Differences in Physicochemical properties and flavor components among different layers of medium-temperature Daqu

doi:10.21203/rs.3.rs-2863429/v1

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Differences in Physicochemical properties and flavor components among different layers of medium-temperature Daqu

https://doi.org/10.21203/rs.3.rs-2863429/v1

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Quality differences among three layers of Daqu were comprehensively studied by analyzing physicochemical indices and main enzyme system and using headspace solid-phase microextraction gas chromatography-mass spectrometry. The water content, fermentation ability and esterifying enzyme activity of Daqu among different layers all rank in the order of core > fire circle > surface, and the sulyase activity ranks as surface > fire circle > core, all showing significant differences. The acidity of Daqu surface is significantly higher and the amino acid nitrogen content is significantly lower compared with the fire circle and core. The starch content of the core is significantly higher and the cellulase activity is significantly lower compared with the surface and fire circle. The volatile flavor component analysis shows the Daqu contains 73 volatile substances, including alcohols, esters and phenols. Especially, the contents of methyl myristate, methyl hexadecanoate, ethyl hexadecanoate, caproic acid, octanoic acid, n-palmitic acid, 2,4,-(di)phenol, n-hexyl alcohol, tetramethylpyrazine, and phenylethanol are relatively high and all differ significantly among different layers of Daqu. The fire circle contains the most diverse volatile flavor components, including 39 types. Principal component analysis demonstrates that the volatile flavor components are largely different among different layers of Daqu.

different layers of Daqu

physicochemical indices

hydrolase

volatile flavor components

Taorong-type

Daqu, a saccharification yeast of liquors (Zheng et al., 2011), contains complex microbial communities and key enzyme systems and is an important source of fermentation and fragrant substances or precursors. Hence, Daqu decides the flavor properties of liquors (Li et al., 2015). During the fermentation with Daqu, it can be divided by the highest product temperature into low-temperature Daqu (≤ 50ºC), medium-temperature Daqu (50–60ºC) and high-temperature Daqu (≥ 60ºC). Endogenous biological heat is the main driving force of Daqu to form microbial communities (Xiao et al., 2017). The Taorong-type medium-temperature Daqu is produced in Yangshao by natural inoculation and indoor cultured fermentation and is exclusive for making Taorong-type baijiu. The quality level of this Daqu directly decides the style and quality of Taorong-type liquors. As a bulky starter, the quality of Daqu differs among different layers owing to microenvironmental differences in ventilation, moisture and temperature (Ming et al., 2015).

There is already research on different layers of Daqu. Ming et al. (2015) compared the physicochemical and biochemical properties of Luzhou-flavor Daqu and found the acidity and ammonia nitrogen content both differed significantly between the surface and core. The Daqu surface quality was maximally affected by starch conversion rate and esterifying ability, and the Daqu core quality was maximally impacted by the esterifying ability and acidity (Ming et al., 2015). Comparison of comprehensive scores of principal components demonstrated that the comprehensive score of Daqu core quality was significantly higher than that of Daqu surface quality (Ming et al., 2015). Yang et al. (2019) analyzed the biochemical indicators at different layers of medium-high temperature Daqu during fermentation and studied the differences and changing rules among different layers. The esterifying ability was maximized in the core, and the microbial quantity and activities of other enzymes were maximized on the surface, while water content and acidity were maximized in the fire circle and other indices were in between those of the core and the surface (Yang et al., 2019). In the primary fermentation period, the fermentation ability rapidly rose, the sulyase activity at the core was rapidly decreased, the acidity increased fast, and moisture at the surface rapidly dissipated (Yang et al., 2019). At the humidity removal phase, the fermentation ability rapidly dropped, the esterification ability and acidic protease activity were enhanced, the acidity was gentle, and the surface moisture content further declined (Yang et al., 2019). At the high-temperature phase, the enzymatic activity and microbial quantity both decreased, and the moisture content and acidity at the core started to drop (Yang et al., 2019). At the terminal high-temperature phase, the esterification ability, acidic protease activity, and the core sulyase activity recovered (Yang et al., 2019). At the cultivation phase, the sulyase activity was further enhanced, the esterifying ability and acidic protease activity slowly declined, and the moisture content at the core further decreased (Yang et al., 2019). Zhou et al. (2019) tested the physicochemical indicators and main enzymatic system dynamics at different layers of site-flavor Daqu pieces during fermentation and found the internal water contents of Daqu increased outside-in. The activities of acidic protease, sulyase, cellulase and amylase differed among different layers of Daqu, but generally were higher in the outer layer than in the fire circle layer and core (Zhou et al., 2019).

In all, the existing studies on quality differences among different layers of Daqu pieces focus mostly on single physicochemical and biochemical indicators, but rarely on the quality at different layers by combining main enzymatic systems and volatile flavor composition. The current studies on Daqu quality focus on Fen-flavor liquor Daqu (Fan et al., 2018; Liu et al., 2019; Hu et al., 2021), Maotai-flavor liquor Daqu (Hao et al., 2021; Li et al., 2021) and Luzhou-flavor liquor Daqu (Sun et al., 2016; Qian et al., 2021). Since the quality of Taorong-typer Daqu has been rarely studied, the differences among various parts of Taorong-type Daqu shall be comprehensively investigated. Herein, the physicochemical indices, main hydrolase system and volatile flavor components at different layers of Yangshao Taorong-type medium-temperature Daqu were comparatively analyzed. The differences in quality at different layers of medium-temperature Daqu were studied. Thereby, the quality of Daqu was comprehensively uncovered to offer a theoretical basis for optimizing the making of Taorong-type liquor fermentation Daqu.

Materials and reagents

Specimen: Taorong-type medium-temperature Daqu was provided by Henan Yangshao Distillery Co., Ltd.

Reagents: NaOH, glucose, anhydrous ethanol (Tianjin Kemiou Chemical Reagent Co.), HCl, H₂SO₄ (Tianjin Yongda Chemical Reagent Co., Ltd.), formaldehyde (Tianjin Sheng'ao Chemical Reagent Co., Ltd.), iodine, KI (Xilong Chemical Co. Ltd.), copper sulfate pentahydrate and potassium sodium tartrate (Chengdu Jinshan Chemical Reagent Co., Ltd.) were all analytically pure. Other reagents included caproic acid (Shanghai Jingchun Biochemical Technology Co., Ltd.), liquification amylase (α-amylase, Nanjing Jiancheng Biology Co., Ltd.), soluble starch (Tianjin Yongcheng Fine Chemicals Co., Ltd.), and 2-octanol (chromatographic, Chengdu Preferred Biotechnology Co., Ltd.).

Instruments

An SW-CJ-2F double two-sided purification workbench (Suzhou Purification Equipment Co. Ltd.), a PHS-25 precision pH meter (Shanghai Lei-ci Co., Ltd.), an HH-6 digital-display thermostat waterbath (Changzhou Fangke Instrument Co. Ltd.), a DNP-9272BS-Ⅲ electro-heating thermostatic cultivator (Shanghai CIMO Medical Instrument Manufacturing Co. Ltd, China), and an LDZX-50KB upright steam sterilization pot (Shanghai Shenan Medical Instrument) were used here. Other instruments included an A590 ultraviolet spectrophotometer (Shanghai Aoyi Instrument Co. Ltd.), fermentation bolts (Xuzhou Dahua Glass Products Co., Ltd.), an FA1104 analytical balance (0.0001 g, Shanghai Shunyu Hengping Scientific Instruments Co., Ltd.), and a GCMS-QP2010Ultra gas chromatograph mass spectrometer (GC-MS, Shimadzu, Japan).

Experimental methods

Liquor Sampling

The Daqu studied here was brick-shaped (27×17×7–8 cm³) and collected at three layers (outer, middle, inner). The 1-cm-thick layer outside-in was defined as the surface layer (surface). The layer between the surface and the core was dark and set as the middle layer (fire circle). The layer from the middle layer by 2 cm to the center was considered as the inner layer (core). Samples collected from each of the three layers were crushed, uniformly mixed and sieved. Then 200 g of samples from each layer was weighed and stored in a freezer at 4ºC (Zhou et al., 2019).

Detection of physicochemical indices and hydrolase system of Daqu

Water, starch, fermentation ability, sulyase activity, and esterifying ability

The water content, starch content, fermentation ability, sulyase activity, and esterifying ability of Daqu were detected according to industry standard Liquor-making Daqu General Analytical Methods (QB/T 4527 − 2011).

Acidity

The acidity of Daqu was detected according to Liquor-Making Analysis and Detection (Wang, 2012) and 1 degree of acidity (mmol/10 g) was defined as the millimoles of NaOH consumed by 10 g of materials during titration.

Cellulase activity

Cellulase activity was detected using the 3,5-dinitrosalicylic acid (DNS) method according to Cellulase Preparation (QB 2583 − 2003). The rationale is that cellulase under certain temperature and pH hydrolyzes the substrate (cellulose or filter paper) to release reducing sugars. Under the alkali and boiling condition, DNS and the reducing sugars chromogenically react, and the color intensity of the reaction system is proportional to the concentration of reducing sugars (calculated as glucose). The concentration of reducing sugars can be determined by detecting the absorbance at 540 nm and thereby the filter paper enzyme activity can be computed. This can be used to express the cellulase activity. Definition of cellulase activity: 1 unit of enzymatic activity (U/g) is defined as 1 g of solid enzyme (or 1 mL of liquid enzyme) hydrolyzes the substrate (sodium carboxymethyl cellulose) for 1 h at 50 ± 0.1ºC and certain pH (pH 4.8 for acidic cellulase, pH 6.0 for neutral cellulase), and produces the amount of reducing sugars that is equal to 1 mg of glucose.

Volatile flavor components of Daqu

Sample pretreatment: the Daqu samples from section 1.3.1 were passed through a 40-mesh screen and then mixed uniformly. Then 2 g of Daqu was put into a 20 mL headspace bottle, which was then added with 30 µL of a 12.60 mg/L 2-octanol internal standard solution (mass concentration), 5 mL of purified water and 2 g of NaCl. After balancing at 60ºC for 20 min and headspace adsorption for 30 min, the resulting solution was desorbed at 250ºC for 5 min and sent to GC-MS (Zhang, 2012).

Qualitative analysis: The unknown compounds were searched on the computer and simultaneously recognized by comparing with the standard spectrum pool. Only the components with matching degree > 90% were reserved.

Semi-quantitative analysis: With 2-octanol as the internal standard, the concentrations of volatile flavor substances identified in the Daqu samples were calculated as per some reported methods (Zhang et al., 2011).

Data processing

Data were analyzed, plotted and statistically analyzed on Excel 2010, SPSS 20.0 and Origin 2020. Also standard deviations were computed. The significance level was 5%.

Physicochemical indices at different layers of Daqu

Water content

The water content of Daqu maximizes to 11.62% in the core and minimizes to 10.56% on the surface, showing significant differences (P < 0.05) (Fig. 1A). In the core which is the innermost layer, microbes grow slowly, moisture is not easily evaporated or consumed, and the relative humidity is higher than in the other two layers. On the surface which is the outmost layer of Daqu, moisture evaporates easily and microbes grow and metabolize severely, which causes large water consumption, so the relative humidity is lower than in the other two layers. The overall water content of Daqu is 10.97%. According to the provisions in the Daqu making shop, the delivery inspection standard states that the water content of Daqu finished products shall be less than 13.00% of total product weight, which meets national standards of China.

Acidity

The measured data of acidity at different layers of Daqu are shown in Fig. 1B. The acidity on the surface is significantly higher than in the fire circle, core and the mixed sample (P < 0.05), but the acidity is not significantly different among the fire circle, core and mixed sample (P > 0.05). During the Daqu making process, the substrate including starch is converted by lactic acid bacteria, Acetobacter aceti and other microbes to organic acids such as lactic acid and acetic acid, which become the main material basis for the acidity of Daqu. Hence, the acidity of Daqu is maximized on the surface (0.79 mmol/10 g), which may be because the surface contains abundant microbes that have strong acid-producing ability.

Starch content

The measured data of starch content at different layers of Daqu are shown Fig. 1C. The starch content in the core is significantly higher than on the surface and the fire circle (P < 0.05), but the starch contents are not significantly different between the surface and the fire circle (P > 0.05). The starch content of Daqu minimizes to 52.32 mg/g on the surface. The starch content of Daqu represents the level of starch consumption by microbes during yeast starter production. A higher starch consumption rate indicates the propagation and metabolism of microbial populations are more active and the metabolites are richer, suggesting the microbial metabolism on the surface is the most active. The starch content of Daqu maximizes to 56.34 mg/g in the core, indicating microbial growth and metabolism in the core are slow and the starch utilization rate is the lowest.

Amino acid nitrogen content

The measured data of amino acid nitrogen content at different layers of Daqu are shown in Fig. 1D. The amino acid nitrogen content on the surface (1.98 g/kg) is significantly lower than in the fire circle, core and the mixed sample (P < 0.05). The amino acid nitrogen contents are not significantly different between the fire circle and the core (P > 0.05). These results suggest the protease activity on the surface in the weakest. The amino acid nitrogen content in the core is the highest (2.95 g/kg). This is because Bacillus, the major functional microbe that secretes protease (Xie et al., 2020), can grow well at high temperature because of low air permeability, high water content and high temperature in the core, leading to a significantly higher amino acid nitrogen content inside Daqu than on the surface.

Hydrolase system analysis at different layers of Daqu

Sulyase activity

The measured data of sulyase activity at different layers of Daqu are shown in Fig. 2A. The sulyase activity of Daqu is maximized to 690.90 U/g on the surface, which is significantly higher than in the fire circle, core and mixed sample (P < 0.05). The sulyase activity reflects the ability of converting starch to sugar, and a higher sulyase activity indicates a higher starch utilization rate, which directly affects the distillation yield of liquors (Gao et al., 2021). The sulyase activity is minimized to 427.95 U/g in the core, which is consistent with the highest starch content in the core.

Fermentation force

The results of fermentation ability at different layers of Daqu are shown in Fig. 2B. The fermentation ability of Daqu is maximized to 0.41 g/(0.5 g·72 h) in the core, which is significantly higher than those of the surface, fire circle, and mixed sample (P < 0.05). The fermentation ability reflects the liquor-making ability of Daqu and indicates the ability of saccharomycetes to convert surgar to liquors. The fermentation ability in the core is high, which may be because the environment in the core is suitable for the growth and metabolism of saccharomycetes. Our results are consistent with a previous study that the fermentation ability is significantly and positively correlated with the moisture content, starch content, yeast content and Daqu surface bacterium count (Yang et al., 2019). The fermentation ability of the mixed sample is 0.38 g/(0.5 g·72 h), which indicates the overall fermentation ability of Daqu is low. The possible reason is that the growth and metabolism of saccharomycetes are lowered by the storage time and storage temperature.

Esterification activity

The results of esterification activity at different layers of Daqu are shown in Fig. 2C. The esterifying activity of Daqu is maximized to 616.71 mg/(50 g·7 d) in the core and is minimized to 242.82 mg/(50 g·7 d) on the surface. The esterifying activity in the core is significantly higher compared with the surface, fire circle and mixed sample (P < 0.05). Rhizopus and Monascus purpureus which possess strong ester synthesizing ability mainly exist on the sections and the middle, so the esterifying ability on the surface is lower than in the fire circle and the core. The ester compounds of Daqu are closely related to flavor formation, and the esterifying ability of Daqu is the power to catalytically produce esters. A higher esterifying ability indicates more esters are produced by Daqu during fermentation, which promotes the flavor formation of liquors (Xu et al., 2022). Our results suggest the core offers the main driving force to promote the formation of flavor substances in liquors.

Cellulase activity

The results of cellulase activity at different layers of Daqu measured using DNS are shown in Fig. 2D. The cellulase activity of different layers ranks as surface > fire circle > core, which is 11.907, 11.906 and 11.891 U/g respectively (Fig. 2D). The cellulase activity of the core is significantly lower than in the fire circle and the surface (P < 0.05), but the cellulase activity is not significantly different between the surface and the fire circle (P > 0.05). Cellulase can degrade cellulose and destroy the structure of mesenchymal cell walls, thereby releasing the starch that is contained in the walls, which is favorable for saccharification (Zhou et al., 2021; Li et al., 2022) and indicate the cellulase activity is positively correlated with the sulyase activity. Results show the sulyase activity is the highest on the surface, which is consistent with the highest cellulase activity on the surface.

Difference analysis of characteristic flavor substances at different layers of Daqu

With headspace solid-phase microextraction (HS-SPME) GC-MS and the findings of flavor substances reported before (Wu et al., 2009; Zhang et al., 2011; Yang et al., 2021), we qualitatively recognized 73 types of volatile substances at different layers of Taorong-flavor medium-temperature Daqu, including alcohols, esters, aldehydes, ketones, pyrazines and phenols. There are 34 types of esters, 5 types of aldehydes/ketones, 11 types of alcohols, 9 types of acids, 3 types of phenols, 9 types of alkyls/alkenes, and 2 types of pyrazines. The diversity and total content of flavor substances in the fire circle are both large, and the total ion current of volatile components is shown in Fig. 3. The components and contents of volatile flavor substances at different layers of Daqu are listed in Table 1. Then the differences in the contents of characteristic flavor substances were compared using one-factor analysis of variance among different layers of Daqu (Table 1).

Table 1

volatile flavor components and contents in different parts of Daqu
characteristic flavor substances		content/(g/ng)
characteristic flavor substances		surface	fire circle	core	mixed sample
Phenols	phenol	2877.61 ± 72.79^b	3424.35 ± 260.44^a	-	-
	2-Methoxy-4-vinylphenol	-	-	251.69 ± 12.58^b	517.46 ± 6.04^a
	2,4,-(Bis)phenol	148.45 ± 13.50^b	121.17 ± 18.53^c	291.69 ± 3.58^a	163.42 ± 3.63^b
Alcohols	Alpha-terpineol	2700.55 ± 23.50^a	97.43 ± 0.89^b	-	-
	1-Pentanol	-	-	144.11 ± 3.84	-
	1-Hexanol	151.77 ± 25.41^c	77.18 ± 3.19^d	843.19 ± 8.61^a	647.64 ± 3.62^b
	1-Octanol	335.91 ± 24.71^a	-	-	52.26 ± 1.62^b
	1-Nonanol	-	-	147.49 ± 4.14^a	87.06 ± 1.72^b
	3,7-Dimethyl-3-octanol	-	-	-	37.99 ± 0.42
	1-Methoxy-2-propanol	-	-	-	184.83 ± 4.48
	2,3-Butanediol	-	-	-	172.44 ± 4.03
	3-Methyl-1-butanol	-	-	228.23 ± 7.79	-
	Benzyl alcohol	-	75.96 ± 2.87^a	-	68.16 ± 1.28^b
	Phenylethanol	723.82 ± 43.47^c	631.24 ± 12.22^d	1178.79 ± 44.93^a	949.09 ± 18.92^b
Esters	(Z)-Methyl 9-hexadecenoate	90.58 ± 3.27^a	68.87 ± 0.43^b	-	-
	Ethyl 9-hexadecenoate	-	26.44 ± 1.61	-	-
	Methyl 10-octadecenoate	-	34.82 ± 1.46	-	-
	(Z,Z)-Methyl 9,12-octadecadienoate	-	-	1404.84 ± 48.68	-
	(Z,Z,Z)-Methyl 9,12,15-octadecatrienoate	69.84 ± 4.99^a	69.84 ± 1.76^a	73.11 ± 1.70^a	-
	methyl tetradecanoate	223.53 ± 23.35^a	185.91 ± 8.47^b	68.90 ± 1.18^d	99.78 ± 3.74^c
	Ethyl myristate	173.01 ± 18.36^b	169.30 ± 9.01^b	283.27 ± 10.64^a	-
	methyl pentadecanoate	170.60 ± 13.31	-	-	-
	Ethyl pentadecanoate	64.06 ± 2.55^a	-	61.65 ± 1.03^a	-
	methyl hexadecanoate	1855.06 ± 78.21^b	2207.20 ± 119.11^a	1078.41 ± 55.08^c	1217.27 ± 19.65^c
	Ethyl hexadecanoate	1688.33 ± 56.04^a	845.67 ± 34.00^b	141.20 ± 4.69^c	860.41 ± 9.22^b
	Ethyl heptadecanoate	-	-	1848.90 ± 44.55	-
	Cetyl 2-ethylhexanoate	-	-	210.18 ± 5.70^a	49.88 ± 2.56^b
	(E)-Methyl 9-octadecenoate	1769.17 ± 101.96^a	1675.80 ± 147.36^a	-	-
	(E)-ethyl 9-octadecenoate	1390.83 ± 231.98^a	762.33 ± 17.56^b	248.72 ± 4.27^c	78.72 ± 0.54^c
	Methyl 11-octadecenoate	-	-	535.35 ± 7.18^b	737.37 ± 9.35^a
	(E)-2-heptenoic acid ethyl ester	-	1767.06 ± 59.72^a	-	68.12 ± 0.47^b
	Octyl acetate	89.19 ± 2.21	-	-	-
	methyl caproate	-	320.05 ± 5.69^b	-	381.39 ± 2.98^a
	Ethyl caproate	-	267.46 ± 13.26	-	-
	Hexyl caproate	-	-	144.75 ± 3.55	-
	Octyl caproate	-	-	245.13 ± 6.89	-
	Methyl octanoate	-	111.65 ± 11.12	-	-
	Ethyl octanoate	-	74.64 ± 1.71	-	-
	methyl pelargonate	-	91.81 ± 1.75	-	-
	Ethyl pelargonate	-	28.07 ± 1.99	-	-
	Methyl 2-formamidobenzoate	-	-	-	76.21 ± 2.85
	methyl phenylacetate	-	328.40 ± 19.76	-	-
	Ethyl phenylacetate	-	151.52 ± 20.90	-	-
	Ethyl phenylpropionate	-	-	72.18 ± 1.71	-
	methyl stearate	-	91.63 ± 3.13	-	-
	Diethyl phthalate	1316.83 ± 163.26	-	-	-
	Isopropyl Palmitate	-	-	28.28 ± 3.39	-
	Ethyl oleate	-	90.09 ± 0.71	-	-
	caproic acid	761.25 ± 19.84^b	373.93 ± 7.44^c	827.42 ± 9.40^a	143.14 ± 2.47^d
	bitter	75.90 ± 1.60^b	82.54 ± 2.78^b	151.24 ± 6.61^a	62.81 ± 1.69^c
	Heptanoic acid	-	-	-	547.13 ± 9.41
	Oleic acid	-	264.72 ± 10.24	-	-
Acids	3-Methyl-butyric acid	-	303.37 ± 5.06^b	208.35 ± 4.65^a	213.94 ± 3.89^b
	n-hexadecanoic acid	1129.25 ± 99.65^c	1655.98 ± 92.74^a	1385.45 ± 35.72^b	1250.20 ± 30.28^bc
	octadecanoic acid	-	760.75 ± 16.69	-	-
	(Z,Z,Z)-8,11,14-eicosatrienoic acid	-	-	-	56.35 ± 3.06
	(Z,Z)-9,12-octadecadienoic acid	-	746.85 ± 10.42	-	-
Alkenes	Caryophyllene	-	73.39 ± 1.29^b	-	168.58 ± 2.98^a
	Caryophyllene oxide	-	-	-	52.16 ± 1.11
	tetradecane	-	-	-	1132.74 ± 49.48
	docosane	-	-	3711.21 ± 59.44	-
	n-tetracosane	-	-	4333.86 ± 121.30	-
	tetradodecane	-	71.23 ± 2.49^b	-	241.50 ± 8.86^a
	Pentadodecane	-	-	149.53 ± 3.86^b	1866.91 ± 21.64^a
	2-Methyleicosane	-	-	3318.45 ± 122.25	-
	Nonanediane	-	-	3031.01 ± 44.45	-
Pyrazines	tetramethylpyrazine	458.05 ± 6.85^b	281.44 ± 10.15^c	766.92 ± 20.57^a	759.80 ± 23.84^a
Pyrazines	Trimethylpyrazine	-	-	198.26 ± 6.55^b	245.90 ± 16.52^a
Aldehydes and ketones	Valeraldehyde	-	243.31 ± 8.72	-	-
	benzaldehyde	-	-	-	70.52 ± 1.73
	4-Hydroxy-3-methylacetophenone	703.42 ± 11.47	-	-	-
	2-(1-Methylethylene)cyclohexanone	710.88 ± 19.69	-	-	-
	6,10,14-Trimethyl-2-pentadecanone	114.47 ± 10.04^b	128.68 ± 4.91^a	-	-
Note: The date were presented as mean ± SD, -: undetected. The different letters in the same row indicate significant differences (P < 0.05).

The contents of total volatiles and volatile substances are both different among different layers of Daqu (Table 1). The contents of methyl myristate, methyl hexadecanoate, ethyl hexadecanoate, caproic acid, octanoic acid, n-palmitic acid, 2,4,-(di)phenol, n-hexyl alcohol, tetramethylpyrazine, and phenylethanol are high and significantly different among different layers (P < 0.05). The most diverse volatile flavor components were detected in the fire circle, including 39 types. The least diverse volatile flavor components were detected on the surface, including 25 types. Among all types of flavor substances, the proportion of esters is the highest (46.58%), followed by alcohols (15.07%), and the proportion of pyrazines is the lowest (2.74%). Analysis shows that among all esters, the difference of hexadecanoate contents is the largest (1547.13 ng/g), and the hexadecanoate contents are significantly different among the surface, fire circle and core.

Esters often present the pleasant fragrant aroma from fruits, and play a critical role in the formation of special flavor and style of liquors (Niu et al., 2020). The aldehydes and ketones in Daqu can be produced by microbial degradation of starch, a raw material of Daqu, during glycolysis especially under the action of yeasts. These aldehydes and ketones together with esters constitute the unique fragrance of Daqu, and ketones and aldehydes can also enter liquors through fermentation, and are involved in liquor aroma formation in the form of precursors of microbial metabolism. Alcohols are the material basis of esters, which together with aromatic compounds constitute the unique fragrance of Daqu. Alcohols are mainly produced from dextrose fermentation by yeasts under aerobic conditions, but are produced from amino acids under anaerobic conditions or from reduction reaction of relevant aldehydes by yeasts (Fan et al., 2011). The style of liquors is decided by whether the alcohol concentrations are consistent with the proportion of other compounds (Ma et al., 2010). Among the alcohols detected in Daqu, the n-hexanol concentrations are significantly different among the three parts of Daqu, and are the highest in the core (843.19 ng/g). n-Hexanol endows liquors with a strong, elegant and fragrant flavor and plays a key role in the aroma of liquors (Fan et al., 2015). Phenylethanol has a cookie and rose aroma, and its concentration is significantly different among different layers of Daqu, with a maximum of 1178.79 ng/g in the core. Noticeably, tetramethylpyrazine detected in Daqu is a N-containing heterocyclic compound and its concentration is significantly different among different layers of Daqu, with a maximum of 766.92 ng/g in the core. Tetramethylpyrazine has a baking and nut fragrance. This compound can also produce special fragrance of liquors and has some medical effects, such as vascular dilation, suppression of thrombocyte conglutination and thrombosis, and beneficial pharmacological effects (Wu et al., 2012).

The relative contents of volatile compounds in different layers of Daqu are shown in Fig. 4. Clearly, the volatile flavor components differ among different layers of Daqu (Fig. 4). The relative contents of volatile compounds rank as esters > alcohols > acids > phenols > alkyls/alkenes > aldehydes/ketones > pyrazines. The relative contents of esters rank among different layers as fire circle > surface > core, and the relative contents of alcohols rank as surface > core > fire circle.

Main influence factors on quality of different layers of Daqu

Principal component analysis (PCA) of physicochemical indices and main hydrolase system among different layers of Daqu

The contribution rates of the first and second principal components are 73.70% and 20.50% respectively, with a sum of 94.20%, and thus they can well reflect the original information at different layers of Daqu and of the mixed sample (Fig. 5). The arrowhead reflects the different physicochemical indices and the main hydrolase system, and its direction indicates the effect orientation of a certain index on the different layers of Daqu, and its length reflects the values of physicochemical indices at different layers and the effect size on the main hydrolase system. Then correlations between the physicochemical biochemical indices and the main enzyme system were analyzed. The fire circle samples and mixed sample of Daqu are located in the first quadrant, and are correlated positively with starch content and fermentation ability and negatively with cellulase activity. The samples of the core are distributed in the second quadrant and are correlated positively with sulyase activity and acidity and negatively with moisture content, amino acid nitrogen content, and esterification ability. The samples of the surface are distributed in the fourth quadrant and are correlated positively with moisture content, amino acid nitrogen content, and esterification ability, and negatively with sulyase activity and acidity.

PCA of characteristic flavor substances among different layers of Daqu

The contribution rates of the first and second principal components are 44.20% and 30.30% respectively, with a sum of 74.50%, and thus they can well explain the information at different layers of the samples (Fig. 6). The score of PC1 is contributed from methyl hexadecanoate, methyl (Z)-9-hexadecenoate, methyl (E)-9-octadecenoate, 10-methyl octadecenoate, (E)-2-ethyl heptenoate, and phenol, and the score of PC2 is made by ethyl tetradecanoate, octanoic acid, caryophyllene, and dotetracontane (Fig. 6). The volatile flavor components are largely different among different layers of Daqu (Fig. 6).

To study the differences in the quality of different layers of Taorong-type medium- temperature Daqu, we measured the basic physicochemical indices of Daqu and found the water contents were significantly different among the surface, fire circle and core of Daqu. The acidity and amino acid nitrogen content at the core are both significantly different from those in the fire circle and the core. The starch contents in the core and the surface are significantly different. Analysis of main enzyme systems among different layers shows that the fermentation ability and esterifying enzyme ability of Daqu both rank as core > fire circle > surface, and the sulyase activity ranks as surface > fire circle > core, all showing significant differences. The cellulase activity of the core is significantly lower than on the surface and the fire circle. The volatile flavor components at different layers of Taorong-type medium-temperature Daqu were studied using HS-SPME-GC-MS. Totally 73 types of volatile substances including alcohols, esters, aldehydes, ketones, pyrazines and phenols were recognized. The most diverse volatile flavor components were detected in the fire circle, including 39 types. The contents of volatile flavor components are largely different among the three layers of Daqu, and the content of the same volatile component significantly differs among different layers.

The moisture content of Daqu in this study was 10.97%, which was lower than the results of Deng et al. (Deng et al., 2020). The higher free water content indicates that the fermentation maturity of Daqu is better (Tang et al., 2022). The internal water content of Daqu is core > Fire Ring > surface, which is consistent with the research results of Zhou Feicheng et al. (Zhou et al. 2019). The starch content was 55.75 mg/g, which was higher than that of Fan et al.'s study on sesame-flavor liquor Daqu (Fan et al., 2020). It may be due to the weak metabolism of microorganisms in Daqu during storage (Liang et al., 2017). The saccharification enzyme activity was 669.75 U/g, and the esterase activity was 458.66 U/g, which were higher than the results of Chen et al. (Chen et al., 2021). The esterase activity significantly affected the flavor characteristics of Daqu. High indicates that Daqu has richer flavors (Liu et al., 2018). Among them, the saccharifying enzyme activity of surface was significantly higher than that of core, which was consistent with the research results of Chen et al. The cellulase activity was 11.889 U/g, which was higher than the research results of Zhou Feicheng et al., and the cellulose content of surface was significantly higher than that of core, which was consistent with the research results (Zhou et al. 2019). A total of 73 volatile flavor compounds were identified in this study, which is higher than the 28 volatile compounds identified by Fan et al. from Daqu samples by HP-SPME-GC-MS (Fan et al., 2020). Among them, the proportion of esters identified was the highest, and important flavor compounds in liquor such as tetramethylpyrazine, phenethyl alcohol, and ethyl caprylate were detected. Contributes sweetness, fruit aroma, floral aroma and important functional substances to liquor. Flavor compounds such as (E)-ethyl 9-octadecenoate, ethyl pelargonate, 2-methoxy-4-vinylphenol were additionally detected in this study. The interaction between various compounds together promotes the unique flavor of Tao rong-type daqu.This study comprehensively studied the different parts of Daqu by combining physical and chemical indicators, main enzymes and volatile flavor components, and comprehensively revealed the characteristics of different parts of Daqu in Tao-rong liquor, and provided a theoretical basis for optimizing the production process of Daqu. Thereby, the quality of liquor can be further improved.

Ethics approval and consent to participate

Not applicable. No animal experiments were involved in this study

Consent for publication

Not applicable.

Competing interests

The authors declare no conflict of interest.

Funding

This work supported by the Key Technologies Research and Development Program of Henan Province of China(202102110130), Major Science and Technology Projects of Henan Province of China(181100211400) and Food Science and Engineering Key Discipline Construction Project of Henan University of Animal Husbandry and Economy (XJXK202203).

Authors' contributions

Conceptualization, Y.L. and C.P.; writing-original draft preparation, Y.L.;

Writing-review and editing, supervision, S.H. and C.P; investigation, H.L.; R.H.,; H.L. and Y.X.; validation, F.L. and X.S.; funding acquisition, H.L. and S.H.; project administration, C.P.; All authors have read and agreed to the published version of the manuscript.

Acknowledgements

Not applicable.

Availability of data and material

The data and material presented in this study are available on request from the corresponding author.

Chen Y, Li K, Liu T, Li R，Fu G，Wan Y，Zheng F. Analysis of difference in microbial community and physicochemical indices between surface and central parts of Chinese special-flavor Baijiu Daqu[J]. Frontiers in microbiology, 2021: 3340.
Deng L, Mao X, Liu D, Ning X，Shen Y，Chen B，Nie H，Huang D，Luo H. Comparative analysis of physicochemical properties and microbial composition in high-temperature Daqu with different colors[J]. Frontiers in microbiology, 2020: 3010.
Fan H, Fan W, Xu Y. Characterization of key odorants in Chinese chixiang aroma-type liquor by gas chromatography–olfactometry, quantitative measurements, aroma recombination, and omission studies[J]. Journal of Agricultural and Food Chemistry, 2015, 63(14): 3660-3668.
Fan G, Sun B, Fu Z, Xia Y，Huang M，Xu C，Li X. Analysis of physicochemical indices, volatile flavor components, and microbial community of a light-flavor Daqu[J]. Journal of the American Society of Brewing Chemists, 2018, 76(3): 209-218.
Fan G, Du Y, Fu Z, Chen M，Wang Z，Liu P, Li X. Characterisation of physicochemical properties, flavour components and microbial community in Chinese Guojing roasted sesame-like flavour Daqu[J]. Journal of the Institute of Brewing, 2020, 126(1): 105-115.
Fan W L, Shen H Y, Xu Y. Quantification of volatile compounds in Chinese soy sauce aroma type liquor by stir bar sorptive extraction and gas chromatography-mass spectrometry[J]. Journal of the science of food and agriculture, 2011, 91(7): 1187-1198.
Gao H, Wang Y, Yang Q, Peng H，Li Y，Zhan D，Wei H, Lu H, M.A.Bakr M，M.EI-Sheekh M，Qi Z，Peng L, Lin X. Combined steam explosion and optimized green-liquor pretreatments are effective for complete saccharification to maximize bioethanol production by reducing lignocellulose recalcitrance in one-year-old bamboo[J]. Renewable Energy, 2021, 175: 1069-1079.
Hao F, Tan Y, Lv X, Chen X，Yang F，Wang H, Du H, Wang L, Xu Y. Microbial community succession and its environment driving factors during initial fermentation of Maotai-Flavor Baijiu[J]. Frontiers in microbiology, 2021, 12: 960.
Hu Y, Huang X, Yang B, Zhang X，Han Y, Chen X，Han B. Contrasting the microbial community and metabolic profile of three types of light-flavor Daqu[J]. Food Bioscience, 2021, 44: 101395.
Li H, Qu Y, Cao K, Xiong J，Hua K，Liu Z，Wang H，Yue L，Tang Y, Ding G. Cigarette Flavouring Regulation by Using Aroma-producing Microorganism Isolated from Maotai Daqu[C]//E3S Web of Conferences. EDP Sciences, 2021, 271: 04031.
Li P, Francis W K A, Yu H, Luo L，Lin W . Characterization of activity and microbial diversity of typical types of Daqu for traditional Chinese vinegar[J]. Annals of Microbiology, 2015, 65(4): 2019-2027.
Li Y, Zhang P, Zhu D, Yao B，Hasunuma T，Kondo A, Zhao X. Efficient preparation of soluble inducer for cellulase production and saccharification of corn stover using in-house generated crude enzymes[J]. Biochemical Engineering Journal, 2022, 178: 108296.
Liang C, Du H, Xu Y. The succession of procaryotic microbial community and the flavor components in the storage process of Daqu[J]. Microbiology China, 2017, 44(2): 384-393.
Liu J, Chen J, Fan Y, Huang X, Han B. Biochemical characterisation and dominance of different hydrolases in different types of Daqu–a Chinese industrial fermentation starter[J]. Journal of the Science of Food and Agriculture, 2018, 98(1): 113-121.
Liu P, Zhang L, Du X, Zhao J，Gao G，Zhang X. Dynamic analysis of physicochemical and biochemical indices and microbial communities of light-flavor daqu during storage[J]. Journal of the American Society of Brewing Chemists, 2019, 77(4): 287-294.
Ma R S, Zhang K, Jiao C Y, Tang S H. Changes of Alcohols During the Storage of Fuqu Liquor[J]. China Brewing, 2010, 29: 91-93.
Ming H M, Chen X X, Liu Q B, Xu D F, Yao X, Zhang H L, Xue D W. Comparative Studies on Physicochemical and Biochemical Indicators Between Surface and Inner about Luzhou Laojiao Daqu[J]. Hubei Agricultural Sciences, 2015, 54(6): 1445-1447+1451.
Niu Y, Zhu Q, Xiao Z. Characterization of perceptual interactions among ester aroma compounds found in Chinese Moutai Baijiu by gas chromatography-olfactometry, odor Intensity, olfactory threshold and odor activity value[J]. Food Research International, 2020, 131: 108986.
Qian Y, Zhang L, Sun Y, Tang Y, Li D，Zhang H, Yuan S, Li J. Differentiation and classification of Chinese Luzhou‐flavor liquors with different geographical origins based on fingerprint and chemometric analysis[J]. Journal of Food Science, 2021, 86(5): 1861-1877.
Tang J, Liu L, Long Y, Zhao Y, Zhao T, Wang F, Li Z. Bacterial community structure and physicochemical characteristics of sauce-flavor Daqu with different sensory characteristics [J].China Brewing, 2022, 41(01): 138-142.
Sun W, Xiao H, Peng Q, Zhang Q，Li X，Han Y. Analysis of bacterial diversity of Chinese Luzhou-flavor liquor brewed in different seasons by Illumina Miseq sequencing[J]. Annals of Microbiology, 2016, 66(3): 1293-1301.
Wang F R. Winemaking Analysis and Testing[M]. Beijing: Chemical Industry Press, 2012.
Wu Q, Xu Y. Transcriptome profiling of heat-resistant strain Bacillus licheniformis CGMCC3962 producing Maotai flavor[J]. Journal of agricultural and food chemistry, 2012, 60(8): 2033-2038.
Wu X H, Zheng X W, Han B Z, Vervoort J，M.J.Robert N. Characterization of Chinese liquor starter,“Daqu”, by flavor type with 1H NMR-based nontargeted analysis[J]. Journal of agricultural and food chemistry, 2009, 57(23): 11354-11359.
Xiao C, Lu Z M, Zhang X J, Wang S T, Ao L, Shen C H，Shi J S, Xu Z H. Bio-heat is a key environmental driver shaping the microbial community of medium-temperature Daqu[J]. Applied and environmental microbiology, 2017, 83(23): e01550-17.
Xie M, Lv F, Ma G, Farooq A, Li H, Du Y, Liu Y. High throughput sequencing of the bacterial composition and dynamic succession in Daqu for Chinese sesame flavour liquor[J]. Journal of the Institute of Brewing, 2020, 126(1): 98-104.
Xu Y, Zhao J, Liu X, Zhang C, Zhao Z, Li X, Sun B. Flavor mystery of Chinese traditional fermented baijiu: The great contribution of ester compounds[J]. Food Chemistry, 2022, 369: 130920.
Yang Y, Wang S T, Lu Z M, Zhang X J, Chai L J, Shen C H, Shi J S，Xu Z H. Metagenomics unveils microbial roles involved in metabolic network of flavor development in medium-temperature daqu starter[J]. Food Research International, 2021, 140: 110037.
Yang Y, Li Y R, Jiang L, Jia Y W, Wang Q B. Differences and Changes of Biochemical Indexes Between Parts of Middle-High-Temperature Daqu Qubei[J]. Food And Fermentation Industries, 2019,45(19): 73-78.
Zhang C, Ao Z, Chui W, Shen C, Tao W, Zhang T. Characterization of volatile compounds from Daqu‐a traditional Chinese liquor fermentation starter[J]. International journal of food science & technology, 2011, 46(8): 1591-1599.
Zheng X W, Tabrizi M R, Nout M J R, Han B Z. Daqu—a traditional Chinese liquor fermentation starter[J]. Journal of the Institute of Brewing, 2011, 117(1): 82-90.
Zhang C, Ao Z, Chui W Q, Shen C, Tao W, Zhang S. Characterization of the aroma-active compounds in Daqu: a tradition Chinese liquor starter[J]. European Food Research and Technology, 2012, 234(1): 69-76.
Zhou C X, Zhen F P, Li H H, Wu, J. H., Huang, M. Q., & Sun, B. G. Research progress on flavor compounds of Baijiu Daqu[J]. China Brewing, 2019, 38(5): 6-12.
Zhou F C, Wu S W, Zhu Q S. Physicochemical indexes and dynamic analysis of main enzymes in different layers of Te-flavor Daqu during fermentation process[J]. China Brewing, 2019, 38(4): 58-64.
Zhou Z, Ju X, Chen J, Wang R, Zhong Y, Li L. Charge-oriented strategies of tunable substrate affinity based on cellulase and biomass for improving in situ saccharification: a review[J]. Bioresource Technology, 2021, 319: 124159.

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Differences in Physicochemical properties and flavor components among different layers of medium-temperature Daqu

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Abstract

Figures

Introduction

Materials and Methods

Materials and reagents

Instruments

Experimental methods

Liquor Sampling

Detection of physicochemical indices and hydrolase system of Daqu

Volatile flavor components of Daqu

Data processing

Results

Physicochemical indices at different layers of Daqu

Water content

Acidity

Starch content

Amino acid nitrogen content

Hydrolase system analysis at different layers of Daqu

Sulyase activity

Fermentation force

Esterification activity

Cellulase activity

Difference analysis of characteristic flavor substances at different layers of Daqu

Main influence factors on quality of different layers of Daqu

PCA of characteristic flavor substances among different layers of Daqu

Discussion

Declarations

References

Supplementary Files

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