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.

Extraction, partial purification and characterisation of vanillic acid decarboxylase from Alicyclobacillus acidoterrestris DSM 3923.

BACKGROUND: Vanillic acid decarboxylase (VAD) is the key enzyme responsible for guaiacol production in Alicyclobacillus acidoterrestris; however, information related to this enzyme is currently unavailable. The aim of this study is to characterise the VAD from A. acidoterrestris. RESULTS: Specific activity of VAD in vanillic acid-induced A. acidoterrestris DSM 3923 cells was highest in the early stage of the log phase, and almost undetectable in the stationary and death phases. Of the four techniques used to extract VAD, sonication was found to be the most effective and recovered 3.23 U mg(-1) of VAD. Through optimisation of the crucial parameters for sonication, the recovery of VAD had more than doubled (6.81 U mg(-1) ). The crude enzyme extract was purified by ammonium sulfate precipitation and a 9.87-fold purification was obtained. The partially purified VAD exhibited optimum activity at pH 6.0-6.5, 45°C and was stable at pH 5.0-7.5, 20-45°C. The Km and Vmax values of the VAD were 0.53 mmol L(-1) and 96 U mg(-1) protein, respectively. VAD activity was stimulated by Co(2+) and Mn(2+) , but was inhibited by Ni(2+) , Cu(2+) , Ba(2+) and Fe(3+) . Cinnamic acid, ferulic acid, resveratrol, quercetin and rutin at the concentration of 1 mmol L(-1) could completely inhibit the activity of VAD. CONCLUSION: The present study provides the first report on the characteristics of the VAD from A. acidoterrestris, which will contribute to the development of more effective control methods to minimise A. acidoterrestris-related spoilage in fruit juices. © 2015 Society of Chemical Industry.

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.

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.

Micronized benzoic acid decreases the concentration necessary to preserve acidic beverages against Alicyclobacillus.

AIMS: The aim of this study was a challenge testing the effect of lower concentrations of micronized benzoic acid against two strains of Alicyclobacillus. METHODS AND RESULTS: The effect of micronized benzoic acid was compared with the usual levels of untreated commercial sodium benzoate and benzoic acid, at the challenge temperature of 45°C. The size of the benzoic acid particles was determined by scanning electron microscopy. The diameter of the micronized particles was around 10 µm with a maximum length of 200 µm, while the untreated preservative structures were irregular with lengths up to 500 µm. A continuous bactericidal effect against two Alicyclobacillus strains, throughout the 28-day period, was observed with 50 mg l(-1) of micronized benzoic acid, but when the untreated preservative was used, the same lethal effect was not achieved even after doubling its concentration. CONCLUSIONS: The antimicrobial activity of benzoic acid was improved by micronization. The process proved to be an effective alternative to reduce the benzoic acid concentration necessary to ensure stability of an orange juice matrix. SIGNIFICANCE AND IMPACT OF THE STUDY: The results proved that the micronization process represents an alternative to reduce the required food preservative concentration; this method increased the stability of the compound, which maintains its bioavailability.