Fermentation-induced changes in the concentrations of organic acids, amino acids, sugars, and minerals and superoxide dismutase-like activity in tomato vinegar

ABSTRACT

Various raw materials are used to produce vinegars that contain functional compounds associated with disease prevention. We evaluated changes in functional compounds during tomato vinegar production and superoxide dismutase-like activity of tomato vinegar. Tomato vinegar contained abundant anti-hypertensive compounds, e.g., γ-aminobutyric acid and potassium derived from tomatoes and acetic acid and pyroglutamic acid produced during fermentation. It had stronger superoxide dismutase-like activity than commercial vinegars because of tomato-derived superoxide dismutase-like compounds, including phenolic acids, flavonoids, and glutathione. These data indicate that tomato vinegar is a candidate dietary supplement with potential preventive effects against cardiovascular diseases due to its anti-hypertensive and superoxide dismutase-like compounds.

KEYWORDS:

• Tomato vinegar
• Fermentation processes
• Anti-hypertensive and superoxide dismutase-like compounds
• Functional food
• Superoxide dismutase-like activity

Introduction

Vinegar is a common seasoning that has long been used as a traditional medicine to regulate blood pressure and blood glucose, promote calcium absorption and fatigue recovery, and stimulate appetite.^[1–4] Acetic acid is an essential and major constituent of vinegar; it has beneficial effects against hypertension,^[5] hyperglycemia,^[6] and dyslipidemia.^[7] In Japan, acetic acid is an active constituent of foods categorized as government-approved Food for Specified Health Uses for people with higher than normal blood pressure (https://hfnet.nih.go.jp/). In addition to acetic acid, vinegar contains various functional compounds derived from its raw material. In a previous study, we reported that apple vinegar made with high quantities of raw material contains abundant apple phenolic compounds with potent superoxide dismutase (SOD)-like activity.^[8] Budak et al.^[9] reported that red wine vinegar contains grape polyphenols that have high SOD-like activity. Horiuchi et al.^[10] developed onion vinegar that was abundant in onion-derived potassium, which has anti-hypertensive effects. These results demonstrate that the health benefits of vinegar may be related to functional compounds derived from raw material as well as products obtained by fermentation.

Tomatoes have the highest production value of any fruits in the world.^[11] Tomatoes and tomato products are rich in chemo-preventive and health-promoting compounds, including organic acids, amino acids, minerals, phenolic compounds, carotenoids, and vitamins.^[12–14] γ-Aminobutyric acid (GABA) is a well-known non-standard amino acid with a blood pressure lowering (BPL) effect^[15] and GABA-rich tomato show a significant BPL effect in spontaneously hypertensive rats.^[16] Potassium, which is abundant in tomato fruits, also has a BPL effect via natriuretic activity.^[17] In addition, phenolic compounds and vitamins exert pharmacological effects via anti-oxidant action. Anti-oxidant compounds trap radical oxidants, such as SOD, which is an important enzyme involved in cellular defense against reactive oxygen species (ROS).^[18] Phenolic compounds, including flavonoids, are potent SOD-like compounds in tomatoes and their products.^[19] Vitamin C is a major SOD-like compound in freshly consumed tomatoes.^[14]

In 2008, tomato vinegar was developed by Mizkan Co., Ltd. (Aichi, Japan). Its production involved two steps: alcohol fermentation of tomato juice to tomato wine and acetic acid fermentation of tomato wine to tomato vinegar. The tomato vinegar was expected to be a functional food containing acetic acid and tomato-derived functional compounds. Despite recent studies of tomato vinegar,^[20,21] their functional compounds and SOD-like activity have not been examined. We quantified the functional compounds in raw tomato juice and during the tomato vinegar fermentation processes. Additionally, we examined SOD-like activity in tomato vinegar and analyzed compounds with SOD-like activity using a liquid chromatography mass spectrometry (LC-MS) and database collation. The results of these analyses provide a basis for determining the health benefits of tomato vinegar. The present study analyzed changes in functional compounds during fermentations, SOD-like activity, and SOD-like compounds to estimate the functionality of the tomato vinegar.

Materials and methods

Chemicals

Formic acid, high-performance liquid chromatography (HPLC)-grade acetonitrile, methanol, p-toluenesulfonic acid, ethylenediamine tetraacetate (EDTA)’ after ‘methanol’ disodium hydrogen phosphate (Na₂HPO₄), sodium dihydrogen phosphate (NaH₂PO₄), citric acid, malic acid, pyroglutamic acid, tyrosine, phenylalanine, glutathione, wogonoside, naringin, chlorogenic acid, and rutin were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). Ninhydrin reagent, uridylic acid, adenylic acid, guanylic acid, and caffeic acid were purchased from Wako Pure Chemical Industries (Osaka, Japan). Trifluoroacetic acid (TFA) was purchased from Watanabe Chemical Industries, Ltd. (Hiroshima, Japan). Apple and rice vinegars were purchased from a local supermarket.

Preparation of tomato vinegar

Tomato vinegar was provided by Mizkan Co., Ltd. (Handa, Japan). The product was made from raw tomato juice by alcohol fermentation and acetic acid fermentation. Tomato fruits (Lycopersicon esculentum “Roma VF”) was crushed with water (2.5 kg tomato/L). After filtration and heat sterilization, the raw clear tomato juice was used for alcohol fermentation with yeast. Saccharomyces cerevisiae (25 g; Oriental Yeast Co., Ltd., Tokyo, Japan) was used as the starter and pectinase was used to promote fermentation; both were added to 20 L of clear tomato juice (Brix 15%), and the mixture was incubated at 30°C for 96 h. After filtration, the alcohol-fermented product with 3.6% (v/v) alcohol was obtained. The interim tomato wine and previously prepared unsterilized tomato vinegar including Acetobacter aceti with 7% acid were mixed in the ratio of 7:3, and then acetic acid fermentation was performed until the alcohol concentration decreased to 0.1% (v/v) in deep fermentation at 32°C for 110 h. The acetic acid fermentation product was added water, and the acid concentration was adjusted to 4%. After filtration and heat sterilization, the final tomato vinegar product was obtained. The unsterilized tomato vinegar was prepared in a similar manner for preparing the tomato vinegar using a usual vinegar starter culture of Acetobacter aceti with 7% acid prepared by Mizkan Co., Ltd., and the final product of tomato vinegar in three lots was prepared using the same tomato vinegar starter. In this study, the raw clear tomato juice (tomato juice), the interim product with 2.5% (v/v) alcohol (tomato wine), and the final product with 4% acid (tomato vinegar) were used for subsequent analyses.

Analysis of functional compounds in the tomato vinegar, interim product, and raw material

The functional compounds in the tomato vinegar, interim product, and raw material were analyzed at Mizkan Co., Ltd. To compare functional compounds among samples, the test volumes of the interim product and raw material were corresponded with that of the tomato vinegar obtained in the production process. Each of the three samples was prepared in triplicate and each preparation was analyzed. The results are expressed as means ± standard error (SE).

Organic acids

Organic acids were analyzed using an HPLC system (Shimadzu Co., Kyoto, Japan) equipped with an electronic conductivity detector. Separation was performed at 50°C on an RSpak KC-811 column (8.0 × 300 mm i.d., Showa Denko K.K., Tokyo, Japan) at a flow rate of 0.6 mL/min using an isocratic method. The mobile phases and injection volume were 4 mM p-toluenesulfonic acid aqueous solution and 20 μL, respectively. Detection was conducted with a postcolumn pH buffered method. Mixtures of 4 mM p-toluenesulfonic acid and 100 μM EDTA were used as postcolumn reagents.

Sugars

Sugars were analyzed using an HPLC system (Jasco Co., Tokyo, Japan) equipped with a refractive index detector. Separation was performed at 25°C on a Shodex NH2P-50 Column (4.6 × 250 mm i.d., Showa Denko K.K.) at a flow rate of 1.0 mL/min using an isocratic method. The mobile phases and injection volume were acetonitrile/water (75:25, v/v) and 5.0 μL, respectively.

Minerals

An atomic absorption spectrometer AA-6800 (Shimadzu) was used for analyses of Ca, Na, K, Mg, and Fe following methods described previously.^[22]

Amino acids

Amino acids in each sample were first separated by ion exchange chromatography, secondary reacted with ninhydrin reagent, and finally detected by visible light absorption detector on a JEOL AminoTac JLC-500V analyzer (JEOL, Tokyo, Japan). Samples without pre-treatment were injected directly to the analyzer.

Identification of SOD-like compounds in tomato vinegar by LC-MS and database collation

Preparation of 10% and 70% MeOH fractions from tomato vinegar

Tomato vinegar (2 mL) was applied to a Sep-Pak Vac C18 2 g Cartridge (Waters Co., Milford, MA, USA) pretreated with 24 mL of methanol/0.1% TFA water (10:90 v/v) at room temperature. The first 12 mL eluted with methanol/0.1% TFA water (10:90 v/v) was collected as the 10% MeOH fraction, and the latter 12 mL eluted with methanol/0.1% TFA water (70:30 v/v) was collected as the 70% MeOH fraction. The eluent composition was determined in preliminary experiments. The 10 and 70% MeOH fractions were lyophilized after evaporation under a vacuum and 174.6 and 9.4 mg were obtained, respectively. The lyophilized fractions were used for the LC-MS analysis and SOD-like activity assay.

LC-MS analysis of the MeOH fractions and database collation

The lyophilized fractions were dissolved in 500 μL of 0.1% formic acid-water and used for the LC-MS analysis. The analysis was performed using the Quattro micro API (MS) with a Waters 2695 LC system (Waters Co.) at the CREFAS (Collaborated Research Center for Food Functions, Faculty of Agriculture, Shinshu University). Separation was performed at 40°C on a CHEMCOBOND 5-ODS-W reversed-phase column (4.6 × 150 mm; Chemco Plus Scientific Co., Ltd. Kyoto, Japan) with an injection volume of 10 μL. Elution was performed at 0.8 mL/min using 0.1% formic acid-water (solvent A) and 0.1% formic acid-acetonitrile (solvent B): 0–6 min, 0–5% solvent B; 6–15 min, isocratic 5% solvent B; 15–30 min, 5–20% solvent B; 30–40 min, isocratic 20% solvent B. The UV detection wavelength was 215 nm. Mass spectra were acquired in electrospray ionization mode using 3500 V capillary voltage, 20 V cone voltage, 350 L/h N₂ gas flow (desolvation), 50 L/h N₂ gas flow (cone), 100°C source temperature, and 350°C desolvation temperature. The mass spectrometer was operated in negative and positive modes. In the analysis, compounds in the tomato vinegar fractions were identified by comparing the observed data (retention time, precursor ion, and fragment ions) with a spectral database, i.e., the Kazusa OMICS (KOMICS) Database: http://webs2.kazusa.or.jp/komicmarket.^[23] Validation of the identified compounds was performed by spiking the fractions with standards and repeating the analysis. The MS data for non-commercially available glycosides were reconfirmed based on literature information combined with experimental data derived from LC-MS-based metabolomics experiments. In each case, the identification was verified.

Measurement of SOD-like activity

SOD-like antioxidant activity of tomato vinegar was measured on 96-well plates using the SOD Assay Kit-WST (Dojindo Laboratories, Kumamoto, Japan) according to a method reported previously^[8] and compared the results with those of rice (typical cereal vinegar) and apple vinegars (typical fruit vinegar). The WST method is an in vitro SOD-like activity assay suitable for food materials.^[8,24] Briefly, a phosphate buffer solution (pH 7.0) was prepared by mixing 39 mL of 0.1 M NaH₂PO₄ solution and 61 mL of 0.1 M Na₂HPO₄. The vinegars were diluted in three dilution series with the phosphate buffer solution. The rice vinegar which is most popular vinegar in Japan and the apple vinegar which is abundant in phenolic compounds with SOD-like activity were employed as the comparison samples. The tomato vinegar fractions were prepared at three final concentrations using the buffer solution. The dilution ratio and test concentration were determined in preliminary experiments. The sample solution (20 μL) and WST working solution (200 μL) were mixed in a well. Enzyme solution (20 μL) was added to the mixture and the plates were incubated at 37°C for 20 min. After incubation, absorbance was measured at 520 nm using a Model 680 Microplate Reader (Bio-Rad Laboratories Ltd., Hercules, CA). The SOD-like activity (inhibition rate, %) for each sample solution was calculated using the following equation: SOD-like activity (inhibition rate, %) = {[(A blank – A blank control) – (A sample – A sample control)] /(A blank – A blank control)} × 100. In the blank, phosphate buffer was used in place of the sample solution. In the sample control, dilution buffer was used in place of the enzyme solution. In the blank control, phosphate and dilution buffers were used in place of the sample and enzyme solutions. All measurements were performed in triplicate for each sample concentration, and the results are expressed as means ± SE. The linear calibration curve for the dilution range using the liquid vinegar samples or the test concentrations for the tomato vinegar fractions (x) and the corresponding inhibition rate (y) was obtained, and the correlation coefficient was >0.98. The 50% inhibitory concentration (IC₅₀) was calculated from the regression curve.

Statistical analysis

All analyses were performed in triplicate. Functional compounds in the tomato vinegar, raw materials, and tomato wine were quantitatively compared by one-way analysis of variance (ANOVA) followed by Tukey’s tests and p < 0.05 was considered statistically significant. Student’s t-tests were performed to compare SOD-like activity between tomato vinegar, rice vinegar, and apple vinegar and the tomato vinegar fractions. The significance levels were p < 0.05 and p < 0.01 for the t-tests.

Results and discussion

We investigated the functionality of tomato vinegar by evaluating changes in functional compounds during fermentation and by analyzing SOD-like activity and SOD-like compounds.

Functional compounds in tomato vinegar and quantitative changes during fermentation

The upper columns of Table 1 show the total amounts of organic acids, amino acids, sugars, and minerals in tomato vinegar (final product), tomato wine (interim product), and tomato juice (raw material). The most abundant component in tomato vinegar was organic acids, followed by amino acids, sugars, and minerals. During tomato vinegar production, the total organic acid and total sugar contents significantly increased (p < 0.01) and decreased (p < 0.01), respectively, consistent with the conversion of sugar to ethanol by yeast during alcohol fermentation and conversion of ethanol to acetic acid by acetic acid bacteria during acetic acid fermentation. The total amino acid contents did not change significantly during fermentations, and most amino acids in the raw tomato juice were observed in the tomato vinegar. These results are consistent with a previous report that vegetable and fruit vinegars have similar amino acid compositions and contents to those of the raw materials.^[25] The total mineral contents were significantly lower (p < 0.05) in tomato wine than in the raw materials, suggesting that microbes metabolized minerals in the alcohol fermentation process.^[26] However, total minerals in the tomato vinegar were maintained at the same high level as that in the tomato wine.

Table 1. Summary of investigated organic acid, amino acid, sugar, and mineral concentrations during the production process of tomato vinegar (mg/100 mL).

	Tomato juice	Tomato wine	Tomato vinegar
Total contents
Organic acids	1388 ± 47^a	2065 ± 33^b	4039 ± 145^c
Amino acids	2619 ± 10^a	2280 ± 44^b	2525 ± 64^ab
Sugars	8311 ± 141^a	519 ± 168^b	418 ± 233^b
Minerals	760 ± 33^a	559 ± 18^b	552 ± 29^b
Organic acids
Acetic acid	7.3 ± 0.8^a	843 ± 9^b	2824 ± 136^c
Citric acid	1211 ± 48^a	1036 ± 25^a	1046 ± 18^a
Malic acid	138 ± 4^a	125 ± 8^a	114 ± 11^a
Succinic acid	8.5 ± 0.5^a	38 ± 2^b	40 ± 2^b
Lactic acid	23 ± 1^a	23.8 ± 0.7^a	16 ± 2^a
Amino acids
Pyro-Glu	367 ± 15^a	429 ± 8^ab	558 ± 45^b
Glu	551 ± 8^a	510 ± 10^a	519 ± 9^a
GABA	422 ± 8^a	348 ± 7^a	398 ± 22^a
Asp	318 ± 7^a	289 ± 6^a	291 ± 5^a
Ala	182 ± 12^a	176 ± 3^a	175 ± 7^a
Asn	188 ± 23^a	119 ± 2^a	162 ± 20^a
Gln	282 ± 23^a	162 ± 3^b	137 ± 8^b
Phe	52 ± 1^a	41 ± 1^b	41 ± 1^b
His	34 ± 3^a	35 ± 1^a	35 ± 5^a
Ser	34 ± 1^a	29 ± 1^a	32 ± 1^a
Cys	22 ± 4^a	13 ± 1^a	27 ± 3^a
Thr	29 ± 1^a	24 ± 1^b	27 ± 1^ab
Arg	27 ± 3^a	26 ± 1^a	26 ± 2^a
Lys	22 ± 2^a	20 ± 1^a	23 ± 1^a
Val	26 ± 6^a	7.9 ± 0.2^a	17 ± 2^a
Tyr	16 ± 1^a	13 ± 1^a	16 ± 1^a
Ile	17 ± 1^a	13 ± 1^b	13 ± 1^b
Leu	15 ± 1^a	12 ± 1^b	13 ± 1^ab
Gly	11 ± 1^a	10 ± 1^a	11 ± 1^a
Pro	1.5 ± 0.8^a	N.D.	4 ± 2^a
Met	3.8 ± 0.4^a	3.1 ± 0.1^ab	1.8 ± 0.2^b
Trp	0.41 ± 0.34	N.D.	N.D.
Sugars
Sucrose	787 ± 25^a	482 ± 198^a	287 ± 218^a
Glucose	3811 ± 122^a	37 ± 30^b	118 ± 49^b
Fructose	3712 ± 16	N.D.	N.D.
Minerals
K	694 ± 29^a	500 ± 16^b	497 ± 26^b
Mg	34 ± 2^a	32 ± 1^a	29 ± 4^a
Na	23 ± 2^a	22 ± 0^a	22 ± 1^a
Ca	8.6 ± 0.9^a	4.1 ± 0.4^b	4.2 ± 0.2^b
Fe	0.31 ± 0.03^a	0.30 ± 0.02^a	0.34 ± 0.02^a

Means within rows followed by the same letter (^{a, b}, or ^C) are not significant different at p < 0.05.

N.D.: not detected.

The individual compounds in organic acids, amino acids, sugars, and minerals during the fermentation processes are summarized in Table 1. The tomato vinegar contained the following organic acids (in decreasing order): acetic acid > citric acid > malic acid > succinic acid > lactic acid (ANOVA; F(3,8) = 1412.0; p = 3.1 × 10⁻¹¹). The acetic acid concentration increased significantly from 7.31 to 2824 mg/100 mL (386-fold, p < 0.01) during the alcohol and acetic acid fermentations. Succinic acid, which is produced as a metabolic byproduct of yeast during alcohol fermentation,^[27] also significantly increased 4.4-fold (from 8.51 to 37.7 mg/100 mL, p < 0.01) during alcohol fermentation. The concentrations of citric, malic, and lactic acids did not change significantly during the fermentation process.

The amino acid contents in the vinegar varied widely and were as follows (from highest to lowest concentration): pyroglutamic acid (pyro-Glu) > Glu > GABA > Asp > Ala > Asn > Gln > Phe > His > Ser > Cys > Thr > Arg > Lys > Val > Tyr > Ile > Leu > Gly > Pro > Met > Trp (ANOVA; F(21,44) = 133.1; p = 4.9 × 10⁻³³). The major amino acids were pyro-Glu, Glu, GABA, and Asp (558, 519, 398, and 291 mg/100 mL, respectively). The same four amino acids were also major components of the raw tomato juice and remained in the tomato vinegar at high levels. During the two-step fermentation, the pyro-Glu, Gln, Ile, Met, Phe, and Trp contents changed significantly (p < 0.05). In particular, pyro-Glu and Gln increased by 51.8% and decreased by 51.6% during alcohol fermentation, respectively. Pyro-Glu is chemically produced from Gln in the alcohol fermentation process.^[28]

There was no significant variability in the sugar components in the tomato vinegar (ANOVA; F(2,6) = 0.76; p = 0.51). However, except for sucrose, the sugar contents changed significantly during the fermentation processes (p < 0.01). Glucose decreased significantly by 96.9% during alcohol fermentation and fructose, which was observed in the raw material, was not detected in the vinegar or wine. These sugars are converted to ethanol during alcohol fermentation, providing a carbon source for acetic acid bacteria during alcohol oxidation.

The minerals in the vinegar varied widely and were observed as follows (in decreasing order): K > Mg > Na > Ca > Fe (ANOVA; F(3,8) = 226.4; p = 4.5 × 10⁻⁸); K constituted 90.0% of all minerals. Although K decreased significantly by 27.9% during alcohol fermentation (p < 0.05), it was the most abundant mineral in the vinegar (497 mg/100 mL). The lowering of K contents during the vinegar production could be caused by removal of yeast cell containing high levels of internal K⁺ in filtration processes. The levels of Mg, Na, and Fe were the same before and after the fermentations.

Collectively, the tomato vinegar contained abundant tomato-derived GABA and potassium, in addition to acetic acid and pyro-Glu produced during the fermentations. Acetic acid intake reduces blood pressure via vasorelaxation in vivo.^[5] GABA has a BPL effect due to the inhibition of norepinephrine release from sympathetic nerves via presynaptic GABA B receptors.^[15] Additionally, pyro-Glu decreases blood pressure by reducing vascular resistance in rat carotid arteries.^[29] Potassium also has a BPL effect due to its natriuretic activity.^[17,30] Hypertension is a multifactorial disorder characterized by persistent increases in blood pressure and involves abnormalities in cardiovascular function.^[31] Based on our results, we inferred that tomato vinegar is likely to have an anti-hypertensive effect owing to the functional compounds derived from raw tomato juice and their increased concentrations during fermentation.

SOD-Like activity of tomato vinegar

SOD-like activity is a well-known indicator of food functionality in tomato fruits.^[32] It is expected that tomato vinegar also have SOD-like anti-oxidant activity. Therefore, we measured the activity using the WST-1 method. The activities of the vinegar samples (tomato, apple, and rice vinegars) are expressed as the dilution that caused 50% inhibition (Fig. 1a).^[8] The IC₅₀ dilutions for tomato, apple, and rice vinegars were 19.6, 4.7, and 1.5 times, respectively. Tomato vinegar demonstrated more potent SOD-like activity (p < 0.01) than commercial apple and rice vinegars. To characterize SOD-like compounds responsible for the SOD-like activity of tomato vinegar, we measured the SOD-like activities of 10 and 70% MeOH fractions prepared from tomato vinegar by ODS solid phase extraction.

Figure 1. SOD-like activities of tomato, apple, and rice vinegars (A), 70% MeOH and 10% MeOH fractions, and lyophilized tomato vinegar (B). Significance (assessed using Student’s t-tests): **p < 0.01.

The lyophilized fractions were assayed, and lyophilized tomato vinegar was used as a control. The activities of the lyophilized samples are expressed as the concentrations that caused 50% inhibition (IC₅₀; Fig. 1b). The IC₅₀ values for the 10% and 70% MeOH fractions and lyophilized tomato vinegar were 9.8, 0.72, and 6.5 mg/mL, respectively. The SOD-like activity of the 70% MeOH fraction was significantly higher (p < 0.01) than that of the 10% MeOH fraction. The activity increased by 9.0 times (p < 0.01) compared with that of lyophilized tomato vinegar. However, the 10% MeOH fraction had moderate SOD-like activity. These results demonstrated that major SOD-like compounds in tomato vinegar were concentrated in the 70% MeOH fraction. Therefore, we investigated the SOD-like compounds contained in both fractions using a database search based on LC-MS.

Identification of SOD-like compounds in the 10 and 70% MeOH fractions of tomato vinegar

Compounds in the 10% and 70% MeOH fractions were identified by collating observed MS data (i.e., the observed peaks) with reference data from the KOMICS tomato derived-compounds database.^[23] Table 2 presents all compounds detected in the 10 and 70% MeOH fractions. Figure 2 shows the LC chromatograms of the fractions. In the 10% MeOH fraction, the major compounds were amino acids and organic acids with moderate SOD-like activity.^[43–45] The 70% MeOH fraction contained phenolic acids, flavonoids and its glycosides, and glutathione. As phenolic acids, caffeic acid (peak N in Fig. 2) and chlorogenic acid (peak P) were identified. Additionally, phenolic acid glycosides, such as caffeic acid-hexose (peak I), p-coumaric acid-hexose (peak J), and sinapic acid-hexose (peak K), were detected. Flavonoids and their glycosides, i.e., naringin (peak O), naringenin chalcone hexose (peak Q), quercetin-hexose-deoxyhexose-pentose (peak R), rutin (peak S), and wogonoside (peak M), were also identified. Glutathione was detected (peak L). Phenolic acids, flavonoids, and their glycosides with at least one aromatic ring bearing one or more hydroxyl groups possess strong SOD-like activity against ROS.^[46,47] Glutathione is an important low-molecular-weight sulfhydryl containing anti-oxidant compounds in vivo.^[48] Vitamins and carotenoids, including vitamin C, lycopene, and β-carotene, were not detected. Thermally unstable Vitamin C might be lost in heat sterilization of the raw tomato juice and final product,^[49] and carotenoids (lycopene and β-carotene) with high lipophilicity might be precipitated and removed as residue by filtration in the vinegar production processes.

Table 2. Mass spectrometry ion data for compounds detected in the tomato vinegar fractions by liquid chromatography electrospray ionization mass spectrometry.

Peak no.	RT (min)	Compound	Observed mass (m/z)	Calculated exact mass	Reference
A	3.5	Malic acid	133.0 [M-H]^−	134.0215	Iijima et al.^[^23^]
B	3.8	Citric acid	191.0 [M-H]^−	192.0270	Iijima et al.^[^23^]
C	4.3	Pyroglutamic acid	128.0 [M-H]^−	129.0426	Iijima et al.^[^23^]
D	5.3	Tyrosine	182.0 [M+H]^+	181.0739	Iijima et al.^[^23^]
E	8.8	Phenylalanine	164.0 [M-H]^−	165.0790	Carrari et al.^[^33^]
F	5.0	Uridylic acid	323.1 [M-H]^−	324.0359	Iijima et al.^[^23^]
			243.1 [M-H2PO3]^−
G	6.7	Adenylic acid	348.0 [M+H]^+	347.0631	Iijima et al.^[^23^]
			268.0 [M-H2PO3]^+
H	7.2	Guanylic acid	362.0 [M-H]^−	363.0580	Iijima et al.^[^23^]
			282.0 [M-H2PO3]^−
I	9.0	Caffeic acid-hexose	340.9 [M-H]^−	342.0951	Winter and Herrmann^[^34^]
			182.0 [M-hexose]^+
J	10.3	p-Coumaric acid-hexose	325.1 [M-H]^−	326.1002	Buta and Spaulding^[^35^]
			166.0 [M-hexose]^+
K	11.5	Sinapinic acid-hexose	387.1 [M+H]^+	386.1213	Schmidtlein and Herrmann^[^36^]
			224.0 [M-hexose]^+
L	12.6	Glutathione	308.0 [M+H]^+	307.0838	Iijima et al.^[^23^]
			251.0 [M-glycine]^+
M	14.6	Tuliposide B	295.0 [M+H]^+	294.0951	Shoji et al.^[^37^]
		Wogonoside	458.9 [M-H]^−	460.1006	Seo et al.^[^38^]
N	20.2	Caffeic acid	179.0 [M-H]^−	180.0423	Le Gall et al.^[^39^]
O	21.2	Naringin	579.3 [M-H]^−	580.1792	Bovy et al.^[^40^]
P	22.5	Chlorogenic acid	353.3 [M-H]^−	354.0951	Le Gall et al.^[^39^]
			161.2 [M-quinic acid]^−
Q	30.0	Naringenin chalcone-hexose	433.0 [M-H]^−	434.1213	Bino et al.^[^41^]
			273.0 [M-hexose]^+
R	31.5	Quercetin-hexose-deoxyhexose-pentose	741.0 [M-H]^−	742.1956	Minoggio et al.^[^42^]
			303.0 [M-hexose-deoxyhexose-pentose]^+
S	32.5	Rutin	611.0 [M+H]^+	610.1534	Minoggio et al.^[^42^]
			303.0 [M-rutinose]^+

Figure 2. Chromatograms from liquid chromatography analyses of tomato vinegar: 10% MeOH (A) and 70% MeOH fraction (B). The detection wavelength was 215 nm.

Based on the LC-MS analyses and the database collation, 19 distinct compounds were identified in the MeOH fractions. The 10 and 70% MeOH fraction contained SOD-like compounds with moderate and potent activity, respectively. The results supported higher SOD-like activity for the 70% MeOH fraction than the 10% MeOH fraction (Fig. 1). Tomato fruits contain abundant non-enzymatic SOD-like compounds, such as phenolic acids and flavonoids.^[12–14] We have observed that the SOD-like activity of vinegar is greatly affected by the raw materials used for its production.^[8] Based on our results, we inferred that the tomato-derived compounds confer potent SOD-like activity in tomato vinegar.

The excessive generation and accumulation of ROS causes the activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in cardiovascular tissue.^[50] NADPH oxidase activation impairs vasodilatory nitric oxide (NO) production and bioavailability in vascular smooth muscle cells,^[51] which causes vascular dysfunction.^[52,53] Therefore, ROS scavenging can prevent vascular dysfunction. SOD converts superoxide anions, including ROS, into hydrogen peroxide.^[54] Accordingly, tomato vinegar, which contained tomato-derived SOD-like compounds, might have preventative effects on vascular dysfunction.

Conclusion

The quantification of organic acids, amino acids, sugars, and minerals as well as the LC-MS database collation analysis revealed existence of BPL and SOD-like compounds in tomato vinegar. Tomato vinegar contained abundant tomato-derived functional compounds with anti-hypertensive effects, such as GABA and potassium, in addition to acetic acid and pyro-Glu, which were produced during fermentation. Moreover, it had potent SOD-like activity, which was attributed to various tomato-derived compounds, including phenolic acids, flavonoids and its glycosides, and glutathione. Hypertension and vascular dysfunction are major risk factors of cardiovascular diseases, including stroke, heart failure, and atherosclerosis.^[55] These observed properties of tomato vinegar suggest that tomato vinegar is a useful functional food with potential preventive effects against cardiovascular diseases via its anti-hypertensive and SOD-like compounds.