Evaluation of temperature, pH and nutrient conditions in bacterial growth and extracellular hydrolytic activities of two Alicyclobacillus spp. strains.

Extremophile bacteria have developed the metabolic machinery for living in extreme temperatures, pH, and high-salt content. Two novel bacterium strains Alicyclobacillus sp. PA1 and Alicyclobacillus sp. PA2, were isolated from crater lake El Chichon in Chiapas, Mexico. Phylogenetic tree analysis based on the 16SrRNA gene sequence revealed that the strain Alicyclobacillus sp. PA1 and Alicyclobacillus sp. PA2 were closely related to Alicyclobacillus species (98% identity and 94.73% identity, respectively). Both strains were Gram variable, and colonies were circular, smooth and creamy. Electron microscopy showed than Alicyclobacillus sp. PA1 has a daisy-like form and Alicyclobacillus sp. PA2 is a regular rod. Both strains can use diverse carbohydrates and triglycerides as carbon source and they also can use organic and inorganic nitrogen source. But, the two strains can grow without any carbon or nitrogen sources in the culture medium. Temperature, pH and nutrition condition affect bacterial growth. Maximum growth was produced at 65 °C for Alicyclobacillus sp. PA1 (0.732 DO600) at pH 3 and Alicyclobacillus sp. PA2 (0.725 DO600) at pH 5. Inducible extracellular extremozyme activities were determined for ß-galactosidase (Alicyclobacillus sp. PA1: 88.07 ± 0.252 U/mg, Alicyclobacillus sp. PA2: 51.57 ± 0.308 U/mg), cellulose (Alicyclobacillus sp. PA1: 141.20 ± 0.585 U/mg, Alicyclobacillus sp. PA2: 51.57 ± 0.308 U/mg), lipase (Alicyclobacillus sp. PA1: 138.25 ± 0.600 U/mg, Alicyclobacillus sp. PA2: 175.75 ± 1.387 U/mg), xylanase (Alicyclobacillus sp. PA1: 174.72 ± 1.746 U/mg, Alicyclobacillus sp. PA2: 172.69 ± 0.855U/mg), and protease (Alicyclobacillus sp. PA1: 15.12 ± 0.121 U/mg, Alicyclobacillus sp. PA2: 15.33 ± 0.284 U/mg). These results provide new insights on extreme enzymatic production on Alicyclobacillus species.

One-pot synthesis of magnetic self-assembled carrageenan-ε-polylysine composites: A reusable and effective antibacterial agent against Alicyclobacillus acidoterrestris.

Epsilon-polylysine (EPL) is a cationic polymer with broad antibacterial activity. Base on this property, anionic carrageenan (CG) was self-assembled with EPL and introduced into the one-pot coprecipitation process to fabricate the magnetic Fe3O4-CG-EPL composites. To demonstrate the successful synthesis of composites, characterization techniques including XRD, FT-IR, TEM, XPS and VSM were employed. The prepared composites exhibited effective antibacterial activity against Alicyclobacillus acidoterrestris with the minimum inhibitory concentration of 10.0 mg mL-1. Investigations into deciphering the antibacterial mechanism demonstrated that the presence of Fe3O4-CG-EPL caused irreversible damage to the cell membrane and serious leakage of intracellular protein, resulting in the inactivation of bacteria. The aim of this work is to develop a new effective control method for A. acidoterrestris in food field.

Characterization and antibacterial properties of epsilon-poly- l-lysine grafted multi-functional cellulose beads.

In recent years, harmful microorganisms in water pose great harm to ecological environment and human health. To solve this problem, epsilon-poly-l-lysine (EPL) grafted cellulose beads were prepared via 2, 2, 6, 6-tetramethylpiperidine-1-oxyl (TEMPO) mediated oxidation and carbodiimide mediated cross-linking reaction. Hydroxyl groups on C6 of cellulose were oxidized to carboxyl groups by TEMPO and grafting reaction was achieved between newly formed carboxyl groups of cellulose and amino of EPL. The beads were characterized by FTIR, SEM, XRD and TGA. The crystalline form of cellulose transformed from cellulose I to cellulose II after being dissolved and regenerated. The grafted cellulose beads showed good antibacterial activities against Gram-negative Escherichia coli, Gram-positive Staphylococcus aureus and Alicyclobacillus acidoterrestris with 10 h. The beads could be biodegraded in soil after 28 days. It is expected that the bio-based composite beads could have potential applications in water purification and food treatment fields.

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.

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.

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.

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.

Characterization and antibacterial effect of quaternized chitosan anchored cellulose beads.

Some thermoduric food spoilage bacteria pose great threat to beverage industry. To tackle the challenge, hydroxypropyl trimethyl ammonium chloride chitosan (HTCC) grafted magnetic cellulose beads have been prepared via a dropping technology. Sodium periodate oxidation process was carried out to form dialdehyde functional groups on the regenerated cellulose beads mixed with maghemite nanoparticles. HTCC was anchored on the beads through Schiff base reaction. The structure and properties of HTCC anchored beads were evaluated by Fourier transform infrared (FTIR) spectra, field emission scanning electron microscope (FESEM), energy dispersive X-ray spectroscopy (EDS), X-ray diffraction (XRD) and vibrating sample magnetometer (VSM). Thermal stability of the beads was estimated by thermogravimetric analysis (TGA) and differential thermal gravity (DTG), and the decomposition temperature of the beads were around 200-300 °C. A long-term antibacterial activities of the beads against Alicyclobacillus acidoterrestris were confirmed caused by the covalent bond between HTCC and the beads. The biodegradable HTCC grafted cellulose beads may provide a novel approach for food safety management.

Transcriptomic Analysis of the Molecular Mechanisms Underlying the Antibacterial Activity of IONPs@pDA-Nisin Composites toward Alicyclobacillus acidoterrestris.

A simple and no-drug resistance antibacterial method was developed by the synthesis of heat-stable and pH-tolerant nisin-loaded iron oxide nanoparticles polydopamine (IONPs@pDA) composites. The composites had a crystal structure and diameters of 25 ± 3 nm, with a saturation magnetization ( Ms) of 43.7995 emu g-1. Nisin was successfully conjugated onto the IONPs@pDA nanoparticles, as evinced by Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy analyses. The novel synthesized material showed good performance in reducing Alicyclobacillus acidoterrestris, a common food spoilage bacterium that represents a significant problem for the food industry. Treatment of A. acidoterrestris cells with composites resulted in membrane damage, as observed by live/dead staining and scanning electron microscopy and transmission electron microscopy analyses. Further, the composites exhibited highly efficient antibacterial activity against cells in only 5 min. Transcriptomic sequencing of culture RNA pools after exposure to composites resulted in a total of 334 differentially expressed genes that were primarily associated with transcriptional regulation, energy metabolism, membrane transporters, membrane and cell wall syntheses, and cell motility. Thus, these results suggested that changes in transcriptional regulation caused by aggregated composites on target cells led to major changes in homeostasis that manifested by decreased energy metabolism, pore formation in the membrane, and repressed cell wall synthesis. Concomitantly, cell motility and sporulation activities were both repressed, and finally, intracellular substances flowed out of leaky cells. The proposed biocontrol method represents a novel means to control microorganisms without inducing drug resistance. Further, these results provide novel insights into the molecular mechanisms underlying the antibacterial activity of composites against microorganisms.

Application of iron oxide nanoparticles @ polydopamine-nisin composites to the inactivation of Alicyclobacillus acidoterrestris in apple juice.

Iron oxide nanoparticles (IONPs) modified with polydopamine were covalently immobilized with nisin to inhibit the growth of Alicyclobacillus acidoterrestris in apple juice. The minimum bactericidal concentration (MBC) of IONPs @ pDA-nisin composites against Alicyclobacillus acidoterrestris cells and spores in three kinds of apple juice were 1.25 mg/mL and 2.5 mg/mL, respectively. The concentration of cells and spores decreased from 106 to 10 CFU/mL in apple juice after pretreatment with the composites at the concentration of 20 mg/mL for 10 min. Meanwhile, the investigation of the content of polyphenols, organic acids, volatile compounds and sugars indicated that there was no significant influence of the composites on the apple juices. The results of cytotoxicity (HepG2, Caco 2, SH-SY5Y, and BV2 cells) and acute toxicity test demonstrated the biosecurity and nontoxicity of the composites, which suggested that it was promising to apply the proposed composites to apple juice industry for the control of Alicyclobacillus acidoterrestris pollution.

The Role of Solid Support Bound Metal Chelators on System-Dependent Synergy and Antagonism with Nisin.

Active packaging can enhance the performance of natural antimicrobials in controlling food spoilage and waste, while addressing consumer demands for cleaner labels. Yet, synergies are system dependent, with some conditions counterintuitively promoting antagonistic effects. In particular, metal chelators can improve performance of certain natural antimicrobials and have been incorporated in nonmigratory metal chelating active packaging technologies. However, the influence of chelating ligand chemistry on antimicrobial efficacy has not been investigated in microbial spoilage models. The effect of three commercial chelating resins on the growth of Alicyclobacillus acidoterrestris ATCC 49025, a thermoduric acidophilic spore-former, in growth media and apple juice was investigated. Dowex MAC-3, Chelex 100, and Lewatit TP260 were used as models for metal chelating active packaging containing carboxylic acid (CA), iminodiacetic acid (IDA), and aminomethylphosphonic acid (AMPA) ligands. Diameters (CA = 472.4 ± 117.2 µm, IDA = 132.93 ± 26.71 µm, and AMPA = 498.3 ± 29.24 µm), dissociation kinetics (CA = 6.44 ± 0.109, IDA = -0.977 ± 9.94, AMPA = 7.43 ± 0.193), and metal chelating capacities (CA = 1.16 × 10-4 mol/g, IDA = 1.52 × 10-3  mol/g, and AMPA = 4.67 × 10-4 mol/g) were used to distinguish differences in antimicrobial efficacies. Growth of A. acidoterrestris in acidified Potato Dextrose Broth over 24 hr with chelating resins indicated early death phase for CA and IDA resins and bactericidal for AMPA resin. However, viability in commercial apple juice with the inclusion of nisin and chelating resins was variable, with IDA resin significantly (P < 0.05) increasing viability while the effect of CA and AMPA resins remained elusive. This work emphasizes the importance of biological repeatability and correct statistical modeling in identifying conditions under which the antimicrobial intervention of nisin in real food systems, such as acidic beverages and juices, are synergistic or antagonistic. PRACTICAL APPLICATION: New technologies to control microbial food spoilage and waste need to be explored to address consumers on-going demands for reducing additive use.  Solid support bound metal chelators can both promote and control microbial growth when used in conjunction with nisin, a natural antimicrobial.  This work explores how system conditions can render a given technology either synergistic or antagonistic, and highlights the importance of sufficient biological replicates in experimental design.

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.

Homogeneous Synthesis of Cationic Chitosan via New Avenue.

Using a solvent formed of alkali and urea, chitosan was successfully dissolved in a new solvent via the freezing⁻thawing process. Subsequently, quaternized chitosan (QC) was synthesized using 3-chloro-2-hydroxypropyl trimethyl ammonium chloride (CHPTAC) as the cationic reagent under different incubation times and temperatures in a homogeneous system. QCs cannot be synthesized at temperatures above 60 °C, as gel formation will occur. The structure and properties of the prepared QC were characterized and quaternary groups were comfirmed to be successfully incorporated onto chitosan backbones. The degree of substitution (DS) ranged from 16.5% to 46.8% and the yields ranged from 32.6% to 89.7%, which can be adjusted by changing the molar ratio of the chitosan unit to CHPTAC and the reaction time. QCs inhibits the growth of Alicyclobacillus acidoterrestris effectively. Thus, this work offers a simple and green method of functionalizing chitosan and producing quaternized chitosan with an antibacterial effect for potential applications in the food industry.

Characterization and long term antimicrobial activity of the nisin anchored cellulose films.

To obtain a safe and biodegradable material for food packaging, this study developed a facile and green process of anchoring nisin onto oxidized cellulose through a simple Schiff base reaction for long-term antimicrobial active food packaging. To the best of our knowledge, this is the first report of such a packaging. The grafting reaction between the amino groups of nisin and the aldehyde groups of oxidized cellulose films was successfully established to obtained a nisin anchored cellulose films (NC) membrane; the structure was characterized with Fourier transform infrared spectroscopy, scanning electron microscopy, and thermogravimetric analysis. The NCs membrane had an outstanding transparent property of above 90% transmittance through 300nm to 800nm; while, the barrier properties against oxygen remain excellent. The thermal stability of the NCs membrane remained excellent and enabled an easier degradation than the original cellulose membrane. The NCs membrane displayed good antimicrobial activity against Alicyclobacillus acidoterrestris DSM 3922T in a long-term way, thus demonstrating good potential as food packaging material. The obtained results are promising and confirm the development of NCs membranes as green materials for food packaging.

Plantaricyclin A, a Novel Circular Bacteriocin Produced by Lactobacillus plantarum NI326: Purification, Characterization, and Heterologous Production.

Bacteriocins from lactic acid bacteria (LAB) are of increasing interest in recent years due to their potential as natural preservatives against food and beverage spoilage microorganisms. In a screening study for LAB, we isolated from olives a strain, Lactobacillus plantarum NI326, with activity against the beverage-spoilage bacterium Alicyclobacillus acidoterrestris Genome sequencing of NI326 enabled the identification of a gene cluster (designated plc) encoding a putative circular bacteriocin and proteins involved in its modification, transport, and immunity. This novel bacteriocin, named plantaricyclin A (PlcA), was grouped into the circular bacteriocin subgroup II due to its high degree of similarity with other gassericin A-like bacteriocins. Purification of PlcA from the supernatant of Lb. plantarum NI326 resulted in an active peptide with a molecular mass of 5,570 Da, corresponding to that predicted from the (processed) PlcA amino acid sequence. The plc gene cluster was cloned and expressed in Lactococcus lactis NZ9000, resulting in the production of an active 5,570-Da bacteriocin in the supernatant. PlcA is believed to be produced as a 91-amino-acid precursor with a 33-amino-acid leader peptide, which is predicted to be removed, followed by joining of the N and C termini via a covalent linkage to form the mature 58-amino-acid circular bacteriocin PlcA. We report the characterization of a circular bacteriocin produced by Lb. plantarum The inhibition displayed against A. acidoterrestris highlights its potential use as a preservative in food and beverages.IMPORTANCE In this work, we describe the purification and characterization of an antimicrobial peptide, termed plantaricyclin A (PlcA), produced by a Lactobacillus plantarum strain isolated from olives. This peptide has a circular structure, and all genes involved in its production, circularization, and secretion were identified. PlcA shows antimicrobial activity against different strains, including Alicyclobacillus acidoterrestris, a common spoilage bacterium, which causes substantial economic losses in the beverage industry every year. In this study, we describe a circular antimicrobial peptide, PlcA, for a Lactobacillus plantarum strain.

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.

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.

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.

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.