Materials
Yeast (Saccharomyces cerevisiae) and pectinase from Aspergillus niger were purchased from Oriental Yeast Co., Ltd. (Tokyo, Japan) and Shin Nihon Chemical Co., Ltd. (Anjyo, Japan), respectively. N-Acethyl-L-cysteine (NAC), N-tert-butyloxycarbonyl-L-cysteine (NBC) and o-phthaldialdehyde (OPA) were from Wako (Tokyo, Japan), Nova Biochemical (Waltham, MA, USA) and Nacalai Tesque (Kyoto, Japan), respectively. D-Amino acid oxidase from porcine kidney and catalase from bovine liver were purchased from Sigma (St. Louis, MO, USA). Lactobacillus and Lactococcus strains were from the Japan Collection of Microorganisms (JCM, Tsukuba, Japan).
Vinegar samples
Eleven vinegars (Table 1) were provided by the Central Research Institute of Mizkan Group Corporation. These samples were stored at 4°C until use. In these 11 vinegars, three kinds of vinegar (high-brix nonglutinous brown rice black vinegar, high-brix nonglutinous brown rice black vinegar matured in barrel and high-brix apple vinegar) have the word “high-brix” in their names. The “high-brix” means that these vinegars were produced using a larger amount of initial material than were used in the production of vinegars lacking the “high-brix”. The initial material of “high-brix nonglutinous brown rice black vinegar” and “high-brix nonglutinous brown rice black vinegar matured in barrel” was nonglutinous brown rice, and one of “high-brix apple vinegar” was apple juice. High-brix nonglutinous brown rice black vinegar matured in barrel and lactic fermented tomato vinegar, which are not currently available on the market, served as test samples for new commodification. High-brix nonglutinous brown rice black vinegar matured in barrel was produced by the maturation of high-brix nonglutinous brown rice black vinegar at 25°C for a month in a 20-L barrel. On the other hand, the lactic fermented tomato vinegar was produced through acetic fermentation using the mixture of alcoholic fermented tomato juice and lactic fermented juice.
D-Amino acids in five different samples collected during the manufacture of lactic fermented tomato vinegar
D-Amino acids in the following five samples produced by Mizkan Group Corporation (Figure 1) were analyzed: sample 1 was tomato juice that served as the source for other samples; sample 2 was alcoholic fermented tomato juice; sample 3 was acetic fermented tomato juice; sample 4 was tomato juice before lactic fermentation; and sample 5 was lactic fermented tomato juice. As shown in Figure 1, these five samples were made on 3-L volumes from a single lot of tomatoes through two separate lines. Sample 1 (tomato juice-source) was prepared by mixing crushed tomatoes with water (2.5 kg-tomato/L). For the preparation of sample 2, the tomato juice-source was incubated at 28°C for 96 h after the addition of 0.5% (g/v) yeast and 0.3% (g/v) pectinase (alcoholic fermentation). After alcoholic fermentation, the tomato juice was filtered through filter paper (Advantec grade No. 2 qualitative filter paper; Advantec, Tokyo) and diatomaceous earth, and then heat-treated at 90°C for 1 min. For the preparation of sample 3, acetic acid bacteria (on the order of 105 cells) were inoculated into the sample 2 (3 L), after which acetic fermentation was proceeded at 32°C for 110 h. The resultant acetic fermented tomato juice was heat-treated and filtered as described above (sample 3). Sample 4 was produced by the addition of 0.3% pectinase to the sample 1 (3 L), followed by incubation at 60°C for 1 h and heat-treatment at 92°C for 30 min. To produce sample 5, lactic acid bacteria (105 cells) were added to sample 4 (3 L), and the mixture was fermented at 30°C for 90 h. The lactic fermented tomato juice was then subjected to heat-treatment at 79°C for 10 min and filtered as described above (sample 5).
D-Amino acids in culture media conditioned by lactic acid bacteria
D-Amino acids were analyzed in the culture medium of eight lactic acid bacterial strains (Table 5). These strains were cultured to the stationary phase in 5 mL of MRS medium (Becton, Dickinson and Co., Franklin Lakes, NJ, USA) at their respective optimum cultivation temperatures (Table 5). An aliquot (1 mL) of the culture medium was then centrifuged (10,000 × g for 1 min at 4°C), and the supernatant was filtered through polytetrafluoroethylene membrane filters (4-mm diameter, 0.2-μm pore size; Merck Millipore, Darmstadt, Germany). The prepared samples were stored at −20°C until use.
Sample preparation for amino acid analyses
To prepare samples for determination of free D- and L-amino acids, an aliquot of each sample (500 μL) was initially neutralized with 10 M NaOH and diluted to 600 μL with purified water. This solution was then filtered through an Amicon Ultra 0.5 mL centrifugal filter 3 K device (Merck Millipore) with centrifugation (14,000 × g for 15 min at 4°C).
Derivatization of amino acids
Amino acids were derivatized using OPA plus NAC or NBC (Aswad, 1984; Hashimoto et al., 1992). Two methanolic solutions containing derivatizing reagents were prepared. Methanolic solution A was prepared by dissolving 8 mg of OPA and 10 mg of NAC in 1 mL of methanol, while methanolic solution B was prepared by dissolving 10 mg of OPA and 10 mg of NBC in 1 mL of methanol. The reaction mixture (250 μL) for the derivatization consisted of 25 μL of sample, 50 μL of methanolic solution (A or B), and 175 μL of 0.4 M borate-NaOH buffer (pH 10.4). After derivatization for 2 min at room temperature in the dark, an aliquot (1 μL) of the reaction mixture was introduced into an ultra-performance liquid chromatography (UPLC) system (Waters, Milford, MA, USA).
Standard solution
The standard solution consisted of 32 kinds of amino acids: D- and L-forms of Asp, Glu, Ser, Ala, Leu, Phe, Val, Met, tryptophan (Trp), Arg, histidine (His), threonine (Thr), Asn, tyrosine (Tyr) and glutamine (Gln), plus L-isoleucine (L-Ile) and D-allo-Ile. For the calibration curves, the standard solution was diluted to 7 concentrations (5, 25, 50, 100, 250, 500 and 1,000 μM) in 0.05 M HCl, after which the solutions were filtered through polytetrafluoroethylene membrane filters (4-mm diameter, 0.2-μm pore size, Merck Millipore).
Operation of the UPLC system
The diastereoisomeric derivatives formed with OPA-NAC or OPA-NBC were applied to a Waters AccQ-Tag Ultra 2.1 × 100 mm column (Waters) in an the ACQUITY UPLC TUV system consisting of a Waters Binary Solvent Manager, a Waters Sample Manager, and a Waters FLR Detector. The excitation and emission wavelengths for fluorescent detection of amino acids were 350 nm and 450 nm, respectively. The data were processed using Empower 2 (Waters). The system was operated with a flow rate of 0.25 mL/min at 30°C. The UPLC gradient system for analysis of OPA-NAC derivatives (A = 50 mM sodium acetate, pH 5.9, and B = methanol) was 10 − 20% B over 3.2 min, 20% B for 1 min, 20 − 40% B over 3.6 min, 40% B for 1.2 min, 40 − 60% B over 3.8 min, 60% B for 1 min, and 60 − 10% B over 0.01 min. The gradient system used for analysis of OPA-NBC derivatives (A = 50 mM sodium acetate, pH 5.9, and B = acetonitrile) was 15 − 21% B over 7 min, 21–27.5% B over 1.5 min, 27.5% B for 2 min, 27.5 − 30% B over 1 min, 30 − 40% B over 2 min, 40% B for 0.5 min, and 40 − 15% B over 0.01 min.
Analysis and quantification of D-amino acids
With the UPLC method, the peak heights were used for quantification of amino acids. In addition, D-amino acid peaks were identified based on their retention times, as verified by comparisons with authentic samples. Food samples usually contain any unidentified amino compounds that give the same retention time-peak as a D-amino acid. Therefore, the peaks of D-amino acids, except for D-Glu, D-Asp, D-Gln and D-Asn, were ascertained from the reduction in fluorescence intensity elicited by treatment with D-amino acid oxidase (DAO) from Sus scrofa (Tosa et al., 1974). This DAO has very broad substrate specificity, oxidizing 12 different D-amino acids, though not D-Glu, D-Asp, D-Gln or D-Asn (D’Aniello et al., 1993). Treatment with DAO removed the 12 susceptible D-amino acids from the food samples, causing the respective peaks to be smaller than those obtained with corresponding untreated samples. To treat samples with DAO, an aliquot of sample solution (200 μL) was mixed with 300 μL of 150 mM sodium pyrophosphate buffer (pH 8.5) containing 182.5 μg of DAO, 67.5 μg of catalase from bovine liver and 2 mM flavin adenine dinucleotide, and incubated for 12 h at 25°C. The sample was then subjected to UPLC analysis after the removal of DAO and catalase by filtration using an Amicon Ultra 0.5 mL centrifugal filter 3 K device (Merck Millipore).
Calibration curves were constructed by plotting peak heights against amino acid concentrations at ranging from 5 to 1,000 μM. For all 16 amino acid types tested, the relation was linear with regression coefficients above 0.999.
When amino acids were derivatized with OPA-NAC or OPA-NBC, any compounds containing an amino group in addition to D- and L-amino acids can also be modified. To decrease the effects of amino compounds other than α-amino acids when assaying D- and L-amino acids, the lower concentrations of amino acids were selected and assessed using in two analyses using NAC and NBC as chiral regents. D-His, L-Arg, D-Trp, and D-allo-Ile were detected using only when OPA-NAC was used as the chiral reagent, whereas L-Glu, D-Glu, L-Asn and D-Asn were detected only using OPA-NBC.