Isolation and characterisation of pink-pigmented facultative methylotrophs bacteria isolated from leafy Malaysian ulam and its applications on beef

Presently, developing new colourants for the food industry is challenging as colourants need to be suitable for many factors, mainly food flavours, safety, and nutritional value, with minimal impact on the price of the product. As compared to natural food colourants, synthetic food colourants are selected due to their stability in long term usage and low cost, even though their safety and nutritional values can be questioned. To tackle this issue, a safe and yet nutritious natural food colourants are the alternatives. Interestingly, pigment of microorganism known as pink-pigmented facultative methylotrophs (PPFM) bacteria have not yet been utilised as a source of natural food colourant. Hence, the aim of this study is to analyse the antioxidant activity of pigments from these bacteria isolated from leafy Malaysian ulam. Initially, the PPFM colonies from three types of ulam leaves Cosmos caudatus Kunth (CC), Oenanthe javanica (Blume) DC. (OJ), and Melicope lunu-ankenda (Gaertn.) T. G. Hartley (ML) were isolated, and their genus and species characteristics were identified. Then, the growth pattern of the selected isolate was studied, followed by mass cell number production, and the pigment was extracted using solvents. The compounds in the extracted pigment were characterised, and their antioxidant activities and total phenolic content were evaluated. Besides, its toxicity level was estimated and its effect on colour, pH, and lipid oxidation in raw beef at certain storage times was observed. The results show a significant difference in PPFM population between the leaf surfaces of CC, OJ, and ML. All the collected isolates belong to Methylobacterium sp. in general, as they demonstrate Gram-negative properties, are motile, and have a rod shape with size (μm) ranging from 7.50 to 2.00 in length and 2.25 to 1.00 in width. They show positive results for catalase activity (all), lipids (most), starch and casein hydrolysis activities (less), citrate utilisation (a few), urease test (a few), oxidase test (most), and nitrate reduction test (a few). However, none of the isolates can degrade gelatin or cellulose as well as glucose fermentation and its metabolism action, or producing indole and hydrogen sulphide gaseous. The species of selected isolates of PPFM were identified as M. radiotolerans strain JCM 2831 (ML8), M. radiotolerans strain DSM 1819 (OJ4), and Methylorubrum salsuginis strain MR (OJ154). The growth study of selected isolate (ML8) display obvious pigmentation occurrence at day 4th and stationary phase is good time for cell harvest. About 5.30 ± 0.21% of pigments can be extracted through methanol higher than ethanol which is only 2.41 ± 0.14% yield. Moreover, a few significant peaks of ML8 pigment extract exist at 460.2, 490.8, 520.4, and 360.6 nm in the UV-visible spectrum and peaks at a range of 3462–990 cm-1 in the fouriertransform infrared spectroscopy (FTIR) spectrum, indicating the presence of carotenoid compounds. Through gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) analysis, several bioactive compound that are responsible for antioxidant activity, including phenolic and carotenoids compounds, were identified. Ethanolic ML8 pigment extract has a higher value than methanolic ML8 pigment extract in all tested antioxidant activities (except ABTS) and phenolic compounds. In addition, both pigment extracts are not toxic. There is improvement in the quality of the colour (redness a*) value, pH value, and lipid oxidation formation of raw minced beef treated with ethanolic ML8 pigment extract, particularly the 1.0% solution. Hence, the bacteria Methylobacterium sp. might be a good candidate for a source of pigment that has natural, safe, and nutritious colourant characteristics for food materials such as beef.

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