Isolation and characterization of lactic acid bacteria isolates that degrade Maillard reaction products from coffee

Abstract

In this study, melanoidin-degrading bacteria were isolated, and the optimal parameters of their degradation ability were examined. Five isolates were inoculated into a GYP broth containing model melanoidin (MM) or coffee melanoidin (CM) and incubated at 30 °C for 1 week. Five strains showed maximum decolorization rates of 31 % for MM and 50 % for CM. 16S rDNA sequencing identified Lactococcus garvieae (3 strains), L. lactis, and Enterococcus faecalis. In DAD-HPLC analysis, substances with molecular weights (MW) greater than 10 000 decreased, and substances with MW less than 500 were observed. Based on the IR spectrum of CM after microbial decomposition, an absorption suggestive of a carbonyl group was detected. microbial decomposition products showed radical scavenging activity. These results indicate that the compounds produced from melanoidin after degradation by strains may increase radical scavenging activity. Thus, conducting further studies is necessary to examine the mechanism of the microbial degradation of melanoidins.

Introduction

The Maillard reaction is a chemical reaction that occurs between the carbonyl groups of reducing sugars and the amino groups of amino acids, peptides, or proteins. This nonenzymatic browning reaction can generate melanoidins, which are the brown polymer and end-product formed during food processing and preservation. Melanoidins are routinely consumed because they are found in everyday foods.

The primary dietary source of melanoidins is bread crust. For bread and dry biscuits, melanoidin intakes in the range of 1.8–15.0 and 3.2–8.5 g/day have been reported (Borrelli et al., 2002). Melanoidins are also the major components of coffee beverages, accounting for 25 % of dry matter. Coffee melanoidin (CM) intake ranges from 0.5 to 2.0 g/day (Fogliano and Morales, 2011). Coffee is a widely consumed product in the world, and melanoidin is a key component that determines its unique flavor, and color (Fogliano and Morales, 2011; Shakoor et al., 2022; Song et al., 2018). In previous studies, the physiological effects of melanoidins have been reported, including their antioxidant properties (Nicoli et al., 1997), dietary fiber-like action (Borrelli and Fogliano, 2005), inhibitory effect on nitrosamine formation (Kato et al., 1987), plasma antioxidant capacity in humans (Natella et al., 2002), and their ability to decrease the synthesis of lipid hydroperoxides and secondary lipoxidation products (Tagliazucchi et al., 2010).

Melanoidins in coffee extract have been demonstrated to be more potent antioxidants than those in other thermally processed foods (Pastoriza et al., 2014). In addition, melanoidins in coffee extract exhibit anticancer (Daglia et al., 2002), antibacterial, anti-inflammatory (Mesías et al., 2017), and antihypertensive effects by modulating the renin–angiotensin–aldosterone system (Rufián-Henares et al., 2007). Thus, melanoidins have various bioactivities, and they exhibit anti-aging properties and prevent lifestyle-related diseases. The Maillard reactions are involved in the quality improvement and deterioration of food products. Although the production of color is the desirable effects of the Maillard reaction, excessive coloration and formation of reaction products are among the undesirable effects (Van Boekel et al., 2016; Rufián-Henares et al., 2006). Thus, developing methods to control this reaction is necessary.

Lactic acid bacteria have been used in the fermentation and brewing fields for a long time, but in recent years, various potentials have been revealed. In various fields such as “food” and “health,” a new method of utilizing lactic acid bacteria and their metabolites transcending the framework of traditional use has been established. The application of lactic acid bacteria to bioremediating distillery effluents has been reported. According to Ohmomo et al. (1988), Lactobacillus hilgardii W-NS decolorized approximately 28 % of molasses melanoidin under optimal conditions. In another study, Lactobacillus plantarum, L. casei, and Pediococcus parvulus decolorized 25 % of sugar beet molasses at pH 6.5 and 30 °C. Lactobacillus plantarum SF5.6 also decolorized molasses melanoidin by 61 % at 30 °C under facultative conditions (Wilk et al., 2019). Most lactic acid bacteria, including Lactobacillus, are generally recognized as safe (GRAS). Based on previous reports, lactic acid bacteria suppress the browning of cheese (Mukherjee et al., 1994). However, few studies have reported on the use of lactic acid bacteria to control food colorization by removing melanoidins from food end-products and controlling the Maillard reaction.

This study aimed to isolate lactic acid bacteria that degrade melanoidin in a model system and CM, as well as to determine the properties of the isolates. The isolates of lactic acid bacteria were collected mainly from a sugarcane farm and mangrove floor soil in the Ryukyu Archipelago. Furthermore, changes in the biological activity of melanoidins after microbial degradation were examined.

Materials and Methods

Preparation of a nondialyzable model melanoidin (MM) and CM  Model melanoidin (MM) samples were prepared by heating an aqueous solution of 2 M D-glucose and 2 M glycine (pH 5.0) at 95 °C for 1 h. The resulting brown stock solution was dialyzed against distilled water using a cellulosic dialysis tube [molecular-weight (MW) cut-off of 10 000 Da] for 1 week. The dialyzed internal solution was freeze dried (Fig. 1A). In preparing CM samples, commercially roasted bean powder (green bean–producing countries: Brazil, Colombia, etc.; coffee type: blend, roast, and medium roast, Camel Coffee Co., Tokyo) treated using a standard coffee grinder was added to hot tap water (95 °C). The ratio between the coffee powder and water was 1:6 (w/w). After the extraction, aqueous solutions were filtered through a filter paper. The resulting coffee extract was dialyzed against distilled water using a cellulosic dialysis tube (MW cut-off of 10 000 Da) for 1 week. The dialyzed internal solution was freeze dried (Fig. 1B). The freeze-dried 100 mg MM and CM were dissolved in 10 mL distilled water and sterilized by filtration using a 0.20 µm PTFE membrane filter (Advantec, Tokyo, Japan) for various tests.

Fig. 1

Nondialyzable model (A) and coffee (B) melanoidin in this study.

Bacterial strains  The lactic acid bacteria isolates were collected mainly from a sugarcane farm and mangrove floor soil in the Ryukyu Archipelago. A total of 22 strains were isolated (Table 1).

Table 1. Bacterial strains used in this study.

Strain name	Isolation place	Isolation source
M14-1-a	Miyakojima	Mangrove floor soil
M14-1-b1
M14-1-b2
M14-1-c
M14-2-a
M14-2-b1
M14-2-b2
Noudai3	Tokyo	Soil*1
Y9-1-a	Yonagunijima	Sugarcane farm soil
Y9-1-b
Y9-2
Y9-3
M4-2-a	Miyakojima	Spring water of mangrove
M4-2-b
M13-3-a		Bagasse*2
M13-3-b
M9-3-a		Seawater of molasses waste disposal site
M9-3-b
Y1-1-a	Yonagunijima	Sugar factory soil
Y1-1-b
M12-1	Miyakojima	Waste of bagasse
M13-2

^*1  Tokyo University of Agriculture, Setagaya Campus grounds.

^*2  Sugar cane fiber waste left after juice extraction from Miyakojima City Resource Recycling Center

Screening of bacteria for MM decolorization  Bacteria that can decolorize MM were screened to select a melanoidin-degrading bacterium. The screened bacteria were inoculated into a GYP medium containing 2 % D-glucose, 0.5 % yeast extract, 0.5 % peptone, 0.2 % sodium acetate, 1 % Tween 80, 0.5 % salt solution (2 % MgSO₄·7H₂O, 2 % MnSO₄·4H₂O, 2 % FeSO₄·7H₂O, and 2 % NaCl; pH 8.0), and 0.1% model melanoidin samples and then incubated at 30 °C for 24 h. The absorbance of culture broth supernatant was measured at 500 nm, and the decolorization ratio was expressed as the decrease in the absorbance at 500 nm against initial absorbance.

Identification of bacteria  Bacteria that can decolorize MM were identified. Taxonomic identifications of genera were performed by using the standard method (observation under a microscope and then Gram staining). Each strain was grown in a de Man–Rogosa–Sharpe (MRS) (Merck Japan, Tokyo) broth at 30 °C for 24 h without shaking. After appropriate dilutions using 0.85 % NaCl, 0.1 mL of the dilute solution was mixed into 15 mL of MRS agar containing 0.5 % calcium carbonate. This agar plate was incubated at 30 °C for 1 week. Consequently, a clear zone formed on the MRS agar plate. In addition, morphology was observed using a tabletop scanning electron microscope (SEM, JASCO International Co., Ltd., Tokyo, Japan). Finally, these strains were identified by 16S rRNA sequencing in accordance with the Central Institute for Experimental Animals ICLAS Monitoring Center (Kanagawa, Japan).

Melanoidin-decolorizing activity  The melanoidin-decolorizing activity was assayed as the decrease in color density at an absorbance of 500 nm. The identified isolates were inoculated into a GYP broth containing 0.1 % MM or CM samples and then incubated at 30 °C for 1 week under aerobic and anaerobic conditions. The decolorization activity was expressed as the decrease at an absorbance of 500 nm against the initial absorbance at the same wavelength (Eq. 1).

  

Effect of pH, temperature, NaCl concentration, and carbon source on the growth of isolates  The growth of isolates was observed at different initial pH values (4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0), incubation temperatures (30 °C, 37 °C, 45 °C, and 50 °C), and NaCl concentrations (0.5 %, 5.0 %, 7.5 %, 8.0 %, 8.5 %, 9.0 %, 9.5 %, 10.0 %, 12.5 %, and 15.0 %). The initial pH values were adjusted with 1 M HCl or 1 M NaOH. The carbon source (glucose) of the GYP medium was changed to fructose, galactose, lactose, maltose, sucrose, and xylose to determine the best carbon source. The concentration of each carbon source was 0.2 %. For each test, 30 µL of the washed cell suspension was inoculated into 3 mL of GYP broth and incubated for 24 h. Growth was determined by measuring OD₆₆₀ after 24 h of incubation. Samples with OD₆₆₀ of 0.5 or more were regarded as tolerant.

Microbial degradation of melanoidins  The ability of melanoidins to degrade bacteria was also confirmed by diode array detector (DAD)-HPLC analysis. DAD-HPLC analyses were performed on the Hitachi Chromaster HPLC System, which includes 5430 DAD (HITACHI, Tokyo, Japan). HPLC was performed using a TSKgel® G3000PW HPLC Column (7.5 mm I.D. × 30 cm, 12 µm, Tosoh Corporation, Tokyo, Japan). The DAD was scanned from 200 to 600 nm (with a monitor wavelength of 400 nm, mobile-phase distilled water, and flow rate of 1.0 mL/min).

Determination of laccase activity  The culture medium was incubated at 30 °C for 1 week and sterilized by filtration using a 0.45-µm PTFE membrane filter (Advantec). Forty microliters of this solution was dispensed into 96-well plates. The substrate solution was obtained by mixing 10 mM 2,2′-azinobis(3-ethyl-2,3-dihydrobenzothiazole-6-sulfonic acid) (ABTS), 10 × McIlvaine buffer, and MilliQ water in a ratio of 1:1:6 and incubated in an incubator at 30 °C for 30 min. The substrate solution was dispensed in 160 µL portions and kept in an incubator at 30 °C for 24 h. The absorbance at 420 nm was measured. A₄₂₀ was measured, and the enzymatic activity (1U = µmol/min) of laccase was calculated by subtracting the average absorbance of the control from that of each sample. The amount of ABTS oxide was determined using the absorption coefficient of 36 mM/cm.

2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay  Each bacterium was inoculated into 3 mL of BHI broth and incubated at 30 °C for 24 h. Ten microliters of each bacterial solution was inoculated into 0.2 mL of GYP broth containing MM or CM solution (final concentration of 0.25 %) and incubated at 30 °C for 1 week. In addition, 0.05 mL of culture broth supernatant was mixed with 1.2 mL of 0.1 M acetic buffer and 0.75 mL of 0.016 % DPPH/ethanol solution. After standing for 10 min, the absorbance of the solution was measured at 510 nm. Ascorbic acid was used as the positive standard for DPPH radical scavenging assay (Eq. 2).

  

FT/IR spectrum  The culture broth obtained by using the same method as described above was centrifuged at 4 000 g for 5 min. The supernatant was sterilized by filtration through a 0.20 µm PTFE membrane filter (Advantec) and then freeze dried. Each test sample and potassium bromide (KBr) were mixed and pressed to prepare tablets of φ 3 mm. The FT/IR spectrum was measured using a Fourier transform infrared spectrophotometer (FT/IR-550; Nippon Bunko Co., Ltd.) in the range of 600–4 000 cm⁻¹ with an integration number of 20. Tablets were prepared by using only KBr for background measurement.

Statistical analysis  Each experiment was repeated at least three times. The results shown in the figures corresponded to the means ± SD. All data were analyzed by using one-way ANOVA, followed by the Dunnett’s test or Tukey–Kramer test. The level of significance was set at p < 0.05 or 0.01.

Results

Screening of bacteria for MM decolorization  The results of the MM decolorization test are shown in Fig. 2. The strains M14-1-c, M14-2-a, Y9-1-a, M9-3-a, and M13-2 showed a decolorizing effect on MM. These five bacterial strains were used for the decolorization of melanoidin.

Fig. 2

Screening of bacteria for the decolorization of model melanoidin (MM). Values represent the mean ± SD for three independent experiments.

Control: no bacteria inoculated. *p < 0.05 versus Control; **p < 0.01 versus Control

Identification of bacteria showing MM decolorization  All isolates were considered Gram positive based on Gram staining and SEM observations. The M14-1-c, M14-2-a, Y9-1-a, and M13-2 strains were diplococci, whereas the M9-3-a strain was cocci (Fig. 3A). All five strains showed the decomposition activity of calcium carbonate (Fig. 3B). Based on 16S rDNA sequencing, these strains belonged to Lactococcus garvieae (M14-1-c, homology 99.9 %; Y9-1-a, 99.9 %; and M13-2, 99.9 %), L. lactis (M9-3-a, 99.8 %), and Enterococcus faecalis (M14-2-a, 99.7 %).

Fig. 3

(A) Scanning electron microscopic (SEM) images. (B) Bacteria decolorizing calcium carbonate and melanoidin.

Melanoidin-decolorizing activity of strains  The decolorizing activity of each strain for MM and CM is shown in Fig. 4. Y9-1-a showed the maximum decolorization rate for MM (28.4 %, Fig. 4A) and CM (50.2 %, Fig. 4B) under aerobic conditions. Even under anaerobic conditions, Y9-1-a showed the highest decolorization rates for MM (30.3 %, Fig. 4C) and CM (25.1 %, Fig. 4D).

Fig. 4

Relative absorbance of supernatants obtained by culturing bacteria in GYP broth with melanoidin.

Decolorization of (A) model melanoidin (MM) and (B) coffee melanoidin (CM) by bacteria under aerobic conditions. Decolorization of (C) MM and (D) CM by bacteria under anaerobic conditions. Values represent the mean ± SD for three independent experiments. Control: no bacteria inoculated. *p < 0.01 versus Control

Effect of pH, temperature, NaCl concentration, and carbon source on the growth of isolates  The results of the salt tolerance test are shown in Fig. 5. All the strains had an optimum initial pH value of 8.0 (Fig. 5A). M14-1-c and M14-2-a exhibit resistance at a high temperature (45 °C, Fig. 5 B). Most strains showed tolerance to 5 % NaCl. M14-1-c and M14-2-a were strongly resistant to 7.5 % NaCl (Fig. 5C). All the strains grew at a temperature range of 30 °C–37 °C. Furthermore, the best carbon source for the growth of M14-1-c, M14-2-a, and M9-3-a was glucose, whereas that for Y9-1-a and M13-2 was sucrose (Fig. 5D).

Fig. 5

Effect of pH, temperature, NaCl concentration, and carbon source on isolate growth.

(A) Initial pH, (B) temperature, (C) NaCl, and (D) carbon source. Each bacterium was incubated in a GYP broth for 24 h before checking the growth. Values represent the mean ± SD for three independent experiments.

DAD-HPLC chromatogram of melanoidins after degradation by strains  Although the rate of decrease in peak area values differed among the strains, all five strains showed a similar DAD-HPLC chromatogram; thus, only the result of strain M14-1-c is shown in Fig. 6. Molecular weight markers were detected for polyethylene oxide (MW: 340 000) at a retention time of 5.2 min, polyethylene glycol (MW: 8 565) at 6.5 min, and polyethylene glycol (MW: 1 000) at 7.8 min. A calibration curve was constructed with log X (MW) on the y-axis and retention time on the x-axis, yielding the formula for the estimated molecular weight. DAD-HPLC analysis before and after CM decolorization revealed a decrease in the peak area (blue arrows) for MWs greater than 10 000 (retention time of 6.4–6.6 min) and the presence of a new peak in the low-MW fraction (red arrow).

Fig. 6

DAD-HPLC analysis of the microbial degradation of melanoidins.

Strain M14-1-c was incubated in a GYP broth containing 0.1 % model melanoidin samples and incubated at 30 °C for 24 h. Control: no bacteria inoculated. (A) HPLC chromatogram (DAD, 400 nm) of Control. (B) HPLC chromatogram at scanning wavelengths of DAD from 200 to 600 nm of Control. (C) HPLC chromatogram (DAD, 400 nm) after microbial degradation. (D) HPLC chromatogram at scanning wavelengths of DAD from 200 to 600 nm after microbial degradation.

Radical scavenging capacity of melanoidins after decolorization by strains with DPPH  The radical scavenging activities of MM and CM after decolorization by strains are shown in Fig. 7. Among the strains, the decolorized melanoidin samples showed about 18 % and 20 % increase in radical scavenging activity, respectively, compared with MM and CM before decolorization.

Fig. 7

The radical scavenging activity of melanoidin after microbial degradation.

CM; coffee melanoidin, MM; model melanoidin, Control: No bacteria inoculated. Values represent the mean ± SD for three independent experiments. *p < 0.01 versus Control

FT/IR spectrum of melanoidin before and after degradation by strains  FT/IR analysis was performed using the M14-1-c strain that produced melanoidin with the highest radical scavenging activity. The IR spectrum of GYP broth (Fig. 8A), and GYP broth with or without CM before microbial degradation is shown in Figs. 8B and C. An absorption showing a C-H stretching of the aromatic rings in the IR spectrum of the GYP broth with CM (without M14-1-c strain) was detected 2 922 and 2 850 cm⁻¹ (Fig. 8C). Based on the IR spectrum of the sample after microbial degradation, a strong stretching band showing a carbonyl group at approximately 1 650 cm⁻¹ was detected (Fig. 8D).

Fig. 8

The FT/IR spectrum of melanoidin after microbial degradation by Strain M14-1-c.

(A) GYP broth without coffee melanoidin (CM). (B) Strain M14-1-c was incubated in a GYP broth without adding CM. (C) Before microbial degradation. (D) After microbial degradation.

Discussion

In this study, bacteria with melanoidin-degrading activity in the strains isolated from a sugarcane farm and mangrove floor soil in the Ryukyu Archipelago was investigated using the decolorization of MM as an indicator. Consequently, five of the 22 isolates (strains M14-1-c, M14-2-a, Y9-1-a, M9-3-a, and M13-2) decolorized MM (Fig. 2). The properties and optimal culture conditions of such bacteria were also determined.

The colonies of these five strains were white, round, and shiny, different from those of basidiomycetes, imperfections, and filamentous fungi that have been reported to decolorize and degrade melanoidins. (Ohmomo et al., 1985). The five isolates were identified by 16S rDNA sequencing as L. garvieae (M14-1-c, Y9-1-a, and M13-2), L. lactis (M-9-3-a), and E. faecalis (M-14-2-a). All of isolates showed calcium carbonate resolution (Fig. 3), which supports the identification results. Based on previous reports, Pediococcus acidilactici (Soni et al., 2013; Tiwari et al., 2013), Paracoccus pantotrophus (Santal et al., 2016), and Lactobacillus plantarum (Wilk and Krzywonos, 2020, Seruga and Krzywonos, 2016, Limkhuansuwan and Chaiprasert, 2010) can decolorize melanoidin. Lactococcus garvieae is a species of lactic acid bacteria isolated from healthy human feces, and it has been reported to be a causative agent of bacterial pathogenesis in fish (Meyburgh et al., 2017). Lactococcus garvieae has also been reported to be frequently detected in Italian Protected Designation of Origin cheese (Fortina et al., 2003). Lactococcus lactis is closely related to Lactococcus garvieae, and it is the most recently related species on the evolutionary tree (Blaiotta et al., 2002). Lactococcus lactis is often detected in fermented foods such as raw milk, cheese, and butter (Li et al., 2020). Enterococcus faecalis improves the aroma, texture, and flavor of fermented dairy products, and it is used as a probiotic, but it is considered as an opportunistic pathogen that can cause infections in immunocompromised patients (Graham et al., 2020). At present, no reports have been found on these three identified strains decolorizing or degrading melanoidin, which indicates that the decolorization and degradation mechanisms may be due to a specific factor other than existing enzymes such as laccases. Lactococcus species (e.g., L. lactis, L. cremoris, and L. diacetylactis) are known for their economic value and probiotic properties. Most members of the Lactococcus genus are GRAS, and they can survive during intestinal transit (Qi et al., 2020). These bacterial species have been widely applied in the food industry; thus, the strains isolated in this study also have great application potential in the control of browning in the processing of food products in which Maillard reactions occur, such as beer, cheese, miso, and soy sauce.

MM and CM decolorization tests were performed by each isolate under aerobic and anaerobic conditions. The results indicated that the isolates were decolorized under both conditions, but the decolorization rate was higher under aerobic conditions than under anaerobic conditions (Fig. 3). However, under anaerobic conditions, the Maillard reaction proceeds more slowly, so the browning of the control does not progress, and the apparent decolorization rate is low. Under aerobic conditions, the decolorization rate of CM, which contains polysaccharides (arabinogalactan and glucomannan) and is formed by proteins and phenolic compounds (mainly hydroxycinnamic acid) (Silván et al., 2010), was higher than that of MM. Therefore, the isolate was highly effective in decolorizing against CM derived from phenolic compounds.

In verifying the characteristics of the identified isolates, their growth was examined in a GYP medium prepared under various conditions (incubation pH: 4.0–8.0; temperature range: 30 °C–50 °C; NaCl concentration: 5.0 %–15.0 %; carbon source: glucose, fructose, galactose, lactose, maltose, sucrose, and xylose). The optimum temperature varied among the strains and media (Fig. 5B), while the optimum pH was 8.0 (Fig. 5A). L. garvieae has been isolated from rivers, sewage (Aguado-urda et al., 2010), and various foods such as vegetables, meat, and dairy products (Ferrario et al., 2012), as well as fecal samples from healthy people, indicating that this microorganism may be part of the human indigenous flora or a transient bacterium ingested with food (Aguado-Urda et al., 2013). L. garvieae are adapted to pH (4.5–9.6), temperature (10 °C–45 °C), and salinity (0–6.5 %) (Kusuda et al., 1991). M14-1c, which is identified as L. garvieae, grew under NaCl concentration of 8.5 % at 45 °C (Figs. 5B and C). In addition, M14-1c was more thermostable and salt tolerant than Y9-1-a and M13-2, which were also identified as Lactococcus garvieae (Fig. 5C). Furthermore, glucose (M14-1-c, M14-2-a, and M9-3-a) and sucrose (Y9-1-a and M13-2) were considered as the best carbon sources for growth (Fig. 5D). Comparison of collection sites showed that M14-1c was collected from a mangrove floor soil; Y9-1-a was obtained from a sugarcane field soil at the site of a former sugar mill, whereas M13-2 was collected from waste of bagasse (sugarcane residue). These findings indicate that the strains varied because of their different environments.

Therefore, the supernatants of these isolates after incubation were analyzed by HPLC-DAD using a gel filtration column to evaluate the presence of CM degradation. DAD-HPLC analysis after CM decolorization showed a decrease in the peak area for MW greater than 10 000 (blue arrows) and a new peak was detected in the low-MW fraction (red arrow) (Fig. 6). No change in the color of the bacteria was observed after CM decolorization (data not shown), suggesting that it is unlikely that the brown pigment was removed by adsorption on the bacteria. These results indicate that the main action of the microbial degradation of these five strains may be the depolymerization of macromolecules, rather than a change in the functional group of the chromophore. Based on previous reports, melanoidin depolymerization was due to the action of laccase activity (Pant and Adholeya, 2007; Bharagava et al., 2009; Yadav and Chandra, 2012). Thus, the isolate was examined for laccase activity, but no activity was found. Therefore, decolorization was not due to the cleavage of conjugated C = C, C = O, and C ≡ N bonds of melanoidins (Chandra et al., 2018) by direct laccase action.

To verify whether the decolorization and degradation of melanoidin was due to intracellular or extracellular enzymes, whether the decolorization and degradation of melanoidin also occurred in the culture supernatant was examined. After incubating each isolate in MRS broth at 30 °C for 24 h, the resulting supernatant was added to MM and CM and reacted for another 24 h at 30 °C. Consequently, no decrease in absorbance was observed (data not shown). These results indicate that the isolates do not produce extracellular enzymes to decolorize and degrade MM and CM.

The DPPH radical scavenging activity test was performed to examine the changes in the bioactivity of melanoidin after microbial degradation. Based on the results, the radical scavenging activity of MM and CM increased after decolorization (Fig. 7). Phenolic compounds have been reported as radical scavenging compounds (Rice-Evans et al., 1997), suggesting that the increase of the radical scavenging activity of MM and CM after decolorization is due to the formation of phenol-like compounds.

In identifying the factors that affect the bioactivity of CM, FT/IR spectral analysis was performed on CM before and after microbial degradation. The M14-1-c strain, which showed the highest antioxidant activity, was used. An absorption specific for alkyl groups around 2 900 cm⁻¹, which was not observed in the IR spectrum (Fig. 8B) of the control (culture supernatant from M14-1-c inoculated with a medium without CM), was only detected in the sample with CM (Fig. 8C). The peaks at 2 922 and 2 850 cm⁻¹ in the FTIR spectrum are attributed to the C-H stretching of the aromatic rings, which might be due to stretching vibrations of the CH₂ and CH₃ groups derived from fatty acids in the melanoidin fraction (Batista et al., 2016). The band in the range of 1 616–1 690 cm⁻¹ is attributed to the N-H bending vibrations from amine or amide groups and C = O stretching vibrations from flavonoids, phenolic acids and their derivatives, quinones, and lipids (Mot et al., 2011; Oliveira et al., 2016). Compounds with endiol structure in CM are considered the main contributors to the increase in antioxidant capacity (Oracz et al., 2019). The intermediate of quinone formation in the antioxidant reaction of compounds with endiol structure is the semiquinone radical, and protoncoupled electron transfer is involved in the antioxidant capacity (Kanzler et al., 2016). The IR spectrum of CM after microbial degradation (Fig. 8D) also contained a strong stretching band at approximately 1 650 cm⁻¹, which is assigned to carbonyl (C = O), C = C, or C = N double-bond stretching (Mohsin et al., 2018; Batista et al., 2016; Mot et al., 2011). The difference in structure around 1 600 cm⁻¹ indicates that melanoidin after decolorization has a more unsaturated structure, which may be the core of melanoidin’s antioxidant capacity by transferring electrons and protons (Vhangani and Van Wyk, 2013). These results indicate that carbonyl compounds and compounds with endiol structure are formed by CM degradation caused by M14-1-c, which may contribute to the increase in antioxidant capacity. In the future, the structure and antioxidant capacity of melanoidins should be systematically recognized.

In this study we identified melanoidin-degrading lactic acid bacteria newly and characterized degraded melanoidin partly. From a point of body and health, it will be crucial to clarify the relationship between melanoidins taken from food and lactic acid bacteria in body.

Acknowledgements  This research was funded by grant from the All Japan Coffee Association. The authors would like to thank Enago (www.Enago.jo) for the English language review.

Conflict of interest  There are no conflicts of interest to declare.

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