Characteristics and Functions of Dominant Yeasts Together with Their Applications during Strong-Flavor Baijiu Brewing.

Yeasts are pivotal brewing microbes that are associated with the flavor and quality of Chinese baijiu, yet research on dominant yeasts in strong-flavor baijiu brewing remains limited. In this study, Saccharomyces cerevisiae, Pichia kudriavzevii, and Kazachstania bulderi were identified as predominated yeasts in strong-flavor baijiu. Each strain showed distinct characteristics in ethanol resistance, thermal tolerance, and lactic acid tolerance, severally. S. cerevisiae FJ1-2 excelled in ethanol and ethyl ester production, P. kudriavzevii FJ1-1 in ethyl acetate, and K. bulderi FJ1-3 in lactic acid generation. Subsequently, the reinforced Fuqu of each yeast were severally prepared for application in baijiu brewing to verify their functions. Results revealed that the relative abundance of fortified yeast in each group rose. Pichia, Kazachstania, and Saccharomyces emerged as the core microbe for each group, respectively, by co-occurrence network analysis, influencing the microbiota to regulate flavor substances. In short, P. kudriavzevii FJ1-1 enhanced ethyl acetate. K. bulderi FJ1-3 improved ethyl caproate production and decreased levels of ethyl acetate and higher alcohols by modulating yeast community between Pichia and Saccharomyces. This is a systematic endeavor to study the functions of yeasts of strong-flavor baijiu, providing a solid basis for improving baijiu quality.

Isolation, identification, and degradation mechanism by multi-omics of mesotrione-degrading Amycolatopsis nivea La24.

Mesotrione is a herbicide used in agricultural production; however, its stability and long-term residues pose ecological risks to soil health and subsequent crops. In this research, the strain Amycolatopsis nivea La24 was identified as capable of completely degrading 50 mg∙L-1 mesotrione within 48 h. It exhibited a broad adaptability to various environment and could degrade three sulfonylurea herbicides (nicosulfuron, chlorimuron-methyl, and cinosulfuron). Non-target metabonomic and mass spectrometry demonstrated that La24 strain broke down the mesotrione parent molecule by targeting the ß-diketone bond and nitro group, resulting in the production of five possible degradation products. The differentially expressed genes were significantly enriched in fatty acid degradation, amino acid metabolism, and other pathways, and the differentially metabolites in glutathione metabolism, arginine/proline metabolism, cysteine/methionine metabolism, and other pathways. Additionally, it was confirmed by heterologous expression that nitroreductase was directly involved in the mesotrione degradation, and NDMA-dependent methanol dehydrogenase would increase the resistance to mesotrione. Finally, the intracellular response of La24 during mesotrione degradation was proposed. This work provides insight for a comprehensive understanding of the mesotrione biodegradation mechanism, significantly expands the resources for pollutant degradation, and offers the potential for a more sustainable solution to address herbicide pollution in soil.

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.

Metagenome-assembled genome reveals species and functional composition of Jianghan chicken gut microbiota and isolation of Pediococcus acidilactic with probiotic properties.

BACKGROUND: Chickens are one of the most widely farmed animals worldwide and play a crucial role in meat and egg production. Gut microbiota is essential for chickens' health, disease, growth, and egg production. However, native chickens such as Jianghan chickens have better meat and egg production quality than centralized chickens, their intestinal microbial diversity is richer, and the potential gut microbial resources may bring health benefits to the host. RESULTS: The bacterial species composition in the gut microbiota of Jianghan chickens is similar to that of other chicken breeds, with Phocaeicola and Bacteroides being the most abundant bacterial genera. The LEfSe analysis revealed significant differences in species composition and functional profiles between samples from Jingzhou and the other three groups. Functional annotation indicated that the gut microbiota of Jianghan chickens were dominated by metabolic genes, with the highest number of genes related to carbohydrate metabolism. Several antibiotic resistance genes (ARGs) were found, and the composition of ARGs was similar to that of factory-farmed chickens, suggesting that antibiotics were widely present in the gut microbiota of Jianghan chickens. The resistance genes of Jianghan chickens are mainly carried by microorganisms of the Bacteroidota and Bacillota phylum. In addition, more than 829 isolates were selected from the microbiota of Jianghan chickens. Following three rounds of acid and bile tolerance experiments performed on all the isolated strains, it was determined that six strains of Pediococcus acidilactici exhibited consistent tolerance. Further experiments confirmed that three of these strains (A4, B9, and C2) held substantial probiotic potential, with P. acidilactici B9 displaying the highest probiotic potential. CONCLUSIONS: This study elucidates the composition of the intestinal microbiota and functional gene repertoire in Jianghan chickens. Despite the absence of antibiotic supplementation, the intestinal microbial community of Jianghan chickens still demonstrates a profile of antibiotic resistance genes similar to that of intensively reared chickens, suggesting resistance genes are prevalent in free-ranging poultry. Moreover, Jianghan and intensively reared chickens host major resistance genes differently, an aspect seldom explored between free-range and pastured chickens. Furthermore, among the 829 isolates, three strains of P. acidilatici exhibited strong probiotic potential. These findings provide insights into the unique gut microbiota of Jianghan chickens and highlight potential probiotic strains offering benefits to the host. Video Abstract.

Effect of simplified inoculum agent on performance and microbiome during cow manure-composting at industrial-scale.

A simplified inoculum agent, only comprising Bacillus subtilis and Aspergillus niger, was utilized for industrial-scale cow-manure composting to investigate its impact on composting performance and microbiome. Inoculants elevated the average and peak temperatures by up to 7 and 10 °C, respectively, during the thermophilic stage, reduced organic matter content, and raised germination index. Inoculation also extended the period of composting above 50 °C from 12 to 26 days. Sequencing unveiled significant shifts in microbial diversity, composition, and function. Aspergillus thrived during the mesophilic phase, potentially initiating composting, whereas Bacillus, Lysinibacillus, and Clostridium were enriched during the thermophilic stage. Metagenomic sequencing revealed an increased abundance of carbohydrate-active enzymes and glycometabolism-related genes responsible for lignocellulose degradation and heat generation after inoculation. These enriched microbes and functional genes contributed to organic matter degradation and temperature maintenance during thermophilic stage, expediting composting. This suggests the effectiveness of this simplified inoculum in industrial-level cow-manure composting.

Developing the Limosilactobacillus reuteri Chassis through an Endogenous Programmable Endonuclease-Based Genome Editing Tool.

Using genetically tractable probiotics to engineer live biotherapeutic products (LBPs) for disease treatment is urgently needed. Limosilactobacillus reuteri is an important vertebrate gut symbiont, which has great potential for developing LBPs. However, in L. reuteri, synthetic biology work is largely limited by the long editing cycle. In this study, we identified a subtype II-A CRISPR-Cas9 system in L. reuteri 03 and found the endogenous Cas9 (LrCas9) recognizing a broad protospacer-adjacent motif (PAM) sequence (3'-NDR; N = A, G, T, C; D = A, G, T; R = A, G). We reprogrammed the LrCas9 for efficient gene deletion (95.46%), point mutation (86.36%), large fragment deletion (40 kb), and gene integration (1743 bp, 73.9%), which uncovered the function of the repeated conserved domains in mucus-binding protein. Moreover, we analyzed the distribution of endogenous endonucleases in 304 strains of L. reuteri and found the existence of programmable endonucleases in 98.36% of L. reuteri strains suggesting the potential to reprogram endogenous endonucleases for genetic manipulation in the majority of L. reuteri strains. In conclusion, this study highlights the development of a new probiotic chassis based on endogenous endonucleases in L. reuteri 03, which paves the way for the development of genome editing tools for functional genetic studies in other L. reuteri. We believe that the development of an endogenous endonuclease-based genetic tool will greatly facilitate the construction of LBPs.

Effect of enzyme-assisted fermentation on quality, safety, and microbial community of black soldier fly larvae (Hermetia illucens L.) as a novel protein source.

Considering the significance and scarcity of quality protein, this study aims to obtain a novel safe protein source through fermenting the black soldier fly larvae (BSFL). Lactobacillus crispatus M1027 and Pichia kudriavzevii DHX19 were added as starters together with neutral protease for enzymolysis during fermentation. The results showed that the low pH value (from 6.60 to 3.99), generated by lactic acid accumulation, created an environment where the pathogen could hardly grow. During fermentation, the flavor compound ethyl acetate content reached up to 406.55 mg/L, and the melanization was effectively inhibited by the starters. The increase of trichloroacetic acid-soluble protein content (from 8.73 % to 17.96 %) contributed to improving the absorbability of product by animals after feeding. Notably, the contents of detrimental substances, including total volatile basic nitrogen and histamine, were both below specified limits after fermentation. Simultaneously, the malonic dialdehyde content remained stable during fermentation. Relative abundance of Lactobacillus and Pichia gradually increased and finally dominated in the culture during fermentation, accompanied by pathogens decline below detection limit (1.0 Log cfu/g). Moreover, there was a close relationship between the dynamics of physicochemical indices and microbial succession. Overall, our studies explored a new process to ferment the BSFL paste which would improve the quality and safety of fermented BSFL paste. This research provided theoretical support for fermented insect as a novel protein source.

Optimizing the scale-up production of fermented astragalus and its benefits to the performance and egg quality of laying hens.

Astragalus is a homologous medicine and food that benefits human beings and poultry rearing. Fermented astragalus (FA) is a valuable product obtained by fermentation, but its scale-up production requires optimization and expansion of solid-state fermentation (SSF). In this study, Lactobacillus pentosus Stm was screened as the most suitable LAB strain for fermenting astragalus due to its excellent capacity. After optimization and expansion of SSF, LAB count and lactic acid content reached 206 × 108 cfu/g and 15.0%, respectively. Meanwhile, the content of bioactive compounds in FA was significantly enhanced. Feeding experiments with laying hens indicated that supplementing FA in the diet significantly improved the performance and egg quality, as evidenced by reduced feed-to-egg ratio and egg cholesterol. This was due to the promotion of intestinal health by shifting intestinal microbiota. Therefore, this is a systematical endeavor of producing scaled-up FA with promising potential as a feed additive in the poultry breeding industry.

Uncovering acid resistance genes in lactic acid bacteria and impact of non-viable bacteria on bacterial community during Chinese strong-flavor baijiu fermentation.

Chinese strong-flavor baijiu (CSFB) brewing is a spontaneously solid-state fermentation process for approximately 60 days. Numerous microorganisms grow, die, and spark a series of metabolic reactions during fermentation. In this study, the microbial community and structure between total and viable bacteria in zaopei from the 5- and 20-year pits of CSFB are revealed by amplicon sequencing. Metagenome sequencing was applied to investigate acid resistance genes in Lactobacillus and predict carbohydrate active enzyme in zaopei. Besides, SourceTracker was conducted to expose bacterial sources. Results revealed that there was no significant difference in the bacterial community and structure between the total and viable bacteria; Lactobacillus was the most dominant bacterium in zaopei of two types of pits. Meanwhile, acid resistance genes argR, aspA, ilvE, gshA, DnaK, and cfa were genes that sustained Lactobacillus survival in the late stages of fermentation with high contents of acid and ethanol, and glycosyltransferases were identified as the predominated enzymes during the CSFB fermentation which catalyzed the process of lactic acid generation via Embden-Meyerhof-Parnas pathway and Hexose Monophosphate Pathway. Moreover, the environment contributed most bacteria to zaopei of the 5- and 20-year pits. These findings will provide a deeper understanding of the microbial community structure of viable and total bacteria and the reason for the dominance of Lactobacillus in the later stages of CSFB fermentation.

The Effect of the Microalgae Chlorella vulgaris on the Gut Microbiota of Juvenile Nile Tilapia (Oreochromis niloticus) Is Feeding-Time Dependent.

Chlorella vulgaris is one of the most commonly used microalgae in aquaculture feeds. It contains high concentrations of various kinds of nutritional elements that are involved in the physiological regulation of aquaculture animals. However, few studies have been conducted to illustrate their influence on the gut microbiota in fish. In this work, the gut microbiota of Nile tilapia (Oreochromis niloticus) (average weight is 6.64 g) was analyzed by high-throughput sequencing of the 16S rRNA gene after feeding with 0.5% and 2% C. vulgaris additives in diets for 15 and 30 days (average water temperature was 26 °C). We found that the impact of C. vulgaris on the gut microbiota of Nile tilapia was feeding-time dependent. Only by feeding for 30 days (not 15 days) did the addition of 2% C. vulgaris to diets significantly elevate the alpha diversity (Chao1, Faith pd, Shannon, Simpson, and the number of observed species) of the gut microbiota. Similarly, C. vulgaris exerted a significant effect on the beta diversity (Bray-Curtis similarity) of the gut microbiota after feeding for 30 days (not 15 days). During the 15-day feeding trial, LEfSe analysis showed that Paracoccus, Thiobacillus, Dechloromonas, and Desulfococcus were enriched under 2% C. vulgaris treatment. During the 30-day feeding trial, Afipia, Ochrobactrum, Polymorphum, Albidovulum, Pseudacidovorax, and Thiolamprovum were more abundant in 2% C. vulgaris-treated fish. C. vulgaris promoted the interaction of gut microbiota in juvenile Nile tilapia by increasing the abundance of Reyranella. Moreover, during the feeding time of 15 days, the gut microbes interacted more closely than those during the feeding time of 30 days. This work will be valuable for understanding how C. vulgaris in diets impacts the gut microbiota in fish.

Postbiotics from Pichia kudriavzevii promote intestinal health performance through regulation of Limosilactobacillus reuteri in weaned piglets.

Postbiotics are attractive as alternatives to antibiotics for use against post-weaning diarrhea. However, their beneficial mechanisms are largely unknown. In the current study, we first demonstrated that supplementation with 0.5% Pichia kudriavzevii FZ12 postbiotics in the diet significantly reduced diarrhea incidence, promoted growth performance, improved gut health performance, and significantly enriched beneficial bacteria, particularly Lactobacillus spp., in the intestines of weaned piglets. Importantly, we identified a heat- and proteinase K-sensitive component, cytochrome c, of the postbiotics that significantly promoted the growth and biofilm formation of Limosilactobacillus reuteri FP13. We demonstrated the importance of P. kudriavzevii FZ12 postbiotics in improving the intestinal health of a model animal and revealed that cytochrome c is one of the important components of yeast postbiotics. These findings may provide new insights into microbe-postbiotics interplay that can be applied to guidelines for dietary modulation to alleviate weaning-induced diarrhea.

Protease or Clostridium butyricum addition to a low-protein diet improves broiler growth performance.

Low-protein (LP) feeds are used in the poultry industry to combat the increasing consumption of protein resources and reduce environmental pollution caused by excessive nitrogen excretion. Dietary supplementation of protease or Clostridium butyricum increases the growth performance of broilers; however, it is unclear whether they counteract the negative effects of LP diets. The effects of protease and C. butyricum on growth performance, intestinal morphology, anti-oxidant capacity, anti-inflammatory response, and microbial community of broilers have not been studied extensively. Here, 450 healthy 1-day-old Cobb500 broilers were allocated to five groups, according to different diets: basal diet (Control); LP diet (LP; 2% less crude protein than the control); LP diet + 200 g/t HuPro protease (LPH); LP diet + 1.0 × 109 CFU/t C. butyricum (LPC); and basal diet + 200 g/t oxytetracycline (Antibiotic). Supplementing both C. butyricum and protease improved the growth performance of broilers. The supplementation of HuPro protease under low-protein conditions could achieve a breeding effect similar to that of the positive control (Antibiotic). Supplementing C. butyricum could maintain intestinal barrier function, alleviate the inflammatory response, and increase ileal and cecal short-chain fatty acid concentrations. Both C. butyricum and protease altered the bacterial diversity in the cecum, increased Bacteroidetes abundance, and resulted in higher abundance of Rikenellaceae RC9 gut spp. and lower abundance of Alistipes spp. in broilers. This study demonstrates the positive effects of proteases and C. butyricum on broilers and serves as a reference for the selection of appropriate supplementation for broilers in the poultry industry. KEY POINTS: • Low-protein diet had a negative effect on growth performance of broilers. • Protease significantly reduced feed conversion rate. • Clostridium butyricum had positive effects on broilers.

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.

Development and application of a rapid all-in-one plasmid CRISPR-Cas9 system for iterative genome editing in Bacillus subtilis.

BACKGROUND: Bacillus subtilis, an important industrial microorganism, is commonly used in the production of industrial enzymes. Genome modification is often necessary to improve the production performance of cell. The dual-plasmid CRISPR-Cas9 system suitable for iterative genome editing has been applied in Bacillus subtilis. However, it is limited by the selection of knockout genes, long editing cycle and instability. RESULTS: To address these problems, we constructed an all-in-one plasmid CRISPR-Cas9 system, which was suitable for iterative genome editing of B. subtilis. The PEG4000-assisted monomer plasmid ligation (PAMPL) method greatly improved the transformation efficiency of B. subtilis SCK6. Self-targeting sgRNArep transcription was tightly controlled by rigorous promoter PacoR, which could induce the elimination of plasmids after genome editing and prepare for next round of genome editing. Our system achieved 100% efficiency for single gene deletions and point mutations, 96% efficiency for gene insertions, and at least 90% efficiency for plasmid curing. As a proof of concept, two extracellular protease genes epr and bpr were continuously knocked out using this system, and it only took 2.5 days to complete one round of genome editing. The engineering strain was used to express Douchi fibrinolytic enzyme DFE27, and its extracellular enzyme activity reached 159.5 FU/mL. CONCLUSIONS: We developed and applied a rapid all-in-one plasmid CRISPR-Cas9 system for iterative genome editing in B. subtilis, which required only one plasmid transformation and curing, and accelerated the cycle of genome editing. To the best of our knowledge, this is the rapidest iterative genome editing system for B. subtilis. We hope that the system can be used to reconstruct the B. subtilis cell factory for the production of various biological molecules.

Functional Comparison of Clostridium butyricum and Sodium Butyrate Supplementation on Growth, Intestinal Health, and the Anti-inflammatory Response of Broilers.

Butyrate has been reported to promote proliferation of colonic epithelial cells and maintain intestinal barrier integrity in broilers. Although supplementation of Clostridium butyricum and sodium butyrate have been shown to confer benefits on broilers, their effects and mechanisms have not been compared. In this study, C. butyricum and sodium butyrate were added into the basal diet of broilers and their effects on growth performance, intestinal health, and anti-inflammatory response were analyzed. It was found that both C. butyricum and sodium butyrate showed good probiotic effects on broilers. Their effects on growth rate and expression of inflammation related genes were superior to that of the antibiotic oxytetracycline. Besides, the two dietary supplements improved intestinal structure integrity and secretion of inflammatory cytokines, whereas the antibiotic had negative effects. Comparison of the two supplements revealed that sodium butyrate more effectively improved the growth and intestinal structure of broilers than C. butyricum. On the contrary, C. butyricum was superior to sodium butyrate in promoting tight junction protein expression and anti-inflammatory response. In summary, this study demonstrates the positive effects of C. butyricum and sodium butyrate on broilers, and will serve as a reference for selection of appropriate butyrate supplementation for broilers in the breeding industry.

Unraveling the microbial community and succession during zha-chili fermentation and their relationships with flavor formation.

Zha-chili is a popular traditional fermented food among people in central and southwest China for its nice flavor. It is produced from grain and fresh chili undergoing spontaneously anaerobic fermentation. In this study, the high-throughput sequencing (HTS), high-performance liquid chromatography (HPLC), and head-space solid-phase microextraction and gas chromatography-mass spectrometry (HS-SPME-GC-MS) were applied to systematically study the dynamic of microbial community and flavor substances during zha-chili fermentation. Simultaneously, the physicochemical factors were investigated to decode the core factors affecting the microbial succession. Results showed that Lactobacillus and Kazachstania were the most dominant bacterium and fungus, respectively, and lactic acid bacteria (LAB), acetic acid bacteria (AAB), and yeasts dominated the whole fermentation. Besides, acidity, ethanol, and temperature were the core factors that shifted the microbial succession. Herein, the acidity and ethanol drove the bacterial succession from Weissella to Lactobacillus, then temperature affected the succession that Lactobacillus was partially replaced by Acetobacter. Meanwhile, acidity and ethanol actuated the fungal succession that Gibberella was superseded by Kazachstania. Moreover, Lactobacillus, Acetobacter, Kazachstania, Wickerhamomyces, Kluyveromyces, and Cyberlindnera were the core microbes associated with the formation of flavor substances including lactic acid, acetic acid, ethanol, ethyl lactate, ethyl acetate, and umami free amino acids. These findings will be helpful to unravel the formation of flavor substances based on microbial community and succession during zha-chili fermentation, providing a solid foundation for the process optimization and quality control of zha-chili.

Unraveling the composition and succession of microbial community and its relationship to flavor substances during Xin-flavor baijiu brewing.

A novel baijiu type, Xin-flavor, has been developed via combining Qu and craftsmanship from three famous baijiu types including light-flavor, sauce-flavor, and strong-flavor. However, the microbial community and its relationship to physicochemical factors and flavor substances were little known during the three-stage brewing processes including saccharification, stacking, and fermentation. In this study, 21 phyla, 407 genera, and 615 species distributed in bacteria and fungi were identified via high-throughput sequencing (HTS). For bacterial community, the dominant bacteria at saccharification stage were Weissella and Leuconostoc, which were superseded by Acetobacter and Gluconobacter at stacking stage, then quickly replaced by Lactobacillus at fermentation stage. For fungal community, Rhizopus, dominant at saccharification stage, was partially replaced by Thermomyces at stacking stage, then was completely superseded by Thermoascus and three yeasts (Saccharomyces, Kazachstania and Apiotrichum) at fermentation stage. The moisture, ethanol, and acidity, as key factors affecting the microbial community, explained this microbial succession. Interestingly, Apiotrichum was firstly detected in fermented grains. Furthermore, the rapid accumulation of ethanol mainly occurred in the first 10 days of fermentation stage, and highly correlated with Saccharomyces, while Kazachstania and Apiotrichum were related to the formation of flavor substances (acetic acid, lactic acid, caproic acid, ethyl lactate and ethyl caproate). Therefore, this study provides fundamental basis for the rational control of baijiu production and improving the craft and quality of Xin-flavor baijiu.

Comprehensive utilization of palm kernel cake for producing mannose and manno-oligosaccharide mixture and yeast culture.

Palm kernel cake (PKC) is an agricultural waste derived from palm kernel oil manufacturing, and its production is increasing year by year. It is very urgent to process this agricultural waste in an environmentally friendly way. Here, PKC was used to produce mannose and manno-oligosaccharides mixture (MMOM) and yeast culture (YC) through enzymolysis and solid-state fermentation (SSF). In enzymolysis, five factors were optimized separately and a response surface methodology analysis was performed. Then, enzymolysis of PKC was carried out at the optimal condition, and the extraction efficiency of mannose and manno-oligosaccharides reached 68.90% with mannose concentration achieving 60.27 g/L. After enzymolysis, the enzymatic hydrolysate was dried by spray drying, and the contents of MMOM reached 42.9%. In SSF, the enzymolysis residues were utilized with inoculating Saccharomyces cerevisiae for yielding YC. After optimization, the cells number of S. cerevisiae reached 2.08 × 109 cells/g and the crude protein content was increased to 27.31%. Therefore, a novel approach to produce feed additives, including MMOM and YC, with high value by comprehensive utilization of PKC was proposed, which has good application prospects in the breeding industry. KEY POINTS: • New idea for the comprehensive utilization of PKC is proposed. • PKC was used to produce mannose and mannan-oligosaccharides mixture (MMOM) by enzymolysis and spray drying. • The enzymolysis residues were reused via SSF for producing yeast culture (YC).

Detection of viable and total fungal community in zaopei of Chinese strong-flavor baijiu using PMA combined with qPCR and HTS based on ITS2 region.

BACKGROUND: Chinese strong-flavor baijiu (CSFB), one of the three major baijiu types, is the most popular baijiu type among consumers in China. A variety of microbes are involved in metabolizing raw materials to produce ethanol and flavor substances during fermentation, which fundamentally determined the quality of baijiu. It is of great importance to study microbial community of fermented grains (zaopei) during baijiu brewing process for improving its quality. In this study, we firstly used propidium monoazide (PMA) to treat zaopei samples from 5-year pit and 20-year pit for removing the interference of non-viable fungi, and analyzed the diversity of total fungi and viable fungi by quantitative PCR (qPCR) and high-throughput sequencing (HTS) based on ITS2 gene. RESULTS: The results showed that total fungi and viable fungi displayed no significant differences at OTU, phylum, or genus levels during fermentation within two kinds of pits. A total of 6 phyla, 19 classes, and 118 genera in fungi were found based on OTUs annotation in zaopei samples from 5-year pit and 20-year pit. Besides, non-viable fungi had little effect on the fungal community diversity during the fermentation cycle. It was found that the most dominant viable fungi belonged to Saccharomyces, Kazachstania, Naumovozyma, and Trichosporon, and Naumovozyma was firstly detected in zaopei samples of CSFB. Moreover, based on the variation of flavor substances in zaopei samples, the quality of CSFB produced from older pit was better than that produced from younger pit. CONCLUSION: The non-viable fungi had little effect on the fungal diversity, structure, and relative abundance in zaopei samples of CSFB, and Naumovozyma was firstly detected in zaopei samples of CSFB. Our findings can be applied as guidance for improving the quality and stability of CSFB.