Effect of dual-frequency thermosonication, food matrix, and germinants on Alicyclobacillus acidoterrestris spore germination.

The off-odors associated with spoilage of acidic beverages are linked to the germination and growth of Alicyclobacillus acidoterrestris (AAT) spores. As a consequence, we determined the influence of nutrients, non-nutrient germinants, dual-frequency thermosonication (DFTS), and food matrix on spore germination. AAT spores in orange juice (OJ), supplemented by L-alanine (L-ala), had the highest germination rate and lowest DPA content at 10 h of incubation. The formation of microscopic pores in cell membranes during DFTS caused irreversible damage in AAT spores in citrate buffer solution (CBS); however, it stimulated AAT spore germination in CBS containing L-ala. Hence, the germination potential was established in the order: L-ala > Calcium dipicolinate > asparagine, glucose, fructose, and potassium ion mixture (AGFK) > L-valine. The conductivity analysis indicated that membrane damage could be a key factor contributing to the artificial germination in CBS. AFM images revealed that after 2 h of adding L-ala, the protein content increased with increased germinated cells. TEM showed that membrane poration and coat detachment were the main pre-germination morphological changes detected after DFTS treatment. This study provides evidence that germination stimulated with DFTS might be an effective strategy for reducing A. acidoterrestris spores in fruit juices.

Producing natural vanilla extract from green vanilla beans using a ß-glucosidase from Alicyclobacillus acidiphilus.

Current methods for the production of natural vanilla extract are long and tedious, and the efficiency of the vanillin extraction is usually conditioned by different factors during the traditional curing process (temperatures and weather conditions). As an important fraction of vanillin is present in the form of glucovanillin in green beans, endogenous ß-glucosidases contribute to its hydrolysis; however, these enzymes lose efficiency during the curing process. The use of extremophilic organisms as a source of an appropriate exogenous enzyme can offer a valid alternative when producing natural vanillin. Here, a ß-glucosidase from the thermo-acidophilic organism Alicyclobacillus acidiphilus (AacGH1) was cloned, expressed in E. coli BL21, and fully characterized in respect to both function and crystal structure. Notably, AacGH1 was stable at a temperature up to 50 °C and exhibited good tolerance to glucose, fructose and organic solvents, in particular it maintained full activity in the presence of up to 20 % (v/v) ethanol. The enzyme was then successfully applied to an ethanol-water (20 % (v/v)) extract of green vanilla beans and the complete hydrolysis of glucovanillin (1.7 mM) to vanillin, and other flavour compounds commonly found in vanilla, was achieved using 0.5 mg/mL of enzyme in just 15 min at 30 °C.

Metabolic extract of the endophytic fungus Flavodon flavus isolated from Justicia brandegeana in the control of Alicyclobacillus acidoterrestris in commercial orange juice.

In this work, the antibacterial activity of a crude extract of the endophytic fungus Flavodon flavus (JB257), isolated from leaves of Justicia brandegeana, was evaluated against both the vegetative and sporulated forms of Alicyclobacillus acidoterrestris. The microdilution technique was performed in order to determine the antibacterial activity of the crude extract alone as well as in combination with the bacteriocin, nisin. The minimum inhibitory concentration (MIC) of the crude extract and nisin alone against A. acidoterrestris vegetative forms were 250 µg/mL and 31.5 µg/mL, respectively, while the minimum bactericidal concentrations (MBC) were 1000 µg/mL and 62.5 µg/mL,respectively. For A. acidoterrestris spores, treatment with the crude extract at a concentration of 500 µg/mL caused a 47% reduction in growth, while nisin at 62.5 µg/mL could reduce 100% of the growth. The in vitro evaluation of the crude extract combined with nisin against A. acidoterrestris by the Checkerboard method showed a synergistic interaction between the two compounds. In addition, greater selectivity towards bacterial cells over host cells, a human hepatocyte cell line, was achieved when the crude extract was combined with nisin, Using scanning electron microscopy, interferences in the cell membrane of A. acidoterrestris could be observed after treatment with the crude extract. The results presented in this study indicate that the crude extract of the endophyte F. flavus has biotechnological potential in the food industry, especially for the treatment of orange juices through the control of A. acidoterrestris.

Antimicrobial photodynamic treatment as an alternative approach for Alicyclobacillus acidoterrestris inactivation.

Alicyclobacillus acidoterrestris is a cause of major concern for the orange juice industry due to its thermal and chemical resistance, as well as its spoilage potential. A. acidoterrestris spoilage of orange juice is due to off-flavor taints from guaiacol production and some halophenols. The present study aimed to evaluate the effectiveness of antimicrobial Photodynamic Treatment (aPDT) as an emerging technology to inactivate the spores of A. acidoterrestris. The aPDT efficiency towards A. acidoterrestris was evaluated using as photosensitizers the tetracationic porphyrin (Tetra-Py+-Me) and the phenothiazinium dye new methylene blue (NMB) in combination with white light-emitting diode (LED; 400-740 nm; 65-140 mW/cm2). The spores of A. acidoterrestris were cultured on YSG agar plates (pH 3.7 ± 0.1) at 45 °C for 28 days and submitted to the aPDT with Tetra-Py+-Me and NMB at 10 µM in phosphate-buffered saline (PBS) in combination with white light (140 mW/cm2). The use of Tetra-Py+-Me at 10 µM resulted in a 7.3 ± 0.04 log reduction of the viability of A. acidoterrestris spores. No reductions in the viability of this bacterium were observed with NMB at 10 µM. Then, the aPDT with Tetra-Py+-Me and NMB at 10 µM in orange juice (UHT; pH 3.9; 11°Brix) alone and combined with potassium iodide (KI) was evaluated. The presence of KI was able to potentiate the aPDT process in orange juice, promoting the inactivation of 5 log CFU/mL of A. acidoterrestris spores after 10 h of white light exposition (140 mW/cm2). However, in the absence of KI, both photosensitizers did not promote a significant reduction in the spore viability. The inactivation of A. acidoterrestris spores artificially inoculated in orange peels (105 spores/mL) was also assessed using Tetra-Py+-Me at 10 and 50 µM in the presence and absence of KI in combination with white light (65 mW/cm2). No significant reductions were observed (p < .05) when Tetra-Py+-Me was used at 10 µM, however at the highest concentration (50 µM) a significant spore reduction (≈ 2.8 log CFU/mL reductions) in orange peels was observed after 6 h of sunlight exposition (65 mW/cm2). Although the color, total phenolic content (TPC), and antioxidant capacity of orange juice and peel (only color evaluation) seem to have been affected by light exposition, the impact on the visual and nutritional characteristics of the products remains inconclusive so far. Besides that, the results found suggest that aPDT can be a potential method for the reduction of A. acidoterrestris spores on orange groves.

Clove Oil (Syzygium aromaticum L.) Activity against Alicyclobacillus acidoterrestris Biofilm on Technical Surfaces.

Acidotermophilic bacteria Alicyclobacillus acidoterrestris is one of the main contaminants in the fruit industry forming biofilms which are difficult to remove from the production line by conventional methods. An alternative approach aims for the use of essential oils to prevent Alicyclobacillus biofilm development. The effect of clove essential oil on A. acidoterrestris biofilms on glass and polyvinyl chloride surfaces under static and agitated culture conditions was investigated by atomic force microscopy and the plate count method. The medium-flow and the type of technical surface significantly influenced A. acidoterrestris biofilm. The PVC was colonized in a greater extent comparing to glass. Clove essential oil in 0.05% (v/v) caused 25.1-65.0% reduction of biofilms on the technical surfaces along with substantial changes in their morphology by a decrease in the biofilm: height, surface roughness, and surface area difference. The oil also induced alteration in individual bacterial cells length and visible increase of their roughness. Clove essential oil seems to release EPS from biofilm and thus induce detachment of bacteria from the surface. Due to anti-A. acidoterrestris biofilm activity, the clove oil may be used in the juice industry to hinder a development of A. acidoterrestris biofilms on production surfaces.

Further Insight into Polycyclization Cascades of Acyclic Geranylfarnesol and its Acetate by Squalene-hopene Cyclase from Alicyclobacillus acidocaldarius.

The enzymatic reactions of geranylfarnesol (8) and its acetate 9, classified as sesterterpenes (C25), using squalene-hopene cyclase (SHC) were investigated. The enzymatic reaction of 8 afforded 6/6-fused bicyclic 20, 6/6/6-fused tricyclic 21, and 6/6/6/6-fused tetracyclic compounds 22 and 23 as the main products (35% yield), whereas that of 9 afforded two 6/6/6-fused tricyclic compounds 24 and 25 in a high yield (76.3%) and a small amount (5.0%) of 26 (the acetate of 22). A significantly higher conversion of 9 indicates that the arrangement of the substrate in the reaction cavity changed. The lipophilic nature and/or the bulkiness of the acetyl group may have changed its binding with SHC, thus placing the terminal double bond of 9 in the vicinity of the DXDD motif of SHC, which is responsible for the proton attack on the double bond to initiate the polycyclization reaction. The results obtained for 8 are different to some extent than those reported by Shinozaki et al. The products obtained in this study were deprotonated compounds; however, the products reported by Shinozaki et al. were hydroxylated compounds.

Antibacterial activity of papain and bromelain on Alicyclobacillus spp.

Alicyclobacillus spp. are spore forming bacteria that are often related to the deterioration of acidic products such as beverages and citrus juices. After the process of industrial pasteurization, the spore produced by the bacteria can germinate and the microorganism can grow, causing sensory abnormalities in the product. Alternative biopreservatives, such as the antimicrobial compounds, are of considerable importance to the food industry. Papain and bromelain are proteolytic enzymes derived frompapaya and pineapple, respectively. These enzymes are widely used in medicine and in the pharmaceutical and food industries, but while some studies have described their antibacterial action, no studies of the Alicyclobacillus spp. exist. The aimof this studywas to analyze the antibacterial effect of papain and bromelain on Alicyclobacillus spp. through 1) determining minimum inhibitory and bactericidal concentration (MIC and MBC); 2) determining the death time curve of the micro-organism in the presence and absence of enzymes; and 3) investigating the enzymatic mechanism on the microorganism. The antibacterial activity of enzymes in combination with nisin was also evaluated. The results showed that for the Alicyclobacillus acidoterrestris strain, the MIC of papain was 0.98 µg/mL and the MBC was 3.91 µg/mL, while theMIC of bromelain was 62.5 µg/mL and the MBCwas 250 µg/mL. The concentration of 4 ×MIC for both the enzymes was sufficient to eliminate 4 logs of the micro-organism after 24 h of incubation. Through the use of enzyme inhibitors specific for cysteine proteases, it was found that the antibacterial activity of papain and bromelain is not related to its proteolytic activity, butmay be related to other activities, such as amidse and esterase. The synergistic activity of the enzymes revealed a fractional inhibitory concentration (FIC) level of 0.16. Combination with nisin revealed an FIC of 0.25 for papain and 0.19 for bromelain, indicating synergism between both compounds. The application of enzymes in reconstituted orange juice contaminated with A. acidoterrestris was found to be effective, as after 48 h of incubation, at three different temperatures, the initial microbial population was eliminated. This study showed that the enzymes papain and bromelain have an antibacterial effect on A. acidoterrestris.

Evaluation of the antibacterial activity of Piperaceae extracts and nisin on Alicyclobacillus acidoterrestris.

Alicyclobacillus acidoterrestris is a gram-positive aerobic bacterium. This bacterium resists pasteurization temperatures and low pH and is usually involved in the spoilage of juices and acidic drinks. The objective of this study was to evaluate the antibacterial activities of nisin and the species Piper (Piperaceae) on A. acidoterrestris. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) were determined by the broth microdilution method. The species Piper aduncum had the lowest MIC and an MBC of 15.6 µg/mL and was selected for fractionation. Six fractions were obtained, and the dichloromethane fraction (F.3) had the lowest MIC/MBC (7.81 µg/mL). The dichloromethane fraction was again fractionized, and a spectral analysis revealed that the compound was prenylated chromene (F.3.7). The checkerboard method demonstrated that the crude extract (CE) of P. aduncum plus nisin had a synergistic interaction (fractional inhibitory concentration [FIC] = 0.24). The bactericidal activity of (F.3.7) was confirmed by the time-kill curve. P. aduncum, nisin, and prenylated chromene exhibited strong antibacterial activity against the spores and vegetative cells of A. acidoterrestris. The results of this study suggest that extracts of the genus Piper may provide an alternative to the use of thermal processing for controlling A. spoilage.

Inactivation of Alicyclobacillus acidoterrestris in orange juice by saponin extracts combined with heat-treatment.

Alicyclobacillus acidoterrestris is a spoilage-causing bacterium in fruit juices. The inactivation of this bacterium by commercial saponin and saponin purified extract from Sapindus saponaria fruits combined with heat-treatment is described. We investigated heat treatment (87, 90, 95, and 99°C) with incubation time ranging from 0 to 50min, in both concentrated and reconstituted juice. Juices were inoculated with 1.0×10(4)CFU/mL of A. acidoterrestris spores for the evaluation of the best temperature for inactivation. For the temperatures of 87, 90, and 95°C counts of cell viability decreased rapidly within the first 10 to 20min of incubation in both concentrated and reconstituted juices; inactivation at 99°C ensued within 1 and 2min. Combination of commercial saponin (100mg/L) with a very short incubation time (1min) at 99°C showed a reduction of 2.34 log cycle for concentrated juice A. acidoterrestris spores (1.0×10(4)CFU/mL) in the first 24h of incubation after treatments. The most efficient treatment was reached with 300, 400 or 500mg/L of purified extract of saponins from S. saponaria after 5days of incubation in concentrated juice, and after 5days with 300 and 400mg/L or 72h with 500mg/L in reconstituted juice. Commercial saponin and purified extracts from S. saponaria had similar inactivation power on A. acidoterrestris spores, without significant differences (P>0.05). Therefore, purified extract of saponins can be an alternative for the control of A. acidoterrestris in fruit juices.

Germination and inactivation of Bacillus coagulans and Alicyclobacillus acidoterrestris spores by high hydrostatic pressure treatment in buffer and tomato sauce.

Acidothermophilic bacteria like Alicyclobacillus acidoterrestris and Bacillus coagulans can cause spoilage of heat-processed acidic foods because they form spores with very high heat resistance and can grow at low pH. The objective of this work was to study the germination and inactivation of A. acidoterrestris and B. coagulans spores by high hydrostatic pressure (HP) treatment at temperatures up to 60°C and both at low and neutral pH. In a first experiment, spores suspended in buffers at pH 4.0, 5.0 and 7.0 were processed for 10min at different pressures (100-800MPa) at 40°C. None of these treatments caused any significant inactivation, except perhaps at 800MPa in pH 4.0 buffer where close to 1 log inactivation of B. coagulans was observed. Spore germination up to about 2 log was observed for both bacteria but occurred mainly in a low pressure window (100-300MPa) for A. acidoterrestris and only in a high pressure window (600-800MPa) for B. coagulans. In addition, low pH suppressed germination in A. acidoterrestris, but stimulated it in B. coagulans. In a second series of experiments, spores were treated in tomato sauce of pH 4.2 and 5.0 at 100 - 800MPa at 25, 40 and 60°C for 10min. At 40°C, results for B. coagulans were similar as in buffer. For A. acidoterrestris, germination levels in tomato sauce were generally higher than in buffer, and showed little difference at low and high pressure. Remarkably, the pH dependence of A. acidoterrestris spore germination was reversed in tomato sauce, with more germination at the lowest pH. Furthermore, HP treatments in the pH 4.2 sauce caused between 1 and 1.5 log inactivation of A. acidoterrestris. Germination of spores in the high pressure window was strongly temperature dependent, whereas germination of A. acidoterrestris in the low pressure window showed little temperature dependence. When HP treatment was conducted at 60°C, most of the germinated spores were also inactivated. For the pH 4.2 tomato sauce, this resulted in up to 3.5 and 2.0 log inactivation for A. acidoterrestris and B. coagulans respectively. We conclude that HP treatment can induce germination and inactivation of spores from thermoacidophilic bacteria in acidic foods, and may thus be useful to reduce spoilage of such foods caused by these bacteria.