Proteomic and high-throughput analysis of protein expression and microbial diversity of microbes from 30- and 300-year pit muds of Chinese Luzhou-flavor liquor

doi:10.1016/j.foodres.2015.06.029

Food Research International

Volume 75, September 2015, Pages 305-314

https://doi.org/10.1016/j.foodres.2015.06.029 Get rights and content

Highlights

•
A protein extraction method suitable for pit mud of Chinese liquor was optimized.
•
iTRAQ technique was used to analyze mixed microbial communities.
•
Microbial diversities were revealed by high-throughput sequencing technique.

Abstract

Luzhou-flavor liquor is fermented based on the metabolism of special microbial communities in pit. In this study, total proteins and DNAs of microbes from 30- and 300-year pit muds were firstly extracted. Meanwhile, an efficient approach for protein extraction with increased protein content was optimized. iTRAQ-based proteomic was then applied to investigate the aroma-forming functional protein expression of microbes from the samples. Furthermore, high-throughput sequencing of 16S rDNA was employed to reveal microbial diversity. We comparatively identified 63 proteins of aroma-forming functional microbes in these samples, and found that 59 of these proteins were highly expressed in the 300-year pit mud. Those aroma-forming functional proteins were found to be involved in methanogenesis, as well as the formation of caproic acid and butyric acid during the liquor fermentation. High-throughput sequencing revealed that the microbes most commonly found in both samples were members of phylum Firmicutes (by 97% sequence similarity), both of which, along with another common Methanobacterium, were important components of aroma-forming functional colonies in the pit muds for the brewing of Chinese liquor. The findings in this study afford us new insight into the different protein expression levels and microbial communities in two pit muds.

Introduction

Luzhou-flavor liquor is one of the foremost traditional distilled liquors in China. Many famous Luzhou-flavor liquors, such as Luzhou Laojiao, Jiannanchuan and Wuliangye, are all fermented from grains such as sorghum and rice in earth cellars. The production of desired liquor is related to either pit mud or pits. The pit mud is the main substance contributing to liquor fermentation. The fermentation refers to a complex metabolic process in which a variety of microbes colonizing the pits or pit mud engage in three phase boundaries of solid, liquid and gas (Luo, Liu, Tian, Liang, & Xiang, 2011). After long-term cultivation and diversification of pit mud microbes, the species were enriched and slowly formed a microecological bacterial system dominated by methanogens and caproic acid forming clostridia (Deng, Tang, & Zhang, 2010). The composite aroma produced by the metabolism would further lead to rich aroma of Luzhou-flavor liquor. Along with different fermentation stages, the bacteria population structure changes as well; thus, the quality of pit mud is changed year after year. Therefore, it is essentially important to elucidate the correlation between the protein expression of microbes from pit mud of different ages and the production of Luzhou-flavor liquor, which would be helpful for the development of Chinese liquor industry.

Systematic identification and quantification of the proteome require high-throughput molecular biology techniques. Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) is a technique launched by Applied Biosystems Inc. in 2004 (Wang, Dai, & Tu, 2010). The technique combines multidimensional liquid chromatography and tandem mass spectrometry, which was widely used for proteomics investigation (Wu, He, & Jiang, 2013). The iTRAQ technique could be employed for relative or absolute quantification of either four or eight different protein samples simultaneously (Liu et al., 2012).

iTRAQ is a powerful method which could be applied to differential proteomics research for profiling protein expression changes, in particular of protein detection and identification in a variety of pit mud microbes. The objective of this study was to determine the correlation between protein function and aroma compounds of liquor by comparing the protein expression levels in the 30- and 300-year pit muds of Luzhou-flavor liquor with iTRAQ technique followedby a bioinformatics analysis.

Section snippets

Sampling and location of pit mud

The pit mud samples were collected from two pits used for production of liquor by the Luzhou Laojiao Group (Luzhou, Sichuan, China, 105°270′86″ E, 28°53′15″ N; April 2014). Samples were taken from two locations (the wall and the bottom of the cellar) of three individual pits with 30 years (PM30) and 300 years (PM300) old, respectively. Each sample plot was divided into five sub-plots. All samples taken from the same year were mixed together. After sampling, the two pit mud samples (PM30 and

Comparison of protein extraction protocols from 30- and 300-year pit muds

Compared with the other three protein extraction protocols, the bands of 30- and 300-year protein samples extracted with the C/S–P–M method in SDS-PAGE gels were clearer. Interestingly, much more low- and high-molecular weight proteins were obtained using this protocol (Fig. 1). The concentration of protein extracted with C/S–P–M method was 29.3 mg/mL from the 300-year pit mud and 25.9 mg/mL from the 30-year pit mud, respectively. The values were higher than those of SDS method (11.6 mg/mL and 11.1

Discussions

The aroma components in Luzhou-flavor liquor are generated during the entire fermentation process, including decomposition of raw materials via microbes and/or enzymes, metabolism of microbes, as well as the complex interactions between all kinds of metabolites from microbes and raw materials (Fan and Qian, 2005, Shen, 2005, Zhuang, 2007). The interactions in microbes in the pit mud are crucial for solid state fermentation of Chinese liquor. There was no report in microbial field in respect to

Conclusions

The aroma-forming functional proteins in 300-year pit mud were highly expressed with much higher content than that of 30-year pit mud, which was contributed to the production of more organic compounds during the fermentation of the 300-year pit mud. High-throughput sequencing of 16S rDNA revealed that aroma-forming functional microorganisms like methanogens and Clostridium were higher in abundance in PM300 sample. This result was in accordance with the result of iTRAQ. The diversification of

Acknowledgment

We wish to thank Dr. Brian McGarvey for proofreading this paper. The present study was jointly funded by grants from the Foundation for Studies on Pit Mud of Vinasse for Liquor by DGGE and PLFA (the project number: ychx00018 (2013); Luzhou, Sichuan, China), and the Funds for Researches by Leading Personnel in Higher Education Institutions (the project number: k8012012a (2012); Fuzhou, Fujian, China).

References (43)

F. Bastida et al.
Metaproteomics of soils from semiarid environment: functional and phylogenetic information obtained with different protein extraction methods
Journal of Proteomics
(2014)
B. Chen et al.
Filamentous fungal diversity and community structure associated with solid state fermentation of Chinese Maotao-flavor liquor
International Journal of Food Microbiology
(2014)
H.B. Ding et al.
Caproate formation in mixed-culture fermentative hydrogen production
Bioresource Technology
(2010)
X.F. Ding et al.
Eubacterial and archaeal community characteristics in the man-made pit mud revealed by combined PCR-DGGE and FISH analyses
Food Research International
(2014)
K.M. Keiblinger et al.
Soil metaproteomics—comparative evaluation of protein extraction protocols
Soil Biology & Biochemistry
(2012)
X.C. Lv et al.
Bacterial community dynamics during the traditional brewing of Wuyi Hong Qu glutinous rice wine as determined by culture-independent methods
Food Control
(2013)
C.J. Martyniuk et al.
DIGE and iTRAQ as biomarker discovery tools in aquatic toxicology
Ecotoxicology and Enviromental Safety
(2012)
C.D. Wang et al.
Long-term batch brewing accumulates adaptive microbes, which comprehensively produce more flavorful Chinese liquors
Food Research International
(2014)
L.F. Wu et al.
A family of homologous substrate-binding proteins with a broad range of substrate specificity and dissimilar biological functions
Biochimie
(1995)
J. Zheng et al.
Characterization of microbial communities in strong aromatic liquor fermentation pit muds of different ages assessed by combined DGGE and PLFA analyses
Food Research International
(2013)

X.W. Zheng et al.

Complex microbiota of a Chinese “Fen” liquor fermentation starter (Fen-Daqu), revealed by culture-dependent and culture-independent methods

Food Microbiology

(2012)

D. Benndorf et al.

Functional metaproteome analysis of protein extracts from contaminated soil and groundwater

ISME Journal

(2007)

B. Bornstein et al.

The nutrition of Clostridium kluyveri

Journal of Bacteriology

(1948)

J.G. Caporaso et al.

QIIME allows analysis of high-throughput community sequencing data

Nature Methods

(2010)

J.G. Caporaso et al.

Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample

Proceedings of the National Academy of Sciences of the United State of America

(2011)

B. Deng et al.

PCR-DGGE analysis on microbial communities in pit mud of cellars used for different periods of time

Journal of the Institute of Brewing

(2012)

Y. Deng et al.

Application of 16S–23S rRNA ITS-AFLP fingerprint in the analysis of prokaryotic microbial diversity in liquor pit mud

Liquor Making Science & Technology

(2010)

X.F. Ding et al.

Characterization of eubacterial and archaeal community diversity in pit mud of Chinese Luzhou-flavor liquor by nested PCR-DGGE

World Journal of Microbiology and Biotechnology

(2014)

W.L. Fan et al.

Headspace solid phase microextraction and gas chromatography–olfactometry dilution analysis of young and aged Chinese “Yanghe Daqu” liquors

Journal of Agricultural and Food Chemistry

(2005)

L. Fu et al.

Ecological diversity and industrial application of methanogens

Chinese Journal of Applied & Environmental Biology

(2009)

J.E. Galagan et al.

The genome of M. acetivorans reveals extensive metabolic and physiological diversity

Genome Research

(2002)

Cited by (61)

Novel insights into flavor formation in whey fermented wine: A study of microbial metabolic networks
2024, LWT
In this study, the alterations in the flavor components of whey fermented wine (WFW) were analyzed using gas chromatography–mass spectrometry (GC-MS). Moreover, the changes in the bacterial community structure of WFW were examined using high-throughput sequencing technology. The results showed that the pH value decreased from 5.61 to 3.51 between 0 d and 28 d of fermentation. Meanwhile, the content of total acids (1.31°T to 7.32°T), alcohol (8.83%–12.57%), flavonoids (33.98–60.52 mgRE/100 mL), and polyphenols (120.24–230.34 mgGAE/100 mL) increased during fermentation. At the early stage of fermentation, Lactobacillus was the predominant bacterial genus in WFW. However, as fermentation progressed, Lactobacillus and Lactococcus gradually became the dominant genera. Using GC-MS, 102 volatile compounds were identified during fermentation. Among them, 15 substances — including ethyl benzoate, phenyl ethyl acetate, and ethyl butyrate — were identified as the important aroma components of WFW. Fourteen amino acids were detected during fermentation, among which leucine (260.78 μg/L) and tyrosine (250.37 μg/L) showed the highest content. Correlation analysis revealed that Lactococcus was positively correlated with most ester compounds, and Lactobacillus was positively correlated with ethyl phenylpropionate, acetaldehyde, and caprylic acid. Meanwhile, Klebsiella exhibited a significant positive correlation with histidine, aspartic acid, and alanine.
Mining the factors driving the succession of microbial community in pit mud used for the production of Nongxiangxing baijiu
2024, LWT
The pit mud (PM) provides a unique anaerobic environment for the fermentation of Nongxiangxing baijiu. Previous research has indicated a close relationship between PM quality and cellar age. However, the molecular basis of microbial dynamics and associated functions of microbial community remain unclear. Here, we explored the microbial communities of PM samples from three different locations with various temporal gradients. Based on the maturity time of PM, all samples were divided into two stages, P1 (cellar age<20) and P2 (cellar age≥20), and 30 microbial genera were identified as biomarkers. Mantel test demonstrated a significant correlation between acetic acid and biomarkers in P1 stage, whereas in P2 stage, hexanoic acid and lactic acid were significantly correlated with the bacterial community. Furthermore, random forest analysis suggested that the main driving factors of the bacterial community succession were NH₄⁺–N, available phosphorus, and moisture, while the fungal community succession were available phosphorus and acetic acid. PICRUSt2 analysis demonstrated that the bacteria in aged PM had a higher potential to synthesize butyric acid compared with the new pit mud. Results presented in this study may provide a basis for the future artificial adjustment of PM quality.
Proteomics and transcriptomics and their application in fermented foods
2023, Indigenous Fermented Foods for the Tropics
Fermentation is a primeval practice to preserve, sometimes, inedible raw materials into safe, shelf-stable, and tasty foods or beverages using the microorganisms. Owing to the partial oxidation of organic molecules, various aromatic compounds and other products are produced. Owing to the diverse microbiome that exists in these fermented products, the properties of these fermented products such as the texture, flavor, acidity, and the associated health benefits are usually affected. Previously, traditional culture-dependent microbiological techniques have been employed to study food microbiota for a long time to understand the subtleties of such products. With the advent of molecular- or culture-independent approaches generally known as omics science, a solid grasp of microbiota dynamics has been established. The use of omics in studying fermented products provides one with metabolic and gene/functional potential knowledge of how microbiomes impact a given product. These include the chemicals that characterize its scent and flavor as well as its nutritional characteristics—metabolomics. On the other hand, analyzing the functional potential—transcriptomics and proteomics—of microbiomes is a difficult undertaking, but tremendous efforts have been put forward toward vital and deeper insights into understanding the underlying transcriptional and proteomic mechanisms of fermented products. Until recently, most omics research studies have been concentrated on commercial products, while traditional fermented foods have been overlooked. This chapter provides reports on the applications of omics, specifically transcriptomics and proteomics in the study of traditional fermented foods.
The differences in carbohydrate utilization ability between six rounds of Sauce-flavor Daqu
2023, Food Research International
Sauce-flavor Daqu is an important source of fermentation power in baijiu brewing. Revealing carbohydrate metabolism will help to explore the underlying reasons for the difference in fermentation performance of Daqu. In this study, metagenomic and metaproteomic technologies were performed to explore the carbohydrate metabolism network and its active functional microorganisms of Sauce-flavor Daqu. The sugar profile was analyzed using LC-MS to confirm the metabolic network. The results showed that 23 fungi and 5 bacteria were involved in carbohydrate metabolism. Starch metabolism, cellulose metabolism, and glucan metabolism were the main metabolic pathways, in which fungi especially Aspergillus were more involved than bacteria. Among these active microorganisms, Saccharomycopsis fibuligera, Aspergillus oryzae, Monascus purpureus, Byssochlamys spectabilis, Lichtheimia ramosa, Thermomyces lanuginosus, and Thermoascus aurantiacus were significant functional microorganisms with the ability to produce multiple enzymes. Lichtheimia ramosa, Lichtheimia corymbifera and Kroppenstedtia eburnea were biomarkers of Daqu in the first round, granting it a better liquefaction ability. β-amylase derived from wheat also played an important role in starch degradation, and the synergistic effect with α-amylase endowed Daqu with higher liquefaction power in the first two rounds. The results of this study are of great significance for the analysis of the mechanism of Daqu fermentation and provide a reliable theoretical basis for strengthening the fermentation performance of Daqu.
Spatiotemporal distribution of environmental microbiota in spontaneous fermentation workshop: The case of Chinese Baijiu
2022, Food Research International
The strong-flavor Baijiu (SFB) brewing workshop is a complex ecosystem with diverse microbiomes. As a potential source of microbiomes in fermentation, microbiota in the environmental microecology may affect the quality and flavor of SFB. Here, we report the collection of environmental microecological samples from three SFB workshops with different usage times (named 70a, 30a, and new, respectively). We used 16S rRNA and internal transcribed spacer (ITS) gene amplicon full-length sequencing to explore the microbial community structure in SFB. The SourceTracker tool was used to investigate links among fermentation samples, raw materials, and the environment and decipher the construction process in the workshop indoor environment. Lactobacillus acetotolerans was the most important bacterial genus in Zaopei after fermentation, whereas other types of samples exhibited different prokaryotic community structures. The composition of the fungal community was similar, with Saccharomycopsis fibuligera, Debaryomyces hansenii, Lichtheimia ramosa, Lichtheimia corymbifera, and Pichia kudriavzevii being the most abundant, and were detected in most samples. Further comparison of the microbiota in the workshop environment showed that the diversity of the microbiota in the indoor environment decreased, showing different clustering patterns under the influence of location. With increasing usage time, the contribution of deterministic processes to the assembly of the prokaryotic community increases, and the community structure tends to stabilize, exhibiting its own characteristics. SFB-fermenting resident functional fungi were the major components of the fungal community, and SourceTracker analysis also highlighted the contributions of Zaopei, Daqu, and tool surfaces as fungal sources. This study is the first to comprehensively monitor the microbial profile of the SFB production environment. This research can be extended to involve more complex spontaneous fermentation environment microbiota and has important implications for the control of spontaneous fermentation.
Flavor mystery of Chinese traditional fermented baijiu: The great contribution of ester compounds
2022, Food Chemistry
Citation Excerpt :
oryzae, Lic. ramosa, Mon. purpureus, Rhizomucor miehei, Rhizomucor pusillus, and Rhizopus microspores are the most frequently isolated ones (Du et al., 2019; Wang et al., 2011; Wang et al., 2017; Yan, Tong, & Guang, 2019; Zhang et al., 2014; Zheng, Yan et al., 2015) (Table S2). Candida tropicalis, Clavispora lusitaniae, Issatchenkia orientalis, Pichia kudriavzevii, Saccharomyces cerevisiae, Saccharomycopsis fibuligera, and Wickerhamomyces anomalus are the most frequently found yeast species (Yan, Tong, & Guang, 2019; Zheng, Yan et al., 2015) (Table S2).
Chinese traditional fermented baijiu is a famous alcoholic beverage with unique flavor. Despite its consumption for millennia, the flavor mystery behind baijiu is still unclear. Studies indicate that esters are the most important flavor substances, and bring health benefits. However, the aroma contribution and formation mechanism of esters still need to be clarified to reveal the flavor profile of baijiu. This review systematically summarizes all the 510 esters and finds 9 ethyl esters contribute greatly to the flavor of baijiu. The 508 different microbial species that have been identified affect the synthesis of esters through fatty acid and amino acid metabolism. The determination of minimum functional microbial groups and the analysis of their metabolic characteristics are crucial to reveal the mechanism of formation of baijiu flavor, and ensure the reproducible formation of flavor substances.

View all citing articles on Scopus

¹: These authors contributed equally to this work.

View full text

Proteomic and high-throughput analysis of protein expression and microbial diversity of microbes from 30- and 300-year pit muds of Chinese Luzhou-flavor liquor

Highlights

Abstract

Introduction

Section snippets

Sampling and location of pit mud

Comparison of protein extraction protocols from 30- and 300-year pit muds

Discussions

Conclusions

Acknowledgment

Journal of Proteomics

International Journal of Food Microbiology

Bioresource Technology

Food Research International

Soil Biology & Biochemistry

Food Control

Ecotoxicology and Enviromental Safety

Food Research International

Biochimie

Food Research International

Food Microbiology

Functional metaproteome analysis of protein extracts from contaminated soil and groundwater

ISME Journal

The nutrition of Clostridium kluyveri

Journal of Bacteriology

QIIME allows analysis of high-throughput community sequencing data

Nature Methods

Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample

Proceedings of the National Academy of Sciences of the United State of America

PCR-DGGE analysis on microbial communities in pit mud of cellars used for different periods of time

Journal of the Institute of Brewing

Application of 16S–23S rRNA ITS-AFLP fingerprint in the analysis of prokaryotic microbial diversity in liquor pit mud

Liquor Making Science & Technology

Characterization of eubacterial and archaeal community diversity in pit mud of Chinese Luzhou-flavor liquor by nested PCR-DGGE

World Journal of Microbiology and Biotechnology

Headspace solid phase microextraction and gas chromatography–olfactometry dilution analysis of young and aged Chinese “Yanghe Daqu” liquors

Journal of Agricultural and Food Chemistry

Ecological diversity and industrial application of methanogens

Chinese Journal of Applied & Environmental Biology

The genome of M. acetivorans reveals extensive metabolic and physiological diversity

Genome Research