Isolation and Properties of New Photosynthetic Bacteria Isolated from Seawater

Three strains of purple nonsulfur photosynthetic bacteria were isolated from seawater. They showed the characteristic properties of Rhodobacter species. Their biochemical, physiological, and chemotaxonomical properties were different from those of previously known species.     

Two Forms of Glucoamylase from Mucor rouxianus 1. Purification and Crystallization

Mucor rouxianus produced two forms (isoenzymes) of glucoamylase which could be separated from each other by polyacrylamide gel electrophoresis or by chromatography on SP-Sephadex C-50, and they were designated glucoamylase I and glucoamylase II. Glucoamylases I and II were isolated in crystalline form, and were homogeneous in poly acrylamide gel electrophoresis and in ultracentrifugation, respectively. The sedimentation coefficient () molecular weight of glucoamylase I were 4.39 S and 59,000, and those of glucoamylase II were 4.29 S and 49,000, respectively.     

苹果浓缩汁中嗜酸耐热菌的分离与鉴定

为了解和控制浓缩苹果汁中的嗜酸耐热菌,采用酸化的凯氏培养基对苹果浓缩汁中的耐热菌进行分离、培养和鉴定,并与标准菌株Aliyclobacillus acidoterrestris DSM3922进行了比较分析.结果表明,分离到的2株污染菌均可以在21℃～55℃温度范围及2.4～6.2的pH值范围内生长,符合脂环酸芽胞杆菌属嗜酸耐热的特点.经与标准菌株的细胞、菌落形态观察、生长条件和生理生化反应等方面的比较表明,2株分离菌与标准菌株Aliyclobacillus acidoterrestris DSM3922有明显的相似特征.     

浓缩苹果汁生产过程中脂环酸芽孢杆菌的分离及初步鉴定

本文对浓缩苹果汁生产过程中的嗜酸耐热菌进行了分离,得到45株纯的嗜酸耐热芽孢杆菌。根据脂环酸芽孢杆菌(Alicyclobacillus)嗜酸的特点,用LB平板进行筛选,结果表明所有的菌株都嗜酸。用抗热性试验研究了这些菌株产生芽孢的培养时间,结果表明,所有考察的菌株中,33株与DSM3922的生长周期一致,48h内产生芽孢;3株菌生长速度较快,培养17h就能产生芽孢;还有3株菌生长速度较慢,需培养48h后才能产生芽孢。在采用16S rDNA PCR-RFLP法对筛选得到的脂环酸芽孢杆菌进行快速鉴定的基础上,选取7株可能是新种的未知菌株与5株已知的参比菌株的19个表型特征进行了试验研究和聚类分析,结果进一步证实了这7株菌都是与已知参比菌株不同的菌株。     

Inactivation of Alicyclobacillus acidoterrestris vegetative cells in model system,apple, orange and tomato juices by high hydrostatic pressure

The objective of this study is to determine the effect of high hydrostatic pressure (HHP) on inactivation of Alicyclobacillus acidoterrestris vegetative cells in a model system (BAM broth) and in orange, apple and tomato juices. The shelf-life stability of pressurized juices is also studied. In general the viability loss was enhanced significantly as the level of pressure and temperature were increased (P < 0.05). 4.70 log cycle reduction was obtained after pressurization at 350 MPa at 50 °C for 20 min in BAM broth whereas thermal treatment at 50 °C for 20 min caused only 1.13 log cycle inactivation showing the effectiveness of HHP treatment on inactivation. The D values for pressure (350 MPa at 50 °C) and temperature (50 °C) treatments were 4.37 and 18.86 min in BAM broth, respectively. All juices were inoculated with A. acidoterrestris cells to 10⁶ c.f.u./ml and were pressurized at 350 MPa at 50 °C for 20 min. More than 4 log cycle reduction was achieved in all juices studied immediately after pressurization. The pressurized juices were also stored up to 3 weeks at 30 °C and the viable cell numbers of A. acidoterrestris in orange, apple and tomato juices were 3.79, 2.59 and 2.27 log cycles, respectively after 3 weeks. This study has indicated that A. acidoterrestris vegetative cells can be killed by HHP at a predictable rate even at temperatures at which the microorganism would normally grow.     

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.     

Effects of high-pressure homogenization on the survival of Alicyclobacillus acidoterrestris in a laboratory medium

AIMS: This study was aimed to investigate the effectiveness of high-pressure homogenization (HPH) against Alicyclobacillus acidoterrestris. METHODS AND RESULTS: The susceptibility of three different strains of A. acidoterrestris (DSMZ 2498, Gamma4 and c8) to HPH (500-1700 bar) was studied. The experiments were performed in a laboratory medium (malt extract broth) on cells and spores. HPH caused a significant reduction of the initial cell number (1-2 log CFU ml(-1) at the highest pressures) in Gamma4 and DSMZ 2498 strains, whereas the effect on the spores was less significant. CONCLUSIONS: This study showed that the susceptibility of A. acidoterrestris to HPH was strain-dependent: DSMZ 2498 seemed the most susceptible strain, whereas c8 was the most resistant one. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of our study will provide useful information on the sensitivity of an emerging spoilage micro-organism, such as A. acidoterrrestris, to HPH.     

Catalytic Function of the Residues of Phenylalanine and Tyrosine Conserved in Squalene-Hopene Cyclases

Site-directed mutagenesis experiments on all the conserved residues of Phe and Tyr in all the known squalene-hopene cyclases (SHCs) were carried out to identify the active site residues of thermophilic Alicyclobacillus acidocaldarius SHC. The following functions are proposed on the basis of kinetic data and trapping of the prematurely cyclized products: (1) The Y495 residue probably amplifies the D376 acidity, which is assumed to work as a proton donor for initiating the polycyclization cascade, but its role is moderate. (2) Y609 possibly assists the function of F365, which has previously been assigned to exclusively stabilize the C-8 carbocation intermediate through cation-π interaction. The Y609A mutant produced a partially cyclized bicyclic triterpene. (3) Y612 works to stabilize both the C10 and C8 carbocations, this being verified by the finding that mono- and bicyclic products were formed with the Y612A mutant. (4) F129 was first identified to play a crucial role in catalysis. (5) The three redsidues, Y372, Y474 and Y540, are responsible for reinforcing the protein structure against thermal denaturation, Y474 being located inside QW motif 3.     

Development of novel Alicyclobacillus spp. isolation medium

AIM: To develop a new isolation medium with higher recovery rates of Alicyclobacillus spp. METHODS AND RESULTS: SK agar was developed with optimized incubation temperature, pH, acidulant, Tween 80 concentration and divalent cation addition. Results indicate that detection of Alicyclobacillus spp. by SK agar was significantly higher (P > 0.05) than those obtained by K agar, orange serum agar, and potato dextrose agar. CONCLUSIONS: Current media used for Alicyclobacillus spp. isolation still resulted in high numbers of false negative products. The sensitivity of SK agar to Alicyclobacillus spp. allows detection of low numbers of Alicyclobacillus spp. and also provides a more higher isolation results compared with currently used media. SIGNIFICANCE AND IMPACT OF THE STUDY: SK agar will be useful to the fruit juice industry to obtain more accurate numbers of contaminant Alicyclobacillus spp. With this media, false negative samples can be reduced, and the likelihood of exported products being rejected can be greatly reduced.     

Structure of the ATPase subunit CysA of the putative sulfate ATP-binding cassette (ABC) transporter from Alicyclobacillus acidocaldarius

CysA, the ATPase subunit of a putative sulfate ATP-binding cassette transport system of the gram-positive thermoacidophilic bacterium Alicyclobacillus acidocaldarius, was structurally characterized at a resolution of 2.0 Angstroms in the absence of nucleotides. In line with previous findings on ABC-ATPases the structures of the two monomers (called CysA-1 and CysA-2) in the asymmetric unit differ substantially in the arrangement of their individual (sub)domains. CysA-2 was found as a physiological dimer composed of two crystallographically related monomers that are arranged in an open state. Interestingly, while the regulatory domain of CysA-2 packs against its opposing domain that of CysA-1 undergoes a conformational change and, in the dimer, would interfere with the opposing monomer thereby preventing solute translocation. Whether this conformational state is used for regulatory purposes will be discussed.