Screening and identification of selected halophilic bacteria producing histamine and alkaline phosphatase

Abstract

Forty-one tetrad-forming halophilic lactic acid bacteria were isolated from 7 kinds of fermented foods using MRS agar with 10% NaCl. These bacteria were Gram positive cocci arranged in tetrad form. On the basis of MboI and AluI digested 16S rRNA gene restriction fragment patterns and 16S rRNA gene sequence analysis of the representative isolates, 41 isolates could be divided into two groups (A, B). Twenty-two isolates (group A) were identified as T. halophilus while another nineteen isolates (group B) were identified as T.muriaticus. Histamine formation was determined using HPLC and the partial gene of histidine decarboxylase (hdc) was sequenced. Only one isolate, KS87-14 identified as T. muriaticus, prolifically formed histamine. It indicated that rare strains produced histamine. However, this strain produced histamine 10 times higher than the reported T. muriaticus strains. A structural gene of pyruvoyl dependent histidine decarboxylase (hdcA gene) found mostly in histamine-producing gram-positive bacteria, was detected in KS87-14. Alignment of the partial sequence of hdcA gene from KS87-14 to hdcA gene previously deposited in the database showed that it was similar to the staphylococcal hdcA gene. Seventy-six moderately halophilic bacteria were isolated from 33 samples of fermented fish and salted fish using halobacterium JCM no.377. Only 17 isolates exhibited extracellular ALP activity. Based on their phenotypic characteristics and 16S rRNA gene sequence analyses, these isolates were identified as Staphylococcus saprophyticus (3 isolates), S. napalensis (3 isolates), S. sciuri (1 isolates), Bacillus vietnamensis (1 isolates), B. pumilus (1 isolates), Virgibacillus halodenitrifican (3 isolates), Oceanobacillus iheyensis (1 isolates), Halobacillus mangrovi (1 isolates), H. dabanensis (2 isolates), and the last isolate was closely related to Idiomarina zobellii. They possessed ALP activities ranging from 10.08-81.50 U/ml. The isolate TPS4-2 showed the highest ALP activity. It grew optimally at 30-37oC, pH 8 and in the presence of 10-15% (w/v) NaCl. The predominant ubiquinone was Q-8 and major cellular fatty acids were iso-C15:0 and iso-C17:0. DNA G+C content was 47.0 % mol. Based on 16S rRNA gene sequence analyses, TPS4-2T was closely related to I. zobellii DSM 15924T at 98.7%. It had low levels of DNA-DNA relatedness to the closest type strain I. zobellii DSM 15924T and its unique (GTG)5-PCR genomic fingerprint pattern was different from all closely phylogenic type strains of Idiomarina, therfore it was proposed as I. piscisalsi nov. sp. TPS4-2T was selected for further study due to its novel specie and high ALP production. TPS4-2T could produce both extracellular and intracellular ALP at the beginning of the exponential phase and the highest production was observed at the early stationary phase. TPS4-2T was cultivated in halobacterium JCM377 pH 8.0 at 37C in rotary shaking at 200 rpm. Since intracellular ALP was found higher than extracellular ALP at every sampling hour, therefore, intracellular ALP was further characterized. The maximum intracellular ALP production was at 36 h. The partially purified ALP presented a molecular mass of 53 kDa, calculated by gel filtration. Using p-nitrophenylphosphate as substrate, the Vmax and Km values were 28.01 μmol/min and 0.09 mM, respectively. It had maximal activity in the presence of 0.3 M NaCl, pH 10.0, at 60°C. Stability was fully at pH 7.0-8.0 and 37–55C. The presence of Mg2+ and Ca2+ could stimulate the ALP activity. In addition, the ALP activity was proportional to NaCl concentration in the range of 0-0.3 M. When NaCl concentration was increased 1-4 M, the ALP activity decreased and ceased at 40%. The EGTA could completely inhibit ALP activity. This study indicated that the ALP of TPS4-2T was metalloenzyme that can be activated by Ca2+and Mg2+ ions.