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Article

The Differences in the Composition of Maillard Components between Three Kinds of Sauce-Flavor Daqu

by
Qi Zhu
1,2,†,
Liangqiang Chen
3,†,
Xiuxin Pu
3,
Guocheng Du
1,2,
Fan Yang
3,
Jianjun Lu
3,
Zheng Peng
1,2,
Juan Zhang
1,2,* and
Huabin Tu
4,*
1
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China
2
Science Center for Future Foods, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China
3
Kweichow Moutai Distillery Co., Ltd., Renhuai 564501, China
4
Kweichow Moutai Group, Renhuai 564501, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Fermentation 2023, 9(9), 860; https://doi.org/10.3390/fermentation9090860
Submission received: 17 August 2023 / Revised: 14 September 2023 / Accepted: 15 September 2023 / Published: 21 September 2023
(This article belongs to the Special Issue Perspectives on Microbiota of Fermented Foods)
Browse Figures
Versions Notes

Abstract

:
Sauce-flavor Daqu is a saccharifying and fermenting agent for sauce-flavor baijiu. Three kinds of Daqu (White Daqu (WQ), Yellow Daqu (YQ), and Black Daqu (BQ)) with different qualities are formed owing to the stacking fermentation. Maillard reaction is an important factor that causes significant differences among the three kinds of high-temperature Daqu, which is also an important source of flavor substances. However, there is a lack of research on the composition differences of Maillard reaction products in the three types of Daqu. In our study, HS-SPME-GC/MS, Py-GC/MS, and high-throughput sequencing were used to investigate the small molecule volatile Maillard substances, melanoidin (macromolecular Maillard substance) composition, and microbial community of three kinds of Daqu. The results showed that there were significant differences in the composition of Maillard products (furans, pyrazines, and melanoidin structure) between the three kinds of Daqu. The melanoidin was mainly composed of furans, pyrrolopyrazines, phenols, and indoles, the proportions of which were different in the three types of Daqu. BQ contained more Maillard products, making the melanoidin more complex. Kroppenstedtia, Monascus, and Thermoascus were the biomarkers of BQ, which contribute to the Maillard reaction. This study is of great significance to further deepen the understanding of the formation mechanism of BQ.

1. Introduction

Sauce-flavor baijiu is one of the most famous traditional Chinese baijiu. It is a product of traditional solid fermentation and is generally described as being complexly flavored, sweet, and refreshing owing to its special production process [1]. Its fermentation process mainly contains two steps: Daqu fermentation and liquor fermentation, both of which are under high-temperature conditions, and the complex process makes it easier to be significantly different from other types of liquors [2].
Sauce-flavor Daqu, a saccharifying and fermenting agent for the brewing of sauce-flavor baijiu, is made from grains, water, and Muqu (mature Daqu). During the solid-state fermentation, grains were degraded by the microbiota, which mainly originated from raw materials and the environment, then the sauce flavors were formed and accumulated through the bio-chemical process and Maillard reaction under high-temperature conditions [3]. However, owing to the stacking fermentation, Daqu in different parts of the fermentation room have significant differences in their appearances. Three types of Daqu are distinguished by sensory evaluation (such as smell and color) and are named White Daqu (WQ), Yellow Daqu (YQ), and Black Daqu (BQ), respectively [4]. The mature Daqu for liquor fermentation is a mixture of three types of Daqu in a certain proportion. Therefore, it is of great significance to ensure the continuity of the yield of three types of Daqu and investigate the differences between them. In the previous study, we concluded that the Maillard reaction was the main reason for the differences among the three Daqu, and on this basis, it is necessary to further study Maillard reaction products, especially melanoidin to deeply explore the differences in the formation of the three Daqu.
In this study, HS-SPME-GC/MS was used to analyze the volatile substances of the three types of Daqu to explore the heterocyclic substances generated by the Maillard reaction. Melanoidin is the final product of the Maillard reaction. Py-GC/MS was used to analyze the pyrolysis compounds of melanoidin to compare the differences of Maillard reaction degree in three kinds of Daqu. High-throughput sequencing was performed to investigate the microbial community to compare the differences in microbial composition. The functional microorganisms contributing to the Maillard reaction in BQ were explored by correlation analysis. This study is of great significance to further deepen the understanding of the formation mechanism of BQ.

2. Materials and Methods

2.1. Samples

The sauce-flavor Daqu was provided by Moutai Baijiu Industry Co., Ltd. (Renhuai, China). At the end of Daqu fermentation on the 40th day, three types of Daqu were classified by experienced workers according to their color. Samples of WQ, YQ, and BQ were collected from the same fermentation room, each of which consisted of three parallel samples. Samples were cooled by liquid nitrogen and ground into powder, transferred to sterile bags, sealed, and stored at −40 °C.

2.2. Determination of Total Reducing Sugar and Amino Acids

The concentration of total reducing sugar was determined by the methods described by Luo [5]. To extract amino acids from samples, 5 g Daqu were treated with 25 mL sterilized water, followed by 1 h of ultrasound and 24 h of refrigeration at 4 °C, then centrifuged at 12,000× g for 10 min (4 °C) and collected the supernatant. The supernatant was filtered through the 0.45 µm organic phase filter membrane. OPA was used as the derivatization reagent for amino acids. The C18 column (4.6 × 250 mm, 5 μm) was used for chromatographic separations using gradient elutions by an Agilent HPLC system. The mixture of twenty amino acids was used as standard. The procedure of HPLC is shown in Table 1.

2.3. Extraction and Quantification of Melanoidin

According to the method described by Luo [5], accurately weigh 2 g of Daqu powder, add 35 mL of 40% ethanol solution, and immerse in a water bath at 70 °C for 3 h. Then, centrifuge at 6000 r.min−1 for 10 min, the supernatant was dried by the rotary evaporator, and dilute the residue with distilled water in the 50 mL volumetric flask. The absorbance was detected at 470 nm. The concentration of melanoidin was calculated by the following formula [6]: C = A/eb, C: melanoidin content (mmol/L), A: absorbance at 470 nm, e: molar extinction coefficient of melanoidin 0.64 L/mmol·cm [7], b: cuvette thickness (cm). The melanoidin solution was mixed with ethanol (100%) in a ratio of 1:2 (v/v) and immersed in a water bath at 40 °C for 3 h. Then, it was centrifuged at 6000 r.min−1 for 10 min, the precipitate was freeze-dried at vacuum degree <1 Pa for 48 h in a freeze dryer, and the crude melanoidin was obtained.

2.4. Profiling of Volatile Compounds by HS-SPME-GC/MS

First, 5 g Daqu powder were added to a glass vial with 20 μL 2-octanol (10 mg/L) used as an internal standard. The volatile compounds were extracted with an SPME fiber (50:30 μm DVB/CAR/PDMS, Supelco Co., Ltd., Bellefonte, PA, USA). To absorb volatiles, the fiber needle was inserted into the headspace of the vial for 30 min in a 60 °C water bath. The SPME fiber was then inserted into the GC injection port for 8 min of desorption at 230 °C. The process of GC-MS and data analysis was performed based on the methods described in our previous study [8]. Each compound was identified by comparing the mass spectra with the standard mass spectrum in the NIST library (Gaithersburg, MD, USA).

2.5. Profiling of Melanoidin Pyrolysis Compounds by Py-GC/MS

Pyrolysis–gas chromatography/mass spectrometry was carried out on the Agilent system. First, 1 mg melanoidin was wrapped in hot foil. The pyrolysis was carried out at 450 °C for 10 s. The chromatographic separation was carried out on a DB-Wax column (30.0 m × 0.25 mm × 0.25 μm, Agilent, Santa Clara, CA, USA). Helium was used as a carrier gas at a constant flow rate of 1.0 mL/min. The oven temperature was maintained at 50 °C for 2 min, programmed at 5 °C/min to 145 °C, then at 15 °C/min to 230 °C, and held for 5 min. The mass spectrometer was operated in electron impact mode with the electron energy set at 70 eV and a scan range of 33–400 m/z. The temperature of the MS source and quadrupole was set at 200 °C and 230 °C, respectively. Each compound was identified using the NIST library (Gaithersburg, MD, USA). The relative content of pyrolysis compounds was calculated by the area normalization method.

2.6. Investigation of the Microbial Community in Three Types of Sauce-Flavor Daqu

The parameters of amplicon sequencing and metagenomic sequencing were performed based on the methods described in our previous study [8]. Total genomic DNA samples were extracted using the OMEGA Soil DNA Kit (Omega Bio Tek, Norcross, GA, USA).
The bacterial 16S rRNA genes V4–V5 region was performed using the forward primer 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and the reverse primer 907R (5′-CCGTCAATTCMTTTRAGTTT-3′) [9]. The fungal ITS1 region was performed using the forward primer ITS1F (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and the reverse primer ITS2R (5′-GCTGCGTTCTTCATCGATGC-3′) [10]. The pair-end 2 × 250 bp sequencing was performed using the Illumina MiSeq platform with MiSeq Reagent Kit v3 at Shanghai Personal Biotechnology Co., Ltd. (Shanghai, China).
Amplicon sequencing analysis was performed with QIIME2 [11]. Briefly, the DADA2 plugin [12] was used for quality control. The feature classifier plugin [13] was used for taxonomic annotation against the SILVA Release 138 (bacteria) (https://www.arb-silva.de/, accessed on 12 September 2023) and the NCBI Database (fungi) [14], respectively.
The raw data have been deposited in the NCBI Sequence Read Archive (SRA) BioProject PRJNA1017164.

2.7. Statistical Analysis

One-way analysis of variance (ANOVA) with Tukey’s test was used to compare the content of melanoidin, reducing sugar, and amino acids of three types of Daqu. The volatile compounds with matching degrees greater than 800 were selected. The semi-quantification of the volatile compounds was determined with the internal standard method. Partial least squares discriminant analysis [15] (PLS-DA) was performed to investigate the differences in volatile compounds and microbial community between three types of Daqu. A Venn diagram was generated to visualize the shared and unique volatile compounds and pyrolysis compounds among samples by R software “venn” packages (https://CRAN.R-project.org/package=venn (accessed on 17 August 2023), version: 1.11). A heatmap was generated to visualize the differences in the content of volatile heterocyclic substances and pyrolysis compounds between samples by R software with “pheatmap” packages (https://CRAN.R-project.org/package=pheatmap (accessed on 17 August 2023), version: 1.0.12). The Spearman correlation indices (ρ) and p values were computed to reveal the relationship between marked microorganisms and Maillard products.

3. Results and Discussion

3.1. The Differences in Melanoidin, Amino Acids, and Reducing Sugar between Three Kinds of Daqu

WQ, YQ, and BQ are three kinds of Daqu produced in the fermentation process of Sauce-flavor Daqu. At present, the formation mechanism of the three types of Daqu has not been fully analyzed. According to the previous research results, the Maillard reaction is the key factor that causes the differences between the three types of Daqu. Melanoidin is the final product of the Maillard reaction. As shown in Figure 1a, the average concentration of melanoidin in BQ was 2.82 mmol/L, which was significantly higher than that in WQ and YQ (p < 0.05). The content of melanoidin in WQ is the lowest, which is 1.28 mmol/L. The concentration of melanoidin is the same as the color difference of the three Daqu and showed that the Maillard reaction degree in the fermentation process of sauce-flavor Daqu was BQ > YQ > WQ. Reducing sugar and amino acids are important precursors of the Maillard reaction. The results showed that among the three kinds of Daqu, the reducing sugar content of WQ was the highest (Figure 1b), with an average of 29.11 mg/g, but there was no significant difference between WQ, YQ, and BQ (p > 0.05), indicating that YQ and BQ actually degrade starch and cellulose in raw materials to a higher degree, and the reducing sugar generated by hydrolysis not only satisfied the growth of microorganisms, but also participated in a large number of Maillard reactions, and produced more melanoidin than WQ. The total content of amino acids in BQ was 10.49 mg/g (Figure 1c), which was significantly higher than that in YQ and BQ (p < 0.05). It indicated that BQ could provide richer amino acids for the Maillard reaction. The total content of amino acids was positively correlated with the content of melanoidin (r > 0.80, p < 0.001). The results showed that amino acids had a greater influence on the formation of melanoidin. The degree of hydrolysis of protein in raw materials of BQ was much higher than that of other Daqu. A large number of amino acids were generated in BQ, which greatly promoted the formation of melanoidin in BQ. Furthermore, the types and concentrations of amino acids in the three kinds of Daqu were compared. The results (Figure 1c) showed that 16 common amino acids were detected from the three kinds of Daqu, among which the concentrations of glutamate, alanine, and proline were higher. The concentration of amino acids such as alanine, phenylalanine, and leucine in BQ was significantly higher than that in WQ and YQ, which provided more precursors for the Maillard reaction in BQ.

3.2. Profiling of Volatile Compounds in the Three Types of Daqu

A total of 87 volatile compounds were detected from BQ, YQ, and WQ. There were 67, 61, and 54 volatile substances detected in BQ, YQ, and WQ, respectively (Table S1). The difference in metabolic composition between the three types of Daqu was analyzed by PLS-DA. According to Figure 2a, nine samples were divided into three groups according to the types of Daqu, indicating that there were significant differences in the composition of volatile substances. Heterocyclic compounds (pyrazines [16,17] and furans [18]) were the main Maillard reaction products in Sauce-flavor Daqu. Three furans and eleven pyrazines were detected from BQ, YQ, and WQ. Pyrazines are important flavor substances in Chinese baijiu, which are baked and nutty. Among the three kinds of Daqu, pyrazines accounted for the highest proportion of volatile components (Figure 2b), all of which were more than 30%. The proportion of pyrazines in BQ was 34% on average, which was higher than that in WQ and YQ. Tetramethylpyrazine [19], trimethylpyrazine [20], 2,6-Dimethylpyrazine [21], etc., (Figure 2c) are active flavor substances in Chinese baijiu, which were high-concentration pyrazines in Daqu, especially in BQ. By calculating the VIP values of each variable in the PLS-DA model, it can be seen that tetramethylpyrazine was a marked volatile substance of BQ (VIP > 1), and the content of which in BQ is significantly higher than that in YQ and WQ. The total concentration of furans in BQ was 23.18 mg/g, which was significantly higher than that in YQ and WQ, and only 2-Pentylfuran was detected in WQ. 3-Phenylfuran and 2-Furfuraldehyde are known Maillard reaction products. Among them, 2-Furfuraldehyde is almond-scented and roasted, which is a common substance in Daqu and baijiu. The VIP values of both furans were greater than 1, and their concentrations in BQ were higher than those in other Daqu, which were marker substances of BQ. This result is consistent with the previous research [5]. The enrichment of Tetramethylpyrazine, 3-Phenylfuran, and 2-Furaldehyde in BQ is an important factor that the baking aroma in BQ is better than that in other types of Daqu. In addition to heterocyclic compounds, amines, the important precursor of the Maillard reaction, and aldehydes, the intermediate product of the Maillard reaction, were also detected from three kinds of Daqu (Table S1). The content of amines in WQ was highest, while that in BQ was the lowest. Among the three kinds of Daqu, the concentration of aldehydes in WQ was the lowest, and that in BQ was the highest. The results all showed that the degree of Maillard reaction of BQ was the highest and that of WQ was the lowest.

3.3. Profiling of Melanoidin Pyrolysis Compounds in the Three Kinds of Daqu

Melanoidin is the final product of the Maillard reaction, and the pyrolysis products of melanoidin were analyzed by pyrolysis at high temperature. According to Table S2, a total of 50 pyrolysis products were detected from three kinds of Daqu. Overall, 50, 39, and 39 kinds of pyrolysis products were detected from BQ, WQ, and YQ, respectively, and the number of pyrolysis products in BQ was 11 more than that in WQ and YQ, respectively (Figure 3a), which indicated that the structure of melanoidin in BQ was more complicated. According to PCA analysis (Figure 3b), there were significant differences in the composition of the pyrolysis products between the three kinds of Daqu, and there were also differences in the composition of the melanoidin pyrolysis products between the parallel samples of each kind of Daqu, indicating that the Maillard reaction in sauce-flavor Daqu is complex. Melanoidin pyrolysis products in sauce-flavor Daqu were mainly composed of pyrrolopyrazines, furans, pyrazines, pyrroles, indoles, phenols, pyrans, and other heterocyclic substances. Among them, pyrrolopyrazines, furans, phenols, and indoles had the highest proportion in melanoidin pyrolysis components, but the proportion in the three types of Daqu is quite different. The melanoidin is formed by polymerizing sugar degradation products and then the cross-linking of carbohydrates, proteins, polyphenols or other macromolecular substances to produce high molecular mass polymers [22,23]. Several reports have elucidated the partial structure of melanoidin were heterocyclic substances such as furan and pyrrole [24,25].
In BQ, 4-amino-1-Pentanol (14.34%), 5,10-Diethoxy-2,3,7,8-tetrahydro-1H,6H-dipyrrolo [1,2-a:1′,2′-d]pyrazine (8.01%), 3,5-dimethoxy-Phenol (4.95%), hexahydro-Pyrrolo[1,2-a]pyrazine-1,4-dione (4.66%), Butanal (4.15%), 9H-Pyrido[3,4-b]indole (4.06%), etc., were the main substances with the high proportion in BQ. Pyropyrazines, indoles, and phenols were the main components of BQ melanoidin. According to the results of the comparison of peak areas, 5,10-Diethoxy-2,3,7,8-tetrahydro-1H,6H-dipyrrolo[1,2-a:1′,2′-d]pyrazine, Indole, Pyrrole and Phenol in BQ were significantly higher than those in YQ and WQ (Figure 3c). The results showed that the Maillard reaction produced a large number of pyrazines in BQ, and even after a large number of pyrazines participated in the formation of melanoidin, high concentrations of pyrazines could still be detected in BQ. Melanoidin components in YQ were mainly pyrrolopyrazines, furans, and phenols, including 5,10-Diethoxy-2,3,7,8-tetrahydro-1H,6H-dipyrrolo[1,2-a:1′,2′-d]pyrazine (11.81%), 4-amino-1-Pentanol (9.63%), p-Cresol (9.48%), hexahydro-Pyrrolo[1,2-a]pyrazine-1,4-dione (7.22%), 3-methyl-1,4-diazabicyclo[4.3.0]nonan-2,5-dione (5.18%), Indole (4.22%), hexahydro-3-(phenylmethyl)-Pyrrolo[1,2-a]pyrazine-1,4-dione (3.78%), Furfural (3.74%). The difference between YQ and BQ in the proportion of pyrolysis compounds was smaller than that of WQ (Figure 3a). The detection peak area of hexahydro-Pyrrolo [1,2-a] pyrazine-1,4-dione was higher than that of WQ and BQ, suggesting that pyrazines in YQ were mainly involved in the formation of melanoidin, so the content of pyrazines detected in YQ was lower than that in WQ and BQ. There was a big difference between WQ and BQ in the proportion of pyrolysis products. The melanoidin components in WQ were mainly composed of p-Cresol (8.45%), Acetamide (5.74%), Furfural (5.48%), Indole (5.18%), hexahydro-Pyrrolo[1,2-a]pyrazine-1,4-dione (4.54%), 5,10-Diethoxy-2,3,7,8-tetrahydro-1H,6H-dipyrrolo[1,2-a:1′,2′-d]pyrazine (4.20%), Phenol (4.08%), 3,5-dihydroxy-2-methyl-4H-Pyran-4-one (3.97%), and 5-methyl-2-Furancarboxaldehyde (3.95%), which were mainly furans, phenols, pyrrolopyrazines, etc. The detection peak areas of Fural, 5-Methyl-2-furancarboxaldehyde, and 2-Furanmanol in WQ were higher than those of YQ and BQ, which indicated that furans in WQ were further involved in the formation of melanoidin, making fewer types and concentration of furans detected in WQ. However, more furans were produced in the fermentation process of YQ and BQ, and even if they participated in the production of melanoidin, more furans could still be detected from these two kinds of Daqu. The Maillard reaction degree of the three kinds of Daqu was different in the fermentation process, and BQ was the most complicated. The composition of amino acids in the three kinds of Daqu was different, which led to the differences in the types and contents of intermediate products such as heterocyclic substances, and finally made huge differences in melanoidin structure between the three kinds of Daqu (Figure 4).

3.4. Composition of the Microbial Community in Three Kinds of Daqu

There were mainly 139 bacterial genera and 22 fungal genera detected from three kinds of Daqu. Most of the bacterial genera belong to Firmicutes, Actinobacteria, and Proteobacteria, and the fungal genera mainly belong to Ascomycota and Mucoromycota. The top 10 dominant bacterial genera and the top 10 dominant fungi genera were shown in (Figure 5a,b). The results showed that the sum of the relative abundance (RA) of the top 10 dominant bacterial genera in YQ, WQ, and BQ was greater than 89.14%, 90.58%, and 98.47%, respectively. The main dominant bacterial genera in YQ (Figure 5a) were Kroppenstedtia (37.32%), Saccharopolyspora (12.52%), Virgibacillus (8.92%), Scopulibacillus (8.63%) and Cupriavidus (8.33%). Kroppenstedtia (55.84%), uncultured_Bacillaceae (13.91%), and Bacillus (13.62%) were the main dominant genera in WQ while the dominant bacterial genera in BQ were Kroppenstedtia (82.88%), Saccharopolyspora (8.80%), Pseudonocardia (1.97%), and Bacillus (1.24%) (Figure 5a).
The sum of the RA of the top 10 dominant fungal genera in YQ, WQ, and BQ was more than 99%. The main dominant fungal genera in YQ (Figure 5b) were Thermomyces (48.71%), Thermoascus (34.04%), Aspergillus (11.13%), and Byssochlamys (5.09%). Thermomyces (89.13%), Thermoascus (3.36%), Hyphopichia (1.72%), Byssochlamys (1.44%), and Rhizopus (1.10%) were dominant fungi in WQ (Figure 5b). The dominant fungi in BQ (Figure 5b) were Thermoascus (49.92%), Thermomyces (42.00%), Monascus (4.92%), and Byssochlamys (2.12%). According to the microbial community composition, there were many kinds of high-temperature-resistant microorganisms (Kroppenstedtia, Saccharopolyspora, Thermoascus, Thermomyces and Aspergillus) in YQ and BQ, and the previous research [26] showed that Saccharopolyspora is positively correlated with the temperature of Daqu, so it is speculated that the temperature of YQ and BQ is higher during fermentation, which intensifies the Maillard reaction and produces more melanoidin, making the appearance of BQ and YQ darker. Meanwhile, the Maillard reaction also provided more flavor substances for high-temperature Daqu.
According to PLS-DA analysis (Figure 5c), the microbial communities of the three kinds of Daqu were significantly different, and the differential microorganisms in the three kinds of Daqu were analyzed by calculating the VIP value. It can be seen that the RA of Virgibacillus, Cupriavidus, Staphylococcus, and Aspergillus in YQ were significantly higher than those in WQ and BQ, and they were the biomarkers in YQ (Figure 5d, VIP > 1). Bacillus, Thermomyces, and Hyphopichia were the biomarkers in WQ (Figure 5d, VIP > 1). Bacillus is an important source of Tetramethylpyrazine in Daqu [27], which is presumably the reason for the high content of Tetramethylpyrazine in WQ. The RA of Kroppenstedtia, Monascus, and Thermoascus in BQ was significantly higher than that in WQ and YQ, which were the marked microorganisms in BQ. It has been reported [28] that Thermoascus, Virgibacillus, and Aspergillus are differential functional microorganisms of three kinds of Daqu, which can be used as marker microorganisms to distinguish the three kinds of Daqu. Kroppenstedtia, Monascus, and Thermoascus, the dominant genera in BQ, were positively correlated with Maillard reaction products. Kroppenstedtia was positively correlated with 3-Phenylfuran, Tetramethylpyrazine, 2,3-Dimethylpyrazine, Phenol, etc. Monascus was positively correlated with Melanoidin, 3-Phenylfuran, Phenol, 3-Isopropyl-2,5-dimethylpyrazine, 4-Ethyl-2-methoxyphenol, and Tetramethylpyrazine. Thermoascus was positively correlated with Melanoidin, 2-Methoxyphenol, 3-Isopropyl-2,5-dimethylpyrazine, 2-Ethyl-6-methylpyrazine, 3-Phenylfuran and Phenol. According to the previous research [29], Aspergillus [30], Monascus [31,32], Byssochlamys, Thermomyces [33], Thermoascus [34], Saccharopolyspora [35,36], and Kroppenstedtia are important functional microorganisms in sauce-flavor Daqu. Although Maillard reaction does not require the participation of microorganisms, these functional microorganisms in BQ, YQ, and WQ participated in the degradation of macromolecular carbohydrates and proteins during Daqu fermentation, which provided a large number of precursors for the Maillard reaction. The functional microorganisms occupied an absolute advantage in the microbial community of BQ, especially Monascus, Thermoascus and Kroppenstedtia, which were also highly positively correlated with heterocyclic substances and melanoidin and were the key factors for the formation of BQ. The degradation of raw materials in Daqu fermentation can be enhanced by strengthening biomarkers of BQ, which can provide a large number of precursors for the Maillard reaction and promote the formation of BQ.

4. Conclusions

Maillard reaction is one of the most important factors resulting in significant differences among BQ, WQ, and YQ. Our research analyzed the composition differences of Maillard reaction products in three types of Daqu. The content and composition of heterocyclic compounds and Melanoidin in BQ were more complicated than YQ and WQ, which endowed BQ with a stronger flavor and deeper color. By strengthening the degradation of raw materials in the fermentation process of Daqu, the Maillard reaction can be promoted, which is ultimately beneficial to the formation of BQ. However, the environmental factors affecting Maillard reaction in the fermentation process of three types of Daqu are still not clear, which needs further study.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/fermentation9090860/s1, Table S1: Contents of volatile substances in three kinds of Sauce-flavor Daqu; Table S2: Proportion of melanoidin pyrolysis compounds in the three types of Daqu; Excel sheet1: The ASVs ID, taxonomy, and sequences of 16S rRNA; Excel sheet2: The ASVs ID and taxonomy of ITS rRNA.

Author Contributions

Conceptualization, data curation, formal analysis, methodology, software, writing—original draft, Q.Z.; data curation, formal analysis, writing—original draft, investigation, methodology, resources, validation, L.C.; validation, methodology, X.P.; visualization, writing—review and editing, G.D.; methodology, resources, project administration, J.L. and F.Y.; conceptualization, methodology, funding acquisition, supervision, writing—review and editing, Z.P., J.Z. and H.T. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Key Research and Development Program of China (2019YFC1605800), a grant from the Key Technologies R & D Program of Jiangsu Province (BE2021624).

Institutional Review Board Statement

Ethics approval was not required for this research.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data that support the findings of this study are available in the Supporting Information of this article.

Conflicts of Interest

Authors L.C., X.P., F.Y., J.L. and H.T. were employed by Kweichow Moutai Distillery Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Fermentation 09 00860 g001
Figure 1. Contents of melanoidin (a), reducing sugar (b) and free amino acids (c) in three types of Daqu.
Figure 1. Contents of melanoidin (a), reducing sugar (b) and free amino acids (c) in three types of Daqu.
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Fermentation 09 00860 g002
Figure 2. Analysis of differences in volatile components between three types of Daqu (a), R2X (cum) = 0.978, R2Y (cum) = 0.996, Q2 (cum) = 0.980; the proportion of volatile substances (b); the heatmap of heterocyclic substances in BQ, WQ, and YQ (c).
Figure 2. Analysis of differences in volatile components between three types of Daqu (a), R2X (cum) = 0.978, R2Y (cum) = 0.996, Q2 (cum) = 0.980; the proportion of volatile substances (b); the heatmap of heterocyclic substances in BQ, WQ, and YQ (c).
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Fermentation 09 00860 g003
Figure 3. VENN diagram of melanoidin pyrolysis products (a); analysis of difference in composition of pyrolysis products between three types of Daqu based on PCA (b); the heatmap of melanoidin pyrolysis products in BQ, WQ, and YQ (c).
Figure 3. VENN diagram of melanoidin pyrolysis products (a); analysis of difference in composition of pyrolysis products between three types of Daqu based on PCA (b); the heatmap of melanoidin pyrolysis products in BQ, WQ, and YQ (c).
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Fermentation 09 00860 g004
Figure 4. Analysis of melanoidin formation pathway in three kinds of Daqu.
Figure 4. Analysis of melanoidin formation pathway in three kinds of Daqu.
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Fermentation 09 00860 g005
Figure 5. Dominant (top10) bacterial genera (a) and fungal genera (b) in three kinds of Daqu; microbial community difference analysis based on PLS-DA (c), R2X (cum) = 0.639, R2Y (cum) = 0.982, Q2 (cum) = 0.937; microbial genera with a VIP value greater than 1 are defined as microbial markers (d) in three kinds of Daqu; Spearman correlation analysis between marked microorganisms and Maillard products in BQ (e), the blue represents the positive correlation (ρ > 0.6), while the red represents the negative correlation (|ρ| > 0.6, ρ < 0), * p < 0.05.
Figure 5. Dominant (top10) bacterial genera (a) and fungal genera (b) in three kinds of Daqu; microbial community difference analysis based on PLS-DA (c), R2X (cum) = 0.639, R2Y (cum) = 0.982, Q2 (cum) = 0.937; microbial genera with a VIP value greater than 1 are defined as microbial markers (d) in three kinds of Daqu; Spearman correlation analysis between marked microorganisms and Maillard products in BQ (e), the blue represents the positive correlation (ρ > 0.6), while the red represents the negative correlation (|ρ| > 0.6, ρ < 0), * p < 0.05.
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Table 1. Mobile phases and gradient condition.
Table 1. Mobile phases and gradient condition.
Time (min)Mobile Phase A (1 L, 5 g Sodium Acetate, 200 μL Triethylamine, 5% Acetic Acid, pH 7.2)Mobile Phase B (1 L, 5 g Sodium Acetate, 20% Water, 40% Acetonitrile, 40% Methanol, pH 7.2)Flow Rate (mL/min)
0.009281.00
27.0040601.00
31.5001001.00
32.0001001.00
34.0001001.00
35.509281.00
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Zhu, Q.; Chen, L.; Pu, X.; Du, G.; Yang, F.; Lu, J.; Peng, Z.; Zhang, J.; Tu, H. The Differences in the Composition of Maillard Components between Three Kinds of Sauce-Flavor Daqu. Fermentation 2023, 9, 860. https://doi.org/10.3390/fermentation9090860

AMA Style

Zhu Q, Chen L, Pu X, Du G, Yang F, Lu J, Peng Z, Zhang J, Tu H. The Differences in the Composition of Maillard Components between Three Kinds of Sauce-Flavor Daqu. Fermentation. 2023; 9(9):860. https://doi.org/10.3390/fermentation9090860

Chicago/Turabian Style

Zhu, Qi, Liangqiang Chen, Xiuxin Pu, Guocheng Du, Fan Yang, Jianjun Lu, Zheng Peng, Juan Zhang, and Huabin Tu. 2023. "The Differences in the Composition of Maillard Components between Three Kinds of Sauce-Flavor Daqu" Fermentation 9, no. 9: 860. https://doi.org/10.3390/fermentation9090860

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