Organic acid (OA) profiling analysis was performed in culture media from Lactobacillus pentosus K34 (L. pentosus K34) and Pediococcus lolli PL24 (P. lolli PL24) by gas chromatography-mass spectrometry (GC-MS) following methoxime/ tert-butyldimethylsilyl derivatives. 12 OAs were positively identified in culture media. Most of OA levels from L. pentosus K34 of hetero lactic fermentation were found to be higher when compared with those from P. lolli PL24 of homo lactic fermentation, which may explain different OA metabolism in each strain. In addition, the distorted dodecagonal star patterns were readily distinguishable, and the characteristics of each strain were well represented. The present study demonstrates that the OA metabolic profiling method by GC-MS combined with star pattern recognition is useful for the monitoring study of characteristic OA metabolism in various microorganisms.
Organic acids (OAs) were well known as the final products in the metabolic process, which therefore supply information for altered biochemical metabolism from various biological samples including microorganisms.1-3 In particular, lactic acid in industrial chemistry fields was reported as important metabolite in fermentation step.4-9 In our previous report,10 phenyllactic acid was produced in high abundance with lactic acid and acetic acid. Phenyllactic acid is a novel antimicrobial compound active against Gram-positive and Gram-negative bacteria.11,12 Phenyllactic acid can be converted from phenylpyruvic acid by NADH dependent lactic acid dehydrogenase (LDH, EC 1.1.1.2.7).14 Therefore, the analysis of OAs in culture media and cells of microorganisms is important in the monitoring and screening of microbial availability. In our previous reports, OA profiling analyses by gas chromatography (GC) and GC-mass spectrometry (GC-MS) were found to be useful for the comparative analysis between control and experimental groups.9,10,14,15
Gram positive
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Strain and culture condition
OA standards including 3,4-dimethoxybenzoic acid as internal standard (IS), triethylamine (TEA) methoxyamine hydrochloride were purchased from Sigma-Aldrich.
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Gas chromatography-mass spectrometry
Derivatized samples were analyzed using an Agilent 6890N gas chromatograph interfaced to an Agilent 5975B mass-selective detector (70 eV, electron ionization source). The mass spectra were scanned in the mass range of 50- 650 u at a rate of 0.99 scans/s. The temperatures of the injector, interface, and ion source were 260, 300, and 230 ℃, respectively. An Ultra-2, cross-linked capillary column coated with 5% phenyl-95% methylpolysiloxane bonded phase (25 m × 0.20 mm I.D., 0.11 mm film thickness, Agilent Technologies, Santa Clara, CA, USA) was used for all analyses. Helium was used as the carrier gas at a flow rate of 0.5 mL/min in the constant flow mode. Samples (1 μL) were introduced in split-injection mode (10:1), and the oven temperature was set initially at 100 ℃ (2 min), then increased to 250 ℃ at rate of 5 ℃/min and finally programmed to 300 ℃ at rate of 20 ℃/min (5 min).
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Sample preparation for measurements of organic acids in cell culture media
Control media (MRS), and culture media from
The mean peak area ratios to IS of OAs confirmed in the control media and culture media from
GC-MS profiles from the control media (A),
Values of organic acids in control media, and cultured media with L. pentosus K34 and P. lolli PL24.
strain.
the various OAs including lactic acid throughout the different metabolic pathway. Typically, hydroxyl acids such as No.4, No.5, and No.6 produced by
The OA levels in the media of control and
OA metabolic profiles in cell growth media from