Revealing the Succession of Spatial Heterogeneity of the Microbial Community during Broad Bean Paste Fermentation.

This study aimed to elaborate the assembly processes and metabolic regulation of the microbial community under the conditions of environmental factors and artificial intervention using broad bean paste (BBP) fermentation as a tractable research object. Spatial heterogenicity of amino acid nitrogen, titratable acidity, and volatile metabolites were observed between upper and lower layers after fermentation for 2 weeks. Amino nitrogen contents in the upper fermented mash reached 0.86, 0.93, and 1.06 g/100 g at 2, 4, and 6 weeks, respectively, which were significantly higher than those of mash located at the lower layer (0.61, 0.79, and 0.78 g/100 g). Moreover, higher concentrations of titratable acidity were accumulated in upper layers (2.05, 2.25 and 2.56 g/100g) than those in lower layers, and the differentiation of volatile metabolites was the greatest (R = 0.543) at 36 days, after which the BBP flavor profiles converged with the fermentation progress. The successive heterogenicity of the microbial community in the mid-late stage was also found during fermentation, and Zygosaccharomyces, Staphylococcus, and Bacillus had heterogeneous characteristics driven by sunlight, water activity, and microbial interactions. This study provided new insights into the mechanisms underlying the succession and assembly of the microbial community of BBP fermentation, which also laid new clues for researches of the microbial communities in complex ecosystems. IMPORTANCE Gaining insights into the community assembly processes is essential and valuable for the elaboration of underlying ecological patterns. However, current studies about microbial community succession in multispecies fermented food usually treat the research object as a whole, are focused exclusively on temporal dimensions, and have ignored the changes of community structure in spatial dimensions. Therefore, dissecting the community assembly process from the view of spatiotemporal dimensions will be a more comprehensive and detailed perspective. Here, we found the heterogenicity of the BBP microbial community under the traditional production technology from spatial and temporal scales, systematically analyzed the relationship between the spatiotemporal succession of community and the difference of BBP quality, and elucidated the roles of environmental factors and microbial interactions to drive the heterogeneous succession of the microbial community. Our findings provide a new insight into understanding the association between microbial community assembly and the quality of BBP.

Transcriptomic and metabolomic analysis reveals genes related to stress tolerance in high gravity brewing.

The fermentation performance of yeast is the key of beer production. High gravity brewing is a commonly used technique in industrial lager beer production and it is environmentally friendly. Therefore, there has been extensive effort toward improving high gravity brewing. In this study, through transcriptomic and metabolomic analysis of two homologous lager yeasts, genes that relate to stress tolerance in high gravity brewing were screened. The results showed EMP pathway and multiple amino acid metabolism pathway were the most enriched pathways, and pyruvate might be the core metabolite. Overexpression and knockdown strains were constructed to verify the genes' functions. The overexpression of MAN2, PCL1 and PFK26 genes were beneficial to fermentation without significantly changes in flavor profiles. The relative intracellular ATP levels can help us understand the change of metabolic flux such as enhancement of sugar consumption. This work is helpful to reveal the stress tolerance mechanism of high gravity brewing and breed yeast strains with improved performance.

Silencing of miR-1246 Induces Cell Cycle Arrest and Apoptosis in Cisplatin-Resistant Ovarian Cancer Cells by Promoting ZNF23 Transcription.

Ovarian cancer (OC) is the most frequent cause of death among patients with gynecologic malignancies. In recent years, the development of cisplatin (DDP) resistance has become an important reason for the poor prognosis of OC patients. Therefore, it is vital to explore the mechanism of DDP resistance in OC. In this study, microRNA-1246 (miR-1246) expression in OC and DDP-resistant OC cells was determined by RT-qPCR, and chemosensitivity to DDP was assessed by the CCK-8 assay. A dual-luciferase reporter assay was performed to confirm the interaction between miR-1246 and zinc finger 23 (ZNF23), while changes in ZNF23 expression were monitored by RT-qPCR, immunofluorescence, and western blot assays. Moreover, cell proliferation, cycle phase, and apoptosis were determined by EdU staining, flow cytometry, TUNEL staining, and Hoechst staining. Our data showed that miR-1246 was highly expressed in DDP-resistant OVCAR-3 and TOV-112D cells. Functionally, overexpression of miR-1246 markedly enhanced DDP resistance and cell proliferation, and suppressed cell cycle arrest and apoptosis of OC cells. Inhibition of miR-1246 expression significantly attenuated DDP resistance and cell proliferation, and increased cell cycle arrest and apoptosis in DDP-resistant OC cells. Furthermore, ZNF23 was identified as a target gene of miR-1246, and ZNF23 protein expression was notably downregulated in DDP-resistant OC cells. Moreover, overexpression of miR-1246 significantly downregulated the ZNF23 levels in OVCAR-3 and TOV-112D cells, and inhibition of miR-1246 upregulated the ZNF23 levels in the DDP-resistant OVCAR-3 and TOV-112D cells. In conclusion, miR-1246 might be a novel regulator of DDP-resistant OC that functions by regulating ZNF23 expression in DDP-resistant cells, as well as cell proliferation, cell cycle progression, and apoptosis.

Genome comparison of three lager yeasts reveals key genes affecting yeast flocculation during beer fermentation.

Yeast flocculation plays an essential role in industrial application. Appropriate flocculation of yeast cells at the end of fermentation benefits the cell separation in production, which is an important characteristic of lager yeast for beer production. Due to the complex fermentation environment and diverse genetic background of yeast strains, it is difficult to explain the flocculation mechanism and find key genes that affect yeast flocculation during beer brewing. By analyzing the genomic mutation of two natural mutant yeasts with stronger flocculation ability compared to the parental strain, it was found that the mutated genes common in both mutants were enriched in protein processing in endoplasmic reticulum, membrane lipid metabolism and other pathways or biological processes involved in stress responses. Further functional verification of genes revealed that regulation of RIM101 and VPS36 played a role in lager yeast flocculation under the brewing condition. This work provided new clues for improving yeast flocculation in beer brewing.

Screening lager yeast with higher ethyl-acetate production by adaptive laboratory evolution in high concentration of acetic acid.

Ethyl-acetate is important for the flavor and aroma of the alcoholic beverages, therefore, there have been extensive efforts toward increasing its production by engineering yeast strains. In this study, we reported a new approach to breed non-genetic modified producing yeast strain with higher ethyl-acetate production for beer brewing. First, we demonstrated the positive effect of higher acetic acid concentration on inducing the expression of acetyl-CoA synthetase (ACS). Then, we applied adaptive laboratory evolution method to evolve strain with higher expression level of ACS. As a result, we obtained several evolved strains with increased ACS expression level as well as ethyl-acetate production. In 3 L scale fermentation, the optimal strain EA60 synthesized more ethyl-acetate than M14 at the same time point. At the end of fermentation, the ethyl-acetate production in EA60 was 21.4% higher than M14, while the other flavor components except for acetic acid were changed in a moderate degree, indicating this strain had a bright prospect in industrial application. Moreover, this study also indicated that ACS1 played a more important role in increasing the acetic acid tolerance of yeast, while ACS2 contributed to the synthesis of cytosol acetyl-CoA, thereby facilitating the production of ethyl-acetate during fermentation.

Evaluation and prediction of the biogenic amines in Chinese traditional broad bean paste.

Biogenic amines (BAs) are a threat to the safety of broad bean paste, and biosynthetic mechanism of BA and its regulation are unknown. This study aimed to assess microbial BA synthesis in Chinese traditional broad bean paste and determine favorable fermentation conditions for BA regulation. The BAs content in 27 pastes was within the safe range. 64 strains with potential decarboxylation were screened in Luria-Bertani Glycerol medium and identified as Bacillus spp. Although Bacillus amyloliquefaciens produced highest levels of BAs (70.14 ± 2.69 mg/L) in LBAA, Bacillus subtilis produced 6% more BAs than B. amyloliquefaciens. Meanwhile, temperature was the most remarkable factor affecting BAs production by B. amyloliquefaciens 1-13. Furthermore, the fermented broad bean paste model revealed that BA content increased by 61.2 mg/kg every 10 days at 45 °C, which was approximately threefold of that at 25 °C. An ARIMA prediction model of BAs content was constructed, and the total BAs content of 40 mg/100 g was set as the critical value. This study not only contributed to understanding the BAs formation mechanism, but also provided potential measures to control the BAs in fermented soybean products.

Screening of thermosensitive autolytic mutant brewer's yeast and transcriptomic analysis of heat stress response.

Brewer's yeast has been widely used in the food industry, and the autolysates thereof are increasingly being studied for their valuable nutritional compositions. Yeast autolysis is most affected by medium composition and temperature. In this study, a thermosensitive autolytic brewer's yeast P-510 was obtained with atmospheric and room temperature plasma mutagenesis plus 5-bromo-chloro-3-indolyl phosphate screening. The mutant rapidly autolyzed at 37 °C and the autolysates contained more active components and showed higher antioxidant activities compared with that of the parental strain, which indicated that the mutant's autolysates can potentially be used as functional food and nutritional ingredients. Transcriptomic analysis of the mutant and parental strains at 28 and 37 °C suggested that thermosensitive autolysis of P-510 was probably caused by mitochondrial disfunction, glycogen metabolic flux of glycolysis and pentose phosphate pathway disorder, as well as hexose transport inhibition. The results revealed the important role of mitochondrial metabolism and glycogen utilization regulation in heat stress response of yeast.

Residue analysis of gibberellic acid isomer (iso-GA3) in brewing process and its toxicity evaluation in mice.

Gibberellic acid (GA3) is an efficient plant growth regulator, which could speed up barley germination in the brewing industry. However, the residue of GA3 in malt gets denatured into an isomer, termed iso-GA3. In this study, the concentration of iso-GA3 and the conversion rate of GA3 to iso-GA3 during the brewing process was studied by high performance liquid chromatography and the potential toxicity of iso-GA3 was evaluated in ICR mice. The concentration of iso-GA3 increased in the saccharification and wort boiling processes while its concentration was stable during fermentation. The maximum conversion rates of GA3 to iso-GA3 in Canadian malt, Australian malt SCO and Australian malt FAQ were 88%, 87% and 87%, respectively. In the acute oral toxicity study, the median lethal dose (LD50) of iso-GA3 was 2.82 g/kg body weight (BW). In the 28-day repeated dose toxicity study, the iso-GA3 could cause weight loss in mice. And the mice of high-dose group showed a slight decrease in food consumption. Moreover, inflammation and cell necrosis were found in kidney and liver tissue, which were alleviated during the recovery phase. These results establish a practical reference for food safety in products, in which GA3 is added as a food additive.

Reverse metabolic engineering in lager yeast: impact of the NADH/NAD+ ratio on acetaldehyde production during the brewing process.

Acetaldehyde is synthesized by yeast during the main fermentation period of beer production, which causes an unpleasant off-flavor. Therefore, there has been extensive effort toward reducing acetaldehyde to obtain a beer product with better flavor and anti-staling ability. In this study, we discovered that acetaldehyde production in beer brewing is closely related with the intracellular NADH equivalent regulated by the citric acid cycle. However, there was no significant relationship between acetaldehyde production and amino acid metabolism. A reverse engineering strategy to increase the intracellular NADH/NAD+ ratio reduced the final acetaldehyde production level, and vice versa. This work offers new insight into acetaldehyde metabolism and further provides efficient strategies for reducing acetaldehyde production by the regulating the intracellular NADH/NAD+ ratio through cofactor engineering.

Engineering the cytosolic NADH availability in lager yeast to improve the aroma profile of beer.

OBJECTIVE: To improve the aroma profile of beer by using metabolic engineering to increase the availability of cytosolic NADH in lager yeast. RESULTS: To alter NADH levels in lager yeast, the native FDH1 (YOR388C) encoding NAD+-dependent formate dehydrogenase was overexpressed in the yeast strain M14, yielding strain M-FDH1. This led to a simultaneous increase of NADH availability and NADH/NAD+ ratio in the M-FDH1 strain during fermentation. At the end of the main fermentation period, ethanol production by strain M-FDH1 was decreased by 13.2%, while glycerol production was enhanced by 129.4%, compared to the parental strain respectively. The production of esters and fusel alcohols by strains M14 and M-FDH1 was similar. By contrast, strain M-FDH1 generally produced less organic acids and off-flavor components than strain M14, improving the beer aroma. CONCLUSIONS: Increased NADH availability led to rerouting of the carbon flux toward NADH-consuming pathways and accelerated the NADH-dependent reducing reactions in yeast, greatly impacting the formation of aroma compounds and improving the beer aroma.

Process optimization of the extraction condition of ß-amylase from brewer's malt and its application in the maltose syrup production.

ß-Amylase is of important biotechnological aid in maltose syrup production. In this study, the extraction condition of ß-amylase from brewer's malt and the optimal dosage of ß-amylase in maltose syrup production were optimized using response surface methodology and uniform design method. The optimal extraction condition of ß-amylase from brewer's malt was composed of 1:17 (g/v) material/liquid ratio, 44°C extraction temperature, pH 6.4 buffer pH, 2.3 H extraction time, and 1.64 g L-1 NaSO3 dosage with a predicted ß-amylase activity of 1,290.99 U g-1 , which was close to the experimental ß-amylase activity of 1,230.22 U g-1 . The optimal dosages of ß-amylase used in maltose syrup production were 455.67 U g-1 starch and its application in maltose syrup production led to a 68.37% maltose content in maltose syrup, which was 11.2% and 28.9% higher than those using ß-amylases from soybean and microbe (P < 0.01). Thus, ß-amylase from brewer's malt was beneficial for production of high maltose syrup.

Rationally designed perturbation factor drives evolution in Saccharomyces cerevisiae for industrial application.

Saccharomyces cerevisiae strains with favorable characteristics are preferred for application in industries. However, the current ability to reprogram a yeast cell on the genome scale is limited due to the complexity of yeast ploids. In this study, a method named genome replication engineering-assisted continuous evolution (GREACE) was proved efficient in engineering S. cerevisiae with different ploids. Through iterative cycles of culture coupled with selection, GREACE could continuously improve the target traits of yeast by accumulating beneficial genetic modification in genome. The application of GREACE greatly improved the tolerance of yeast against acetic acid compared with their parent strain. This method could also be employed to improve yeast aroma profile and the phenotype could be stably inherited to the offspring. Therefore, GREACE method was efficient in S. cerevisiae engineering and it could be further used to evolve yeast with other specific characteristics.

Simultaneous determination of diethylacetal and acetaldehyde during beer fermentation and storage process.

BACKGROUND: Acetaldehyde is an important flavor component in beer which is possibly carcinogenic to humans. Owing to the limitations of present detection methods, only free-state acetaldehyde in beers has been focused on, while acetal in beers has hardly been reported so far. RESULTS: A sensitive headspace gas chromatography method was developed for the determination of diethylacetal and acetaldehyde in beer. The column DB-23 was chosen with a total run time of 22.5 min. The optimal addition amount of NaCl, equilibrium temperature and equilibrium time were 2.0 g, 70 °C and 30 min respectively. For both diethylacetal and acetaldehyde analyses, the limit of detection was 0.005 mg L-1 with relative standard deviation < 5.5%. The recoveries of acetaldehyde and diethylacetal were 95-110 and 95-115% respectively. The diethylacetal and acetaldehyde average contents in 24 beer products were 11.83 and 4.36 mg L-1 respectively. The Pearson correlation coefficient between diethylacetal and acetaldehyde was the highest (0.963). Both diethylacetal and acetaldehyde contents increased to a peak value after fermentation for 3 days and then decreased to a lower value. During both normal and forced aging storage, the diethylacetal content decreased and the acetaldehyde content increased gradually over time. When beers were forced aged for 4 days, the increased ratio of acetaldehyde could be above 40.00%. CONCLUSION: The newly established method can be used to assess acetaldehyde level and flavor quality in beer more scientifically. © 2018 Society of Chemical Industry.

Cell wall polysaccharides: before and after autolysis of brewer's yeast.

Brewer's yeast is used in production of beer since millennia, and it is receiving increased attention because of its distinct fermentation ability and other biological properties. During fermentation, autolysis occurs naturally at the end of growth cycle of yeast. Yeast cell wall provides yeast with osmotic integrity and holds the cell shape upon the cell wall stresses. The cell wall of yeast consists of ß-glucans, chitin, mannoproteins, and proteins that cross linked with glycans and a glycolipid anchor. The variation in composition and amount of cell wall polysaccharides during autolysis in response to cell wall stress, laying significant impacts on the autolysis ability of yeast, either benefiting or destroying the flavor of final products. On the other hand, polysaccharides from yeast cell wall show outstanding health effects and are recommended to be used in functional foods. This article reviews the influence of cell wall polysaccharides on yeast autolysis, covering cell wall structure changings during autolysis, and functions and possible applications of cell wall components derived from yeast autolysis.

Nomogram Predicting Overall Survival in Operable Cervical Cancer Patients.

OBJECTIVE: Nomograms are widely used as predictive tools to predict oncological outcomes intuitively and precisely. The aim of our study was to develop a nomogram for predicting overall survival (OS) of patients with early stage cervical cancer. METHODS: In this retrospective study, the clinical, pathological, and hematological data and prognosis of 795 cervical cancer patients were investigated. We identified and incorporated independent significant prognostic factors for OS to develop a nomogram. The predictive accuracy and discriminative ability were measured by concordance index. RESULTS: By univariable analysis and subsequent multivariable analysis, we identified body mass index, albumin, platelet, leukocyte, tumor differentiation, and the status of the pelvic lymph node (PLN) (all P < 0.05) as independent prognostic factors. The concordance index of the nomogram integrating these 6 variables was 0.74. The calibration curves for probability of 3- and 5-year OS also demonstrated ideal agreement between nomogram prediction and actual observation. CONCLUSIONS: We developed a novel nomogram that can provide prediction of OS for patients with early stage cervical cancer individually. Furthermore, studies are required to validate whether it can be applied to other cohorts.

Long non-coding RNA LINC00261 inhibits cell growth and migration in endometriosis.

AIM: A previous study reported that LINC00261 is significantly downregulated in human ectopic endometrial tissues. The present study aimed to explore whether LINC00261 is functional in endometriosis cell proliferation, apoptosis and migration. METHODS: By transfecting human endometriosis cell line CRL-7566 with plasmids containing LINC00261, we successfully established the cell CRL-7566/LINC00261 with a high LINC00261 expression level. Cell-counting kit-8 and colony formation assays were conducted to evaluate the effect of LINC00261 on cell proliferation, and flow cytometry analysis and transwell migration assay were conducted to evaluate its effect on cell apoptosis and cell migration, respectively. RESULTS: Cell-counting kit-8 and colony formation assays both indicated that LINC00261 could inhibit cell proliferation in CRL-7566. Flow cytometry analysis confirmed that LINC00261 mediated inhibition of cell proliferation, which might be a consequence of inducting apoptosis. Furthermore, transwell migration assay indicated that LINC00261 could inhibit cell migration in endometriosis. CONCLUSION: LncRNA LINC00261 is capable of inhibiting cell growth and migration in endometriosis.

Assessment of ADMA, estradiol, and progesterone in severe preeclampsia.

BACKGROUND: Decreased nitrous oxide (NO) levels are crucial factors in severe preeclampsia (sPE), and asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of NO synthetase. Steroid hormones are closely related to the vascular endothelium. This study determined the levels of and correlations between ADMA, estradiol (E2), and progesterone (Pg) in sPE to investigate the roles of these factors in this disease. METHODS: Sixty-two sPE patients (sPE group) were divided into the sPE1 subgroup (28(+1)-32(+0) weeks of pregnancy), the sPE2 subgroup (32(+1)-36(+0) weeks), and the sPE3 subgroup (36(+1)-40(+0) weeks) and 75 normal pregnant women (NC group) were divided into the NC1 subgroup (28(+1)-32(+0) weeks of gestation), the NC2 subgroup (32(+1)-36(+0) weeks), and the NC3 subgroup (36(+1)-40(+0) weeks). Serum and placental ADMA levels were measured by enzyme-linked immunosorbent assay (ELISA), and serum E2 and Pg concentrations were determined by the chemilumineseent immunoassay (CLIA). RESULTS: ADMA concentrations in both the placenta and the maternal serum were significantly higher in the sPE group (p < 0.05). Higher ADMA contents were observed in the placenta relative to the maternal serum (p < 0.05). Serum E2 levels were significantly lower in the sPE group (p < 0.05). For Pg, the only significant difference was observed between the sPE1 and NC1 subgroups (p < 0.05). The Pg/E2 ratios in the sPE groups were significantly higher, with a significant high positive correlation between Pg/E2 ratios and serum ADMA levels. CONCLUSION: Increased serum levels of ADMA in sPE may result from increased secretion from the placenta, and the increased Pg/E2 ratio may play a role in the development of sPE by aggravating ADMA.

The staining patterns of 53BP1 nuclear foci and 53BP1 mRNA level are associated with cervical cancer progression and metastasis.

p53-binding protein 1 (53BP1) plays a key role in DNA damage response mechanism, which protects genome integrity and guards against cancer. Although abnormal DNA damage response type of 53BP1 nuclear foci (NF) have been indicated to be associated with many types of malignancies, how the staining pattern of 53BP1 NF and the mRNA level of 53BP1 correlate with the clinicopathologic characteristics of cervical cancer is still unclear. In this study, we examined the staining pattern and mRNA level of 53BP1 in cervical premalignant and malignant lesions and normal cervical tissue by immunofluorescence staining and quantitative real-time polymerase chain reaction. We found that the level of 53BP1 NF increased in the following order: normal cervical tissues, cervical intraepithelial neoplasia (CIN) 1, CIN2/3, and cervical cancers, indicating that the level of 53BP1 NF increases as cervical cancer initiates and progresses. In addition, we also found that abnormal DNA damage response type of 53BP1 NF and low mRNA level of 53BP1 was significantly correlated with high histologic grade of cervical cancer, and low mRNA level of 53BP1 was also significantly associated with positive lymph node metastasis of cervical cancer.

Active management of the third stage of labor with and without controlled cord traction: a systematic review and meta-analysis of randomized controlled trials.

OBJECTIVE: To determine the specific effect of controlled cord traction in the third stage of labor in the prevention of postpartum hemorrhage. DATA SOURCES: We searched PubMed, Scopus and Web of Science (inception to 30 October 2013). STUDY SELECTION: Randomized controlled trials comparing controlled cord traction with hands-off management in the third stage of labor were included. RESULTS: Five randomized controlled trials involving a total of 30 532 participants were eligible. No significant difference was found between controlled cord traction and hands-off management groups with respect to the incidence of severe postpartum hemorrhage (relative risk 0.91, 95% confidence interval 0.77-1.08), need for blood transfusion (relative risk 0.96, 95% confidence interval 0.69-1.33) or therapeutic uterotonics (relative risk 0.94, 95% confidence interval 0.88-1.01). However, controlled cord traction reduced the incidence of postpartum hemorrhage in general (relative risk 0.93, 95% confidence interval 0.87-0.99; number-needed-to-treat 111, 95% confidence interval 61-666), as well manual removal of the placenta (relative risk 0.70, 95% confidence interval 0.58-0.84) and duration of the third stage of labor (mean difference -3.20, 95% confidence interval -3.21 to -3.19). CONCLUSIONS: Controlled cord traction appears to reduce the risk of any postpartum hemorrhage in a general sense, as well as manual removal of the placenta and the duration of the third stage of labor. However, the reduction in the occurrence of severe postpartum hemorrhage, need for additional uterotonics and blood transfusion is not statistically significant.

Cushing's syndrome secondary to ectopic ACTH secretion from carcinoid tumor within an ovarian mature teratoma: a case report and review of the literature.

A 46-year-old woman with Cushing's syndrome secondary to ectopic adrenocorticotropic hormone (ACTH) secretion caused by primary ovarian mature teratoma with carcinoid components was presented in our case. The patient manifested sustained hypercortisolemia without circadian rhythm and a lack of suppression of either low-dose dexamethasone suppression test (LDDST) or high-dose dexamethasone suppression test (HDDST). There was no evidence of a pituitary mass or secretion of other hormones. After careful clinical evaluation, no other tumor masses were found. Resection of the ovarian tumors led to sharp reduction of serum ACTH and cortisol concentrations. Immunohistochemistry showed positivity in CgA, Syn, CK, NSE. To the best of our knowledge, there are rare reports of an ACTH-secreting carcinoid components located in an ovarian mature teratoma, and bilateral ovarian mature teratoma makes it rarer.