Rongqing Zhou

Progress in engineering acid stress resistance of lactic acid bacteria

Lactic acid bacteria (LAB) are widely used for the production of a variety of fermented foods, and are considered as probiotic due to their health-promoting effect. However, LAB encounter various environmental stresses both in industrial fermentation and application, among which acid stress is one of the most important survival challenges. Improving the acid stress resistance may contribute to the application and function of probiotic action to the host. Recently, the advent of genomics, functional genomics and high-throughput technologies have allowed for the understanding of acid tolerance mechanisms at a systems level, and many method to improve acid tolerance have been developed. This review describes the current progress in engineering acid stress resistance of LAB. Special emphasis is placed on engineering cellular microenvironment (engineering amino acid metabolism, introduction of exogenous biosynthetic capacity, and overproduction of stress response proteins) and maintaining cell membrane functionality. Moreover, strategies to improve acid tolerance and the related physiological mechanisms are also discussed.

An Improved and Validated Sample Cleanup Method for Analysis of Ethyl Carbamate in Chinese Liquor

Ethyl carbamate (EC) is a potential human carcinogen widely existing in fermented foods and alcoholic beverages. The solid-phase extraction (SPE) coupled to gas chromatography mass spectrometry is a widely-used method to determine EC levels, but the accuracy varies with sample matrix and the effects of operation parameters are rarely examined. In this study, the influence factors involved in EC determination were investigated using Chinese liquor as sample matrix, and the improved method was further applied. Three types of SPE columns, including diatomite, Florisil, and primary-secondary amine, were compared in extraction efficiency, and the diatomite column exhibited the highest extraction efficiency. The optimal volumes of elution solvents with diatomite column were 15 mL for 3-mL samples solution loaded. In addition, the alcoholic strength for EC determination should be diluted below 20% (v/v) to avoid the enhancement of matrix-induced chromatographic response. Moreover, the pH neutralization could help improve EC recovery and peak resolution, reducing interfering effects. Based on these results, the improved method showed that the limit of detection, the limit of quantification, and average recoveries were 1.10 μg/L, 3.65 μg/L, and 93.06%, respectively. To further elucidate the underlying factors related to EC accumulation, partial least square regression analysis was conducted, and the results suggested that EC levels had the closest relationship with alcoholic strength among the remaining precursors.

Changes in Volatile Compounds of Chinese Luzhou-Flavor Liquor during the Fermentation and Distillation Process.

UNLABELLED The aim of this study was to investigate the dynamic of volatile compounds in the Zaopei during the fermentation and distillation process by headspace solid-phase microextraction-gas chromatography mass spectrometry (HS-SPME-GCMS). Physicochemical properties analysis of Zaopei (fermented grains [FG], fermented grains mixed with sorghum [FGS], streamed grains [SG], and streamed grains mixed with Daqu [SGD]) showed distinct changes. A total number of 66 volatile compounds in the Zaopei were identified, in which butanoic acid, hexanoic acid, ethyl hexanoate, ethyl lactate, ethyl octanoate, hexyl hexanoate, ethyl hydrocinnamate, ethyl oleate, ethyl hexadecanoate, and ethyl linoleate were considered to be the dominant compounds due to their high concentrations. FG had the highest volatile compounds (112.43 mg/kg), which significantly decreased by 17.05% in the FGS, 67.12% in the SG, and 73.75% in the SGD. Furthermore, about 61.49% of volatile compounds of FGS were evaporated into raw liquor, whereas head, heart, and tail liquor accounted for 29.84%, 39.49%, and 30.67%, respectively. Each volatile class generally presented a decreasing trend, except for furans. Especially, the percentage of esters was 55.51% to 67.41% in the Zaopei, and reached 92.60% to 97.67% in the raw liquor. Principal component analysis based ordination of volatile compounds data segregated FGS and SGD samples. In addition, radar diagrams of the odor activity values suggested that intense flavor of fruit was weakened most from FG to SGD. PRACTICAL APPLICATION The dynamic of volatile compounds in the Zaopei during the fermentation and distillation process was tested by SPME-GCMS. The result of this study demonstrated that both volatile compounds of Zaopei and thermal reaction during distillation simply determined the unique feature of raw liquor. This study was conducted based on the real products from liquor manufactory, so it is practicable that the method can be used in an industry setting.

Difference of microbial community stressed in artificial pit muds for Luzhou‐flavour liquor brewing revealed by multiphase culture‐independent technology

Artificial pit muds (APMs) is produced by peats, aged pit muds, yellow and black clays etc. and is one of essential factors for Luzhou‐flavour liquor production. The microbial community of APMs significantly influence the quality of Luzhou‐flavour liquor. The aim of this study was to investigate the differences in bacterial, archaeal and fungal community of APMs, starters and materials.

Formation of ethyl carbamate in Goji wines: Effect of Goji fruit composition

Ethyl carbamate (EC) is a multisite carcinogen widely occurring in alcoholic beverages. In this investigation, solid-phase extraction combined with gas chromatography mass spectrometry was employed to determine EC contents during the fermentation and storage processes, and the effects of Goji varieties on its formation were also examined. The results indicated that natural and potential EC contents were significantly affected by the varied composition of Goji fruits. The analysis of chemical properties showed differences in hundred-grain weight, water contents, amino acids, and nitrogen-to-carbon ratio for Goji berries. Citrulline was completely degraded although it is routinely considered as a non-preferred nitrogen for yeasts. Due to compositional differences, Goji wines accumulated distinct urea levels that positively correlated with the potential EC contents. Furthermore, the temperature in both the production processes highly influenced EC formation. These results contribute to a more comprehensive understanding of EC formation, and in turn, controlling EC in the Goji wine matrix.

Characterizing Soy Sauce Moromi Manufactured by High-Salt Dilute-State and Low-Salt Solid-State Fermentation Using Multiphase Analyzing Methods

Present study was to characterize the physiochemical properties, free amino acids (FAAs), volatiles and microbial communities of various moromi, respectively sampled from different stages of high-salt dilute-state (HSDS) and low-salt solid-state (LSSS) fermentation, using multiphase analyzing methods. Phospholipid fatty acids (PLFA) analysis indicated that Gram-positive bacteria were dominant bacteria and fungi were principal microbes. For DGGE analysis, dominant microbes in moromi were mainly fell into Weissella, Tetragenococcus, Candida, Pichia, and Zygosaccharomyces. During fermentation, the dominant microbes shifted from nonhalophilic and less acid-tolerant species to halophilic and acid-tolerant species. Total of 15 FAAs and 44 volatiles were identified in moromi, mainly Glu, Asp, Tyr, and acids, alcohols, esters, aldehydes, respectively. Odor activity values analysis suggested that the final moromi of LSSS fermentation had more complicated odors than that of HSDS fermentation. Conclusively, technological parameters, microbial communities, raw materials and fermentation process may result in the discrepancy of HSDS and LSSS moromi.

Evaluating the feasibility of fermentation starter inoculated with Bacillus amyloliquefaciens for improving acetoin and tetramethylpyrazine in Baoning bran vinegar

Fermentation starters (Daqu) used in present study included traditional herb Daqu (C Daqu), modified Daqu without herbs (M Daqu) and S Daqu fermented by inoculating acetoin and tetramethylpyrazine high-producing bacterium Bacillus amyloliquefaciens into M Daqu. To evaluate the feasibility of S Daqu combined with M Daqu applied for improving contents of acetoin and tetramethylpyrazine in Baoning bran vinegar without remarkably changing the original microbial community and the other volatiles contents compared with C Daqu, vinegar Pei C, M, M1, M2 and S were correspondingly prepared in lab scale using C Daqu, M Daqu, M1 Daqu (S Daqu: M Daqu=1:9, w/w), M2 Daqu (S Daqu: M Daqu=5:5) and S Daqu. PCR-DGGE suggested that Bacillus, Lactobacillus, Oceanobacillus, Acetobacter, Pichia, Geotrichum and Trichoderma were dominant microbes. Microbial community of M were similar with M1, while that of the others were similar. Differences in physicochemical properties among samples may be ascribed to different enzymes activities of Daqu and bioactivities of microbial metabolism during fermentation. Moreover, total contents of organic acids in M, M1, M2 and S increased by 33.10%, 25.77%, 4.32% and 7.74% relative to C, respectively. Volatiles and PLS-DA analysis suggested that volatile profiles of M were similar with M1, that of M2 were similar with C, while that of S were significantly different with the others. Both M2 Daqu and S Daqu facilitated the formation of acetoin and tetramethylpyrazine. However, M2 Daqu was more efficient for enhancing acetoin and tetramethylpyrazine contents by 191.84% and 123.17% respectively, without significantly changing the other volatiles contents.

Metabolic response of Tetragenococcus halophilus under salt stress

In this study, the effect of salt stress on metabolic response of Tetragenecoccus halophilus was investigated, and the metabolic alternations were analyzed using liquid chromatography-mass spectrometry according to the metabolomics approach. A total of 81 intracellular metabolites were identified, and significant differences were observed in the levels of metabolites mainly participating in central carbon metabolism, fatty acid metabolism, and amino acid metabolism. Analysis of the membrane fatty acid distribution showed that higher proportions of unsaturated fatty acid were observed in salt-treated cells. Additionally, salt-stressed cells exhibited higher amounts of compatible solutes including proline, glycine, citrulline, and N-acetyltyrptophan, and lower amounts of branched-chain amino acids. Interestingly, higher amounts of indole, salicylic acid, and coronatine, which are regarded as signaling molecule and suggested to combat osmotic stress, were detected in salt-shocked cells compared with the untreated cells. Taken together, these results suggested that increased unsaturated membrane fatty acids, accumulation of compatible solutes, and up-regulation of signaling molecule may be potential mechanisms employed by T. halophilus during salt stress.

PLFA Fingerprint Method for Assessing Microbial Communities of Strong Aromatic Liquor Pit Mud

In the view of the specificity of microbial communities in pit mud during brewing process,different types of soil samples and common strains in liquor brewing were investigated to assess the fingerprints of phospholipid fatty acid(PLFA) of microbial communities in Luzhou flavor pit mud.The results suggested the composition and content of PLFA significantly affected by physiological and environmental conditions which resulted in the significant differences(P0.05) of PLFA fingerprints among the samples(the ratio of fungal/bacterial PLFA in garden and roadside soils were 0.30 and 0.66,respectively,which were both less than 1,the ratio in pit mud however was 1.17).The content of special PLFA changed significantly(P0.05) when five pure cultured microorganisms were added.The content of a14:0 and a16:0 in pit mud adding SICC1.13 and CICC20633 were 1.44 and 9.92 times higher than those in blank respectively,while 18:2ω6,9 in SICC3398 and SICC31482 were 82.58 and 90.33 times higher than that in the blank.Furthermore,cy17:0 was identified in the pit mud adding DH5α.Principal component analysis(PCA) was performed to evaluate the changes of special PLFA’s composition and content in pit mud after adding the microorganisms.The results of this study demonstrated that PLFA fingerprint method was a fast biochemical culture-independent method,which was beneficial to characterize the fingerprints of microbial communities during liquor process.

Development of a Rapid Discrimination Tool for Luzhou-flavor Pit Mud Classification by the Kohonen Artificial Neural Network Model

Understanding the volatile composition of pit mud (PM) samples and discriminating these samples has become a highly necessary task, owing to the fact that volatile profiling of PM can significantly affect Chinese Luzhou-flavor liquor quality. In this study, the volatile constitutions of 13 Luzhou-flavor liquor PM samples from four typical producing regions were investigated by gas chromatography–mass spectrometry (GC-MS). Owing to their high concentrations, compounds such as ethyl hexanoate, butyric acid, hexanoic acid, ethyl pentadecanoate, ethyl palmitate, ethyl (9E)-9-octadecenoate, ethyl (9E,12E)-9,12-octadecadienoate, and palmitic acid were considered to be predominant volatiles. A promising artificial neural network model, the Kohonen self-organizing map (SOM), was applied to rapidly discriminate the PM samples in terms of differences based on the quantitative information of volatile compounds. After Kohonen SOM training, 13 distinct clusters, corresponding to PM samples, were clearly visualized on a uniform distance matrix (U-matrix). The influence of volatile compounds on the classification of the PM could be displayed using component panels, which can give quantitative insight. GC-MS coupled with the Kohonen SOM model not only presented the volatile constitution of PM but also provided promising information for discrimination between different PM samples, even in other fermented foods.