed between growth predicted by the kinetic model and the observed kinetics of A. acidoterrestris in fruit drinks at both static and dynamic temperature conditions.

Bacteria, mould and yeast spore inactivation studies by scanning electron microscope observations.

Spores are the most resistant form of microbial cells, thus difficult to inactivate. The pathogenic or food spoilage effects of certain spore-forming microorganisms have been the primary basis of sterilization and pasteurization processes. Thermal sterilization is the most common method to inactivate spores present on medical equipment and foods. High pressure processing (HPP) is an emerging and commercial non-thermal food pasteurization technique. Although previous studies demonstrated the effectiveness of thermal and non-thermal spore inactivation, the in-depth mechanisms of spore inactivation are as yet unclear. Live and dead forms of two food spoilage bacteria, a mould and a yeast were examined using scanning electron microscopy before and after the inactivation treatment. Alicyclobacillus acidoterrestris and Geobacillus stearothermophilus bacteria are indicators of acidic foods pasteurization and sterilization processes, respectively. Neosartorya fischeri is a phyto-pathogenic mould attacking fruits. Saccharomyces cerevisiae is a yeast with various applications for winemaking, brewing, baking and the production of biofuel from crops (e.g. sugar cane). Spores of the four microbial species were thermally inactivated. Spores of S. cerevisiae were observed in the ascus and free form after thermal and HPP treatments. Different forms of damage and cell destruction were observed for each microbial spore. Thermal treatment inactivated bacterial spores of A. acidoterrestris and G. stearothermophilus by attacking the inner core of the spore. The heat first altered the membrane permeability allowing the release of intracellular components. Subsequently, hydration of spores, physicochemical modifications of proteins, flattening and formation of indentations occurred, with subsequent spore death. Regarding N. fischeri, thermal inactivation caused cell destruction and leakage of intracellular components. Both thermal and HPP treatments of S. cerevisiae free spores attacked the inner membrane, altering its permeability, and allowing in final stages the transfer of intracellular components to the outside. The spore destruction caused by thermal treatment was more severe than HPP, as HPP had less effect on the spore core. All injured spores have undergone irreversible volume and shape changes. While some of the leakage of spore contents is visible around the deformed but fully shaped spore, other spores exhibited large indentations and were completely deformed, apparently without any contents inside. This current study contributed to the understanding of spore inactivation by thermal and non-thermal processes.

Analysis of the germination proteins in Alicyclobacillus acidoterrestris spores subjected to external factors.

The presence of Alicyclobacillus acidoterrestris, a thermoacidophilic and spore-forming bacterium, in pasteurized acidic juices poses a serious problem for the processing industry. Therefore, the use of other more effective techniques, such as high hydrostatic pressure (HHP) and supercritical carbon dioxide (SCCD), is considered for preserving juices in order to inactivate these bacteria, while reducing the loss of nutrients and sensory quality of juices. On the other hand, HHP and SCCD when combined with a moderately elevated temperature can induce germination of bacterial spores, making them more vulnerable to inactivation. The spore germination can be also induced by nutrients, such as L-alanine or a mixture of asparagine, glucose, fructose and potassium ions (AGFK). The aim of this work was to determine whether applying activating agents: HHP, SCCD and nutrient germinants (L-alanine and the AGFK mixture), could influence the number of spores which start to germinate and how this affects the proteins involved in the spore germination. SDS-PAGE was used to resolve proteins isolated from the A. acidoterrestris spores. The results that were obtained indicate that the germination of A. acidoterrestris spores treated with HHP, SCCD and nutrient germinants reflect the number of spores which start to germinate. The SDS-PAGE data indicated changes in the level of selected proteins occurring when subjected to the germination activating factors as well as noticeable differences in those proteins' molecular weights.

Continuous ohmic heating of commercially processed apple juice using five sequential electric fields results in rapid inactivation of Alicyclobacillus acidoterrestris spores.

Spores of Alicyclobacillus acidoterrestris, a spoilage bacterium, cause problems for the apple juice industry because they are resistant to thermal treatment. Here, we examined the sporicidal effect of an ohmic heating (OH) system with five sequential electric fields and compared it with that of conventional heating. Apple juice product (50kg) inoculated with A. acidoterrestris spores were subjected to OH (electric field strength=26.7V/cm; frequency=25kHz) at 85-100°C for 30-90s. The effect of conventional heating was also examined under these conditions. OH treatment at 100°C for 30s resulted in total inactivation of the inoculum, with no recovery of viable cells (initial population=4.8-4.9logCFU/ml), whereas 3.6-4.9logCFU/ml of the spores survived conventional heating. OH did not alter the quality (°Brix, color, and pH) of commercial apple juice (p>0.05). These results suggest that the OH system is superior to conventional heating for rapid sterilization (30s) of apple juice to assure microbiological quality in the absence of chemical additives.

Application of bacteriocin RC20975 in apple juice.

Alicyclobacillus acidoterrestris is one of the most spoilage-causing bacteria in fruit juices. In this paper, controlling A. acidoterrestris in apple juice by bacteriocin RC20975 was described. Twenty-one strains of A. acidoterrestris were used to investigate the activity spectrum of bacteriocin RC20975 in apple juice with the result that 16 strains were sensitive . The ratio of activity in apple juice to the activity in laboratory medium was 42%. The reduction of antimicrobial activity in apple juice might be due to problems related to its interaction with food components. Adsorption of bacteriocin RC20975 to A. acidoterrestris cells varied according to the strains and the testing conditions (pH and temperatures). In an acid environment (pH 3 and pH 5), the adsorption was higher than that of the neutral environment. Dynamic model of killing bacteria was built under the condition of different temperatures with the addition of bacteriocin. Electron microscopy examination of vegetative cells revealed substantial cell damage and bacterial lysis after bacteriocin treatment. Although the endospores could not be killed, the addition of bacteriocin RC20975 contributed to the reduction of the thermal resistance of A. acidoterrestris spores in apple juice. In sum, bacteriocin RC20975 was proved to have a good effect on killing A. acidoterrestris in apple juice.

UV Tolerance of Spoilage Microorganisms and Acid-Shocked and Acid-Adapted Escherichia coli in Apple Juice Treated with a Commercial UV Juice-Processing Unit.

The enhanced thermal tolerance and survival responses of Escherichia coli O157:H7 in acid and acidified foods is a major safety concern for the production of low-pH products, including beverages. Little is known about this phenomenon when using UV light treatments. This study was conducted to evaluate the effects of strain (E. coli O157:H7 strains C7927, ATCC 35150, ATCC 43895, and ATCC 43889 and E. coli ATCC 25922) and physiological state (control-unadapted, acid adapted, and acid shocked) on the UV tolerance of E. coli in apple juice treated under conditions stipulated in current U.S. Food and Drug Administration regulations. A greater than 5-log reduction of E. coli was obtained under all tested conditions. A significant effect of strain (P < 0.05) was observed, but the physiological state did not influence pathogen inactivation (P ≥ 0.05). The UV sensitivity of three spoilage microorganisms (Aspergillus niger, Penicillium commune, and Alicyclobacillus acidoterrestris) was also determined at UV doses of 0 to 98 mJ/cm(2). Alicyclobacillus was the most UV sensitive, followed by Penicillium and Aspergillus. Because of the nonsignificant differences in UV sensitivity of E. coli in different physiological states, the use of an unadapted inoculum would be adequate to conduct challenge studies with the commercial UV unit used in this study at a UV dose of 14 mJ/cm(2). The high UV tolerance of spoilage microorganisms supports the need to use a hurdle approach (e.g., coupling of refrigeration, preservatives, and/or other technologies) to extend the shelf life of UV-treated beverages.

Degradation of phenolic compounds by the lignocellulose deconstructing thermoacidophilic bacterium Alicyclobacillus Acidocaldarius.

Alicyclobacillus acidocaldarius, a thermoacidophilic bacterium, has a repertoire of thermo- and acid-stable enzymes that deconstruct lignocellulosic compounds. The work presented here describes the ability of A. acidocaldarius to reduce the concentration of the phenolic compounds: phenol, ferulic acid, ρ-coumaric acid and sinapinic acid during growth conditions. The extent and rate of the removal of these compounds were significantly increased by the presence of micro-molar copper concentrations, suggesting activity by copper oxidases that have been identified in the genome of A. acidocaldarius. Substrate removal kinetics was first order for phenol, ferulic acid, ρ-coumaric acid and sinapinic acid in the presence of 50 µM copper sulfate. In addition, laccase enzyme assays of cellular protein fractions suggested significant activity on a lignin analog between the temperatures of 45 and 90 °C. This work shows the potential for A. acidocaldarius to degrade phenolic compounds, demonstrating potential relevance to biofuel production and other industrial processes.

Development of a Polyclonal Antibody-Based Sandwich Enzyme-Linked Immunosorbent Assay for the Detection of Spores of Alicyclobacillus acidoterrestris in Various Fruit Juices.

A polyclonal rabbit antibody-based sandwich ELISA for the rapid and specific detection of spores of Alicyclobacillus acidoterrestris was established. The reactivity of the antisera with spores was confirmed by immunofluorescence. For a thorough evaluation of the ELISA, 61 strains and isolates of Alicyclobacillus spp. were characterized regarding their guaiacol production ability and genetic variability. The ELISA was highly sensitive, the detection limits were isolate-dependent and ranged from 2.1 × 10(3) - 3.8 × 10(4) spores/mL, except for one isolate, for which a slightly lower sensitivity (5 × 10(5) spores/mL) was observed. Inclusivity tests revealed that the ELISA reacts with all tested A. acidoterrestris, while no cross-reactions with spores of 30 strains of Bacillus spp. and Clostridium spp. were observed. Further on, the assay applicability was tested with orange, apple (clear and unfiltered), tomato, pink grapefruit, pear, and white grape juices. Juices were inoculated with 1 or 10 spores/mL of A. acidoterrestris. After enrichment for 48 h, the established ELISA enabled the reliable and reproducible detection of contaminated samples. The enriched samples could be applied directly to the assay, underlining the robustness of the developed ELISA method.

Effects of preservatives on Alicyclobacillus acidoterrestris growth and guaiacol production.

Alicyclobacillus acidoterrestris can survive the pasteurization process, multiply in pasteurized juices and produce guaiacol which causes medicinal or antiseptic off-flavors. Chemical preservatives have the potential to suppress outgrowth of surviving populations during subsequent storage of fruit juices. In the present study, the individual effects of potassium sorbate, sodium benzoate, potassium metabisulfite, dehydroacetic acid, ethyl 4-hydroxybenzoate, cinnamic acid and ε-polylysine on A. acidoterrestris growth and guaiacol production were firstly evaluated in a laboratory medium. Of the seven preservatives investigated, only dehydroacetic acid, cinnamic acid and ε-polylysine were effective both in controlling growth and guaiacol formation by A. acidoterrestris. Then, these three antimicrobials were applied to apple juice. Through the addition of 270 mg/L dehydroacetic acid, 108 mg/L cinnamic acid or 100 mg/L ε-polylysine, the A. acidoterrestris counts were reduced by 3.43, 3.17 and 4.78 log colony forming unit(CFU)/mL, respectively, and no guaiacol was detected after 14 days of storage. Sensory evaluation revealed that the addition of these three preservatives did not affect the organoleptic properties of the apple juice. Results obtained in this paper could be very useful for a better control of A. acidoterrestris-related spoilage in the fruit juice/beverage industry.

Effects of pomegranate and pomegranate-apple blend juices on the growth characteristics of Alicyclobacillus acidoterrestris DSM 3922 type strain vegetative cells and spores.

The present study examined the growth characteristics of Alicyclobacillus acidoterrestris DSM 3922 vegetative cells and spores after inoculation into apple, pomegranate and pomegranate-apple blend juices (10, 20, 40 and 80%, v/v). Also, the effect of sporulation medium was tested using mineral [Bacillus acidoterrestris agar (BATA) and Bacillus acidocaldarius agar (BAA)] and non-mineral containing media [potato dextrose agar (PDA) and malt extract agar (MEA)]. The juice samples were inoculated separately with approximately 10(5)CFU/mL cells or spores from different sporulation media and then incubated at 37°C for 336 h. The number of cells decreased significantly with increasing pomegranate juice concentration in the blend juices and storage time (p<0.001). Based on the results, 3.17, 3.53, and 3.72 log cell reductions were observed in 40%, 80% blend and pomegranate juices, respectively while the cell counts attained approximately 7.17 log CFU/mL in apple juice after 336 h. On the other hand, the cell growth was inhibited for a certain time, and then the numbers started to increase after 72 and 144 h in 10% and 20% blend juices, respectively. After 336 h, total population among spores produced on PDA, BATA, BAA and MEA indicated 1.49, 1.65, 1.67, and 1.28 log reductions in pomegranate juice; and 1.51, 1.38, 1.40 and 1.16 log reductions in 80% blend juice, respectively. The inhibitory effects of 10%, 20% and 40% blend juices varied depending on the sporulation media used. The results obtained in this study suggested that pomegranate and pomegranate-apple blend juices could inhibit the growth of A. acidoterrestris DSM 3922 vegetative cells and spores.