Characterisation of aroma compounds, sensory characteristics, and bioactive components of a new type of huangjiu fermented with Chinese wild rice (Zizania latifolia).

Chinese wild rice (CWR) is a nutritious and healthy whole grain, worth developing. To develop and use its value, a new type of huangjiu was brewed with CWR, and the flavour characteristics, sensory quality, functional and bioactive components were evaluated. CWR (67 flavour substances) and glutinous rice (GR)-CWR huangjiu (62 flavour substances) had a better flavour than GR huangjiu (54 flavour substances), and the overall style of GR-CWR huangjiu was more skewed towards GR. The fruity, honey, caramel-like, herb and smoky aroma attributes of CWR huangjiu were higher than those of GR huangjiu (P < 0.05), while only the alcoholic was weaker (P < 0.05) due to the lower alcohol content. The huangjiu brewed using CWR had a better taste than that brewed using only GR. Furthermore, CWR huangjiu had the highest content of total dietary fiber (732.0 ± 15.2 mg/100 g), followed by GR-CWR (307.0 ± 8.5 mg/100 g), and GR (127.0 ± 2.3 mg/100 g). CWR huangjiu also had the highest total phenolic compounds (3.32 ± 0.05 mg/100 g/%vol) and total saponins (2.46 ± 0.03 mg/100 g/%vol) contents, followed by GR-CWR and GR. This study provides guidance for exploring further possibilities for CWR in the future.

Enzymatic properties and inhibition tolerance analysis of key enzymes in ß-phenylethanol anabolic pathway of Saccharomyces cerevisiae HJ.

Huangjiu is known for its unique aroma, primarily attributed to its high concentration of ß-phenylethanol (ranging from 40 to 130 mg/L). Phenylalanine aminotransferase Aro9p and phenylpyruvate decarboxylase Aro10p are key enzymes in the ß-phenylethanol synthetic pathway of Saccharomyces cerevisiae HJ. This study examined the enzymatic properties of these two enzymes derived from S. cerevisiae HJ and S288C. After substrate docking, Aro9pHJ (-24.05 kJ/mol) and Aro10pHJ (-14.33 kJ/mol) exhibited lower binding free energies compared to Aro9pS288C (-21.93 kJ/mol) and Aro10pS288C (-12.84 kJ/mol). ARO9 and ARO10 genes were heterologously expressed in E. coli BL21. Aro9p, which was purified via affinity chromatography, showed inhibition by l-phenylalanine (L-PHE), but the reaction rate Vmax(Aro9pHJ: 23.89 µmol·(min∙g)-1) > Aro9pS288C: 21.3 µmol·(min∙g)-1) and inhibition constant Ki values (Aro9pHJ: 0.28 mol L-1>Aro9pS288C 0.26 mol L-1) indicated that Aro9p from S. cerevisiae HJ was more tolerant to substrate stress during Huangjiu fermentation. In the presence of the same substrate phenylpyruvate (PPY), Aro10pHJ exhibited a stronger affinity than Aro10pS288C. Furthermore, Aro9pHJ and Aro10pHJ were slightly more tolerant to the final metabolites ß-phenylethanol and ethanol, respectively, compared to those from S288C. The study suggests that the mutations in Aro9pHJ and Aro10pHJ may contribute to the increased ß-phenylethanol concentration in Huangjiu. This is the first study investigating enzyme tolerance mechanisms in terms of substrate and product, providing a theoretical basis for the regulation of the ß-phenylethanol metabolic pathway.

Saccharomyces cerevisiae strains with low-yield higher alcohols and high-yield acetate esters improve the quality, drinking comfort and safety of huangjiu.

Higher alcohols (HAs) and acetate esters (AEs) produced by yeasts are two important volatile flavor substances in fermented alcoholic beverages (FABs). To improve the FABs overall quality, lab-scale huangjiu brewing and systematic evaluation were performed using 171 Saccharomyces cerevisiae strains. Finally, two S. cerevisiae strains that produced lower HAs and higher AEs were obtained and named jiangnan1# and jiangnan3#, respectively. The results of production-scale huangjiu fermentation indicated that HAs produced by jiangnan1# sample decreased by 24.99 %, and AEs produced by jiangnan1# increased by 36.35 %. Sensory evaluation showed that the acidic taste, honey aroma attribute intensity were higher in 85# huagnjiu, and the fruity aroma attribute intensity was higher in jiangnan1# huangjiu (P < 0.01). Moreover, urea and ethyl carbamate produced by jiangnan1# strain were degraded by 13.89 % and 45.51 % compared with those of the control strain 85#, indicating the positive effects of jiangnan1# strain on health and safety. Thus, the obtained S. cerevisiae strains in this study can better enhance the flavor and improve the drinking safety and comfort of huangjiu.

Combined effects of fermentation temperature and Saccharomyces cerevisiae strains on free amino acids, flavor substances, and undesirable secondary metabolites in huangjiu fermentation.

Fermentation temperature (FT) is one of the most critical factors, which can be used to control the growth conditions of S. cerevisiae to obtain excellent final products in winemaking. In this study, we analyzed the responses of six S. cerevisiae strains with different temperature preferences to FT (20 °C, 30 °C, and 35 °C) in huangjiu fermentation. The flavor substances, free amino acids and undesirable secondary metabolites related to huangjiu quality were determined. Results indicated that both S. cerevisiae strains and FT had significant effects on huangjiu fermentation, while the effects were strain-independent and differentiated temperature preferences for different fermentation characteristics. We found that the effects of FT were greater than that of S. cerevisiae strains under the premise of satisfying fermentation completion. Low temperature (20 °C) and high temperature (35 °C) fermentation were possible in the production of differentiated industrial huangjiu styles, while the effects of FT on undesirable secondary metabolites needed to be considered before industrial application. The results showed that a combination of FT with one or more S. cerevisiae strains could be used as a fermentation starter in huangjiu production for different types of products.

Replicon-Based Typing of IncI-Complex Plasmids, and Comparative Genomics Analysis of IncIγ/K1 Plasmids.

IncI-complex plasmids can be divided into seven subgroups IncI1, IncI2, IncIγ, IncB/O, IncK1, IncK2, and IncZ. In this study, a replicon-based scheme was proposed for typing IncI-complex plasmids into four separately clustering subgroups IncI2, IncI1/B/O, IncIγ/K1 and IncK2/Z, the last three of which were combined from IncI1 and IncB/O, IncIγ and IncK1, and IncK2 and IncZ, respectively. Four IncIγ/K1 plasmids p205880-NR2, p14E509-CTXM, p11011-CTXM and p61806-CTXM were fully sequenced and compared with IncIγ/K1 reference pCT, IncI2 reference R721, IncI1/B/O reference R64 and IncK2/Z reference pO26-CRL-125. These plasmids shared conserved gene organization in the replication and conjugal transfer regions, but displaying considerable sequence diversity among different subgroups. Remarkable modular differences were observed in the maintenance and transfer leading regions. p205880-NR2 contained no resistance genes or accessory modules, while the other seven plasmids acquired one or more accessory modules, which harbored mobile elements [including unit transposons, insertion sequence (IS)-based transposition units and individual IS elements] and associated resistance markers (especially including those involved in resistance to ß-lactams, aminoglycosides, tetracyclins, phenicols, streptomycins, trimethoprims, sulphonamides, tunicamycins and erythromycins). Data presented here provided a deeper insight into diversification and evolution of IncI-complex plasmids.

Comparative analysis of bla KPC-2- and rmtB-carrying IncFII-family pKPC-LK30/pHN7A8 hybrid plasmids from Klebsiella pneumoniae CG258 strains disseminated among multiple Chinese hospitals.

BACKGROUND: We recently reported the complete sequence of a blaKPC-2- and rmtB-carrying IncFII-family plasmid p675920-1 with the pKPC-LK30/pHN7A8 hybrid structure. Comparative genomics of additional sequenced plasmids with similar hybrid structures and their prevalence in bla KPC-carrying Klebsiella pneumoniae strains from China were investigated in this follow-up study. METHODS: A total of 51 bla KPC-carrying K. pneumoniae strains were isolated from 2012 to 2016 from five Chinese hospitals and genotyped by multilocus sequence typing. The bla KPC-carrying plasmids from four representative strains were sequenced and compared with p675920-1 and pCT-KPC. Plasmid transfer, carbapenemase activity determination, and bacterial antimicrobial susceptibility test were performed to characterize resistance phenotypes mediated by these plasmids. The prevalence of pCT-KPC-like plasmids in these bla KPC-carrying K. pneumoniae strains was screened by PCR. RESULT: The six KPC-encoding plasmids p1068-KPC, p20049-KPC, p12139-KPC and p64917-KPC (sequenced in this study) and p675920-1 and pCT-KPC slightly differed from one another due to deletion and acquisition of various backbone and accessory regions. Two major accessory resistance regions, which included the blaKPC-2 region harboring blaKPC-2 (carbapenem resistance) and blaSHV-12 (ß-lactam resistance), and the MDR region carrying rmtB (aminoglycoside resistance), fosA3 (fosfomycin resistance), bla TEM-1B (ß-lactam resistance) and bla CTX-M-65 (ß-lactam resistance), were found in each of these six plasmids and exhibited several parallel evolution routes. The pCT-KPC-like plasmids were present in all the 51 K. pneumoniae isolates, all of which belonged to CG258. CONCLUSION: There was clonal dissemination of K. pneumoniae CG258 strains, harboring bla KPC-2- and rmtB-carrying IncFII-family pKPC-LK30/pHN7A8 hybrid plasmids, among multiple Chinese hospitals.

Comparative genomics of type 1 IncC plasmids from China.

AIM: This study dealt with genomic characterization of type 1 IncC resistance plasmids, capable of spreading across taxonomic borders, from China. MATERIALS & METHODS: p112298-tetA was sequenced and compared with type 1 IncC reference plasmid pR148 and two available sequenced type 1 IncC plasmids pHS36-NDM and pVAS3-1 from China. RESULTS: These plasmids contained one or more exogenous resistance islands, which included the ARI-A islands, the ARI-B islands, the ISEcp1-blaCMY units and the bla KPC-2 region and were inserted at various sites in the IncC backbone and thus represented three distinct lineages. CONCLUSION: Complex rearrangement and homologous recombination events have occurred during evolution of p112298-tetA, making it significantly differ modularly from the other three plasmids with respect to both plasmid backbone and exogenous resistance regions.

p1220-CTXM, a pKP048-related IncFIIK plasmid carrying bla CTX-M-14 and qnrB4.

AIM: This study aimed to characterize plasmid-mediated antimicrobial resistance in clinical Klebsiella pneumoniae 1220 carrying bla CTX-M-14 and qnrB4. MATERIALS & METHODS: Plasmid p1220-CTXM was transformed from the 1220 isolate into Escherichia coli through conjugal transfer and then fully sequenced. Antimicrobial susceptibility was determined by VITEK. RESULTS: p1220-CTXM was an IncFIIK plasmid genetically closely related to pKP048 and carried resistance markers including bla CTX-M-14, bla DHA-1, qnrB4, sul1 and qacEΔ1, all of which were harbored in a 35.7-kb multidrug-resistant region. bla CTX-M-14 was located in a truncated ISEcp1-bla CTX-M-14-orf477 transposition unit, and qnrB4 and bla DHA-1 were in a truncated qnrB4-bla DHA-1 region. CONCLUSION: This study provided the insight into the co-occurrence of bla CTX-M-14 and qnrB4 and the evolution of pKP048-related IncFIIK plasmids.