2021 年 27 巻 3 号 p. 405-416
In this study, the dynamics of the microbial communities and metabolites for soy sauce were investigated. The succession of microbiota was divided into three phases in process. Dynamics of physicochemical properties coincided with the succession of dominant genera in the first 3 months, hereafter changed slightly. The contents of L-malic acid, pyroglutamic acid, and lactic acid were increased by 11.79, 11.39, and 3.19 times (p < 0.05), respectively, compared with that in the initial moromi. The contents of volatiles were changed in the later phase significantly, and dominant constituents were positive correlated with the dominant genera. The ethyl carbamate (EC) and seven kinds of biogenic amines (BAs) reached the maximum in the third month, then their contents decreased. The EC forming involved in two phases, the metabolic pathway was partly shared with that of putrescine biosynthesis. These results laid an important foundation in reducing the endogenous hazards by bioturbating.
Soy sauce, one of the fermented soybean seasonings, is widely consumed all over the world, especially in most Asian countries due to its salty taste and sharp flavor (Feng et al., 2014). In recent years, soy sauce has received more attention because of its health care function (Kobayashi, 2005). The technique of Chinese soy sauce major involved in low-salt solid-state fermentation pattern (LSFP) and high-salt liquid-state fermentation pattern (HLFP) (Feng et al., 2014). HLFP is widely applied due to the unique taste and aroma of soy sauce, and the process involves three-phase which are koji manufacture, moromi fermentation, as well as presses (extraction), and sterilization (Yang et al., 2017). In general, the koji manufacture and moromi fermentation were completed in an open or semi-open environment, and the latter lied on the indigenous microbes, composed of fungi, yeasts, and bacteria (Devanthi and Gkatzionis, 2019).
Tetragenococcus halophilus and Zygosaccharomyces rouxii, as starter inoculated into moromi, could significantly accelerate some key flavors forming, including 2-methyl-1-propanol, 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF), and 3-hydroxy-2-methyl-4H-pyran-4-one (Maltol) (Devanthi and Gkatzionis, 2019). But, it resulted in the higher content of biogenic amines (BAs) in moromi if the abundance of some LAB's species or strains, such as Streptococcus and Leuconostoc, were higher (Papageorgiou et al., 2018). BAs were produced through decarboxylating free amino acids by decarboxylase (Santos, 1996), and were bioactive endogenous constituents, which played important physiological roles, such as cell proliferation, differentiation, signal transduction, etc (Galgano et al., 2012). However, they can also cause adverse reactions, for example, nausea, headaches, rashes, and change in blood pressure if intaken excessively (Qiu et al., 2018). Ethyl carbamate (EC), a probable carcinogen, often occurred in process of soy sauce and was produced by alcoholysis reaction of ethanol and EC precursors, including urea, citrulline, and carbamyl phosphate (Wu et al., 2014). Of them, citrulline is a major component produced by degrading arginine through the arginine deiminase (ADI) pathway (Fang et al., 2018). As we know, the quality and these endogenous hazards content during the soy sauce fermentation depended on the microbial community diversity and their succession (Liu et al., 2018).
Therefore, the technology of degrading or eliminating EC and BAs with special strain had been widely concerned in the last decades. For example, the content of EC could be reduced by inoculating Bacillus amelloliquefaciens (Wu et al., 2012). The isolates originated from soy sauce samples could degrade BAs (Cheng et al., 2020). The contents of BAs and EC were influenced by various factors during the process. There were not document on the succession of EC, BAs, volatiles, and their correlation with dominant microbes during the process, so far.
Hence, the dynamics of various metabolites and the microbial community diversities during the process were investigated by multiphase detection approaches. The dominant metabolites were correlated with the genus by multivariate statistical methods. Aim to lay an important foundation to optimize fermentation regulating parameters and reducing the endogenous hazards by bioturbating.
Samples collection The high-salt liquid-state fermentation soy sauce samples were taken from Qianhe Condiment Co., Ltd (Meishan, Sichuan Province, China). These samples were taken from the same tank every one-month interval from the initial phase to the sixth month. All samples were loaded into sterile polyethylene bottles, transported to our lab immediately, and stored at 4 °C until analysis and at –80 °C for sequence.
Chemicals 1,7-Diaminoheptane dansyl chloride, BA standards, 9-Hydroxyxanthene, EC standard, organic acid standards, and free amino acid standards were purchased from Sigma-Aldrich. Methanol, acetonitrile (HPLC grade), and other chemical reagents (AR) were purchased from local chemical stores.
Determination of total acid, ammonia nitrogen, reducing sugar, NaCl, and saltless soluble solids The contents of total acid (TA), ammonia nitrogen (AN), reducing sugar (RS), NaCl, and saltless soluble solids were determined according to the National Standards of the People's Republic of China (GB 18186-2000 and GB 5009.7-2016). Ethanol content was determined by the potassium dichromate method reported by Sumbhate et al. (2012).
Determination of organic acid and free amino acid The samples were pretreated by the protocols described by Liang et al. (2019) with some modifications. Briefly, samples were extracted by 9 mM H2SO4 with the ultrasound-assisted, then filtered by the C18 SPE column (Swell scientific instruments Co., Ltd. Chengdu, China) and 0.22 µm filter (Micron Separation Inc., Westborough, MA). Organic acids (OAs) were analyzed by HPLC (Agilent 1260 system equipped with an Alltech OA-1000 organic acid column, 300 × 7.8 mm) with a UV detector (215 nm). Degassed H2SO4 (9 mM) was used as a mobile phase and the column temperature was maintained at 75 °C. The mobile phase was set at a constant flow rate of 0.60 mL/min (Zhang et al., 2017).
Free amino acids (FAAs) were determined according to the protocols described by Cui et al. (2014) with some modifications. Briefly, samples were extracted and purified by HCl (0.01 mol/L) and 20% sulfosalicylic acid solution for at least 1 h, respectively, and then determined by Amino acid analyzer (A300, membraPure GmbH, Germany) after filtered through 0.22 µm filter.
Volatiles analyzing Extraction and determination of volatiles were performed by headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry (HS-SPME-GC-MS). Moromi samples were extracted using a DVB/CAR/PDMS fiber (Supelco, Inc., Bellefonte, PA, USA). For extraction, 1.000 g soy sauce moromi and 10 µL internal standard (methyl caprylate, 0.0079 g/100 mL) were added to a 20-mL headspace vial. Then the vial was placed into a thermostatic bath with stirring at 500 r/min to pre-equilibrium for 15 min at 60 °C, subsequent, the fiber was inserted into the bottle and extracted for another 50 min.
The adsorbed volatiles in the extracted sample was analyzed by using a GC–MS system according to the protocols described by Zheng et al. (2013). The system consists of a Thermo trace 1300 gas chromatography instrument equipped with a J&WVF-WAXms column (30.0 m × 0.25 mm, 0.25 µm, Agilent, Santa Clara, USA), and a Thermo TSQ 9000 mass spectrometer detector (Thermo Fisher Scientific, USA).
Determination of biogenic amines and ethyl carbamate The pretreatment of BAs in the sample was completed by the process described in the previous document (Saarinen, 2002). BAs were determined with the Agilent 1260 HPLC system equipped with a C18 column (5 um, 4.6 × 250 mm, Agilent, USA) according to the National Standards of the People's Republic of China (GB 5009. 208-2016).
The samples of EC pretreating were described according to the methods described by Xia et al. (2014). EC was also determined using Agilent 1260 HPLC system, which was equipped with an Eclipse XDB-C18 column (5 um, 4.6 × 250 mm, Agilent, USA) and a fluorescence detector. The process was similar to that described by Zhou et al. (2017).
Microbial community analysis Total genomic DNA from the soy sauce moromi was extracted using the Fast DNA SPIN extraction kits (MP Biomedicals, Santa Ana, CA, USA) according to the manufacturer's instructions. The content and purity of extracted DNAs were measured employing a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and 0.8% agarose gel electrophoresis, respectively. The specific primers including 338F/806R and ITS5/ITS1 (Ai et al., 2019) with the Illumina barcodes were used to amplify the V3–V4 regions of bacterial 16S rRNA gene and ITS regions of fungal rRNA gene, respectively.
Sample-specific 7-bp barcodes were incorporated into the primers for multiplex sequencing. The specific procedures for PCR were conducted based on a previous method (Li et al., 2014). PCR amplicons were purified using Agencourt AMPure Beads (Beckman Coulter, Indianapolis, IN) and quantified according to the PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). After the individual quantification step, amplicons were pooled in equal amounts and were processed to high-throughput sequencing for pair-end 2 × 300 bp sequencing with MiSeq Reagent Kit v3(Shanghai Personal Biotechnology Co., Ltd, Shanghai, China).
The sequencing data were processed with Quantitative Insights Into Microbial Ecology pipeline as formerly described (Caporaso et al., 2010). Briefly, original sequencing reads that exactly match the barcode were assigned to the corresponding samples and identified as effective sequences. After the detection of chimera, the high-quality sequences were clustered into operational taxonomic units (OTUs) at 97% sequence identity with UCLUST (Edgar, 2010). OTU taxonomy classification was performed by BLAST searching against representative sequences in the Greengenes database (DeSantis et al., 2006).
Metabolic pathway prediction of biogenic amines and ethyl carbamate The mapping of metabolic pathways of BAs and EC was based on the Kyoto encyclopedia of genes and genomes (KEGG) database (https://www.kegg.jp/). The information of enzyme abundance was obtained according to PICRUSt2 (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States), based on high-quality sequences.
Data analysis Data were subjected to one-way analysis of variance (ANOVA) and followed with Tukey's multiple range test to compare means using the Statistical Package for Social Sciences (SPSS for window: SPSS Inc., Chicago, IL, USA). Values were considered significantly different when p < 0.05. Origin 9.0 (Origin Lab Inc., Hampton, MS, United States) was used to draw figures. The experiments of the determination of physicochemical characteristics, OAs, FAAs, volatile compounds, BAs, and EC were processed in triplicate.
Difference in the microbial community during the process Chao1 and Shannon were calculated to describe the alpha-diversity of the microbial community (Chao and Yang, 1993; Simpson, 1949) in soy sauce fermentation (Table 1). Chao1/Observed_species of bacteria increased in the first to fifth month gradually, but that of fungi reduced in the fourth month and then increased again. The change of Shannon and Simpson of both bacteria and fungi was also similar to that of the Chao1/Observed_species. These results suggested that the species richness and uniformity were increased during the process except the fourth month for fungi, which would be resulted in inhibiting some fungal species or strains since the acidity increased and high salt for a long time.
| Abundance index | Diversity index | |||||||
|---|---|---|---|---|---|---|---|---|
| Samples | Chao1 | Observed_species | Simpson | Shannon | ||||
| Bacteria | Fungi | Bacteria | Fungi | Bacteria | Fungi | Bacteria | Fungi | |
| 0th month | 371.35 | 38.03 | 362.00 | 37.20 | 0.56 | 0.42 | 2.65 | 0.91 |
| 1st month | 500.49 | 89.08 | 493.60 | 88.90 | 0.88 | 0.67 | 4.29 | 1.94 |
| 2nd month | 681.37 | 105.03 | 661.50 | 105.00 | 0.89 | 0.71 | 4.33 | 2.18 |
| 3rd month | 957.40 | 271.92 | 934.70 | 271.90 | 0.94 | 0.84 | 5.24 | 4.25 |
| 4th month | 1631.10 | 81.37 | 1585.70 | 81.00 | 0.96 | 0.79 | 6.60 | 3.33 |
| 5th month | 2066.89 | 199.87 | 2032.40 | 199.60 | 0.99 | 0.83 | 8.18 | 3.35 |
| 6th month | 1997.27 | 612.61 | 1973.20 | 611.70 | 0.98 | 0.93 | 8.07 | 6.38 |
It indicated that the microbiota composition was significantly different during the processes based on the results of the principal component analysis (PCA) for the sequenced OTU (Fig. 1a and b, genus level). The microbiota of the initial moromi, located in the 3rd quadrant, was far away from that of the moromi fermented. The bacterial microbiota in the moromi of the initial-medium phase ( 1–3 months) were all located in the 4th quadrant, but there was distance among the 1–3 month's moromi. The 4th month, 5th month, as well as 6th month were located in the 1st and 2nd quadrants, respectively. The fungal microbiota varied with the process but was slightly different from that of bacteria. The fungal microbiota in the 1st and 2nd month's moromi were located in the 4th quadrant, and that of the 3rd, 4th, 5th, as well as 6th month's moromi were in the 2nd quadrant and the 1st quadrant, respectively. These results indicated that the community structure was divided into three-phase during the process, i.e initial, middle and later phases. There was a significant difference in microbiota structure between the initial and middle phases.
Principal component analysis (PCA) was displayed to evaluate the ecological distances of different moromi samples. When the distance between samples was closer, the difference in community structure between them was smaller. PCA of bacteria (a) and fungi (b) based on OTU and microbial compositions (OTU > 1%) of bacteria (c) and fungi (d) at the genus level in moromi.
Changes of the major microbial community profile during the process The changes of dominant genera abundance (OTU abundance > 1%) during the process were shown in Fig. 1c and d. Staphylococcus, Weissella, and Tetragenococcus were dominant bacteria, and their abundance was changed significantly during the process. For example, the abundance of Weissella was reduced from 72.83% in the 0th moromi to 2.08% in the finished phase due to weaker acid resistance (Hao and Sun, 2020). As one of the dominant bacteria, the abundance of Staphylococcus increased from 20.32% in the initial moromi to 57.24% and 60.18% in the 1st and 2nd month's moromi, respectively. However, Tetragenococcus became dominant bacteria when fermented for 3 months and 4 months, and its abundance was 40.27% and 39.20%, respectively. Similarly, the abundance of Aspergillus, Zygosaccharomyces, and Issatchenkia was significantly changed with fermenting. Of them, the abundance of Aspergillus was 99.79% in the initial moromi, major originated from Koji (Yan et al., 2013), and then was reduced non-monotonously to 35.34% in the ended moromi. The abundance of Zygosaccharomyces was enhanced by 71.25% in the first month's moromi because the pH of moromi was decreased to about 6.0 (Data was not shown) which was very suitable for yeast propagating (Yong and Wood, 1976). However, Zygosaccharomyces was inhibited as the acidity with the process, and its abundance decreased to 3.97% in the 3rd's moromi, meanwhile, Millerozyma increased to 12.88%. And then, the abundance of Issatchenkia and Thermoascus was increased from 0.72% to 27.12%, and 5.65% to 15.37% when the process was changed from the 3rd month to the 4th month. The sum of four dominant abundance of yeast (Zygosaccharomyces, Issatchenkia, Millerozyma, and Candida) was 71.53% in moromi of the 1st month but reduced to 7.67% in the ended moromi. It was the more important concern that more than 45% of sequenced OTU in the moromi of the 6th month, whether bacteria and fungi, were not matched to the accurate level of the taxonomy. It may have resulted in unrecoverable DNA nearly or the template broken down to very small pieces by stresses of high salt and higher acidity during the process. Yet the speculation needs to be verified furtherly.
Changes of physicochemical characteristics and non-volatile metabolites during the process The change of physicochemical characteristics during the process was shown in Fig. 2. The content of ethanol increased in the initial phase, while that of reducing sugar (RS) decreased, these two parameters changed alternately during the latter process. The contents of total acid (TA), amino nitrogen (AN), and saltless soluble solids increased until the 3rd month. These results indicated that the dynamics of physicochemical factors were closely related to the succession of dominant genera and species, as well as the strength of crude enzyme in the koji. For example, the content of RS in the initial moromi lied on the amylolytic activity secreted by Aspergillus (Poutanen et al., 2009), while that of ethanol major related to the abundance of Zygosaccharomyces in the moromi (Devanthi and Gkatzionis, 2019). AN was the metabolites of various microbes in the moromi, so that the content was closely correlated with the abundance of microbial community diversity.
Changes of physicochemical factors during the fermentation process. TA: total acid, RS: reducing sugar, AN: amino nitrogen.
Seven kinds of organic acids (OAs) were examined in the moromi (Table 2). The contents of OAs were significantly (p < 0.05) changed from the 0th to 3rd months. Of them, the content of succinic acid did not change significantly in the ended moromi, while it showed a trend of first decreasing and then increasing during fermentation, which may be converted to other OAs. Lactic acid increased from 82.66 mg/100g to 910.81 mg/100g up to the 3rd month (Table 2). The result was consistent with the changes of bacteria acid-producing, such as Staphylococcus, Weissella, and Tetragenococcus which organic acids were one of the major metabolites (Fig. 1c). The contents of L-malic acid, pyroglutamic acid, and lactic acid were increased by 11.79, 11.39, and 3.19 times (p < 0.05), respectively, during the process. Furthermore, the dominant constituents were converted from succinic acid and acetic acid in the initial moromi to succinic acid, lactic acid, and L - malic acid in the ended moromi.
| Types of organic acids | Content of organic acid (mg/100g) | ||||||
|---|---|---|---|---|---|---|---|
| 0th month | 1st month | 2nd month | 3rd month | 4th month | 5th month | 6th month | |
| Citric acid | 191.88±1.95c | 179.31±3.20c | 141.91±0.19d | 140.03±12.05d | 109.79±2.16e | 247.92±10.01b | 269.38±5.04a |
| Tartaric acid | 22.04±0.43bc | 19.84±0.94c | 27.04±0.73ab | 22.41±0.13bc | 29.70±3.87a | 30.71±1.54a | 26.61±2.40ab |
| L - malic acid | 54.62±3.97f | 61.18±1.09f | 148.86±0.85e | 394.16±3.41c | 350.62±9.43d | 532.35±5.89b | 698.68±0.78a |
| Succinic acid | 2038.63±38.66c | 2395.08±15.04a | 1264.36±7.74e | 475.31±6.67f | 1977.79±18.09d | 2329.42±10.08b | 2439.83±7.47a |
| Lactic acid | 82.66±3.58f | 252.13±3.37e | 539.71±20.77c | 910.81±5.26a | 761.45±9.56b | 375.66±2.48d | 346.55±12.78d |
| Acetic acid | 689.10±14.53a | 692.67±3.28a | 703.37±50.52a | 282.12±27.93c | 163.66±9.92d | 432.06±15.49b | 414.23±13.11b |
| Pyroglutamic acid | 24.13±2.33f | 156.45±5.34e | 253.59±21.79c | 219.28±13.37d | 221.24±3.33d | 368.71±15.70a | 299.07±1.29b |
Values are expressed as averages of three independent experiments ± SD.
a–e Different letters in the same row indicate a significant difference (p < 0.05, different fermentation time).
17 kinds of free amino acids (FAAs) were identified (Table 3) which can be divided into 4 flavor types, namely MSG-like (Asp, Glu), sweet (Ala, Gly, Ser), sweet, bitter (Thr, Pro, Lys), and bitter (Arg, His, Ile, Leu, Met, Phe, Tyr, Val) (Wang et al, 2014). The contents of FAAs increased by 78.91% after fermented for 1 month, which was resulted in proteolyzing or synthesizing by some microbial strains, such as Aspergillus, Zygosaccharomyces, etc (Liang et al., 2020). The contents of FAAs increased significantly (p < 0.05) in the moromi of the first month, except for Cys, His, and Tyr, the order of dominant constituents were Glu > Leu > Arg > Asp > Lys. Their contents were almost unchanged from 2nd month to 5th month. While FAAs' contents were increased by 24.20% (p < 0.05) due to the contribution of enhanced Gly, Pro, and Tyr content when the fermentation finished. Compared with the 2nd month, there was no difference in the total contents of the 3rd month, while the contents of Asp, Pro, Gly, and Glu increased, that of Tyr, Arg, and His decreased.
| Taste description | Content (mg/100g) | |||||||
|---|---|---|---|---|---|---|---|---|
| 0th month | 1st month | 2nd month | 3rd month | 4th month | 5th month | 6th month | ||
| Asp | MSG-like | 327.88±14.35d | 642.31±5.31c | 720.62±58.96c | 953.12±14.47b | 865.04±4.99b | 883.02±2.77b | 1112.51±50.53a |
| Glu | MSG-like | 475.68±15.45e | 959.94±5.98d | 1139.81±85.01bc | 1328.04±20.14ab | 1251.73±38.41a | 1011.02±34.52cd | 1357.1±27.17a |
| Percentage (%) | 21.07 | 23.48 | 27.11 | 32.48 | 29.99 | 27.13 | 28.75 | |
| Ser | sweet | 242.7±9.66d | 439.68±6.42dc | 454.72±23.1bc | 490.71±6.19b | 477.65±3.55bc | 469.98±3.81bc | 560.16±14.15a |
| Gly | sweet | 114.13±6.93e | 206.84±3.65d | 245.3±15.15c | 292.13±2.17b | 261.57±0.5c | 244.67±1.98c | 379.21±3.64a |
| Ala | sweet | 191.59±7.06d | 360.64±3.16c | 365.91±25.31bc | 406.48±6.78b | 382.84±0.72bc | 371±1.16bc | 465.96±8.48a |
| Thr | sweet, bitter | 142.64±6.51d | 266.6±1.65dc | 277.78±18.56bc | 291.37±4.46bc | 299.11±5.54b | 286.36±2.69bc | 345.08±3.76a |
| Lys | sweet, bitter | 332.21±4.43d | 516.98±5.94b | 457.72±7.38c | 505.27±3.76bc | 476.2±23.99bc | 471.21±16.92bc | 576.75±10.34a |
| Pro | sweet, bitter | 142.17±15.15c | 290.33±1.63b | 295.11±8.47b | 361.58±19.48b | 331.11±26.26b | 329.5±10.63b | 549.63±37.11a |
| Percentage (%) | 30.56 | 30.5 | 30.55 | 33.42 | 31.57 | 31.12 | 33.49 | |
| Val | bitter | 230.11±50.54c | 408.07±1.42b | 407.41±42.55b | 454.61±0.86ab | 433.64±4.9ab | 437.13±6.68ab | 526.37±2.05a |
| Met | bitter | 73.15±1.31a | 104.72±9.53a | 107.27±33.91a | 96.32±11.96a | 87.81±30.58a | 94.5±17.52a | 106.83±16.67a |
| Ile | bitter | 190.2±1.55c | 415.4±5.88b | 413.72±9.41b | 448.23±9.1b | 430.3±13.81b | 428.93±13.96b | 529.99±2.08a |
| Leu | bitter | 385.35±6.21c | 702.14±3.83b | 683.33±30.07b | 713.07±7.4b | 700.11±20.22b | 690.43±12.35b | 801.64±24.87a |
| Tyr | bitter | 238.33±7.73bc | 294.65±7.93ab | 260.18±2.16abc | 28.85±1.27d | 238.83±13.01bc | 221.02±17.83c | 303.72±32.05a |
| Phe | bitter | 215.88±19.33b | 408.41±0.93a | 417.25±43.39a | 388.06±5a | 406.24±34.92a | 382.7±8.79a | 506.03±72.01a |
| His | bitter | 146.84±62.82a | 172.86±15.03a | 155.62±17.39a | 125.76±32.39a | 146.71±11.4a | 172±5.32a | 191.62±13.8a |
| Arg | bitter | 304.93±11.72d | 573.66±17.93a | 401.4±23.47c | 85.56±2.44f | 218.94±8.52e | 488.9±16.27b | 278.46±6.4de |
| Percentage (%) | 46.8 | 45.14 | 41.48 | 33.42 | 37.72 | 41.75 | 37.76 | |
| Cys | 59.95±20.02a | 59.75±3.55a | 58.71±1.59a | 54.41±2.52a | 50.85±5.31a | 0.00±0.00b | 0.00±0.00b | |
| Percentage (%) | 1.57 | 0.88 | 0.86 | 0.68 | 0.72 | 0 | 0 | |
Values are expressed as averages of three independent experiments ± SD.
a–e Different letters in the same row indicate a significant difference (p < 0.05, different fermentation time).
Dynamics of volatiles during the process As for volatile components, volatile identified were grouped into seven classes according to their chemical structure, including alcohols, acids, esters, phenols, aldehydes, ketones, and others (Fig. 3a). The dynamics of volatiles during the process were divided into two phases. The first phase was from the initial moromi to the 3rd month, the volatile species increased to 64, and their content increased from 2.4 mg/kg to 73.7 mg/kg (Fig. 3a). The second phase was from the 3rd month to the 6th month, in which the contents of various volatile increased to 232.3 mg/kg of the moromi in the 4th month, and then increased to 881.6 mg/kg non-monotonically (Fig. 3a).
The changes of volatile substances in moromi during the process. (a) The changes in the total amount of volatile substances at different fermentation times. (b) OAV profile analysis of moromi during the fermentation process. The OAV profile was expressed as the log of OAV from main volatiles (OAV > 1).
Odour activity values (OAVs), calculated by dividing the concentrations with their respective odour thresholds (Giri et al., 2010) of dominant flavor constituents, characterized the effect of volatility on food flavor. The OAV of 17 kinds of different compounds, significantly influenced (OAV > 1) the flavor of moromi, were determined (Table 4). These constituents including in 5-ethyl-4-hydroxy-2-methyl-3(2H)-furanone (HEMF), 3-(methylthio)-1-propanol, 1-octen-3-ol, 4-ethyl-2-methoxy-phenol (4-EG), 2-methoxy-4-vinylphenol (4-VG), 3-methyl-1-butanol, acetic acid, octanoic acid, ethyl ester and benzeneacetaldehyde were higher more than 100 (Table 4). An aroma-like profile was constructed to compare the effect of characteristic flavor in moromi (Fig. 3b). Of them, HEMF, 3-(methylthio)-1-Propanol, and acetic acid increased with the process. 1-octen-3-ol was partially originated from koji and was mostly bio-synthesized during the later process. OAV of 3-methyl-1-butanol, increased to 346.73 (Table 4) after fermented for 1 month, was always enhanced. While ethyl palmitate was increased alternately during the process. Although maltol and 4-ethyl-phenol were higher content, it was a nonsignificant effect because OAV was too low. The dominant phenols of HEMF, 4-EG, and 4-VG, endowed soy sauce to smoke and soy sauce flavor, were related to the abundance of functional yeast and bacteria (Liang et al., 2019).
| Compounds | Threshold a(µg/L) | OAV b | ||||||
|---|---|---|---|---|---|---|---|---|
| 0th month | 1st month | 2nd month | 3rd month | 4th month | 5th month | 6th month | ||
| 1-Butanol | 459.20 | 0.02 | 0.49 | 0.31 | 0.23 | 3.17 | 0.81 | 1.72 |
| 2-Methyl-1-Propanol | 16.00 | 0.55 | 14.69 | 8.72 | 10.03 | 25.60 | 32.28 | 46.89 |
| 3-Methyl-1-Butanol | 4.00 | 18.36 | 346.73 | 239.08 | 302.38 | 639.80 | 874.42 | 1008.65 |
| 1-Octen-3-ol | 1.50 | 100.13 | 94.71 | 199.59 | 411.66 | 405.64 | 400.23 | 384.76 |
| 3-(Methylthio)-1-Propanol | 0.50 | 54.62 | 399.54 | 330.94 | 777.62 | 1327.68 | 1310.38 | 2952.17 |
| Phenylethyl alcohol | 546.20 | 0.21 | 7.54 | 6.14 | 11.81 | 26.45 | 27.48 | 50.89 |
| Acetic acid | 5.50 | 20.39 | 112.33 | 252.90 | 974.11 | 703.04 | 737.09 | 735.49 |
| Octanoic acid, ethyl ester | 19.40 | 0.66 | 52.82 | 53.39 | 54.60 | 95.77 | 152.74 | 110.71 |
| Ethyl palmitate | 2000.00 | 0.16 | 5.94 | 6.30 | 4.90 | 23.66 | 21.15 | 26.27 |
| Maltol | 1200.00 | 0.02 | 0.20 | 0.46 | 1.30 | 1.63 | 1.84 | 2.61 |
| 4-Ethyl-2-methoxy-Phenol | 89.30 | 0.44 | 11.58 | 62.72 | 46.23 | 385.92 | 30.06 | 209.92 |
| 4-Ethyl-Phenol | 1010.10 | 0.01 | 0.27 | 1.18 | 0.80 | 6.89 | 0.47 | 4.67 |
| 2-Methoxy-4-vinylphenol | 12.00 | 20.71 | 196.81 | 131.38 | 55.64 | 130.55 | 130.22 | 125.15 |
| 2,4-Di-tert-butylphenol | 2.00 | 42.47 | 205.85 | 149.16 | 168.15 | 154.17 | 344.40 | 103.55 |
| Benzaldehyde | 750.90 | 0.23 | 0.61 | 0.91 | 2.41 | 4.29 | 3.26 | 5.00 |
| 5-Ethyl-4-hydroxy-2-methyl-3(2H)-furanone | 1.15 | 0.00 | 1314.90 | 4853.52 | 5856.37 | 9855.56 | 10796.21 | 14648.86 |
| Benzeneacetaldehyde | 4.00 | 48.73 | 111.28 | 233.86 | 831.18 | 1379.03 | 1153.09 | 2104.31 |
Mechanism of biogenic amines and ethyl carbamate forming during the process The changes in the content of BAs and EC were also investigated in the process (Table 5), which was endogenous hazards for the traditional fermentation foods. The contents of total BAs identified ranged from 80.38 mg/kg to 2826.52 mg/kg. BAs in the initial moromi were composed of tryptamine (20.1%), putrescine (35.4%), and tyramine (44.4%) (Table 5). The content of tyramine increased up to 2073.27 mg/kg when fermented for the 3rd month (Table 5), meanwhile, that of tyrosine decreased to the lowest concentration (Table 3) as tyrosine converted into tyramine (Santos, 1996). The contents of 2-phenethylamine and spermine were increased gradually during the processes. Cadaverine was only detected until the moromi of the 3rd month. The contents of histamine and 2-phenethylamine significantly (p < 0.05) increased up to the maximum value, which was 521.21 mg/kg and 136.83 mg/kg, respectively, in the moromi of the 3rd month (Table 5). The contents of His and Phe, precursors of corresponding biogenic amine (histamine and 2-phenethylamine), decreased to the lowest (Table 3). The contents of seven kinds of BAs were decreased after that of these BAs increased up to the highest concentration in the 3rd moromi (Table 5). These results suggested that most BAs were converted from the corresponding FAA by decarboxylases, such as Arg could be converted into putrescine and spermine through the arginine decarboxylase pathway (Benerroum, 2016). However, tyramine accounted for 75.2% of BAs in the moromi of the 6th month (Table 5) due to Tetragenococcus can inhibit the synthesis of histamine and cadaverine (Kuda et al., 2012; Kim et al., 2019). In fact, it was influenced by multiple factors that the corresponding FAA converted into BAs. The accumulation of BAs depended on the activity of corresponding decarboxylases that had strain specificity although lactic acid bacteria, such as Weissella, Staphylococcus, Leuconostoc, etc were considered as BAs producers (Aymerich et al., 2006; Özoğul et al., 2012). LAB, such as Weissella, Staphylococcus, Leuconostoc, etc, were dominant (> 96.8%) in the initial moromi (Fig. 1c), then the abundance of three dominant genera (Weissella, Staphylococcus, and Leuconostoc) ranged between 36.89% and 58.89% during the initial-and middle-phase (1–3 month) (Fig. 1c). OAs, which was one of the important metabolites produced by these genera, resulted in enhancing the acidity, and then Tyr, His, and Phe were converted into corresponding BAs by the corresponding decarboxylase for a response to acid stress.
| BAs (mg/kg) | 0th month | 1st month | 2nd month | 3rd month | 4th month | 5th month | 6th month |
|---|---|---|---|---|---|---|---|
| TRY | 16.16±1.03c | 17.79±1.19c | 10.66±0.04d | 25.15±0.70a | 2.70±0.24e | 22.56±0.60b | 1.97±1.71e |
| PHE | ND | 13.78±0.08c | 17.00±0.06b | 136.83±1.75a | 14.92±0.09c | 16.56±0.23b | 14.15±0.37c |
| PUT | 28.42±0.02d | 33.41±0.17c | 26.42±0.04e | 49.2±0.06a | 40.27±0.04b | 15.33±0.01g | 23.44±0.03f |
| CAD | ND | ND | ND | 7.39±4.29b | 3.93±0.03c | 1.48±0.04d | 15.71±1.46a |
| HIS | 0.07±0.05e | 1.71±0.03e | 40.24±0.02c | 521.21±11.33a | 174.57±0.15b | 17.89±0.03d | 8.21±0.03de |
| TYR | 35.72±3.37g | 48.24±0.39f | 466.30±0.5d | 2073.27±0.42a | 656.55±0.07b | 574.52±0.45c | 255.29±0.25e |
| SPM | ND | 6.47±1.17bc | 8.22±1.80bc | 15.94±8.59ab | 15.09±3.94ab | 12.72±1.83ab | 20.53±2.88a |
| TBAs | 80.38±2.72g | 121.4±2.48f | 568.84±2.31d | 2826.52±12.55a | 908.04±4.35b | 661.06±2.79c | 339.31±1.75e |
| EC(ng/kg) | 19.5±1.37e | 13.44±1.88e | 205.94±28.37d | 822.51±58.06a | 559.77±39.74b | 561.78±11.61b | 326.29±31.73c |
Values are expressed as averages of three independent experiments ± SD.
a–g Different letters in the same row indicate significant differences (p < 0.05, different fermentation time).
ND: Not Detected. TRY, tryptamine; PHE, 2-phenethylamine; PUT, putrescine; CAD, Cadaverine; HIS, histamine; TYR, tyramine; SPD, spermidine, TBAs, total biogenic amines. EC, ethyl carbamate.
As shown in Table 5, the content of EC increased to peak values (822.51 µg/kg) in the moromi of the 3rd month. Conversely, the contents of ethanol and Arg, considered as precursors, were decreased from highest in the moromi of the first month to minimum value of 0.70 g/100g (Fig. 2) and 85.56 mg/100g (Table 3) respectively in the moromi of the 3rd month. Meanwhile, Staphylococcus was also dominant bacteria and could accelerate the metabolic rate of Arg to Cit and then accelerated the formation of EC (Fang et al., 2018), resulted in reducing Arg from 573.66 mg/100g to 85.56 mg/100g (Table 3). Yet, the abundance of Staphylococcus, Weissella, etc decreased by increasing the acidity in the later phase (Hao and Sun, 2020). However, EC content was not reduced with the contents of ethanol and Arg increased in the subsequent process. Therefore, we speculated that EC had other metabolic pathways in the late fermentation period.
Metabolic pathways of BAs and EC during the process The effect of metabolic pathways difference on dynamics of BAs and EC based on the database KEGG and MetaCys was shown in Fig. 4. Compared with the first month, the relative abundance of the aromatic-l-amino-acid decarboxylase (EC: 4.1.1.28) in the moromi of the 3rd month was higher more than 2 times (Table 6). Therefore, the contents of tyramine and phenylethylamine were enhanced from 1st month to 3rd month, which was closely related to the higher strength of decarboxylase. Similarly, the higher abundance of histidine decarboxylase (EC: 4.1.1.22) in the 3rd month (Table 6) caused histamine content was as high as 521.21 mg/kg (Table 5), meanwhile the histidine dropped to 125.76 mg/100g (Table 3). Monoamine oxidase (EC: 1.4.3.4) could act on BAs to oxidize and decompose them (Dapkevicius et al., 2000). Therefore, as the monoamine oxidase (EC: 1.4.3.4) in the 6th month was 131.06% greater than that in the 3rd month (Table 6), the BAs content decreased in the late fermentation period.
Correlation pathways of EC and putrescine and metabolic pathways of other BAs during the process.
| Enzyme abundance | 0th month | 1st month | 2nd month | 3rd month | 4th month | 5th month | 6th month | |
|---|---|---|---|---|---|---|---|---|
| EC: 4.1.1.18 | 6.0 | 13.5 | 20.2 | 29.8 | 24.0 | 53.8 | 75.8 | |
| EC: 4.1.1.28 | 1.2 | 1.8 | 2.2 | 3.8 | 4.3 | 13.1 | 13.2 | |
| EC: 4.1.1.22 | 0.6 | 0.4 | 0.0 | 0.6 | 0.5 | 0.4 | 1.2 | |
| EC: 1.4.3.21 | 1.8 | 2.4 | 2.2 | 5.8 | 5.2 | 10.8 | 15.7 | |
| up-regulated | EC: 3.5.3.1 | 819.8 | 821.0 | 833.9 | 439.9 | 307.0 | 392.7 | 406.5 |
| EC: 3.5.3.6 | 481.3 | 58.9 | 67.4 | 80.1 | 69.8 | 132.9 | 124.3 | |
| EC: 4.1.1.17 | 27.6 | 63.8 | 76.0 | 87.0 | 88.4 | 179.9 | 203.9 | |
| EC: 2.1.3.3 | 1909.0 | 1324.4 | 1358.4 | 1204.6 | 1165.6 | 717.3 | 583.6 | |
| EC: 2.5.1.6 | 1504.5 | 1387.7 | 1403.9 | 1387.3 | 1420.7 | 1242.6 | 1176.0 | |
| EC: 2.6.1.82 | 3.5 | 5.7 | 8.2 | 13.7 | 12.7 | 17.2 | 20.5 | |
| EC: 1.4.3.4 | 25.6 | 64.2 | 97.2 | 46.9 | 48.2 | 119.4 | 108.3 | |
| EC: 1.2.1.5 | 0.0 | 0.0 | 0.1 | 0.0 | 0.8 | 2.5 | 5.7 | |
| down-regulated | EC: 6.3.4.5 | 1483.9 | 1378.4 | 1390.8 | 1363.5 | 1389.3 | 1195.3 | 1120.8 |
| EC: 4.3.2.1 | 1489.6 | 1432.8 | 1433.1 | 1461.8 | 1477.9 | 1331.4 | 1334.6 | |
| EC: 1.4.3.10 | 2.5 | 1.4 | 1.0 | 2.4 | 1.1 | 3.9 | 3.5 | |
| EC: 3.5.1.5 | 541.2 | 648.2 | 877.3 | 793.7 | 632.7 | 1150.4 | 1376.5 | |
| EC: 1.5.99.6 | 3.5 | 38.7 | 14.8 | 66.1 | 46.5 | 68.1 | 138.0 |
The urea cycle can regulate and control EC content by regulating EC precursors including Arg, urea, citrulline, etc. During the moromi fermentation process, the content of ethanol, Arg, and EC changed alternately. At prophase of moromi fermentation (1–3months), the abundance of ornithine carbamoyltransferase (EC: 2.1.3.3) and arginase (EC: 3.5.3.1) was 57.61% and 89.37% greater than those in the later stage (4 - 6 months), respectively (Table 6). Therefore, this speeded up the formation of EC from citrulline and urea in the 3rd month. Interestingly, EC and BAs are not unrelated, putrescine can be synthesized by ornithine, one of the important precursors of EC (Fig. 4), with a similar result obtained for another soy sauce manufacturer latterly (Data was not shown). At the later stage of fermentation, urease (EC: 3.5.1.5) abundance increased by about 77.15% (Table 6), which increased the metabolic rate of urea into ammonia and CO2. Therefore the increment of urease (EC: 3.5.1.5) reduced the precursors of EC, thereby reducing the content of EC, which was consistent with the previous report by Kim et al. (1995) and Miyagawa et al. (1999).
In the present study, the change of BAs, EC, other metabolites, and the microbial community diversity, was investigated during the moromi fermentation process. It indicated that Weissella, Staphylococcus, Tetragenococcus, Aspergillus, Zygosaccharomyces, and Issatchenkia were dominant although their abundance various significantly with the process. BAs were mostly converted from their precursor free amino acids, while the formation of EC was not only related to ethanol but also related to Arg. The formation process of EC is related to the synthesis pathway of putrescine. Weissella, Staphylococcus, and Leuconostoc, acid-producing bacteria, were directly related to the generation of BAs and EC in the early stage of fermentation. At the later stage of fermentation, monoamine oxidase was secreted to reduce the content of BAs in the moromi. The content of EC was also closely related to the urea cycle, in which the activity of urease was closely related to the content of urea and Arg, etc, thereby influenced EC's content. Succinic acid, malic acid, Glu, and Asp were the dominant taste components, HEMF, phenylacetaldehyde, 4-EG, and 4-VG were the main flavor substances in the moromi.
Acknowledgements This work was supported by the National Key R&D Program of China [grant number SQ2018YFC160049].
