Detection of risk areas in dairy powder processes: The development of thermophilic spore forming bacteria taking into account their growth limits.

Anoxybacillus flavithermus, Geobacillus stearothermophilus and Bacillus licheniformis are the main contaminants found in dairy powders. These spore-forming thermophilic bacteria, rarely detected in raw milk, persist, and grow during the milk powder manufacturing process. Moreover, in the form of spores, these species resist and concentrate in the powders during the processes. The aim of this study was to determine the stages of the dairy powder manufacturing processes that are favorable to the growth of such contaminants. A total of 5 strains were selected for each species as a natural contaminant of dairy pipelines in order to determine the minimum and maximum growth enabling values for temperature, pH, and aw and their optimum growth rates in milk. These growth limits were combined with the environmental conditions of temperature, pH and aw encountered at each step of the manufacture of whole milk, skim milk and milk protein concentrate powders to estimate growth capacities using cardinal models and the Gamma concept. These simulations were used to theoretically calculate the population sizes reached for the different strains studied at each stage in between two successive cleaning in place procedures. This approach highlights the stages at which risk occurs for the development of spore-forming thermophilic bacterial species. During the first stages of production, i.e. pre-treatment, pasteurization, standardization and pre-heating before concentration, physico-chemical conditions encountered are suitable for the development and growth of A. flavithermus, G. stearothermophilus and B. licheniformis. During the pre-heating stage and during the first effects in the evaporators, the temperature conditions appear to be the most favorable for the growth of G. stearothermophilus. The temperatures in the evaporator during the last evaporator effects are favorable for the growth of B. licheniformis. In the evaporation stage, low water activity severely limits the development of A. flavithermus.

Resistance, removal, and bioaccumulation of Ni (II) and Co (II) and their impacts on antioxidant enzymes of Anoxybacillus mongoliensis.

In this study, it was hypothesis that A. mongoliensis could be used as bioindicator for Ni (II) and Co (II). Thus, Ni (II) and Co (II) resistance, removal, bioaccumulation, and the impacts of them on antioxidant enzyme systems of thermophilic Anoxybacillus mongoliensis were investigated in details. The bioaccumulation of Ni (II) and Co (II) on the cell membrane of thermophilic A. mongoliensis, variations on surface macrostructure and functionality by FT-IR and SEM, and determination of antioxidant enzyme activities were also tested. The highest bioaccumulation values of Co (II) and Ni (II) were detected as 102.0 mg metal/g of dry bacteria at 10 mg/L for the 12th h and 90.4 mg metal/g of dry bacteria for the 24th h, respectively, and the highest Ni (II) and Co (II) cell membrane bioaccumulation capacities of A. mongoliensis were determined as 268.5 and 274.9 mg metal/g wet membrane, respectively at the 24th h. In addition, increasing on SOD and CAT activities were observed on depend of concentration of Ni (II) and Co (II) with respect to control. The antioxidant enzyme activity results also indicated that A. mongoliensis might be used as a bioindicator for Ni (II) and Co (II) pollution in environmental water specimens.

Comparing membrane and spacer biofouling by Gram-negative Pseudomonas aeruginosa and Gram-positive Anoxybacillus sp. in forward osmosis.

Bacteria of different Gram-types have inherently different outer cell structures, influencing cell surface properties and bacterial attachment. Dynamic biofouling experiments were conducted over four days in a bench-scale forward osmosis (FO) system with Gram-negative Pseudomonas aeruginosa or Gram-positive Anoxybacillus sp. Biofouling resulted in ∼10% decline in FO permeate water flux and was found to be significant for Anoxybacillus sp. but not for P. aeruginosa. Additionally, a stronger permeate water flux decline for P. aeruginosa in experiments with a superhydrophilic feed spacer demonstrated that mitigation methods require testing with different bacterial Gram-types. It was found that although permeate water flux decline can be affected by bacterial Gram-type the stable performance under enhanced biofouling conditions highlights the potential of FO for wastewater reclamation.

Thermal treatment of skim milk concentrates in a novel shear-heating device: Reduction of thermophilic spores and physical properties.

Endospores of thermophilic bacilli are a major concern for producers of dairy powders. In this study, we heat treated 10 different spore suspensions at 110 °C in skim milk and skim milk concentrate (36% dry matter) of the species Geobacillus stearothermophilus (10 min) and Anoxybacillus flavithermus (5 min) in a new shear-heating device. The highest log reduction in skim milk concentrate was 3.5. The death behavior of the spores was strain dependent. Particle formation and Maillard reaction were observed. By increasing the shear-rate up to 1500 s-1 the particle size was reduced for both heating times (D90 reduction: 57.4 and 77.0%, respectively). The particle size was lessened by a reduction of dry matter of 27%, compared to 36%. This work emphasizes, that heat treatment of concentrated dairy products represents a technological option to reduce thermophilic spores in skim milk concentrate and powders produced thereof.

[Adaptation of Anoxybacillus flavithermus ssp. yunnanesis E13(T) to toluene at the level of fatty acid composition of membrane].

OBJECTIVE: Anoxybacillus flavithermus subsp. yunnanensis is now the only species of thermophilic bacteria able to tolerate toxic solvents at high temperature. The adaptive responses of A. flavithermus subsp. yunnanensis E13(T) to toluene on the level of fatty acid composition of membrane were studied in detail. METHODS: The extraction of fatty acids was performed according to the method described in the Sherlock Microbial Identification System manual. The fatty acid compositions were analyzed by gas chromatography mass spectrometry (GC-MS). RESULTS: In presence of 0.3% (V/V) toluene, key moment to adapt the saturated straight-chain fatty acids was that when cells grew from the lag phase to the initial growth phase in liquid. The saturated straight-chain fatty acids were continuously decreased as the strain E13(T) to grow. In survival of the cells in 100% toluene, the saturated straight-chain fatty acids increased significantly. CONCLUSION: A. flavithermus ssp. yunnanesis E13(T) alters its membrane fluidity via fatty acid composition to become more rigid when it is exposed to solvent, which is consistent that commonly found in mesophilic organic solvent-tolerant bacteria. However, it adapted its membrane by increasing straight-chain saturated fatty acids, rather than unsaturated fatty acids, which was demonstrated in mesophilic organic solvent-tolerant bacteria.

Characterization of aluminum resistant Anoxybacillus sp. SK 3-4 isolated from a hot spring.

The Anoxybacillus sp. SK 3-4, previously isolated from a hot spring, was screened for its heavy metals resistance (Al(3+), Mn(2+), Cu(2+), Co(2+), Zn(2+), and Ni(2+)) and the strain was found to be most resistant to aluminum. Significant growth of the strain was observed when it was grown in medium containing aluminum (200 mg L(-1)-800 mg L(-1)) with relative growth rates ranging between 77% and 100%. A gene encoding the aluminum resistance protein (accession number: WP_021095658.1) was found in genome of strain SK 3-4, which revealed high sequence identity (>95%) to its homologues from Anoxybacillus species. Sequence comparisons with two functionally characterized aluminum resistance proteins, namely G2alt and ALU1-P, showed 97% and 81% of sequence identity, respectively. Four putative metal binding sites were detected in SK 3-4 aluminum resistance protein and G2alt at same amino acid residue positions of 186, 195, 198, and 201. Strain SK 3-4 was found to be able to remove aluminum from aqueous solution. This study demonstrated that Anoxybacillus sp. SK 3-4 could be applied in the treatment of aluminum contaminated wastewater.

Purification and characterization of thermostable α-amylase from thermophilic Anoxybacillus flavithermus.

This study reports on the purification and characterization of thermostable α-amylase (α-1-4 D-glucan glucanohydrolase EC 3.2.1.1) from a newly isolated Anoxybacillus flavithermus. A. flavithermus was used, which was isolated from hot water springs of Ömer, Afyonkarahisar, Turkey. The gram-positive, spore-forming, motile, moderately thermophilic bacteria were found to be a strain of A. flavithermus analysed by 16S rRNA comparison. The optimal conditions for bacterial growth were determined to be at 20 thh, 55 °C and pH 6.0. Maximum α-amylase activity was obtained at 55 °C at pH 7.0 after 24h of incubation. Thermostable α-amylase from A. flavithermus was purified by 70% (NH4)2SO4 and ion-exchange chromatography (5.2-fold; 65.8% yield). The molecular weight of α-amylase was 60 kDa, as estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The α-amylase hydrolyzed soluble starch at 55 °C with Km: 0.005 mM and Vmax: 3.5 µmol min(-1).

Anoxybacillus suryakundensis sp. nov, a moderately thermophilic, alkalitolerant bacterium isolated from hot spring at Jharkhand, India.

Four closely related facultative anaerobe, moderately thermophilic, Gram positive rods (JS1(T), JS5, JS11, and JS15) were isolated from sediment samples from a hot spring at Suryakund, Jharkhand, India. Colonies were pale yellow, rough surface with uneven edges on TSA after 72 h incubation. Heterotrophic growth was observed at 40-60°C and pH 5.5-11.5; optimum growth occurred at 55°C and pH 7.5. 16S rRNA gene sequence analysis revealed the strains belong to genus Anoxybacillus. DNA-DNA homology values among strains were above 70% and showed distinct ERIC and REP PCR profile. On the basis of morphology and biochemical characteristics, strain JS1(T) was studied further. Strain JS1(T) showed 99.30% sequence similarity with A. flavithermus subsp. yunnanensis, 99.23% with A. mongoliensis, 99.16% with A. eryuanensis, 98.74% with A. flavithermus subsp. flavithermus, 98.54% with A. tengchongensis, 98.51% with A. pushchinoensis, 97.91% with A. thermarum, 97.82% with A. kaynarcensis, 97.77% with A. ayderensis and A. kamchatkensis, 97.63% with A. salavatliensis, 97.55% with A. kestanbolensis, 97.48% with A. contaminans, 97.27% with A. gonensis and 97.17% with A. voinovskiensis. In 16S rRNA secondary structure based phylogenetic comparison, strain JS1(T) was clustered with Anoxybacillus eryuanensis, A. mongoliensis, and A. flavithermus subsp. yunnanensis and showed 15 species specific base substitutions with maximum variability in helix 6. Moreover, DNA-DNA relatedness between JS1(T) and the closely related type strains were well below 70%. The DNA G+C content was 42.1 mol%. The major fatty acids were C(15:0 iso), C(16:0 iso) and C(17:0iso). The polar lipids were a phosphatidylgylycerol, a diphosphatidylglycerol, a phosphatidylethnolamine, a phosphatidylcholine, a phosphatidyl monomethylethanolamine and four unknown lipids. Based on polyphasic approach, strain JS1(T) represent a novel species of the genus Anoxybacillus for which Anoxybacillus suryakundensis sp. nov. is proposed. The type strain is JS1(T) (= DSM 27374(T) = LMG 27616(T) =JCM19211(T)).

Influence of cations on growth of thermophilic Geobacillus spp. and Anoxybacillus flavithermus in planktonic culture.

Free ions of Na(+), K(+), Ca(2+), and Mg(2+) influenced the optical density of planktonic cultures of thermophilic bacilli. Anoxybacillus flavithermus E16 and Geobacillus sp. strain F75 (milk powder manufacturing plant isolates) and A. flavithermus DSM 2641 and G. thermoleovorans DSM 5366 were studied. Ca(2+) and Mg(2+) were associated with increases in optical density more so than Na(+) and K(+). Overall, it appeared that Ca(2+) and/or Mg(2+) was required for the production of protein in thermophilic bacilli, as shown by results obtained with A. flavithermus E16, which was selected for further study.

Isolation and characterization of a novel organic solvent-tolerant Anoxybacillus sp. PGDY12, a thermophilic Gram-positive bacterium.

AIMS: To isolate and characterize new bacteria capable of tolerating high concentrations of organic solvents at high temperature. METHODS AND RESULTS: A solvent-tolerant, thermophilic bacterium was isolated from hot spring samples at 55°C. The strain PGDY12 was characterized as a Gram-positive bacterium. It was able to tolerate 100% solvents, such as toluene, benzene and p-xylene on plate overlay and high concentrations of these solvents in liquid cultures. A comparison of growth showed that 0·2% (v/v) benzene and 0·15% (v/v) p-xylene were capable of enhancing the final cell yields. Transmission electron micrographs showed the incrassation of electron-transparent intracellular material and the distorted cytoplasm in case of the cells grown in toluene. A phylogenetic analysis based on 16S rRNA sequence data indicated that the strain PGDY12 was member of the genus Anoxybacillus. CONCLUSIONS: The thermophilic, Gram-positive Anoxybacillus sp. PGDY12 exhibited a unique and remarkable ability to tolerate solvents at 55°C. SIGNIFICANCE AND IMPACT OF THE STUDY: The solvent tolerance properties are less known in thermophilic bacteria. The Anoxybacillus sp. PGDY12 is the first strictly thermophilic bacterium able to tolerate a broad range of solvents. This strain is a promising candidate for use as a high temperature biocatalyst in the biotechnological applications.

Thermophilic bacilli and their importance in dairy processing.

The thermophilic bacilli, such as Anoxybacillus flavithermus and Geobacillus spp., are an important group of contaminants in the dairy industry. Although these bacilli are generally not pathogenic, their presence in dairy products is an indicator of poor hygiene and high numbers are unacceptable to customers. In addition, their growth may result in milk product defects caused by the production of acids or enzymes, potentially leading to off-flavours. Dairy thermophiles are usually selected for by the conditions during dairy manufacture. These bacteria are able to grow in sections of dairy manufacturing plants where temperatures reach 40-65°C. Furthermore, because they are spore formers, they are difficult to eliminate. In addition, they exhibit a wide temperature growth range, exhibit a fast growth rate (generation time of approximately 15-20 min) and tend to readily form biofilms. Many strategies have been tested to remove, prevent and/or delay the formation of thermophilic bacilli biofilms in dairy manufacture, but with limited success. This is, in part, because little is known about the structure and composition of thermophilic bacilli biofilms in general and, more specifically, in milk processing environments. Therefore, new cleaning regimes often do not target the problem optimally. A greater understanding of the structure of thermophilic biofilms within the context of the milk processing environment and their link with spore formation is needed to develop better control measures. This review discusses the characteristics and food spoilage potential, enumeration and identification methods for the thermophilic bacilli, as well as their importance to dairy manufacture, with an emphasis on biofilm development and spore formation.