Metabolomics mechanism of traditional soy sauce associated with fermentation time

Li Zhu, Siyu H Ying Lu, Jianhong Gan, Ningping Tao,Xichang Wang, Zaoli Jiang, Yuanxiang Hong*, Changhua Xu,c,,f,*

a College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China

b Shanghai Engineering Research Center of Aquatic-Product Processing & Preservation, Shanghai 201306, China

c Laboratory of Quality and Safety Risk Assessment for Aquatic Products on Storage and Preservation (Shanghai), Ministry of Agriculture, Shanghai 201306, China

d Department of Pharmacology, Yale University, New Haven, CT, 06520, US

e Xiamen Gulong Food Co., Ltd., Xiamen 361000, China

f National R&D Branch Center for Freshwater Aquatic Products Processing Technology (Shanghai), Shanghai 201306, China

Keywords:

Soy sauce

Fermentation

Amino acids

Volatile compounds

Flavor

Metabolomics

A B S T R A C T

Given that fermentation time has significant impact on quality and flavor of soy sauce, a global understanding of the metabolic mechanism as fermentation time prolonged is essential for producing satisfactory and consistent quality of traditional soy sauce. Herein, the metabolic compounds changes of soy sauce associated with fermentation time up to 10 years were comprehensively investigated by using Chinese traditional fermented soy sauce (CTFSS) as a demonstration. Results showed that formaldehyde nitrogen, total soluble nitrogen (TSN), non-salt soluble solids, amino acids, free 5’-nucleotides and volatile compounds in CTFSS changed obviously during fermentation. Specifically, glutamic acid and aspartic acid were prominent in CTFSS. Continuous decrease in content of hypoxanthine (Hx) was found from 1M (1-month soy sauce) to 7M (7-months soy sauce). Furthermore, a significant opposite tendency for changes between some volatile compounds and amino acids was indicated that there was a transformation between these two components.Therefore, a better understanding to the influence of fermentation time on soy sauce had been proposed. As the formation and conversion of amino acids and sugars might be mainly responsible for flavor formation in CTFSS, the ratio of these two reactions rate led the metabolism to be divided into three steps, degradation,conversion and balance.

1. Introduction

As a widely used condiment in East and Southeast Asia for hundreds of years, soy sauce has abundant annual output and is popular among consumers owing to its unique and pleasant flavor [1].Specifically, Chinese traditional fermented soy sauce (CTFSS), an intangible cultural heritage which has a long history in manufacture development, is generally produced through a completely natural fermentation [2,3]. With the steady improvement of economy and quality of life, higher requirements were pursued for the quality and flavor of soy sauce. Therefore, the flavor improvement of CTFSS is essential to obtain competitiveness in soy sauce market over the world.

Although the formation mechanism of flavor composition in soy sauce is complex, it has been widely accepted that protein degradation, sugar hydrolysis and Maillard reaction are the most principal biochemical reactions in soy sauce preparation [2]. The degradation products, amino acids and sugars, not only have a considerable influence on the taste activity value, but also contribute directly to the taste and odor characteristics, in some cases serving indirectly as precursors of aromatic products [4-8]. These chemical composition changes during fermentation directly influence the chemical, biological and sensory properties of soy sauce and provide necessary information to improve its sensory quality.

Quality and flavor of soy sauce can be affected by many factors,and fermentation time is one of the key factors [9,10]. The metabolic process and rate in soy sauce with different fermentation times were varied [11], and the concentration of chemical composition in soy sauce may also change with the different fermentation periods. Up to date, there is no universal standard for the fermentation time.The studied fermentation time of general researches varies from 3 months to 1 year [4,12,13]. As the flavor of soy sauce was a result of the interaction and balance among flavor substances, it was believed that the longer fermentation time, the better quality and flavor in soy sauce [14,15]. However, there are very limited scientific evidences about how and why the soy sauce flavor (odors and taste) changes as the fermentation time increasing.

In this study, content of taste and volatile substances in CTFSS with different fermentation times (from 1-month soy sauce to 10-years soy sauce) were systematically studied, and the changes of these substances were thoroughly investigated to reveal the metabolomic mechanism of soy sauce produced by the factory in different fermentation stages. The aim of this study is to reveal the mechanism and effect of fermentation time on the quality and flavor of soy sauce,and propose suggestions for improving fermentation process and flavor of CTFSS.

2. Materials and methods

2.1 Materials and chemicals

Soybean, wheat starch and edible salt were obtained from Gulong food Co., Ltd. (Xiamen, China).Aspergillus oryzaewas used in CTFSS production. CTFSS samples from the same batch of fermenters with different fermentation times (1–10 month, 1–10M;1–10 year, 1–10Y) were also directly obtained from Gulong Food Co., Ltd. (Xiamen, China), a traditional soy sauce company owning inherited ancient sauce technology. All the chemicals used in the analysis were at least analytical grade, and they were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

2.2 Preparation of CTFSS

Cooked soybean was mixed with roasted wheat, inoculated with 0.2% ofA. oryzae, and incubated in fermenter (pottery, 200 kg) to make koji. The resulting koji was subsequently mixed with 20% (m/m)brine, resulting in moromi mash. Then the fermenters were covered tightly with straw hat at natural condition for different fermentation times. CTFSS was processed by pressed, filtered, and pasteurized moromi mash that had fermented for different times. Fig. S1 showed the manufacturing process for fermenting soy sauce. All samples(n= 3) were filtered through filter papers, then kept in polyethylene vials and stored in a refrigerator at −20 °C until analysis.

Fig. 1 Contents of TSN, formaldehyde nitrogen and non-salt soluble solids in soy sauce with different fermentation times. Each value was expressed as mean ± standard deviation (n = 3). Values with different superscripts were significantly different (P ＜ 0.05).

2.3 Analytical methods

2.3.1 Chemical analysis

Formaldehyde nitrogen was determined by titration for three times [15]. Diluted samples (20 mL) were mixed with 60 mL H2O and titrated to pH 9.6 with 0.05 mol/L NaOH before 10 mL formalin solution (37% ) was added. The samples were finally titrated to pH 9.2 with 0.05 mol/L NaOH. Total soluble nitrogen (TSN) content of samples (using 6.25 as the conversion factor) were measured according to AOAC method 992.23 [16]. Salt content (expressed as NaCl, g/100 mL) was determined using a volumetric titration with AgNO3[17]. Non-salt soluble solids content (g/100 mL) was calculated as the total solids content (dry in 105 °C until constant weight) minus salt content. All analyses were carried out in triplicate.

2.3.2 Free amino acids (FAA) analysis

A trichloroacetic acid method was used to extract the FAA in soy sauces [18], and the diluted samples were filtered using the micropore filter (0.22 µm pore size, Sangon Biotech, Shanghai, China), then they were analyzed by an automatic amino acid analyzer (L 8800; Hitachi Ltd., Japan) equipped with a UV detector and a BioBasic SCX cation exchange column (4.6 mm × 60 mm, 5 µm). FAAs were detected at 440 and 570 nm with injection volume 20 µL. The flow rate was 40 mL/min and the column temperature was 60 °C. Identification and quantification of the each FAAs were evaluated by comparing the peak areas with those of amino acids standards. All analyses were carried out in triplicate.

2.3.3 5’-Nucleotides analysis

Measurements of 5’-guanosine monophosphate (GMP),5’-inosine monophosphate (IMP), 5’-adenosine monophosphate(AMP), hypoxanthine (Hx) and hypoxanthine riboside (HxR) were completed [19]. Nucleotides were extracted by 75% methanol. After filtration through 0.22 µm filters, the solution was collected and analyzed on an HPLC device (2695e; Waters Ltd., Milford, MA)equipped with a Waters SunfireTMC18column (250 mm × 4.6 mm,5 µm). The detection parameters were as follows: eluents, (A)0.05 mol/L KH2PO4(PH 4.6) and (B) methanol (HPLC grade);elution mode, gradient elution; column temperature, 30 °C; flow rate,1 mL/min. All analyses were detected in triplicate.

2.3.4 Headspace solid-phase micro-extraction gas chromatography-mass spectrometry (HS-SPME-GC-MS) analysis

The volatile compounds of soy sauce were detected by HS-SPME-GC-MS method [20]. The SPME fiber, Carboxen/polydimethylsiloxane (CAR/PDMS, Supelco, Inc., Bellefonte, PA,USA) was used for the extraction of volatile compounds in samples.Soy sauce (5 mL) in a glass vial (15 mL) was heated at 50 °C for 40 min. Analyses were performed on a 7890B gas chromatograph(Agilent) coupled to a 5977A inert XL series mass spectrometer(Agilent), and the HP-5MS column (30 m × 0.25 mm × 0.25 µm,Agilent Technologies, USA) was used for separation. The column was employed with the following oven temperatures: 40 °C (1 min),5 °C/min to 100 °C, 2 °C/min to 180 °C, 5 °C/min to 240 °C, and 20 °C/min to 260 °C (2 min). The carrier gas was helium (99.999% purity) at a constant flow-rate of 2 mL/min. The detector temperature was 250 °C. The mass spectrometer was operated in the electron impact ionization mode (70 eV). Interface, source, and quadrupole temperatures were 250, 230 and 150 °C, respectively, and the scan range was 35–350 amu. All analyses were carried out in triplicate.

2.3.5 Identification and semi-quantification analysis of volatile compounds

Identification was based on retention indices (RI) and mass spectra (MS) of reference standards matching in the National Institute of Standards and Technology (NIST) mass spectral library. A positive identification of volatile compounds was achieved by matching RI and MS with those of standard reference compounds analyzed under the same experimental conditions. All volatile compounds were reported with their positive and negative matching scores ＞ 800.Literature RI values were sourced from the NIST standard reference database. RI values of each compound was calculated using the C6to C30n-alkane series under the same experimental conditions. An aliquot (5mL) of the soy sauce containing 300 µg/kg 2,4,6-trimethyl pyridine was an internal standard material to quantified volatile compounds. Quantification measurements were carried out by GC-MS on the basis of the internal standard material (response factor = 1).

2.3.6 Statistical analysis

All the assays in this study were performed in triplicate. All the statistics were the averages of three tests. Statistical analysis was performed by origin version 8.6. Differences between samples and the effects of treatments were evaluated by Duncan’s multiple range test(P＜ 0.05). The heat map was generated using MATLAB (version 2016a, The MathWorks, Inc., Natick, Massachusetts, USA).

3. Results and discussion

3.1 Changes of TSN, formaldehyde nitrogen and non-salt soluble solids contents

The TSN and formaldehyde nitrogen contents were objective indexes used to classify the quality of soy sauce [21]. As excepted,contents of TSN and formaldehyde nitrogen (Fig. 1) occurred conspicuous increase (P＜ 0.05) along the first 3 months and decreased from 5M to 9M, and thereafter the contents of these two indicators increased, of which TSN reached the highest contents in 5Y and formaldehyde nitrogen in 10Y. The changes of non-salt soluble solids content were similar to the changes in TSN. High molecular weight materials such as starches and proteins in soybean and wheat were hydrolyzed by the koji enzymes to release amino acids and sugars [22]. The increase of these indicators in the first 3 months could be attributed to the fact that the effective degradation rates of proteolysis and amylolysis by proteases and amylases produced by microorganisms in moromi were greater than those of amino acids and sugars to volatile substances [23]. A similar increase in the first 3 months was reported by a previous study on a traditional fermented fish sauce [24]. The following reasons might explain the decline in these indicators between 5M and 9M: i) the conversion of amino acids and sugars to volatile or Maillard products at a faster rate than proteins degraded to amino acids and starch hydrolyzed to sugars; ii) the utilization by microorganisms as energy; iii) the decrease of enzyme activity in brine solution [2,13,25]. Later, these indicators elevated from 1Y to 5Y while the growth rate of the formaldehyde nitrogen content gradually declined, indicating that the key influencing factor might be the conversion rates of amino acids and sugars slower than the degradation and hydrolysis rates of the soy bean and starch, rather than the evaporation of water in the fermentation process. Finally,the decrease of TSN and non-salt soluble solids contents from 5Y to 10Y was mainly caused by the deficiency in nutrition for strain. In a word, the same change trend of these indicators was closely related to Maillard and volatile transformation reactions and degradation of the substrates in soy sauce.

3.2 Changes of FAA contents

Glutamic acid and aspartic acid were prominent in soy sauce samples of different fermented times, but glycine, histidine,methionine and tyrosine contents were negligible (Fig. 2). Total FAAs contents were rapidly increased from 64.5 g/L to 86.23 g/L during the first 3 months, then decreased markedly from 3M to 9M, later the contents gradually increased from 61.29 g/L of 9M to 90.22 g/L of 5Y, finally decreased slightly from 5Y to 10Y and reached the similar value of 5M.

Fig. 2 Heat map for the significantly changed FAA in soy sauce with various fermentation times. The abscissa represents different fermentation times; the ordinates were the name of amino acids; the value in the picture represents the average content of amino acids (n = 3; g/L).

It was reported that FAAs were regarded as an important contributor to the unique taste of soy sauce, it constituted a potential source of volatile compounds as follows: i) production of 2-methylpropanal, 2-methylbutanal and 3-methylbutanal through Strecker degradation of valine, isoleucine and leucine [26];ii) generation of phenolic compounds from aromatic amino acids,such as tyrosine and phenylalanine; generation of Sulphur-containing compounds from sulfur-containing amino acids, such as cystine,methionine and cysteine [27]; iii) generation of several pyrazines from Maillard reactions [26]; iv) generation of furans from cystine,methionine and cysteine by carbohydrate or lipid oxidation [28].Therefore, changes of FAAs contents in soy sauce depended on the balance between the formation and degradation of FAAs and volatile compounds. FAAs’ increasement was mainly due to the fact that the faster proteolysis reaction rate of aminopeptidase and protease than the conversion between amino acids and volatile substances or Maillard reactions products, while the decrease of FAAs was principally related to the formation of flavor compounds [29].

Evidences could be found in Fig. 2 and Fig. 3. For example,3-methyl-butanal increased by 272 µg/kg from 5M to 9M, meanwhile,the contents of valine, isoleucine and leucine decreased significantly,indicating that valine, isoleucine and leucine were rapidly converted into 3-methylbutanal during this period. 4-methylphenol increased by 1 613 µg/kg from 5M to 9M, similarly, the contents of aromatic amino acids such as tyrosine and phenylalanine tended to decrease,demonstrating that aromatic amino acids rapidly changed into aromatic volatile substances during this period.

Fig. 3 Changes in the content of special odor substances and amino acids from 5M soy sauce to 9M soy sauce. (A) Change of 3-methyl-butanal content detected by SPME-GC-MS. (B) Change in the contents of valine, isoleucine and leucine detected by automatic amino acid analyzer. (C) Change of 4-ethylphenol content detected by SPME-GC-MS. (D) Change in the contents of tyrosine and phenylalanine detected by automatic amino acid analyzer. Each value was expressed as mean ± standard deviation (n = 3). *P ＜ 0.05.

3.3 Changes of non-enzymatic browning index and 5’-nucleotides

The increase in absorbance at 420 nm (Fig. 4A) was used as an indicator of browning development of the browning reaction [30]. The increasing browning contributed significantly to color development of CTFSS. Since glucose was the dominant sugar in soy sauce, it could be assumed that the browning in soy sauce was mainly caused by Maillard reaction, rather than sugar caramelization [2]. An increase in browning was observed from 1M to 5Y suggesting that brown pigment continually increased along with the fermentation time.The decrease from 5Y to 10Y could be attributed to the fact that the degradation rate of Maillard reaction brown products was faster than that of formation.

Fig. 4 Contents of non-enzymatic browning index (A) and Hx (B) in soy sauce with different fermentation times; Metabolic process of Hx (C). Each value was expressed as mean ± standard deviation (n = 3).

It was known that yeast-derived products contribute to food flavor in a number of ways as added ingredients of food components thereby yielding a variety of products with desirable features. Hence, the addition of yeast during the fermentation of soy sauce can generate many nucleotides [31]. In general, soy sauce brewed in natural does not contain AMP, GMP, IMP and HxR, while the end product Hx derived from ATP metabolism can be measured in most products[13,32]. The components of free 5’-nucleotides, only Hx was detected in this study, and this was thought to be the soy sauce samples were naturally fermented only withA. oryzae. Specially, concentrations of Hx had a sharply decrease within the first 7 months and it tended to be stable after 7 months (Fig. 4B). The sharp decrease could be attributed to an increase ofXanthine oxidasecontent (Fig. 4C) [33].

3.4 Changes of volatile compounds

A heat map was generated to investigate the relative quantification of identified volatile compounds and their relationship to the characteristics of soy sauce during fermentation (Fig. 5). Darker red squares indicate higher concentrations of volatile compounds. A total of 66 volatile compounds were identified in the soy sauce samples by HS-SPME-GC-MS (Table S1), but the compounds differed according to the fermentation time-point. These compounds were grouped as phenols, esters, alcohols, benzenes, aldehydes, acids, ketones and others based on their chemical structure. Phenols, esters and alcohols mainly produced in fermented soy sauce from 5M to 9M, meanwhile,aldehydes, ketones and acids concentrated from 1Y to 5Y, and the contents of these substances gradually increased in the corresponding period. It could be seen that there was an opposite tendency for changes between some volatile compounds and amino acids, which was led by the transformation between these two components.Specifically, because of the degradation of aromatic amino acids,4-ethyl-2-methoxyphenol and 4-ethylphenol had a significant increase from 5M to 9M in phenols (Fig. 3) [28]. The increase of most esters and alcohols during this period was due to esterification of sugar metabolites and alcohol fermentation, especially ethanol, which was considered to arise from the fermentation of carbohydrates by the Embden-Meyerhof pathway [34,35]. Branched-chain amino acids,derived from soybean protein, converted intoα-keto acids through deamination or transamination during metabolic processes, and then they could generated branched-chain aldehydes and branchedchain acids through decarboxylation and oxidation [36]. This is also the reason for the increase in acids from 1M to 4Y. Meanwhile,an increase of branched short-chain aldehydes (2-methylpropanal,3-methylbutanal and 2-methylbutanal) from 5M to 9M were mainly generated from the degradation of the corresponding amino acids (valine,isoleucine, and leucine) through the Strecker degradation [26,37].Benzaldehyde and benzeneacetaldehyde could be detected in the consecutive 10 years of samples. These compounds were the degradation products of the aromatic amino acid phenylalanine [38].Futhermore, other components, such as pyrazine and furans(2,5-dimethyl-furan and 2-ethyl-6-methpyrazine) increased from 5Y to 10Y were attributed to sugar dehydration or fragmentation from Maillard reaction during sterilization of soy sauce [27,39]. In a word, changes of volatile substances were strong evidences for the conversion of amino acids or sugars in soy sauce. And it could explain why there was a subtle balance between conversion of sugar and amino acid and degradation of the soybean substrates in soy sauce.

Fig. 5 Heatmap visualization of the volatile compounds in soy sauce with various fermentation times based on GC-MS data. N.D., not detected; the ordinate was the name of volatile compounds; the value in the picture represents the average content of volatile compounds (n = 3; µg/kg).

3.5 Metabolomic mechanism of fermentation

As the increase or decrease of TSN, formaldehyde nitrogen,non-salt soluble solids and FAAs contents in soy sauce in different fermentation times were mainly depended on the reaction rate of proteins degraded into amino acids, starch hydrolyzed into sugars and amino acids and sugars converted into Maillard reaction products or other volatile substances. To explain the change rules of various indexes of CTFSS in detail, the metabolomic mechanism in soy sauce associated with fermentation time was divided into 3 steps (Fig. 6):step 1, hydrolysis of starch and degradation of proteins into sugars and amino acids, respectively; step 2, conversion of amino acids and sugars into Maillard reaction products or other volatile substances;step 3, conversion, promotion or suppression of step 1 and step 2 for a subtle balance. Specifically, the change of 3-methyl acetone content in Fig. 3A represented the reaction rate in step 1, and the contents change in valine, isoleucine and leucine in Fig. 3B represented the reaction rate in step 2. It was found that while the reaction rate in step 1 increased, the reaction rate in step 2 decreased, and the same trend was observed in Figs. 3C and 3D, indicating that step 3 should be defined as step 1 and step 2 promoted or suppressed each other to maintain a subtle balance. In general, metabolomic mechanism existed in the process of soy sauce fermentation.

Fig. 6 Metabolomic mechanism during fermentation in CTFSS.

4. Conclusion

In conclusion, this study has clearly demonstrated the changes of taste and volatile substances of CTFSS with different fermentation times. Variation trends of content in soy sauce showed that the concentrations of the TSN, formaldehyde nitrogen, non-salt soluble solids and amino acids were increased in the first 3 months and decreased gradually from 3M to 9M and later slowly increased as the fermentation year increased to 5Y. In addition, the changes of various odor substances with different fermentation times were different,and their changes were closely related to sugars and amino acids metabolism. Consequently, there was a dynamic balance between conversion of sugars and amino acids and degradation of the soybean substrates in soy sauce. From the above, a metabolomic process based on nutrient and flavor metabolites in CTFSS could be divided into three steps, degradation, conversion and balance.

This study also explained some common concerns. The better soy sauce was not exactly depended on the longer time of fermentation.For improvement of the fermentation process to enhance the quality of soy sauce, 3M–5M of fermentation time was the highest for taste compositions, and 9M–10M was the highest for volatile substances.With the fermentation years increasing, the taste and odor substances contents increased, especially when the fermentation time was 5 years, the content of some indicators was even higher than that of 3M and 5M. However, it was not recommended because of its timeconsuming. Thus for the overall quality of soy sauce, the optimal fermentation time is 5 months, and it was recommended to reinoculate new strains after 5 months of soy sauce fermentation for improving flavor.

In conclusion, the above findings provided understandings leading to the metabolic mechanism of traditional fermented soy sauce, and meaningful reference data for the selection of fermentation time that contribute to enhancing the quality and flavor of CTFSS, furtherly made scientific contribution to the inheritance and improvement of Chinese traditional brewing culture.

Acknowledgements

This work is financially supported by the National Key Research and Development Program of China (2016YFD0401501) and Shanghai Pujiang Program (18PJ1432600).

Conflict of Interest

The authors have no conflicts of interest to declare.

Appendix A. Supplementary data

Supplementary data associated with this article can be found in the online version, at http://doi.org/10.1016/j.fshw.2021.11.023.