Degradation of histamine by the halotolerant Staphylococcus carnosus FS19 isolate obtained from fish sauce

Histamine is found in many fermented food products and may have detrimental effects on the health of its consumers. Histamine and other amines are degraded by the oxidative deamination activity of certain microorganisms. In this study, the growth characteristics and histamine-degrading activity of a Staphylococcus carnosus FS19 isolate derived from fish sauce were investigated. This bacterium exhibits optimal growth at 35 °C, pH 8 and 9% sodium chloride when cultivated in tryptic soy broth. The histamine-degrading activity of the S. carnosus FS19 isolate was optimised at 40 °C and pH 6 in 9% buffered sodium chloride. When added to fish sauce samples, this bacterium exhibits remarkable histamine-degrading activity. The histamine concentration was reduced by approximately 15.1% and 13.8% in the fish sauce samples that contained 18% and 21% salt, respectively. However, no histamine degradation was observed in samples with a salt content greater than 21%. In addition, a slight degradation of other amines, including putrescine and cadaverine, was also observed in some of the samples. In contrast, tyramine degradation did not occur in any of the samples. Therefore, S. carnosus FS19 is a culture that could potentially reduce the histamine content of fermented fish products

Development of a microbial bioassay system for detection of boric acid using Paecilomyces variotii.

Boric acid is a water soluble chemical preservative that has been used as food preservative by some local manufacturers. This chemical is used to preserve food products such as noodle and fish ball in order to inhibit the growth of microorganism, so that the preserved food can stay fresh and longer. However, its usage is prohibited by government of Malaysia as boric acid is considered harmful to human health if consumed in a considerably large quantity. Therefore, the detection method for boric acid is important. To date, no study has been performed to detect boric acid by using microorganism as sensing element. Hence, this study was aimed to develop a simple, fast and environmental friendly bioassay system incorporated with Paecilomyces variotii as bioreceptor for detection of boric acid in food. This detection system was based on the measurement of the changes of β-glucosidase produced by the microorganisms in response to the presence of boric acid. The changes of β-glucosidase concentration were assayed spectrophotometerically and correlated to the concentration of boric acid. In this system, P. variotii was grown in cellobiose medium for two days before its mycelia were entrapped in calcium alginate in bead form. In order to optimize the best condition for β-glucosidase production, the important factors such as initial pH, temperature, amount of cell loading, concentration of sodium alginate and calcium chloride were determined. The system was found to show optimum β-glucosidase production when 2% (w/v) sodium alginate and 0.25 Molar calcium chloride were used. Maximum enzyme production was also obtained with initial pH 7 and temperature 45 °C, using 6% (w/v) mycelia after three hours of incubation. By using these optimum operating conditions, a lower detection limit of 0.037% (w/v) was obtained from a linear range of 0% to 0.215% (w/v). The reproducibility of the system was acceptable with an observed relative standard deviation of 4.96% (n=10) and 4.81% (n=10) in the presence of 0.2% (w/v) boric acid and absence of boric acid, respectively. The bioassay system was then applied to determine boric acid in fish ball and the results of recovery ranging from 61% – 86% were recorded for boric acid spiked at different concentrations of boric acid from 0.05% to 0.20% (w/v). The developed microbial bioassay system not only represents a simple, inexpensive and environmental friendly alternative for determination of boric acid, but also offers a new idea and promising approach to detect boric acid

Identification and functional analysis of cyotochrome P450 from Bacillus lehensis G1

Cytochrome P450s (CYPs) are a superfamily of heme monooxygenases which catalyze a wide range of biochemical reactions. The reactions involve the introduction of an oxygen atom into an inactivated carbon of a compound which is essential to produce an intermediate of a hydroxylated product. Vitamin D 25- hydroxylase catalyses the first step in vitamin D biosynthetic pathway, essential in the activation of vitamin D. Several types of CYPs had been found as potential 25-hydroxylases. However, most of them originate from eukaryotes and are membrane associated proteins. A putative gene sequence encoding a CYP, termed CYP107CB2 was found in the genome of a new isolate Bacillus lehensis G1, and this gene shared sequence identity with the bacterial vitamin D hydroxylase (Vdh) from Pseudonocardia autotrophica. In order to deepen the understanding on the properties and biological function of CYP in B. lehensis G1, the objective of this study was to mine for a novel CYP from B. lehensis G1 with hydroxylase activity on vitamin D metabolites. Computational methods to search for the novel CYP from CYP structural databases were employed to identify the conserved pattern, functional domain and sequence properties of the uncharacterized CYP from B. lehensis G1. The CYP107CB2 gene was isolated and amplified using PCR and the CYP107CB2 protein was over-expressed in E. coli Rosetta-gami (DE3) followed by enzyme purification via single step affinity chromatography. The biological properties and possible functions of CYP107CB2 were characterized through absorption spectral analysis and were assayed for vitamin D hydroxylation activity. Optimization and CYP characterization were conducted to increase the turnover of hydroxylated products with an NADPH-regenerating system. Crystallization trials on CYP107CB2 protein were conducted via preliminary screening with Crystal Screen I and II through vapour-diffusion sitting drop method. Sequence analysis studies indicated that CYP107CB2 contained the fingerprint heme binding sequence motif FxxGxxxCxG at amino acid position 336-345 as well as other highly conserved motifs characteristic of CYP proteins. Docking studies showed several potential substrates, including vitamin D3, 25-hydroxyvitamin D3 and 1α- hydroxyvitamin D3, were located proximally to the enzyme’s heme center. The over-expressed CYP107CB2 protein was dominantly in cytosolic and the purified fraction showed a protein band at approximately 62 kDa on SDS-PAGE, representative for CYP107CB2. Spectral analysis demonstrated that the protein was properly folded and it was in its active form. HPLC and MS analysis on the product from a reconstituted enzymatic reaction confirmed that CYP107CB2 converted vitamin D3 and 1α-hydroxyvitamin D3 into 25-hydroxyitamin D3 and 1α,25-dihydroxyvitamin D3, respectively. CYP107CB2 formed crystal in formulation No. 38 from Crystal Screen II comprising 20% (v/v) PEG 10 000 and 0.1 mM HEPES buffer pH 7.5. In conclusion, a novel CYP107CB2 was identified from B. lehensis G1 and these findings proved that CYP107CB2 is a biologically relevant vitamin D3 25-hydroxylase

Effect of boric acid on the growth and production of β-glucosidase in Paecilomyces variotii

Boric acid was examined for antifungal activity against several species of fungi. The growth and production of β-glucosidase in several strains of fungi were tested in the media containing various concentrations of boric acid. The results indicated that the growth of Trichoderma strains was reduced with increasing amount of boric acid up to 0.3% (w/v). However, Paecilomyces variotii was able to tolerate boric acid above 0.3% (w/v) by increasing its growth. Trichoderma also showed low production of β-glucosidase as compared to P. variotii. Present study revealed that boric acid works as effective fungicide by inhibiting the production of β-glucosidase and the growth of fungi. This suggests that boric acid might be useful for the treatment of fungal infection caused by P. variotii or Trichoderma strains

Risk and health effect of boric acid

Problem statement: Boric acid is a pesticide usually used to kill mites, fungi, plants and insect including fleas, termites, cockroaches and wood decay fungi. Besides, it was also used in many fields such as food preservative, in newborn baby’s nurseries and antiseptic. Many reports indicated that boric acid poisoning occurred due to the misuse of household product and illegal use of boric acid in food product. In this study, the concern issue was the usage of boric acid that may lead to boric acid poisoning. Approach: This review had shown some information for boric acid such as its usage, the existent method for detection of boric acid in food. Besides, this review also discussed about the toxicology and pharmacokinetic of boric acid and the health impact of boric acid on human and animal. Result: Previous studies showed that food products such as yellow noodles contain boric acid. The boric acid level in most foods was different among the factory and the production period. It is due to the lack of standard measurement during the processing. Conclusion: Since boric acid was harmful to human health and may cause poisoning, hence, the control and the awareness of the usage of boric acid especially in food should be increased. There are numerous methods available for quantification of boric acid such as mannitol titration technique, colorimetric method. Accordingly, the analysis of boric acid is essential