Unveiling the complete genome sequence of Alicyclobacillus acidoterrestris DSM 3922T, a taint-producing strain.

Several species from the Alicyclobacillus genus have received much attention from the food and beverages industries. Their presence has been co-related with spoilage events of acidic food matrices, namely fruit juices and other fruit-based products, the majority attributed to Alicyclobacillus acidoterrestris. In this work, a combination of short and long reads enabled the assembly of the complete genome of A. acidoterrestris DSM 3922T, perfecting the draft genome already available (AURB00000000), and revealing the presence of one chromosome (4,222,202 bp; GC content 52.3%) as well as one plasmid (124,737 bp; GC content 46.6%). From the 4,288 genes identified, 4,004 sequences were attributed to coding sequences with proteins, with more than 80% being functionally annotated. This allowed the identification of metabolic pathways and networks and the interpretation of high-level functions with significant reliability. Furthermore, the additional genes of interest related to spore germination, off-flavor production, namely the vdc cluster, and CRISPR arrays, were identified. More importantly, this is the first complete and closed genome sequence for a taint-producing Alicyclobacillus species and thus represents a valuable reference for further comparative and functional genomic studies.

Insight into CAZymes of Alicyclobacillus mali FL18: Characterization of a New Multifunctional GH9 Enzyme.

In the bio-based era, cellulolytic and hemicellulolytic enzymes are biocatalysts used in many industrial processes, playing a key role in the conversion of recalcitrant lignocellulosic waste biomasses. In this context, many thermophilic microorganisms are considered as convenient sources of carbohydrate-active enzymes (CAZymes). In this work, a functional genomic annotation of Alicyclobacillus mali FL18, a recently discovered thermo-acidophilic microorganism, showed a wide reservoir of putative CAZymes. Among them, a novel enzyme belonging to the family 9 of glycosyl hydrolases (GHs), named AmCel9, was identified; in-depth in silico analyses highlighted that AmCel9 shares general features with other GH9 members. The synthetic gene was expressed in Escherichia coli and the recombinant protein was purified and characterized. The monomeric enzyme has an optimal catalytic activity at pH 6.0 and has comparable activity at temperatures ranging from 40 °C to 70 °C. It also has a broad substrate specificity, a typical behavior of multifunctional cellulases; the best activity is displayed on ß-1,4 linked glucans. Very interestingly, AmCel9 also hydrolyses filter paper and microcrystalline cellulose. This work gives new insights into the properties of a new thermophilic multifunctional GH9 enzyme, that looks a promising biocatalyst for the deconstruction of lignocellulose.

Characterization of EstZY: A new acetylesterase with 7-aminocephalosporanic acid deacetylase activity from Alicyclobacillus tengchongensis.

Deacetyl-7-aminocephalosporanic acid (D-7-ACA) is required for producing of many semisynthetic ß-lactam antibiotics; therefore, enzymes capable of converting 7-aminocephalosporanic acid (7-ACA) to D-7-ACA present a valuable resource to the pharmaceutical industry. In the present study, a putative acetylesterase, EstZY, was identified and characterized from a thermophilic bacterium Alicyclobacillus tengchongensis. Sequence alignment showed that EstZY was an acetylesterase which belonged to carbohydrate esterase family 7 (CE7), with substrate preference for short-chain acyl esters p-NPC2 to p-NPC8. Maximum enzyme activity was recorded at pH 9.0 and 50 °C, where Km and Vmax were calculated as 1.9 ± 0.23 mM and 258 ± 18.5 µM min-1, respectively. The residues Ser185, Asp274, and His303 were identified as the putative catalytic triad by homology modelling, site-directed mutagenesis and molecular docking. Moreover, EstZY can remove the acetyl group at C3' position of 7-ACA to form D-7-ACA; this is the first report of a 7-ACA deacetylase from CE7 family in A. tengchongensis and may represent a new enzyme with industrial values.

Diversity and guaiacol production of Alicyclobacillus spp. from fruit juice and fruit-based beverages.

Alicyclobacillus acidoterrestris is an acido-thermophilic, spore-forming bacterial species that can spoil acidic fruit juice and fruit-based beverages. The metabolism of taint compounds by this bacterial species has led to its status as a targeted microorganism in the fruit juice industry. This study aims to assess the genetic diversity of Alicyclobacillus spp. including A. acidoterrestris and its correlation to spoilage taint metabolism. Alicyclobacillus cultures, which were previously isolated from a wide range of domestic and international products including fruit juice, fruit drinks and fruit juice concentrates, were subjected to DNA fingerprint analysis by using randomly amplified polymorphic DNA (RAPD) - polymerase chain reaction. Isolates were classified on the basis of their RAPD profile and the results were used to select representative strains to undergo taint production assessment. The taint guaiacol produced by Alicyclobacillus spp. was measured by headspace gas chromatography and mass spectrometry. From produced RAPD profiles, two genotypic groups and two sub-groups were identified. The groups were independent of product types and geographical origins. A significant number of isolates were clustered in genotypic group I, including A. acidoterrestris ATCC 49025. These isolates produced significant levels of guaiacol, 8.7 mg/L on average. A smaller number of isolates was found in genotypic group II including A. acidocaldarius and they produced no guaiacol. Primer F-64 was useful to identify Alicyclobacillus at the species level, and permitted rapid identification of strains producing fruit juice taint compounds such as guaiacol.

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

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

Synthesis of multifunctional fluorescent magnetic nanoparticles for the detection of Alicyclobacillus spp. in apple juice.

An approach based on multifunctional fluorescent magnetic nanoparticles was proposed for the enrichment and identification of Alicyclobacillus spp. in apple juice simultaneously. The prepared Fe3O4 magnetic particles (MNPs) were modified by the sol-gel process and a silica shell was formed to improve the reactivity, and then the obtained MNPs@SiO2-SH nanoparticles were conjugated with Thioglycolic acid functionalized CdTe/CdS QDs via thiols chemistry. The characteristic evaluation results indicated that the MNPs-QD nanocomposites exhibited good magnetic properties and optical characterization. The polyclonal anti-Alicyclobacillus IgG antibody was immobilized onto the surface of MNPs-QD materials via esterification reactions. The maximum antibody immobilization capacity was 119.62 µg/mg and the adsorption reaction could be accomplished in 60 min. The adsorption process could be represented by Langmuir model and pseudo-second order kinetics equation, respectively. Based on the high immunocapture efficiency and sensitive fluorescence characteristics, the obtained MNPs-QDs-antibody conjugates could be applied to recognize the contamination of Alicyclobacillus spp. and a quantitative analysis method was established for target cells detection. The minimum quantitative limit was 104 CFU (colony forming unit)/mL and the testing process could be completed in 90 min. The results indicated that the MNPs-QDs-antibody conjugates can be successfully applied for immunocapture and detection of Alicyclobacillus spp. in apple juice. That is to say, the developed MNPs-QDs-antibody conjugates have exhibit more attractive and great potential for the immunocapture and recognition of target bacteria, fully demonstrated a new method for enrichment and rapid detection of Alicyclobacillus spp. in fruit juices.

The direction of protein evolution is destined by the stability.

Protein evolution is potentially governed by protein stability. Here, we investigated the relationship between protein evolution and stability through the random mutational drift of a thermophilic bacterial protein, an esterase of Alicyclobacillus acidocaldarius (Aac-Est), at high and low temperatures. In the first random mutation of Aac-Est, few proteins exhibit increased activity at 65 °C, indicating that the wild-type (WT) Aac-Est is located on the peak of a mountain in a fitness landscape for activity at high temperature. To obtain higher active variants than those of WT, it must go down the mountain once and climb another, higher mountain. In the second and third generations from lower active templates, the evolvability (the proportion of variants with higher activity in all the variants obtained in a given generation than a parent protein) depended on the stability of the template proteins. Compared to WT, the stability-maintaining template could recover the activity more. Thus, a low-activity variant with high stability is able to drift vastly in sequence space and reach the foot of a higher mountain. Meanwhile, random mutations in stability-loss templates produced several variants with higher activity at 40 °C than those produced by WT, via cold adaptation. Our results indicate that maintaining protein stability enables the protein to search sequence space and evolve in the original environment, and proteins with lost stability use a cold adaptation path.

Comparison of Alicyclobacillus acidocaldarius o-Succinylbenzoate Synthase to Its Promiscuous N-Succinylamino Acid Racemase/ o-Succinylbenzoate Synthase Relatives.

Studying the evolution of catalytically promiscuous enzymes like those from the N-succinylamino acid racemase/ o-succinylbenzoate synthase (NSAR/OSBS) subfamily can reveal mechanisms by which new functions evolve. Some enzymes in this subfamily have only OSBS activity, while others catalyze OSBS and NSAR reactions. We characterized several NSAR/OSBS subfamily enzymes as a step toward determining the structural basis for evolving NSAR activity. Three enzymes were promiscuous, like most other characterized NSAR/OSBS subfamily enzymes. However, Alicyclobacillus acidocaldarius OSBS (AaOSBS) efficiently catalyzes OSBS activity but lacks detectable NSAR activity. Competitive inhibition and molecular modeling show that AaOSBS binds N-succinylphenylglycine with moderate affinity in a site that overlaps its normal substrate. On the basis of possible steric conflicts identified by molecular modeling and sequence conservation within the NSAR/OSBS subfamily, we identified one mutation, Y299I, that increased NSAR activity from undetectable to 1.2 × 102 M-1 s-1 without affecting OSBS activity. This mutation does not appear to affect binding affinity but instead affects kcat, by reorienting the substrate or modifying conformational changes to allow both catalytic lysines to access the proton that is moved during the reaction. This is the first site known to affect reaction specificity in the NSAR/OSBS subfamily. However, this gain of activity was obliterated by a second mutation, M18F. Epistatic interference by M18F was unexpected because a phenylalanine at this position is important in another NSAR/OSBS enzyme. Together, modest NSAR activity of Y299I AaOSBS and epistasis between sites 18 and 299 indicate that additional sites influenced the evolution of NSAR reaction specificity in the NSAR/OSBS subfamily.

Concurrent metabolism of pentose and hexose sugars by the polyextremophile Alicyclobacillus acidocaldarius.

Alicyclobacillus acidocaldarius is a thermoacidophilic bacterium capable of growth on sugars from plant biomass. Carbon catabolite repression (CCR) allows bacteria to focus cellular resources on a sugar that provides efficient growth, but also allows sequential, rather than simultaneous use when more than one sugar is present. The A. acidocaldarius genome encodes all components of CCR, but transporters encoded are multifacilitator superfamily and ATP-binding cassette-type transporters, uncommon for CCR. Therefore, global transcriptome analysis of A. acidocaldarius grown on xylose or fructose was performed in chemostats, followed by attempted induction of CCR with glucose or arabinose. Alicyclobacillus acidocaldarius grew while simultaneously metabolizing xylose and glucose, xylose and arabinose, and fructose and glucose, indicating that CCR did not control carbon metabolism. Microarrays showed down-regulation of genes during growth on one sugar compared to two, and occurred primarily in genes encoding: (1) regulators; (2) enzymes for cell wall synthesis; and (3) sugar transporters.

PAM-Dependent Target DNA Recognition and Cleavage by C2c1 CRISPR-Cas Endonuclease.

C2c1 is a newly identified guide RNA-mediated type V-B CRISPR-Cas endonuclease that site-specifically targets and cleaves both strands of target DNA. We have determined crystal structures of Alicyclobacillus acidoterrestris C2c1 (AacC2c1) bound to sgRNA as a binary complex and to target DNAs as ternary complexes, thereby capturing catalytically competent conformations of AacC2c1 with both target and non-target DNA strands independently positioned within a single RuvC catalytic pocket. Moreover, C2c1-mediated cleavage results in a staggered seven-nucleotide break of target DNA. crRNA adopts a pre-ordered five-nucleotide A-form seed sequence in the binary complex, with release of an inserted tryptophan, facilitating zippering up of 20-bp guide RNA:target DNA heteroduplex on ternary complex formation. Notably, the PAM-interacting cleft adopts a "locked" conformation on ternary complex formation. Structural comparison of C2c1 ternary complexes with their Cas9 and Cpf1 counterparts highlights the diverse mechanisms adopted by these distinct CRISPR-Cas systems, thereby broadening and enhancing their applicability as genome editing tools.

Process performance and bacterial community dynamics of partial-nitritation biofilters subjected to different concentrations of cysteine amino acid.

Partial-nitritation processes are used for the biological treatment of high nitrogen-low organic carbon effluents, such as anaerobic digestion reject water. The release of certain products generated during the anaerobic digestion process, such as amino acids, could potentially reduce the performance of these partial-nitritation bioprocesses. To investigate this, four partial-nitritation biofilters were subjected to continuous addition of 0, 150, 300, and 500 mg L-1 cysteine amino acid in their influents. The addition of the amino acid had an impact over the performance of the partial-nitritation process and the bacterial community dynamics of the systems analyzed. Ammonium oxidation efficiency decreased with the addition of the amino acid, and a net nitrogen elimination occurred in presence of cysteine through the operation period. Bacterial community dynamics showed a decrease of Nitrosomonas species and a proliferation of putative heterotrophs with nitrification capacity, such as Pseudomonas, or denitrification capacity, such as Denitrobacter or Alicycliphilus. The addition of cysteine irreversible affected the bioreactors, which could not achieve the performance obtained before the addition of the amino acid. A mathematical predictive equation of the process performance depending on cysteine concentration added and operational time under such concentration was developed. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1254-1263, 2016.

Culture dependent and independent genomic identification of Alicyclobacillus species in contaminated commercial fruit juices.

Alicyclobacillus is a genus of thermo-acidophilic, endospore-forming, bacteria species which occasionally cause spoilage of heat-processed fruit juices by producing guaiacol taint. In this study, Alicyclobacillus contamination of commercial fruit juices in West Africa was investigated using culture-dependent and -independent approaches. Firstly, a total of 225 fruit juice products from Ghana (n = 39) and Nigeria (n = 186) were enriched with yeast-starch-glucose (YSG) broth (pH 3.7) following heat shock at 80 °C for 10 min. Alicyclobacillus was detected in 11.6% (26) of samples. Isolates were identified to the genus taxonomic level by genus-specific PCR which targeted the squalene-hopene-cyclase (shc) gene followed by analysis of the almost-complete 16S ribosomal RNA (rRNA) gene sequences that identified 16 Alicyclobacillus acidoterrestris, 7 Alicyclobacillus acidocaldarius and 3 Alicyclobacillus genomic species 1 (Alicyclobacillus sp. 1). Whole-genome fingerprinting using PCR-RAPD primers Ba-10, F-61 and F-64 grouped the 16 A. acidoterrestris isolates into two genetic clusters. Furthermore, high performance liquid chromatographic (HPLC) analyses revealed the activity of vanillic-acid decarboxylase (vdc) in all A. acidoterrestris isolates due to guaiacol production from vanillic-acid. Lastly, species-specific PCR-DGGE targeting the 16S rRNA gene clearly discriminated between the guaiacol-producing A. acidoterrestris and the non-spoilage A. acidocaldarius group. Information provided by this study is fundamental to the development of effective strategies for the improvement of quality and shelf-life of processed tropical fruit juices in W. Africa.

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.

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.

Identification and Characterization of a New 7-Aminocephalosporanic Acid Deacetylase from Thermophilic Bacterium Alicyclobacillus tengchongensis.

UNLABELLED: Deacetylation of 7-aminocephalosporanic acid (7-ACA) at position C-3 provides valuable starting material for producing semisynthetic ß-lactam antibiotics. However, few enzymes have been characterized in this process before now. Comparative analysis of the genome of the thermophilic bacterium Alicyclobacillus tengchongensis revealed a hypothetical protein (EstD1) with typical esterase features. The EstD1 protein was functionally cloned, expressed, and purified from Escherichia coli BL21(DE3). It indeed displayed esterase activity, with optimal activity at around 65°C and pH 8.5, with a preference for esters with short-chain acyl esters (C2 to C4). Sequence alignment revealed that EstD1 is an SGNH hydrolase with the putative catalytic triad Ser15, Asp191, and His194, which belongs to carbohydrate esterase family 12. EstD1 can hydrolyze acetate at the C-3 position of 7-aminocephalosporanic acid (7-ACA) to form deacetyl-7-ACA, which is an important starting material for producing semisynthetic ß-lactam antibiotics. EstD1 retained more than 50% of its initial activity when incubated at pH values ranging from 4 to 11 at 65°C for 1 h. To the best of our knowledge, this enzyme is a new SGNH hydrolase identified from thermophiles that is able to hydrolyze 7-ACA. IMPORTANCE: Deacetyl cephalosporins are highly valuable building blocks for the industrial production of various kinds of semisynthetic ß-lactam antibiotics. These compounds are derived mainly from 7-ACA, which is obtained by chemical or enzymatic processes from cephalosporin C. Enzymatic transformation of 7-ACA is the main method because of the adverse effects chemical deacylation brought to the environment. SGNH hydrolases are widely distributed in plants. However, the tools for identifying and characterizing SGNH hydrolases from bacteria, especially from thermophiles, are rather limited. Here, our work demonstrates that EstD1 belongs to the SGNH family and can hydrolyze acetate at the C-3 position of 7-ACA. Moreover, this study can enrich our understanding of the functions of these enzymes from this family.

Precursors and metabolic pathway for guaiacol production by Alicyclobacillus acidoterrestris.

Alicyclobacillus acidoterrestris has recently received much attention due to its implication in the spoilage of pasteurized fruit juices, which was manifested by the production of guaiacol. Vanillic acid and vanillin have been accepted as the biochemical precursors of guaiacol in fruit juices. The purpose of this study was to try to find other precursors and elucidate details about the conversion of vanillic acid and vanillin to guaiacol by A. acidoterrestris. Four potential substrates including ferulic acid, catechol, phenylalanine and tyrosine were analyzed, but they could not be metabolized to guaiacol by all the thirty A. acidoterrestris strains tested. Resting cell studies and enzyme assays demonstrated that vanillin was reduced to vanillyl alcohol by NADPH-dependent vanillin reductase and oxidized to vanillic acid by NAD(P)(+)-dependent vanillin dehydrogenases in A. acidoterrestris DSM 3923. Vanillic acid underwent a nonoxidative decarboxylation to guaiacol. The reversible vanillic acid decarboxylase involved was oxygen insensitive and pyridine nucleotide-independent.

Effect of extrinsic factors on the production of guaiacol by Alicyclobacillus spp.

Alicyclobacillus spp. is of significance to the fruit juice industry due to the production of guaiacol. Studies on Alicyclobacillus regarding guaiacol focus mainly on novel ways to detect guaiacol or evaluate guaiacol-producing potential of isolated Alicyclobacillus. Basic studies on factors that induce or affect the production of guaiacol and the conversion pathway of vanillic acid to guaiacol are not available. The goal of this study was to evaluate how extrinsic factors can affect the production of guaiacol by Alicyclobacillu s isolates. Guaiacol-producing Alicyclobacillus isolates 1016 and 1101 were used in this study and the effects of temperature (25 to 55 °C), pH (3.0 to 5.5), and oxygen concentration on guaiacol production in laboratory media was investigated. Maximum production of guaiacol by isolate 1016 was detected within 9 h when incubated at 43 °C, pH 4.0, under microaerophilic conditions. Isolate 1101 produced detectable amounts of guaiacol within 8 h at pH 5.0. However, maximum guaiacol production was achieved within 14 h by isolate 1101 when incubated at 50 °C. Our results indicate that the production of guaiacol, contrary to common belief, is a rapid reaction under desirable conditions specific to each isolate. The results of this study can be useful for developing rapid guaiacol monitoring methods for Alicyclobacillus-related spoilage or be applied to more detailed enzyme-related studies.

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.

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

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

Graft copolymerization of ethyl acrylate onto tamarind kernel powder, and evaluation of its biodegradability.

In the present study, tamarind kernel powder and ethyl acrylate were reacted by free radical polymerization to synthesize a grafted copolymer soluble in water. The grafted copolymer was analyzed by Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (NMR); FTIR showed a shift of the vibration of R-CO-OR' from 1258 cm(-1) to 1253 cm(-1). This shift appeared because of the grafting copolymerization. Films were prepared to study the mechanical properties and the biodegradation of this material. The mechanical properties of the grafted copolymer were found to lie between those of the parent polymers, suitable for disposable products. The new grafted copolymer manifested a steady process of biodegradation under incubation with the bacterial strain Alicycliphilus sp. BQ1; this was proved by scanning electron microscopy (SEM) and near infrared spectroscopy (NIR).