Variations of the quantities of K. marxianus L1-1 in the fermentation process of rice-acid
K. marxianus, which is able to utilize various sugars, may be a suitable microbe for lignocellulose hydrolysis and grain matrix at 30 °C [13]. In this study, the fermentation temperature was determined as 30 °C based on our previous study. The number of K. marxianus L1-1 changed significantly in the fermentation process. The number of K. marxianus L1-1 gained the most significant increase rate from 0 d to 1 d (Fig. 1a) and decreased from 1d to 2 d. Interestingly, it increased from 2 d to 3 d. The above variations may be related to the oxygen content in the fermentation tank. The limited supply of oxygen (the terminal electron acceptor) also initiated the synthesis of some esters, but it primarily forced ethanol production during the growth of K. marxianus DSM 5422 [14]. We explored the formation of ethyl acetate, other esters and organic acids based on the growth of K. marxianus L1-1 in this study. In the study, we investigated the key fermentation process of rice-acid with K. marxianus L1-1 in 1 d and 3 d and analyzed the formation mechanism of flavors in K. marxianus L1-1.
Variations of key volatile compounds in the fermentation process of rice-acid
The variations of flavor compounds in 1 d and 3 d were explored. The basic conditions for the formation of ethyl acetate are acetic acid, ethanol and some key enzymes which were discussed in this study. In the obtained volatile compounds, 5 key acids, 13 key alcohols and 12 key esters were found (Table 1). From 1 d to 3 d, ethyl acetate content increased from 162.98 ± 5.02 to 241.37 ± 6.20 g/kg; ethanol content increased from 36.11 ± 4.54 to 52.68 ± 14.45 g/kg; acetic acid content increased from 0.21 ± 0.06 to 32.67 ± 1.57 g/kg. Acetic acid, 2-phenylethyl ester, 2-methyl-propanoic acid, ethyl ester and 9 other esters were also found. Interestingly, ethyl acetate, ethanol and acetic acid are important volatile compounds because of their high contents and low odor thresholds [15]. Ethyl acetate made a significant contribution to the formation of the fruity flavor and promoted the overall flavor balance in rice-acid. Moreover, ethyl acetate exhibits probiotic properties such as being closely linked to the antioxidant function in the fruit [16]. The formation of esters in the alcoholization stage was closely related to the enzyme activity of yeasts. Therefore, it is necessary to explore the formation mechanism of ethyl acetate and other esters.
Table 1
The key volatile compounds (mg/L) and organic acids (mg/L) in fermented rice-acid inoculated with K. marxianus L1-1 at 1 d and 3 d.
volatile Compound
|
RT
|
RI
|
RIL
|
1 d
|
3 d
|
Acids (5)
|
|
|
|
|
|
Acetic acid
|
12.69
|
1508
|
1449
|
0.21 ± 0.06
|
32.67 ± 1.57
|
Propanoic acid, 2-methyl-
|
14.69
|
1671
|
1570
|
0.55 ± 0.13
|
1.12 ± 0.21
|
Butanoic acid, 2-methyl-
|
16.05
|
1791
|
1662
|
0
|
0.33 ± 0.19
|
Butanoic acid, 3-methyl-
|
16.22
|
1806
|
1666
|
0.09 ± 0.01
|
0.93 ± 0.32
|
Hexanoic acid
|
18.17
|
1991
|
1846
|
0.36 ± 0.05
|
0.73 ± 0.29
|
Alcohol (13)
|
|
|
|
|
|
Ethanol
|
3.76
|
937
|
932
|
36.11 ± 4.54
|
52.68 ± 14.45
|
1-Propanol
|
5.52
|
1043
|
1036
|
1.30 ± 0.76
|
1.13 ± 0.25
|
1-Propanol, 2-methyl-
|
6.47
|
1096
|
1092
|
20.89 ± 6.43
|
38.77 ± 10.50
|
1-Butanol, 3-methyl-
|
8.39
|
1209
|
1209
|
17.04 ± 4.79
|
31.84 ± 5.27
|
1-Pentanol
|
9.07
|
1252
|
1250
|
0.36 ± 0.11
|
0.89 ± 0.15
|
1-Hexanol
|
10.60
|
1355
|
1355
|
5.57 ± 0.35
|
3.38 ± 0.92
|
1-Octen-3-ol
|
11.92
|
1450
|
1450
|
0.89 ± 0.31
|
0.43 ± 0.08
|
1-Heptanol
|
12.01
|
1457
|
1453
|
1.49 ± 0.51
|
0.81 ± 0.30
|
1-Hexanol, 2-ethyl-
|
12.46
|
1490
|
1491
|
4.23 ± 1.12
|
4.87 ± 1.79
|
2-Nonanol
|
12.83
|
1519
|
1521
|
0.03 ± 0.00
|
0.64 ± 0.19
|
1-Octanol
|
13.33
|
1559
|
1557
|
1.79 ± 0.67
|
1.69 ± 0.11
|
1-Nonanol
|
14.57
|
1661
|
1660
|
2.26 ± 1.10
|
1.75 ± 0.48
|
Phenylethyl Alcohol
|
17.43
|
1919
|
1906
|
12.22 ± 4.65
|
13.68 ± 3.62
|
Esters (12)
|
|
|
|
|
|
Ethyl Acetate
|
3.05
|
886
|
888
|
162.98 ± 5.02
|
241.37 ± 6.20
|
Propanoic acid, ethyl ester
|
4.06
|
956
|
953
|
5.03 ± 1.44
|
3.06 ± 0.69
|
Propanoic acid, 2-methyl-, ethyl ester
|
4.19
|
964
|
961
|
6.43 ± 2.84
|
8.83 ± 1.17
|
Butanoic acid, methyl ester
|
4.51
|
984
|
982
|
0.75 ± 0.25
|
0.65 ± 0.17
|
Isobutyl acetate
|
4.98
|
1013
|
1012
|
3.56 ± 0.72
|
9.26 ± 1.53
|
1-Butanol, 3-methyl-, acetate
|
6.88
|
1120
|
1122
|
2.36 ± 0.76
|
3.30 ± 0.84
|
1-Butanol, 2-methyl-, acetate
|
6.90
|
1121
|
1125
|
0
|
4.74 ± 6.70
|
Acetic acid, pentyl ester
|
7.73
|
1169
|
1176
|
0.06 ± 0.01
|
0.83 ± 0.16
|
2-Propenoic acid, ethenyl ester
|
8.42
|
1211
|
|
0
|
6.85 ± 1.68
|
Acetic acid, hexyl ester
|
9.37
|
1271
|
1272
|
0.29 ± 0.11
|
0.39 ± 0.11
|
Acetic acid, 2-phenylethyl ester
|
16.41
|
1823
|
1813
|
28.29 ± 7.93
|
38.11 ± 4.26
|
Propanoic acid, 2-methyl-, 2-phenylethyl ester
|
17.11
|
1889
|
1896
|
1.61 ± 0.21
|
2.26 ± 0.62
|
Organic acids
|
|
|
|
1d
|
3d
|
L-lactic acid
|
|
|
|
3.01 ± 0.61
|
6.02 ± 1.67
|
Acetic acid
|
|
|
|
0.007 ± 0.003
|
0.51 ± 0.06
|
Malic acid
|
|
|
|
0.77 ± 0.06
|
0.14 ± 0.08
|
Succinic acid
|
|
|
|
0.022 ± 0.004
|
0.072 ± 0.006
|
Citric acid
|
|
|
|
0
|
0.005 ± 0.001
|
Oxalic acid
|
|
|
|
0.036 ± 0.005
|
0.43 ± 0.03
|
Tartaric acid
|
|
|
|
0.029 ± 0.007
|
0.043 ± 0.005
|
RI: the linear retention indices calculated from a series of n-alkanes (C6-C26). |
RIL: retention indices referred to the literature value with same or equivalent chromatographic column shown on http://www.flavornet.org/flavornet.html and NIST Chemistry WebBook (http://webbook.nist.gov/chemistry/). |
Method of identification: (A) by comparison of the mass spectrum with the NIST/Wiley mass spectral library; (B) by comparison of RI (Kovats indices) with RI of an authentic compound; and (C), by comparison of retention time and spectrum of an authentic compound. |
Variations of organic acids in the fermentation process of rice-acid
Seven organic acids were found in rice-acid, including L-lactic acid, acetic acid, malic acid, succinic acid, citric acid, oxalic acid and tartaric acid (Table 1). Among 7 organic acids, L-lactic acid had the highest content. The content of L-lactic acid increased from 3.01 ± 0.61 g/kg in Day 1 to 6.02 ± 1.67 g/kg in 3 d. The contents of the other 6 organic acids did not show the significant increase during the fermentation process. However, although the contents of the 6 other organic acids were low, they interacted with each other to promote the formation of the sourness and taste of rice-acid. In our study, both volatile components and organic acids affected the formation of the flavor of rice-acid. Lactic acid exists in two isomeric forms which include L-(+) and D-(−)-Lactic acid. It is produced by microbial fermentation and chemical synthesis and used in food, cosmetic, pharmaceutical, and chemical industries [17]. In the study, we mainly focused on the increase in L-(+) -Lactic acid caused by microbial fermentation. L-(+)- Lactic acid with a high enantiomeric purity is required in many industries, especially in medical, pharmaceutical and food industries, since D-(−)- Lactic acid is harmful to humans and can cause decalcification or acidosis [18]. L-(+)-Lactic acid not only promoted the formation of the flavor in rice-acid, but also had an important effect on the health. We will further explore the genes, proteins and enzymes associated with the formation of organic acids.
Transcriptomic analysis of K. marxianus L1-1 during the key fermentation period of rice-acid
In the fermentation process of rice-acid, Y 1 d and Y 3 d initiated the changes in the expressions of a series of genes. The two independent cDNA libraries (Y 1 d and Y 3 d) constructed for high-throughput sequencing respectively yielded 19,617,191 ~ 27,627,279 pair-end reads and 5,885,157,300 ~ 8,288,183,700 clean reads after stringent quality check and data filtering (Q20 bases > 97.28%, Q30 bases > 92.01%, G + C 42.63%~43.03%) (Table 2). All of high-quality clean reads were used for gene comparison and more than 88% of total reads of clean reads were mapped to the database by software Bowtie 2. By using |log2FC| > 1.5 and FDR < 0.05, we identified 1390 DEGs (788 up-regulated and 602 down-regulated) between Y 1 d and Y 3 d (Fig. 2a).
Table 2
Quality assessment results of RNA sequencing in K. marxianus L1-1 when inoculated rice-acid.
Sample ID
|
Read Sum
|
Base Sum
|
GC (%)
|
Q20 (%)
|
Q30 (%)
|
Y1d-1
|
19,617,191
|
5,885,157,300
|
42.90%
|
97.33%
|
92.07%
|
Y1d-2
|
24,816,214
|
7,444,864,200
|
42.91%
|
97.36%
|
92.19%
|
Y1d-3
|
22,852,031
|
6,855,609,300
|
42.63%
|
97.28%
|
92.01%
|
Y3d-1
|
27,627,279
|
8,288,183,700
|
42.99%
|
97.44%
|
92.34%
|
Y3d-2
|
22,987,221
|
6,896,166,300
|
42.95%
|
97.35%
|
92.18%
|
Y3d-3
|
25,331,202
|
7,599,360,600
|
43.03%
|
97.36%
|
92.18%
|
GO analysis of the DEGs showed the enrichment of three major cellular components, biological processes and molecular functions (Fig. 3). In terms of cellular components, most of the DEGs were enriched in nucleolus, small-subunit processome and preribosome, and large-subunit precursor. In terms of biological processes, most of the DEGs were enriched in endonucleolytic cleavage in ITS1 to separate SSU-rRNA from 5.8S rRNA and LSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, and LSU-rRNA). In terms of molecular functions, these DEGs were enriched in structural constituent of ribosome, snoRNA binding and rRNA binding. All DEGs were subjected to KEGG pathway enrichment analysis. KEGG analysis assigned the DEGs of Y1-d and Y3-d to metabolic pathways. At least 4231 genes were identified, including 1390 DEGs annotated to 279 KEGG pathways (Table S1). The significantly enriched pathways (p-value < 0.05 and q-value < 0.05) were ribosome, cytosolic DNA-sensing pathway, RNA polymerase, DNA replication, ribosome biogenesis in eukaryotes, pyrimidine metabolism and purine metabolism. Importantly, the crucial KEGG pathway related to the ethyl acetate and organic acids included amino sugar and nucleotide sugar metabolism, starch and sucrose metabolism, glycolysis/gluconeogenesis, pyruvate metabolism and TCA cycle. These pathways showed the different roles of K. marxianus L1-1 in the formation of flavor and taste of rice-acid.
Ethyl acetate and organic acids promoted the maturity of flavor and taste of rice-acid inoculated with K. marxianus L1-1. Key genes were found to be involved in the ethyl acetate metabolism process in glycolysis, including GLK1 (K00844), GPD2 (K00134), 3 genes of ALD family (K00129 and 2 K00128), 6 genes of ADH family (K13953, K13953 and 4 genes of K13953) and ATF1 protein (BAO42650, BAO42650 and BAO42650) (Table 3). The genes of GLK1, GPD2, ALD family, ADH family and ATF1 encode glucokinase-1, glyceraldehyde 3-phosphate dehydrogenase, aldehyde dehydrogenase, alcohol dehydrogenase and alcohol O-acetyltransferase, respectively. In addition, the gene ERG10 encoding acetyl-CoA C-acetyltransferase was found to be involved in the ethyl acetate metabolism process in pyruvate metabolism. The key genes related to organic acids found in pyruvate metabolism included CYB2 (K00101), DLD1 (K00102), 2 genes of MDH family (2 K00026), and FUM1 (K01679) respectively encoding L-lactate dehydrogenase, D-lactate dehydrogenase, malate dehydrogenase and fumarate hydratase. The key genes related to organic acids found in the citrate cycle included 2 genes of CIT family (2 K01647), 3 genes of SDH family (K00234, K00236 and K00237) and 2 genes of MDH2 (K00026) respectively encoding citrate synthase succinate dehydrogenase (ubiquinone) flavoprotein subunit and malate dehydrogenase. The key KEGG pathways related to the formation mechanism of the flavor based on the DEGs are discussed below.
Table 3
The different expression of genes and proteins (DEGs and DEPs) in key KEGG pathways of K. marxianus L1-1 when inoculated rice-acid.
Seq_ID
|
Description
|
KO/Gene_ID
|
KEGG/ Genename
|
Log2FC/
Transcription
|
Log2FC/ Proteomics
|
Starch and sucrose metabolism
|
KLMA_10051
|
glucokinase-1,hexokinase [EC:2.7.1.1]
|
K00844
|
GLK1, HK
|
1.83
|
13.384
|
KLMA_30608
|
glucan 1,3-beta-glucosidase [EC:3.2.1.58]
|
K01210
|
SCW4
|
1.53
|
6.986
|
Amino sugar and nucleotide sugar metabolism
|
KLMA_10051
|
glucokinase-1,hexokinase [EC:2.7.1.1]
|
K00844
|
GLK1
|
1.83
|
13.384
|
Glycolysis / Gluconeogenesis
|
KLMA_10051
|
glucokinase-1 [EC:2.7.1.1]
|
K00844
|
GLK1
|
1.83
|
13.384
|
KLMA_10836
|
alcohol dehydrogenase, propanol-preferring [EC:1.1.1.1]
|
K13953
|
ADH2
|
1.47
|
None
|
KLMA_20673
|
aldehyde dehydrogenase (NAD(P)+) [EC:1.2.1.5]
|
K00129
|
ALD2
|
-2.15
|
None
|
KLMA_30203
|
alcohol O-acetyltransferase [EC:2.3.1.84]
|
K00664
|
ATF1
|
2.90
|
None
|
KLMA_40102
|
alcohol dehydrogenase, propanol-preferring [EC:1.1.1.1]
|
K13953
|
ADH2
|
-1.77
|
1.65
|
KLMA_40404
|
aldehyde dehydrogenase (NAD+) [EC:1.2.1.3]
|
K00128
|
ALD5
|
-1.65
|
None
|
KLMA_40624
|
alcohol dehydrogenase
|
None
|
ADH
|
None
|
2.047
|
KLMA_50012
|
aldehyde dehydrogenase (NAD+) [EC:1.2.1.3]
|
K00128
|
ALD4
|
-1.07
|
None
|
KLMA_70007
|
alcohol dehydrogenase, propanol-preferring [EC:1.1.1.1]
|
K13953
|
ADH2
|
2.10
|
None
|
KLMA_70462
|
alcohol dehydrogenase, propanol-preferring [EC:1.1.1.1]
|
K13953
|
ADH2
|
3.50
|
None
|
KLMA_80306
|
alcohol dehydrogenase, propanol-preferring [EC:1.1.1.1]
|
K13953
|
ADH3
|
-1.42
|
None
|
KLMA_80339
|
NADP-dependent alcohol dehydrogenase 6
|
none
|
ADH6
|
none
|
1.539
|
KLMA_80427
|
alcohol dehydrogenase, propanol-preferring [EC:1.1.1.1]
|
K13953
|
ADH1
|
2.83
|
None
|
Pyruvate metabolism
|
KLMA_10621
|
L-lactate dehydrogenase (cytochrome) [EC:1.1.2.3]
|
K00101
|
CYB2
|
2.93
|
None
|
KLMA_10649
|
D-lactate dehydrogenase (cytochrome) [EC:1.1.2.4]
|
K00102
|
DLD1
|
-1.20
|
None
|
KLMA_10771
|
homocitrate synthase [EC:2.3.3.14]
|
K01655
|
LYS21
|
1.58
|
2.073
|
KLMA_30013
|
L-lactate dehydrogenase (cytochrome) [EC:1.1.2.3]
|
K00101
|
CYB2
|
-3.88
|
None
|
KLMA_40055
|
malate dehydrogenase [EC:1.1.1.37]
|
|
MDH3
|
None
|
1.523
|
KLMA_40341
|
L-lactate dehydrogenase (cytochrome) [EC:1.1.2.3]
|
K00101
|
CYB2
|
1.13
|
None
|
KLMA_40583
|
D-lactate dehydrogenase (cytochrome) [EC:1.1.2.4]
|
K00102
|
DLD1
|
-2.06
|
None
|
KLMA_60086
|
acyl-coenzyme A, acyl-coenzyme A thioester hydrolase
|
K01068
|
TES1
|
-1.13
|
1.517
|
KLMA_60167
|
malate dehydrogenase
|
K00026
|
MDH1
|
1.12
|
2.984
|
KLMA_60383
|
acetyl-CoA C-acetyltransferase [EC:2.3.1.9]
|
K00626
|
ACCT
|
1.07
|
None
|
KLMA_80028
|
fumarate hydratase
|
K01679
|
FUM1
|
1.03
|
2.034
|
TCA cycle
|
KLMA_10253
|
pyruvate carboxylase [EC:6.4.1.1]
|
K01958
|
PYC2
|
1.50
|
None
|
KLMA_20105
|
citrate synthase [EC:2.3.3.1]
|
K01647
|
CIT1
|
2.28
|
2.47
|
KLMA_20466
|
succinate dehydrogenase (ubiquinone) flavoprotein subunit [EC:1.3.5.1]
|
K00234
|
SDH1
|
1.25
|
1.75
|
KLMA_30124
|
succinate dehydrogenase (ubiquinone) cytochrome b560 subunit
|
K00236
|
SDH4
|
1.28
|
None
|
KLMA_40055
|
malate dehydrogenase
|
None
|
MDH3
|
None
|
1.523
|
KLMA_40391
|
malate dehydrogenase
|
K00026
|
MDH2
|
2.07
|
3.539
|
KLMA_50550
|
succinate dehydrogenase (ubiquinone) membrane anchor subunit
|
K00237
|
SDH4
|
1.36
|
None
|
KLMA_60167
|
malate dehydrogenase [EC:1.1.1.37]
|
K00026
|
MDH1
|
1.12
|
None
|
KLMA_60167
|
malate dehydrogenase [EC:1.1.1.37]
|
K00026
|
MDH2
|
1.12
|
2.984
|
KLMA_80028
|
fumarate hydratase, class II [EC:4.2.1.2]
|
K01679
|
FUM1
|
1.03
|
2.034
|
KLMA_80408
|
succinate dehydrogenase [ubiquinone] iron-sulfur subunit
|
None
|
SDH2
|
None
|
1.651
|
Note: FC value represented the differential expression multiple of mRNA and protein. |
Proteomics Characterization
The total proteins were extracted from the Y1-d and the Y3-d at filling stage and subjected to 4D label-free proteomics analysis to complement the transcriptome analysis. According to the abundance levels of proteins, 610 proteins were identified as DEPs at p-value < 0.05, including 135 proteins with increased abundance levels and 475 proteins with decreased abundance levels (Fig. 2b), and the difference ratio reached > 1.5. The number of up-regulated proteins was smaller than that of down-regulated proteins at the filling stage since the growth of K. marxianus L1-1 was inhibited due to the acid environment in the later fermentation stage of rice-acid.
To obtain a global diagram of proteomic changes, at least 2937 proteins were identified and 187 DEPs were annotated with GO analysis and KEGG analysis (Table S2). In GO functional analysis, 187 proteins were annotated to 59 GO terms. The results of GO analysis showed that the distributions of DEPs in functional classification were consistent with the distributions of transcription levels of DEGs (Fig. 4). In terms of cellular components, most of the up-regulated DEPs were enriched in mitochondrion, tricarboxylic acid cycle enzyme complex and mitochondrial matrix. In terms of the biological process, most of the up-regulated DEPs were enriched in citrate metabolic process, tricarboxylic acid metabolic process and galactose catabolic process. In terms of molecular functions, these up-regulated DEPs were enriched in oxidoreductase activity, L-malate dehydrogenase activity, malate dehydrogenase activity and alcohol dehydrogenase activity. The 6 GO terms involved in alcohol dehydrogenase activity had the smallest p-value (p-value < 0.01) and were related to the formation of ethyl acetate. The 187 DEPs were annotated to 30 KEGG pathways (Table S2). Most up-regulated KEGG pathways analyzed by proteomics were related the formation of ethyl acetate and organic acids, which explained reasonably the formation of flavor and taste in rice-acid inoculated with K. marxianus L1-1. Meanwhile, the down-regulated KEGG pathways obtained by proteomics analysis were most related to the growth of K. marxianus L1-1 (Fig. 5b), verifying that it was reasonable to select the third day as the ending of rice-acid fermentation. According to the pathway analysis (Fig. 5a), we could conclude that many proteins took part in various metabolic pathways including amino sugar and nucleotide sugar metabolism, starch and sucrose metabolism, glycolysis/gluconeogenesis, pyruvate metabolism and citrate cycle, which might affect many aspects of the metabolism of K. marxianus L1-1 during the key fermentation period of rice-acid. However, the different KEGG pathways obtained by proteomics analysis played different roles in the formation of flavor and taste in rice-acid.
Ethyl acetate is the most important volatile compound in rice-acid inoculated with K. marxianus L1-1. Four key proteins were found to be involved in the ethyl acetate metabolism process in glycolysis, including GLK1(BAO37673), GAP1 (BAO40242), 2 ADH1 (BAO40648 and BAO40126) and ADH6 (BAO42650) (Table 2). GLK1, GAP1, ADH1 and ADH6 encode glucokinase-1, glyceraldehyde-3-phosphate dehydrogenase 1, alcohol dehydrogenase and NADP-dependent alcohol dehydrogenase, respectively. In addition, four key proteins were found to be involved in organic acids metabolic process in pyruvate metabolism, including 2 MDH family (BAO41458 and BAO40079), FUM1 (BAO42339) and LYS21 (BAO38393). MDH, FUM1 and LYS21 encode malate dehydrogenase, fumarate hydratase and homocitrate synthase, respectively. Four key proteins were found to be involved in the metabolism process of organic acids in citrate cycle, including CIT1 (BAO38563), SDH1 (BAO38924), MDH2 (BAO40415) and FUM1 (BAO42339), which respectively encode citrate synthase, succinate dehydrogenase (ubiquinone) flavoprotein subunit, malate dehydrogenase and fumarate hydratase. The proteins related to the metabolism of organic acids were consistent with the genes, indicating that the combination of transcriptomics and proteomics were useful tools to analyze the formation of ethyl acetate and organic acids during the key fermentation period of rice-acid inoculated with K. marxianus L1-1.
Correlation Analysis Of Transcriptome And Proteome Data
Transcriptomic and proteomic analysis results are shown in Fig. 1b-1d. At least 4231 genes were identified and 1390 DEGs were annotated to 279 KEGG pathways by using transcriptomic analysis. In addition, 2937 proteins were identified and 610 DEPs were annotated to 30 KEGG pathways by using proteomics analysis. Pearson correlation coefficient was 0.3761 and the results of the two analysis methods were significantly different. Therefore, the combination of transcriptome and proteome data could be an effective way to reveal the formation mechanism of the flavor in rice-acid inoculated with K. marxianus L1-1. The 5 KEGG pathways related to the synthesis of ethyl acetate and organic acids are shown in Table 3.