Distribution of Pyrrolothiazolate, a Pigment Formed through the Maillard Reaction between Cysteine and Glucose, in Foods and Beverages and Some of Its Properties

Abstract

Pyrrolothiazolate is a Maillard reaction product isolated from a model solution containing cysteine and glucose. This compound is a yellow pigment and it has a unique pyrrolothiazole ring, which has never been reported in the various heterocyclic compounds formed through the Maillard reaction. To clarify the distribution of this pigment in foods and some of its biological properties, we measured it in various foods and beverages and examined its antibacterial and mutagenic activities. As a result, this compound was found in soy sauce, miso, and beer. The contents of the pigment in these foods ranged from 1 to 106 µg/100 mL or 100 g. Thus, pyrrolothiazolate was formed during food processing and storage as well as in the model systems. Pyrrolothiazolate did not show antibacterial activity nor mutagenicity with the Ames test.

Introduction

The Maillard reaction is a reaction between proteins/amino acids and reducing sugars. Almost all foods contain these substances, meaning that the Maillard reaction usually occurs during the processing and storage of foods. This reaction has crucial effects on a wide variety of foods, such as browning and the formation of reaction flavor.

The brown color of foods is often attributed to this reaction. The major brown pigments formed by the Maillard reaction are called melanoidins, which are high-molecular-weight polymers derived from amino acids, proteins, reducing sugars, and its decomposed carbonyl compounds. Because of its complexity and heterogeneity, the elucidation of the chemical structures of melanoidins is theoretically and methodologically very hard. On the other hand, low-molecular-weight colored compounds formed through the Maillard reaction can be chemically identified. In fact, several pigments have been isolated and identified from model Maillard reaction systems between amino acids and sugars (Hoffman, 1998; Hayase et al., 1999; Ames et al., 1999; Frank and Hoffman, 2000; Shirahashi et al., 2009). Our group has also reported several pigments, such as furpipate (Murata et al., 2007; Totsuka et al., 2009), dilysyldipyrrolones (Sakamoto et al., 2009; Nomi et al., 2011, 2013), pyrrolothiazolate (Noda et al., 2015, 2016), and pyrrolooxazolates (Noda and Murata, 2017). Almost all of these pigments were isolated from model reaction systems, and it remains unclear if these pigments exist in foods and beverages.

Our group isolated 2,4-dihydroxy-2,5-dimethyl-3(2H)-thiophenone from soy sauce as a Maillard pigment, and showed its formation during soy sauce production (Sato et al., 2011; Furusawa et al., 2013). This compound is derived from cysteine. On the other hand, pyrrolothiazolate, a yellow pigment identified in a model Maillard reaction system containing cysteine, lysine and glucose, is formed by the condensation of cysteine and an intermediate of the Maillard reaction derived from hexoses (Fig. 1; Noda et al., 2016). Although this pigment might be formed in foods containing sufficient amounts of cysteine, free amino acids, and reducing sugars, its formation in foods has not been examined yet. Pyrrolothiazolate has a unique pyrrolothiazole ring, which has never been reported in the various heterocyclic compounds formed through the Maillard reaction. Pyrrolothiazolate shows antioxidative activities (Noda et al., 2016). However, its other biological properties have not been reported. The aim of this study is to clarify the existence of pyrrolothiazolate in foods and some of its biological properties, such as antibacterial activity and mutagenicity.

Fig. 1.

Chemical structure of pyrrolothiazolate and its formation scheme from L-cysteine and D-glucose.

Materials and Methods

Materials    Soybean, wheat, soy sauce (18 brands), fish sauce (3 brands), black vinegar, mirin (rice wine), miso (fermented soybean paste; 9 brands), bread, cocoa powder, kinako (flour of roasted soybean), natto (fermented soybean), onion, beer (6 brands), barley tea powder, and soluble coffee powder were purchased at a local market (Tokyo, Japan). Malt was purchased from Brewland Co. (Nishio, Japan), and wort was prepared as follows. Reverse osmosis water (50 mL) was added to pale malt powder (12.5 g) ground with a coffee mill (SKR-M070-SF, Tiger Co., Kadoma, Japan) and mixed. This suspension was incubated with stirring at 50 °C for 30 min, 60 °C for 30 min, and 70 °C for 30 min, successively, and then heated with stirring at 100 °C for 30 min. The heated suspension was centrifuged at 1 700 × g for 5 min, and the obtained supernatant was used as a wort sample. Authentic pyrrolothiazolate was prepared according to the method of Noda et al. (2016).

Preparation of soy sauce    Soy sauce was prepared at a laboratory scale. Soybeans (Glycine max cv. Toyo-masari; 2.5 L) were soaked in about 5 L of tap water at room temperature (about 25 °C) for 24 h, then boiled for 6 h. Wheat (Triticum aestivam cv. Yukichikara; 2.5 L) was roasted on a pan for 10 min, then crushed with a wooden hammer. Spores of Aspergillus oryzae (about 8 g, Kojiya Mizaemon, Toyohashi, Japan) were added to a mixture of the boiled soybeans and the roasted and crushed wheat, mixed by hand, then incubated at 30 °C for 3 days to form koji. The koji was mixed with about 7.5 L of brine containing 3.3 kg NaCl before the mixture was fermented for 9 months at room temperature (20 °C–25 °C) with occasional stirring. The mixture fermented for 0 to 9 months was centrifuged at 1 700 × g for 15 min, and the obtained supernatant was used for measurements of pyrrolothiazolate.

Sample preparation for analyses    Liquid samples (10 mL) were washed with the same volume of ethyl acetate three times. The pH of the washed samples was adjusted to less than 2 by adding 6 M HCl before pyrrolothiazolate was extracted with the same volume of ethyl acetate three times. The combined ethyl acetate layer was concentrated in vacuo and dissolved in methanol before being applied to high performance liquid chromatography (HPLC) analysis.

For solid samples, each sample (50 g) was homogenized in 250 mL of 80% methanol (v/v) with an ULTRA-TURRAX disperser (Yamato, Tokyo), then the mixture was filtrated. This extraction procedure was repeated three times. The combined filtrate was concentrated in vacuo to remove methanol before being applied to the same procedure as the liquid samples.

Liquid chromatography-mass spectrometry (LC-MS) analysis for the detection of pyrrolothiazolate in soy sauce    The ethyl acetate layer of soy sauce, which was prepared as described above, was analyzed using a mass spectrometer (Triple TOF 4600, AB Sciex, Foster City, CA) coupled with HPLC to detect pyrrolothiazolate. The ionization mode was set to electro spray ionization (ESI+). Pyrrolothiazolate (M.W. 229) was detected using the precursor ion scan of m/z 230.048 (C₉H₁₂NO₄S, (M+H)⁺). The HPLC conditions were as follows: system, Prominence (Shimadzu, Kyoto, Japan); column, Inertsil ODS-3 (2.1 mm i.d. × 150 mm, 3 µm, GL Science, Tokyo); eluent, 0.2% HCOOH-water, v/v; flow rate, 0.2 mL/min; column temperature, 50 °C. Pyrrolothiazolate was detected at a retention time of about 21 min under this condition.

HPLC analysis for the quantification of pyrrolothiazolate    Each sample was analyzed using a reversed-phase HPLC system equipped with diode-array detection (DAD) under the following conditions: system, Agilent 1100 series (Palo Alto, CA); column, TSK gel ODS-100 V (4.6 mm i.d. × 250 mm, 5 µm, Tosoh, Tokyo); eluent, 0.1% HCOOH-water, v/v; flow rate, 1.0 mL/min; column temperature, 50 °C; detection, 220–500 nm, 370 nm for quantification. Pyrrolothiazolate was detected at a retention time of about 18 min. The detection limit of pyrrolothiazolate was 0.8 µg/100 mL or 0.5 µg/100 g of sample in this condition. The quantification limit was 2.5 µg/100 mL and 1.5 µg/100 g of sample in this condition.

Measurement of the absorbance of retail soy sauce    Samples of retail soy sauce were appropriately diluted with reverse osmosis water, and their absorbances at 400 nm were measured using a V-530 UV-visible spectrophotometer (JASCO, Tokyo).

Antibacterial assay    The antibacterial activities of pyrrolothiazolate against three kinds of bacteria were examined by a paper disk method on brain heart infusion agar plates (for Staphylococcus aureus FDA-209P and Escherichia coli NIHJ) or Davis minimum medium agar plates (for Bacillus subtilis PCI-219). Paper disks (8 mm i.d., Toyo Roshi Kaisya, Tokyo) containing solutions (30 µL) of streptomycin (0.025-100 µg/mL, positive control for B. subtilis PCI-219), ampicillin (0.025-100 µg/mL, positive control for S. aureus FDA-209P and E. coli NIHJ), or pyrrolothiazolate (25-1600 µg/mL) were placed on agar plates inoculated with each bacteria (10⁷ colony forming units per plate), then incubated at 37 °C for 24 h, and the diameters of the inhibitory zones were measured.

Ames mutagenicity test for pyrrolothiazolate    The mutagenic activities of pyrrolothiazolate on Salmonella Typhimurium TA98 and TA100 were examined with and without the S9 mix (Kikkoman, Tokyo) by the Ames test (McCann et al., 1975). Dimethyl sulfoxide (DMSO; 100 µL, negative control) or a solution (100 µL) of pyrrolothiazolate (50 mg/mL DMSO), MeIQX (0.3 µg/mL, positive control for S. Typhimurium TA98 with S9 mix), 2-nitrofluorene (20 µg/mL, positive control for S. Typhimurium TA98 without S9 mix), sodium azide (10 µg/mL, positive control for S. Typhimurium TA100 with S9 mix), or 2-aminoanthracene (10 µg/mL, positive control for S. Typhimurium TA100 without S9 mix), 500 µL of S9 mix or PBS, and 100 µL of a suspension of S. Typhimurium TA98 or TA 100 were mixed. Each mixture was incubated at 37 °C for 20 min before 20 mL of soft agar containing 0.5 mM histidine and 0.5 mM biotin was added to the mixture. Each agar plate was incubated at 37 °C for 48 h, then the number of revertant colonies was counted.

Results and Discussion

Detection of pyrrolothiazolate in soy sauce    As our previous study showed that this pigment was formed by the reaction between cysteine and a C6-intermediate of the Maillard reaction derived from sugars, pyrrolothiazolate seems to be formed during the heating, processing, and storage of foods containing enough amounts of those substances. To examine this, soy sauce, which is a fermented food rich in amino acid and reducing sugars, was analyzed using DAD-HPLC. Figure 2A shows a chromatogram of soy sauce. A peak showing absorption shoulders or maxima at 300 and 360 nm was detected at a retention time of about 18 min (Fig. 2B). This spectrum and the retention time corresponded to those of the authentic pyrrolothiazolate. To confirm that this peak is pyrrolothiazolate, the sample was further analyzed using MS coupled with HPLC. Figure 2C shows an ion chromatogram of m/z 230.048. A peak was detected at a retention time of about 21 min, and the product ion spectrum of the peak also corresponded to that of the authentic pyrrolothiazolate (Fig. 2D). This result showed that the detected compound was pyrrolothiazolate, and that pyrrolothiazolate existed in soy sauce. The amount of pyrrolothiazolate in soy sauce measured using DAD-HPLC was almost the same as that using LC-MS (data not shown).

Fig. 2.

Typical HPLC patterns of soy sauce and spectra of pyrrolothiazolate in soy sauce. A, DAD-HPLC of soy sauce. B, UV-Vis spectrum at a retention time of about 14 min. C, Ion chromatogram of LC-MS at m/z 230.048. D, Product ion spectrum of m/z 230.048 at a retention time of about 21 min. Soy sauce (10 mL) was analyzed as described in the Materials and Methods section.

Distribution of pyrrolothiazolate in soy sauce    At first, we quantified pyrrolothiazolate in various soy sauces using DAD-HPLC. The spectrum was checked for qualification. Table 1 shows the contents of pyrrolothiazolate in retail samples of soy sauce. This pigment was detected in soy sauce at the maximal concentration of 99 µg/100 mL (mean ± standard deviation (SD), 27.8 ± 27.9 µg/100 mL). The absorbances at 400 nm of the samples were measured as an index of their browning or color. A weak relationship between the contents of pyrrolothiazolate and the absorbance at 400 nm of soy sauce was apparent (correlation coefficient (r) = 0.68). The correlation coefficient increased (r = 0.77; Fig. 3) when the data of saishikomi (fermented twice) soy sauce were excluded. Shiro (very pale color) soy sauce, in which a larger amount of salt is added and fermentation and the Maillard reaction are repressed, showed the palest color among the soy sauces and contained the least amount of pyrrolothiazolate. Tamari (soybean only) soy sauce, in which only soybean is used and wheat is not used as a raw material, showed the darkest color and contained the highest amount of pyrrolothiazolate. Saishikomi soy sauce, which is soy sauce that has been fermented twice, showed a darker color than koikuchi (standard) soy sauce. During the second fermentation, the pigment might be decomposed or polymerized.

Table 1. Concentration of pyrrolothiazolate in retail soy sauce and its absorbance at 400 nm.

Soy sauce (type)	n*	Pyrrolothiazolate (µg/100 mL)		Absorbance at 400 nm
		Mean	Min.-max.	Mean	Min.-max.
Koikuchi (standard)	6	28.1	12.4–50.4	28.1	14.5–33.8
Saishikomi (twice-fermented)	3	16.3	14.6–17.9	62.4	39.4–104.8
Shiro (very pale-colored)	3	4.9	n.d.–11.5	2.1	1.9–2.4
Tamari (soybean only)	3	76.3	36.5–99.0	135.6	77.6–188.4
Usukuchi (pale-colored)	3	12.9	(1.4)**–21.5	7.3	6.3–8.8

n.d., not detected (< 0.8 µg/100 mL).

*  the number of analyzed brands or samples.

**  <limit of quantification.

Fig. 3.

Relationship between the contents of pyrrolothiazolate and the absorbance at 400 nm (A₄₀₀) of retail soy sauce (n = 15) except for saishikomi soy sauce.

Next, we prepared a standard type of soy sauce (koikuchi) and examined in which step of soy sauce preparation pyrrolothiazolate was formed. A mixture of soybeans and wheat (koji) with brine was fermented at room temperature for 9 months at a laboratory scale. We could not detect the pigment until after 6 months of fermentation. After 9 months of fermentation, a small amount of pyrrolothiazolate was detected (about 8 µg/100 mL). When retail non-pasteurized soy sauce was heated at 80 °C for 10 to 30 min, the content of pyrrolothiazolate did not increase (data not shown). These results showed that the pigment was gradually formed during maturation after enough starch and protein had been hydrolyzed by koji enzymes, including amylase and peptidase. Soy sauce contains about 0 to 17 mg/100 mL of free cysteine (Uchida, 1988).

Distribution of pyrrolothiazolate in various foods and beverages    Tables 2 and 3 show the contents of pyrrolothiazolate in various foods and beverages. Pyrrolothiazolate was detected in fish sauce, miso (salted and fermented soybean paste), and beer, but was not detected in black vinegar, mirin (Japanese sweet seasoning or alcoholic beverage), bread crust, cocoa powder, roasted onion, barley tea, and coffee. Although kinako (flour of roasted soybean) and natto (fermented soybean) are made of the same material as soy sauce and miso, pyrrolothiazolate was not detected in those foods.

Table 2. Contents of pyrrolothiazolate in fish sauce, miso, and other foods.

Sample	n*	Pyrrolothiazolate (µg/100mL for liquid; µg/100g for solid)
		Mean	Min.-max.
Fish sauce (liquid)
Nam pla (Thai fish sauce)	1	24.8
Oyster sauce	1	101.4
Shottsuru (Japanese fish sauce)	1	n.d.
Other seasoning (liquid)
Black vinegar	1	n.d.
Mirin (rice wine)	1	n.d.
Miso (type) (solid)
Kome (rice and soybean)	3	5.7	5.0–6.3
Mame (soybean only)	3	60.2	26.3–105.6
Mugi (barley and soybean)	3	10.5	6.4–16.5
Other foods (solid)
Bread crust	1	n.d.
Cocoa powder	1	n.d.
Kinako (flour of roasted soybean)	1	n.d.
Natto (fermented soybean)	1	n.d.
Roasted onion	1	n.d.

n.d., not detected (< 0.8 µg/100 mL or < 0.5 µg/100 g).

*  the number of analyzed brands or samples.

Table 3. Contents of pyrrolothiazolate in beer, malts, and other beverages.

Sample	n*	Pyrrolothiazolate (µg/100 mL for liquid; µg/100 g for solid)
		Mean	Min.-max.
Beer and its relatives
Pale beer (liquid)	3	14.2	5.2–26.4
Black beer (liquid)	3	7.2	3.1–12.2
Pale malt(solid)	1	n.d.
Chocolate malt (solid)	1	n.d.
Wort (liquid)	1	3.3
Other beverages
Barley tea powder(solid)	1	n.d.
Soluble coffee powder(solid)	1	n.d.

*n.d., not detected (<0.5 µg/100 g).

*  the number of analyzed brands or samples.

Regarding fish sauce (Table 2), pyrrolothiazolate was detected in nam pla (Thai fish sauce, 25 µg/100 mL) and oyster sauce (101 µg/100 mL), while it was not detected in shottsuru (Japanese fish sauce). According to the labels of nam pla and oyster sauce, these samples contained sugar or starch. On the other hand, shottsuru is made from only fish and salt. It does not seem that shottsuru contains a sufficient amount of reducing sugars for the formation of pyrrolothiazolate. In general, fish sauce contains no sugars (Lopetcharat et al., 2001). The concentrations of free cysteine in retail shottsuru are reported to be between 0.5 and 6.4 mg/100 mL (Fujii et al., 1992).

For miso (Table 2), pyrrolothiazolate was detected in the range of 5 to 106 µg/100 g (25.5 ± 24.6 µg/100 g; Table 2). Miso is made from soybean with or without rice or barley, and is classified as “kome” (rice and soybean), “mugi” (barley and soybean) or “mame” (soybean only) depending on the raw materials used. Among the miso, mame miso contained the highest concentration of pyrrolothiazolate. Mame miso showed the darkest color among the three types of miso, because mame miso contains the largest amount of proteins. The relationship between a darker miso and a higher pyrrolothiazolate content is similar to that seen for soy sauce.

Table 3 shows the contents of pyrrolothiazolate in beer samples and malts. Beer contained pyrrolothiazolate in the range of 3 to 26 µg/100 mL (10.8 ± 7.7 µg/100 mL). The color of beer is formed by the Maillard reaction during the roasting of malts, preparation of wort, and fermentation (Shellhammer and Bamforth, 2008). Pyrrolothiazolate was not detected in pale or chocolate malts, but it was detected in wort. These results showed that pyrrolothiazolate was mainly formed after wort preparation by the Maillard reaction between cysteine and glucose. After protein and starch in malts and ingredients are fully decomposed by hydrolytic enzymes, such as peptidase and amylase, the pigment should be formed during fermentation and maturation. Black beer is darker than pale beer, meaning that the Maillard reaction occurs more intensively or rapidly during dark beer production than during pale beer production. However, pale beer contained more pyrrolothiazolate than black beer. This result did not seem to be consistent with the results of soy sauce and miso. However, the color of beer is mainly formed during malt preparation or kilning malts. Different types of malts are used to produce pale and dark beers. For pale beer production, pale malts are used, while for dark beer production, dark colored or special malts are used in addition to pale malts. The dark colored or special malts are roasted at more than 100 °C during kilning. More cysteine might be decomposed during the intensive roasting. As a result, more pyrrolothiazolate seems to be formed in pale beer than in black beer. Pale beer (not detected to 8.0 mg/L) contains more free-cysteine than dark beer (not detected to 2.3 mg/L; Poveda, 2019).

Pyrrolothiazolate was not detected in some foods and beverages, such as black vinegar, mirin, coffee, barley tea, bread crust, roasted onion, and cocoa, although the brown color of these samples is attributed to the Maillard reaction. It does not seem that these foods or beverages contain enough amounts of cysteine for pyrrolothiazolate formation.

As the concentration of pyrrolothiazolate described here was less than that of the visual detection limit (600 µg/mL; Noda et al., 2015), the contribution of this pigment to the total color of foods and beverages is practically very low. However, considering that a wide variety of pigments are formed through the Maillard reaction, and that we recognize cumulatively pigments as total color, it would be necessary to determine quantitatively the various unknown pigments present in foods to understand and regulate the color of foods.

Antibacterial and mutagenic activities of pyrrolothiazolate    Pyrrolothiazolate is an antioxidant showing radical scavenging activity (Noda et al., 2016). Its antibacterial activity and mutagenicity were examined, because some Maillard reaction products have been reported to show these activities (Jägerstad et al., 1991; Chevalier et al., 2001; Morttram et al., 2002).

In antibacterial assays, pyrrolothiazolate did not show any growth inhibition zone, not even at 1 600 µg/mL, while 6.25 µg/mL streptomycin and 3.13 µg/mL ampicillin, the positive controls, showed clear growth inhibition zones (>10 mm i.d.) against B. subtilis PCI-219 and against S. aureus FDA-209P and E. coli NIHJ, respectively. These results showed that pyrrolothiazolate had no antibacterial activity.

Figure 4 shows the results of the Ames test of pyrrolothiazolate and its metabolite. The numbers of revertant colonies of the positive controls (MeIQX, in S. Typhimurium TA98 without S9 mix; 2-nitrofluorene in S. Typhimurium TA98 without S9 mix; sodium azide in S. Typhimurium TA100 with S9 mix; 2-aminoanthracene in S. Typhimurium TA100 without S9 mix) were clearly higher than that of the negative control (DMSO). The numbers of revertant colonies of the samples with pyrrolothiazolate (5 mg/plate) or S9 mix-added pyrrolothiazolate were almost the same as that of the negative control. These results showed that pyrrolothiazolate and its metabolites had no mutagenicity in both S. Typhimurium TA98 and TA 100 with the Ames test.

Fig. 4.

Mutagenicity responses of S. Typhimurium TA98 and TA100 to pyrrolothiazolate. Negative control, DMSO; positive control, MeIQX (0.03 µg/plate) in S. Typhimurium TA98 without S9 mix, 2-nitrofluorene (2.5 µg/plate) in S. Typhimurium TA98 without S9 mix, sodium azide (1.0 µg/plate) in S. Typhimurium TA100 with S9 mix, and 2-aminoanthracene (1.0 µg/plate) in S. Typhimurium TA100 without S9 mix; pyrrolothiazolate, 5 mg/plate (n = 2).

Some pyrrolothiazole derivatives have been reported to have bioactivity, such as antineoplastic activity (Larezari and Schwartz, 1988) and antitubercular activity (Karthikeyan, 2010). Pyrrolothiazolate having an enol structure showed antioxidative activities (Noda et al., 2016), but did not have any antibacterial activity or mutagenicity. Pyrrolothiazolate does not seem to have a reactive structure for these activities.

Conclusion

Pyrrolothiazolate was isolated and identified from a model Maillard reaction system containing cysteine and glucose. Here, we showed that this pigment existed in soy sauce, miso, and beer (1 to 106 µg/100 mL or 100 g). In these fermented foods, proteins and starch in raw materials are hydrolyzed by enzymes from koji or malts, then the Maillard reaction gradually occurs between the formed free amino acids and reducing sugars, which leads to the formation of this pigment as well as color formation. We also showed that this pigment had no mutagenicity with the Ames test.

Acknowledgements    This study was supported by a Grant-in-Aid for Scientific Research (B) [grant number 17H01958] from the Japan Society for the Promotion of Science.

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