Deciphering the microbial succession and color formation mechanism of "green-covering and red-heart" Guanyin Tuqu.

"Green-covering and red-heart" Guanyin Tuqu (GRTQ), as a type of special fermentation starter, is characterized by the "green-covering" formed on the surface of Guanyin Tuqu (SQ) and the "red-heart" in the center of Guanyin Tuqu (CQ). However, the mechanisms that promote temporal succession in the GRTQ microbial ecology and the formation of "green-covering and red-heart" characteristics remain unclear. Herein, we correlated the temporal profiles of microbial community succession with the main environmental variables (temperature, moisture, and acidity) and spatial position (center and surface) in GRTQ throughout fermentation. According to the results of high-throughput sequencing and culture-dependent methods, the microbial communities in the CQ and SQ demonstrated functional complementarity. For instance, the bacterial richness index of the CQ was greater than that of SQ, and the fungal richness index of the SQ was greater than that of CQ at the later stage of fermentation. Furthermore, Saccharomycopsis, Saccharomyces, Aspergillus, Monascus, Lactobacillus, Bacillus, Rhodanobacter, and Chitinophaga were identified as the dominant microorganisms in the center, while the surface was represented by Saccharomycopsis, Aspergillus, Monascus, Lactobacillus, Acetobacter, and Weissella. By revealing the physiological characteristics of core microorganisms at different spatial positions of GRTQ, such as Aspergillus clavatus and Monascus purpureus, as well as their interactions with environmental factors, we elucidated the color formation mechanism behind the phenomenon of "green" outside and "red" inside. This study provides fundamental information support for optimizing the production process of GRTQ.

Effects of sorghum varieties on microbial communities and volatile compounds in the fermentation of light-flavor Baijiu.

Light-flavor Baijiu (LFB) fermentation is a representative spontaneous mixed-culture solid-state fermentation process in which sorghum is used as the raw material. Raw materials and microorganisms are crucial to the flavor formation and quality of LFB. However, the microbial and physicochemical dynamics of different sorghum varieties during LFB fermentation, as well as their impact on flavor compounds are still largely unknown. Herein, PacBio single-molecule real-time (SMRT) sequencing and headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry (HS-SPME-GC-MS) were applied to investigate microbial community succession and volatile flavor formation in glutinous/non-glutinous sorghum-based fermented grains during LFB fermentation. Fermented grains made of glutinous sorghum Liangnuo No. 1 (GLN) had higher bacterial α-diversity and lower fungal α-diversity than those with fermented grains prepared with non-glutinous red sorghum (NRS) (p < 0.05). The dominant microbial species were Saccharomyces cerevisiae, Acetobacter pasteurinus, and Lactobacillus helveticus, the latter two of which were the predominant bacteria observed at the end of fermentation in GLN and NRS, respectively. Moisture content and reducing sugar had a more significant impact on the microorganisms in GLN, while amino acid nitrogen, total free amino acids, and residual starch were the main driving factors driving the microbial community in NRS. The correlation network and discriminant analysis indicated that a relatively high content of 4-vinylguaiacol showed a significant positive association with significant differential microbial species in GLN. These results provided valuable insights for improving the quality of LFB.

Species-level understanding of the bacterial community in Daqu based on full-length 16S rRNA gene sequences.

Daqu is used as the fermentation starter of Baijiu and contributes diversified functional microbes for saccharifying grains and converting sugars into ethanol and aroma components in Baijiu products. Daqu is mainly classified into three types, namely low (LTD), medium (MTD) and high (HTD) temperature Daqu, according to the highest temperatures reached in their fermentation processes. In this study, we used the PacBio small-molecule real-time (SMRT) sequencing technology to determine the full-length 16 S rRNA gene sequences from the metagenomes of 296 samples of different types of Daqu collected from ten provinces in China, and revealed the bacterial diversity at the species level in the Daqu samples. We totally identified 310 bacteria species, including 78 highly abundant species (with a relative abundance >0.1% each) which accounted for 91.90% of the reads from all the Daqu samples. We also recognized the differentially enriched bacterial species in different types of Daqu, and in the Daqu samples with the same type but from different provinces. Specifically, Lactobacillales, Enterobacterales and Bacillaceae were significantly enriched in the LTD, MTD and HTD groups, respectively. The potential co-existence and exclusion relationships among the bacteria species involved in all the Daqu samples and in the LTD, MTD and HTD samples from a specific region were also identified. These results provide a better understanding of the bacterial diversity in different types of Daqu at the species level.

Developing defined starter culture for reproducible profile of flavour compound in Chinese xiaoqu baijiu fermentation.

Defined starter cultures, containing selected microbes could reduce the complexity of natural starter, are beneficial for controllable food fermentations. However, there are challenges in identifying key microbiota and constructing synthetic microbiota for traditional food fermentations. Here, we aimed to develop a defined starter culture for reproducible profile of flavour compounds, using Chinese Xiaoqu Baijiu fermentation as a case. We classified all microbes into 4 modules using weighted correlation network analysis. Module 3 presented significant correlations with flavour compounds (P < 0.05) and the highest gene abundance related with flavour compound production. 13 dominant species in module 3 were selected for mixed culture fermentation, and each species was individually deleted to analyse the effect on flavour compound production. Ten species, presenting significant effects (P < 0.05) on flavour compound production, were selected for developing the starter culture, including Rhizopus oryzae, Rhizopus microsporus, Saccharomyces cerevisiae, Pichia kudriavzevii, Wickerhamomyces anomalus, Lactobacillus acetotolerans, Levilactobacillus brevis, Weissella paramesenteroides, Pediococcus acidilactici, and Leuconostoc pseudomesenteroides. After optimising the structure of the starter culture, the profile similarity of flavour compounds produced by the starter culture reached 81.88% with that by the natural starter. This work indicated feasibility of reproducible profile of flavour compounds with defined starter culture for food fermentations.

Unveiling the microbiota of sauce-flavor Daqu and its relationships with flavors and color during maturation.

This study investigated the microbial community in three-color sauce-flavor Daqu (black, yellow, and white) throughout their maturation processes, together with their physicochemical factors, culturable microbes, flavor components, and fermenting vitalities. Results from high-throughput sequencing revealed distinct microbial diversity, with more pronounced variations in bacterial community than in fungal community. Firmicutes and Ascomycota emerged as the most dominant bacterial and fungal phyla, respectively, during maturation. Genus-level analysis identified Kroppenstedia, Virgibacillus, and Bacillus as dominant bacteria in black Daqu, yellow Daqu, and white Daqu, severally, while Thermoascus was shared as the core dominant fungi for these Daqu. Physicochemical factors, particularly acidity, were found to exert a significant impact on microbial community. Kroppenstedtia was the key bacteria influencing the color formation of these Daqu. Furthermore, correlations between dominant microbes and flavor compounds highlighted their role in Daqu quality. Molds (Aspergillus, Rhizomucor, and Rhizopus), excepting Bacillus, played a crucial role in the formation of pyrazine compounds. Consequently, this study offers innovative insights into the microbial perspectives on color and pyrazine formation, establishing a groundwork for future mechanized Daqu production and quality control of sauce-flavor baijiu.

Screening of ß -damascenone-producing strains in light-flavor Baijiu and its production optimization via response surface methodology.

As a C13-norisoprenoid aroma substance, ß-damascenone is a highly important aromatic compound and an active constituent. The purpose of this study was to investigate the change law of ß-damascenone during the light-flavor Baijiu brewing process, and screen the indigenous microbial strains that produce this compound and optimize fermentation parameters for improving ß-damascenone production using a statistical approach. In this project, Wickerhamomyces anomalus YWB-1 exhibited the highest producing activity of ß-damascenone. Fermentation conditions were optimized for ß-damascenone production using a one-factor-at-a-time (OFAT) approach. A Plackett-Burman design was subsequently adopted to assess the effects of initial pH, incubation temperature, inoculum size, fermentation period, and original Brix degree. Analysis of variance (ANOVA) showed that the correlation coefficient (R 2) of the executive model was 0.9795, and this value was significant (p < 0.05). Three significant variables were optimized at three different coded levels using a Box-Behnken design (BBD) of response surface methodology (RSM). Here, 7.25 µg/L ß-damascenone was obtained under the following optimum conditions: initial pH of 3.31, original Brix degree of 10.53%, and fermentation period of 52.13 h. The yield was increased 3.02-fold compared with that obtained under unoptimized conditions. This information is conducive to the control of flavor production by regulating variable parameters in Baijiu fermentation.

Tetramethylpyrazine in Chinese baijiu: Presence, analysis, formation, and regulation.

Traditional Chinese fermented baijiu is one of the six major distilled spirits consumed worldwide. It plays an important role in people's daily life and social interactions because of its taste, nutritional value, and various health functions. Tetramethylpyrazine (TMP), also known as ligustrazine, is not only an important compound related to the flavor of Chinese baijiu but also has special pharmacological effects. It gives the baijiu a nutty and baked aroma and provides baijiu with important health benefits. Recently, the nutritional, drinking, and health aspects of baijiu have attracted significant attention. Therefore, the study of TMP in baijiu is an important aspect of baijiu health research. This mini novel review summarizes the formation mechanism of TMP, along with the current research progress, analytical methods used, and regulation strategies associated with TMP in Chinese baijiu in recent years.

Study on microbial community of "green-covering" Tuqu and the effect of fortified autochthonous Monascus purpureus on the flavor components of light-aroma-type Baijiu.

"Green-covering" Tuqu (TQ), as one of Xiaoqu, is a special fermentative starter (also known as Jiuqu in Chinese) that originated in southern China and is characterized by a layer of green mold covering (Aspergillus clavatus) the surface and (sometimes) with a red heart. It plays a vital role in producing light-aroma-type Baijiu (LATB). However, to date, the microbiota that causes red heart of TQ remain largely unexplored, and it is still unclear how these microbiota influence on the quality of LATB. In this study, two types of TQ, one with a red heart (RH) and another with a non-red heart (NRH), were investigated by high throughput sequencing (HTS) and directional screening of culture-dependent methods. The obtained results revealed the differences in the microbial communities of different TQ and led to the isolation of two species of Monascus. Interestingly, the results of high performance liquid chromatography (HPLC) detection showed that citrinin was not detected, indicating that Monascus isolated from TQ was no safety risk, and the contents of gamma-aminobutyric acid in the fermented grains of RH were higher than that of NRH during the fermentation. Selecting the superior autochthonous Monascus (M1) isolated from the TQ to reinoculate into the TQ-making process, established a stable method for producing the experimental "red heart" Tuqu (ERH), which confirmed that the cause of "red heart" was the growth of Monascus strains. After the lab-scale production test, ERH increased ethyl ester production and reduced higher alcohols production. In addition, Monascus had an inhibitory effect on the growth of Saccharomyces and Aspergillus. This study provides the safe, health-beneficial, and superior fermentation strains and strategies for improving the quality of TQ and LATB.

Microbial succession and its effect on key aroma components during light-aroma-type Xiaoqu Baijiu brewing process.

Light-aroma-type Baijiu is a Chinese distilled alcoholic beverage produced from fermented sorghum. Microbial composition and dynamics during Baijiu production have a great influence on the flavor and quality of Chinese Baijiu. However, the microbial changes that occur during brewing of Xiaoqu Baijiu are poorly understood. In this study, the microbial composition of light-aroma-type Xiaoqu Baijiu at the saccharification and fermentation stages was investigated to explore microbial dynamics and their effects on aroma components using high-throughput sequencing and gas chromatography-flame ionization detection (GC-FID). Rhizopus, Pichia, Wickerhamomyces, Saccharomyces, Acinetobacter, Lactobacillus, and Weissella constituted the core microbes for Xiaoqu Baijiu production. Microbial succession during brewing could be divided into two phases: at the saccharification and early fermentation stages (F-0d to F-4d), Rhizopus and Acinetobacter were identified as the predominant microbes, accounting for 78.2-90.8% and 53.9-89.5% of the fungal and bacterial communities, respectively, whereas at the middle and late stages of fermentation (F-5d to F-14d), the abundance of Pichia, Wickerhamomyces, Saccharomyces, and Lactobacillus increased. Redundancy analysis (RDA) and Mantel tests indicated that the water, amino acid nitrogen, acid, and reducing sugar contents were significantly correlated with the fungal and bacterial communities in grains (p < 0.05). Pichia, Rhizopus, Saccharomyces, and Wickerhamomyces, especially Saccharomyces, were closely related to the contents of major alcohols, esters and aldehydes, and these microbes had an important functional role in the formation of Xiaoqu Baijiu flavor. This work provides insights into the microbial succession that occurs during brewing of light-aroma-type Xiaoqu Baijiu and the microbial contribution to flavor, which have potential for optimizing production and enhancing the flavor of Baijiu.

Effects of ultra-long fermentation time on the microbial community and flavor components of light-flavor Xiaoqu Baijiu based on fermentation tanks.

Microbial structure and succession of fermented grains play a significant role in Baijiu's flavor and quality. In this study, high-throughput sequencing (HTS) coupled with headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) were used to analyze the microbial community structures and flavor components in the fermented grains at the end of fermentation from different fermentation time of light-flavor Xiaoqu Baijiu. HTS results showed that Lactobacillus acetotolerans, Lactobacillus helveticus, Lactobacillus buchneri, Wickerhamomyces, Saccharomyces, and Condenascus were identified as the dominant microbes, but Lactobacillus (96.28%) exhibited obvious advantages at the end of ultra-long fermentation time (day 98). HS-SPME-GC-MS analysis revealed that esters and alcohols had the most abundance in fermented grains of day 98, containing high concentrations of ethyl acetate, diethyl succinate, phenylethyl alcohol, isoamyl alcohol, and n-propanol, which were related to the succession of Lactobacillus and yeast communities. Interestingly, the content of n-propanol in the ultra-long fermentation time samples (day 98) was 6 times of that in normal fermented grains (day 14), which may be caused by higher abundance of Lactobacillus in day 98 samples. Monte Carlo permutation test showed residual starch, acidity, and amino nitrogen (p < 0.05) were important factors affecting the microbial community. Together, these results shed light on the physicochemical changes, microbial dynamics, and key flavor components of fermented grains at the end of fermentation from different fermentation time and provide a strategy for further improvement of Baijiu quality.

Comparison of Potent Odorants in Traditional and Modern Types of Chinese Xiaoqu Liquor (Baijiu) Based on Odor Activity Values and Multivariate Analyses.

Predominant odorants in modern and traditional types of Chinese xiaoqu liquor (Baijiu) were identified and compared by the combined use of gas chromatography-olfactometry, odor activity values (OAVs), and multivariate analyses. A total of 79 aroma compounds were identified in a typical modern type xiaoqu Baijiu (M) and a typical traditional type xiaoqu Baijiu (T), 42 of them had OAV > 1 in both M and T samples. The main differences between the two samples were obtained for the concentration of 23 aroma-active compounds. A total of 22 samples made by different brewing processes were analyzed to confirm the differences. Partial least squares discriminant analysis confirmed that 20 compounds could be used as potential markers for discrimination between modern type xiaoqu Baijiu and traditional type xiaoqu Baijiu. Their difference in content is between 1.5 and 17.9 times for modern type xiaoqu Baijiu and traditional type xiaoqu Baijiu. The results showed the aroma characteristics of modern and traditional type xiaoqu Baijiu clearly and comprehensively, which will provide guidance for modern Baijiu quality control and evaluation.

Deciphering Succession and Assembly Patterns of Microbial Communities in a Two-Stage Solid-State Fermentation System.

Although the importance of microbiota in the natural environment and in industrial production has been widely recognized, little is known about the formation and succession patterns of the microbial community, particularly secondary succession after disturbance. Here, we choose the Xiaoqu liquor brewing process as an experimental model in which sorghum grains were first aerobically saccharified and then anaerobically fermented after being stirred and acidified to explore multistage community succession patterns. We analyzed microbial composition, physicochemical factors, and metabolites of brewing grains inoculated with two different starters, pure starter and traditional starter, respectively. Two groups showed similar succession patterns where the saccharification microbiota was mainly derived from starters, while environmental microorganisms, mainly Lactobacillaceae and Saccharomyces, dominated the fermentation microbiota regardless of the original saccharification community composition. Species replacement shaped the bacterial community, while species replacement and loss both contributed to fungal community succession in both groups. Grain acidification and hypoxia led to the succession of bacterial and fungal communities during fermentation, respectively. Despite inoculation with starters containing different microorganisms, similar microbial communities during the fermentation stage of the two groups exhibited similar metabolite composition. However, higher abundance of Rhizopus in the saccharification of the pure starter group led to more alcohols, while higher abundance of Monascus and Saccharomycopsis in the traditional starter group promoted acid and ester metabolism. These results revealed the microbial succession patterns of two-stage liquor brewing and its influence on flavor metabolism, which could be used to regulate the microbial community in food fermentation to further promote the modernization of the fermented food industry. IMPORTANCE Revealing formation and assembly mechanisms of microbiota can help us to understand and further regulate its roles in the ecosystems. The Xiaoqu liquor brewing system is a tractable microbial ecosystem with low complexity. This two-stage microbial ecosystem can be used as an experimental model to analyze the multistage temporal succession pattern of microbial communities. Our results demonstrated the dynamic composition and succession pattern of a microbial community in the two-stage liquor brewing system. The results also revealed the microbial origins determining community composition, the ecological processes dominating microbial community succession patterns, the determinants affecting microbial community successions, and the effect of microbial community changes on metabolite synthesis. Overall, our study not only provides an insight into multistage succession patterns of microbial communities in liquor brewing systems but also provides reference for optimizing the quality of fermented products, which will be helpful to understand the succession patterns of microbial communities in other natural ecosystems.

Modeling and Regulation of Higher Alcohol Production through the Combined Effects of the C/N Ratio and Microbial Interaction.

Too large of a higher alcohol content has negative effects on the liquor taste and health. Revealing the key microbes and their key driving forces is essential to regulate the higher alcohol content in spontaneous liquor fermentation. Herein, we used high-throughput sequencing associated with a multivariate statistical algorithm to reveal the contributing microbes for higher alcohol production in Chinese light-aroma-type liquor and identified that Saccharomyces and Pichia were the main contributors. In addition, the C/N ratio and microbial interaction were found to significantly affect the production of higher alcohols. Herein, we used response surface methodology to establish a predictive model for higher alcohol production with the regulating factors, and the content of total higher alcohols decreased significantly from 328.80 ± 24.83 to 114.88 ± 5.02 mg/L with the optimized levels of the regulators. This work would facilitate the control of flavor production via regulating microbial communities in food fermentation.

Hawaiienols A-D, Highly Oxygenated p-Terphenyls from an Insect-Associated Fungus, Paraconiothyrium hawaiiense.

Four new highly oxygenated p-terphenyls, hawaiienols A-D (1-4), have been isolated from cultures of Paraconiothyrium hawaiiense, a fungus associated with the Septobasidium-infected insect Diaspidiotus sp.; their structures were elucidated primarily by NMR experiments. The absolute configurations of 1 and 2-4 were assigned by single-crystal X-ray diffraction analysis using Cu Kα radiation and via electronic circular dichroism calculations, respectively. Compound 1 incorporated the first naturally occurring 4,7-dioxatricyclo[3.2.1.03,6]octane unit in its p-terphenyl skeleton and showed cytotoxicity toward six human tumor cell lines.

Two new ramulosin derivatives from the entomogenous fungus Truncatella angustata.

Two new ramulosin derivatives, 7α-hydroxy-8-dihydroramulosin (1) and 7-ketoramulosin (2), along with three known metabolites, (+)-ramulosin (3), 6-hydroxyramulosin (4), and 8-dihydroramulosin (5), were isolated from the crude extract of Truncatella angustata, an entomogenous fungus isolated from the Septobasidium-infected insect Aspidiotus sp. The structures of 1 and 2 were elucidated by nuclear magnetic resonance experiments, and 1 was further confirmed by X-ray crystallography. The absolute configuration of 1 was assigned by single-crystal X-ray diffraction analysis using Cu Kα radiation, whereas that of 2 was determined by electronic circular dichroism (ECD) calculations. Compounds 1-5 were tested for cytotoxicity against four human carcinoma cell lines, HeLa, A549, MCF-7, and T24. Compound 4 showed weak cytotoxic effects against A549 and T24.

Four new tetramic acid and one new furanone derivatives from the plant endophytic fungus Neopestalotiopsis sp.

Four new tetramic acid analogues neopestalotins A-D (1-4), one new furanone derivative neopestalotin E (6), and the known compound hymenosetin have been isolated from the solid cultures of the plant endophytic fungus Neopestalotiopsis sp. The structures of the new compounds were determined mainly by nuclear magnetic resonance (NMR) experiments. The absolute configurations of 1 and 2 were assigned by circular dichroism (CD) data, whereas those of 3 and 4 were deduced by a combination of CD and heteronuclear long range coupling (HETLOC) data. Compound 2 showed modest antimicrobial activity against the Gram-positive bacteria, Bacillus subtilis, Staphylococcus aureus col, and Streptococcus pneumoniae.

Hawaiinolides E-G, cytotoxic cassane and cleistanthane diterpenoids from the entomogenous fungus Paraconiothyrium hawaiiense.

Hawaiinolides E-G (1-3), three additional new secondary metabolites including two cassane (1 and 2) types of diterpene lactones and one cleistanthane (3) diterpenoid, were isolated from the scale-up fermentation extract of Paraconiothyrium hawaiiense, an entomogenous fungus isolated from the Septobasidium-infected insect Diaspidiotus sp. The structures of 1-3 were elucidated by nuclear magnetic resonance experiments, and 1 and 3 were further confirmed by X-ray crystallography. The absolute configurations of 1 and 3 were assigned by single-crystal X-ray diffraction analysis using Cu Kα radiation, whereas that of 2 was deduced via the circular dichroism data. Compound 1 showed significant cytotoxic effects against the human tumor cell line T24, with an IC50 value (9.32 µM) comparable to that of the positive control cisplatin.

Trichocladinols I-K, oxatricyclic and oxabicyclic polyketides from Trichocladium opacum.

Three new polyketides, trichocladinols I-K (1-3) with oxabicyclic (1 and 3) and oxatricyclic (2) skeletons, together with three knovwnl metabolites, massarilactone G (4), massarigenin D (5), and rosigenin (6), were isolated from the solid-substrate fermentation cultures of the ascomycete fungus Trichocladium opacum. The structures of the new compounds were elucidated primarily by analysis of NMR data. The absolute configurations of 1-3 were deduced from circular dichroism (CD) data. Compounds 1-3 were tested for cytotoxicity against five human carcinoma cell lines, HeLa, A549, MCF-7, HCT116, and T24. Compound I showed a weak cytotoxic effect against HeLa cells, but 2 and 3 were inactive.

Cytotoxic cleistanthane and cassane diterpenoids from the entomogenous fungus Paraconiothyrium hawaiiense.

Hawaiinolides A-D (1-4), four new secondary metabolites including three cleistanthane (1, 3, and 4) and one cassane (2) type of diterpene lactones, were isolated from the crude extract of Paraconiothyrium hawaiiense, a fungus entomogenous to the Septobasidium-infected insect Diaspidiotus sp. The structures of 1-4 were elucidated by nuclear magnetic resonance experiments, and 1 and 3 were further confirmed by X-ray crystallography. The absolute configuration of 1 was assigned via single-crystal X-ray diffraction analysis using Cu Kα radiation, whereas that of 2-4 was deduced via the circular dichroism data. Compound 1 showed significant cytotoxicity against a small panel of five human tumor cell lines, A549, T24, HeLa, HCT116, and MCF-7.

Exserolides A-F, new isocoumarin derivatives from the plant endophytic fungus Exserohilum sp.

Six new isocoumarin derivatives, exserolides A-F (1-6), were isolated from solid cultures of the plant endophytic fungus Exserohilum sp., together with four known metabolites (7-10). The structures of 1-6 were elucidated primarily by NMR experiments. The absolute configuration of the C-3 methine carbon in 1-5 was deduced via the circular dichroism data, whereas that of the 1,3-diol moiety in 6 was assigned from the (1)H NMR data of its (R)- and (S)-MTPA diesters. Compounds 3 and 9 showed antifungal activity against the plant pathogen Fusarium oxysporum, whereas 6 displayed significant inhibitory effects against a small panel of bacteria.