Metagenomic analysis revealing the metabolic role of microbial communities in the free amino acid biosynthesis of Monascus rice vinegar during fermentation

Free amino acid(FAA)is the important component of vinegar that infl uences quality perception and consumer acceptance.FAA is one of the major metabolites produced by microorganisms;however,the microbial metabolic network on FAA biosynthesis remains unclear.Through metagenomic analysis,this work aimed to elucidate the roles of microbes in FAA biosynthesis during Monascus rice vinegar fermentation.Taxonomic profiles from functional analyses showed 14 dominant genera with high contributions to the metabolism pathways.The metabolic network for FAA biosynthesis was then constructed,and the microbial distribution in different metabolic pathways was illuminated.The results revealed that 5 functional genera were closely involved in FAA biosynthesis.This study illuminated the metabolic roles of microorganisms in FAA biosynthesis and provided crucial insights into the functional attributes of microbiota in vinegar fermentation.

Microbiomics and metabolomics insights into the microbial regulation on the formation of flavor components in the traditional fermentation process of Chinese Hongqu aged vinegar

This study aimed to investigate microbial succession and metabolic dynamics during the traditional fermentation of Hongqu aged vinegar,and explore the core functional microbes closely related to the formation of flavor components.Microbiome analysis demonstrated that Lactobacillus,Acetobacter,Bacillus,Enterobacter,Lactococcus,Leuconostoc and Weissella were the predominant bacterial genera,while Aspergillus piperis,Aspergillus oryzae,Monascus purpureus,Candida athensensis,C.xylopsoci,Penicillium ochrosalmoneum and Simplicillium aogashimaense were the predominant fungal species.Correlation analysis revealed that Acetobacter was positively correlated with the production of tetramethylpyrazine,acetoin and acetic acid,Lactococcus showed positive correlation with the production of 2-nonanone,2-heptanone,ethyl caprylate,ethyl caprate,1-hexanol,1-octanol and 1-octen-3-ol,C.xylopsoci and C.rugosa were positively associated with the production of diethyl malonate,2,3-butanediyl diacetate,acetoin,benzaldehyde and tetramethylpyrazine.Correspondingly,non-volatile metabolites were also detected through ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry.A variety of amino acids and functional dipeptides were identified during the traditional brewing of Hongqu aged vinegar.Correlation analysis revealed that Lactobacillus was significantly associated with DL-lactate,indolelactic acid,D-(+)-3-phenyllactic acid,pimelic acid,pregabalin and 3-aminobutanoic acid.This study is useful for understanding flavor formation mechanism and developing effective strategies for the suitable strains selection to improve the flavor quality of Hongqu aged vinegar.

Effect of vinegar supplementation on patients with esophageal lesions lightly stained with Lugol’s iodine solution:Prospective single-centre trial

BACKGROUND Esophageal chromoendoscopy with iodine solution is important for detecting early esophageal cancer.The effect of routine treatment for lesions lightly stained with Lugol’s iodine solution is limited,and the addition of natural substances to a regular diet is becoming increasingly common.Vinegar has antitumor effects as reported in previous studies.AIM To evaluate whether vinegar supplementation could improve the prognosis of patients with lightly stained esophageal lesions.METHODSThis prospective single-centre trial included consecutive patients with lightly stained lesions between June 2020 and April 2022.Patients in the experimental group received increased amounts of vinegar for 6 months.The primary outcome of the study was the clinical therapeutic effect.Complications related to vinegar ingestion and adverse events were also recorded in detail.RESULTS A total of 166 patients were included in the final analysis.There was no significant difference in the baseline data between the two groups.Intention-to-treat(ITT)analysis demonstrated that the rates at which endoscopic characteristics improved were 33.72%in the experimental group and 20.00%in the conventional group(P=0.007);and the rates at which biopsy pathology improved were 19.77%and 8.75%,respectively(P=0.011).Additional vinegar consumption had a statistically protective effect on the rate at which endoscopic characteristics improved[hazard ratio(HR)_(ITT)=2.183,95%CI:1.183-4.028;HR_(per-protocol(PP))=2.307,95%CI:1.202-4.426]and biopsy pathology improved(HR_(ITT)=2.931,95%CI:1.212-7.089;HR_(PP)=3.320,95%CI:1.295-8.507).No statistically significant effect of increased vinegar consumption on preventing high-grade intraepithelial neoplasia or early cancer was observed(HR_(ITT)=0.382,95%CI:0.079-1.846;HRPP=0.382,95%CI:0.079-1.846).The subgroup analyses indicated that the overall therapeutic improvement of endoscopic characteristics and biopsy pathology seemed more obvious in older(age>60)male patients with small lesions(lesion size≤0.5 cm).Three patients in the experimental group reported acid regurgitation and heartburn.No adverse event during gastroscopy were recorded during follow-up.CONCLUSION A moderately increased ingestion of vinegar could not directly reduce the risk of esophageal cancer in the mucosa dysplasia population,but it improved the endoscopic characteristics and ameliorated the biopsy pathology to a certain extent.Further research is needed to verify the effect of nutritional intervention on precancerous esophageal lesions.

Impacts of dietary supplementation of bamboo vinegar and charcoal powder on growth performance,intestinal morphology,and gut microflora of large-scale loach Paramisgurnus dabryanus

Although accumulating data demonstrate that dietary supplementation of bamboo vinegar and charcoal powder(BVC)can significantly strengthen the immunity and boost the growth of domestic animals,its application potency still awaits verification in commercial fish species such as the large-scale loach Paramisgurnus dabryanus.The impacts of 90-day dietary supplementation of 1%and 2%BVC on survival and growth performance of the loach,and on the intestinal morphological characteristics and gut microflora were analyzed.Our data show that the large-scale loach supplied with BVC at the experimental doses had significant higher survival rates and better growth performance(indicated by greater weight gain(1.13–1.14 times),higher specific growth rate(1.04 times),and lower feed conversion ratio(0.88–0.89 times))compared to that of the control(P<0.05).Histological examination revealed significant longer villus(3.22–5.54 times),deeper crypt(1.77–1.87 times),and thicker muscle(1.59–3.17 times)in the intestines of large-scale loach fed with BVC(P<0.05).Furthermore,we found that the gut microflora consisted of significantly fewer proportions of potential pathogenic bacterial species(Aeromonas veronii and Escherichia coli),but significantly greater proportions of beneficial microbes(Lactococus raffinolactis and Faecalibacterium prausnitzii).Therefore,dietary intake of BVC can promote intestinal tract development and optimize gut microflora,by which the survival and growth of large-scale loach may be improved.

Production of Vinegar Mango Using Acetobacter tropicalis CRSBAN-BVA1 and CRSBAN-BVK2 Isolated from Burkina Faso

Production and quality of vinegar from mango juice was evaluated using a two steps production procedure. The first step of fermentation was done using Saccharomyces cerevisae KVL013 for 7 days. The second step, an acetic fermentation was realized using two acetic acid bacteria: A. tropicalis CRSBAN-BVA1 and A. tropicalis CRSBAN-BVK2 for 21 days. Several parameters of the vinegar produced such as physico-chemical and sensory properties were determined. Microbial density during each step was monitored. Results showed that pH, alcoholic and acetic acid contents of vinegar were respectively 2.97%, 7% and 4.54% g/ml respectively by using A. tropicalis CRSBAN-BVA1 and 3.02%, 7% and 4.32% g/ml with A. tropicalis CRSBAN-BVK2. Sensory evaluation revealed that the vinegar was acceptable to the panellists. Results of microbial density showed that the maximum concentration of cell biomass produced was 4.32 × 108 and 4.25 × 108 CFU/ml respectively for CRSBAN-BVA1 and CRSBAN-BVK2.

Effects of Biochar and Wood Vinegar on Labile Phosphorus Pool in Soda Saline-Alkaline Soil

A pot experiment was conducted to research the effect of biochar and wood vinegar on labile phosphorus fractions in saline-alkali soil.There were eight treatments,including CK(0 kg•hm-2 biochar+0 kg•hm-2 wood vinegar),C1(0.6 t•hm-2 biochar),C2(0.6 t•hm-2 wood vinegar),C3(1.2 t•hm-2 wood vinegar),C4(1.8 t•hm-2 wood vinegar),C5(0.6 t•hm-2 biochar+0.6 t•hm-2 wood vinegar),C6(0.6 t•hm-2 biochar+1.2 t•hm-2 wood vinegar),and C7(0.6 t•hm-2 biochar+1.8 t•hm-2 wood vinegar).The results showed that biochar without wood vinegar and the co-application of biochar and wood vinegar significantly increased soil total phosphorus content.Meanwhile,compared with CK,all of treatments increased resin phosphorus and sodium bicarbonate-extracted inorganic phosphorus(NaHCO3-Pi)contents in saline-alkali soil.Especially,the contents of resin phosphorus and NaHCO3-Pi under C5,C6,and C7 treatments were higher than those of C2,C3,and C4 treatments,respectively,indicating that the increases of labile phosphorus contents under the co-application of biochar and wood vinegar were better than those of the alone application of biochar and wood vinegar.Each treatment increased the proportion of labile phosphorus pool in saline-alkali soil and the proportion of labile phosphorus pool increased with the increase of the amount of wood vinegar.In addition,the application of biochar and wood vinegar increased the 100-grain weight of rice,and C6 treatment had the best effect,increasing the 100-grain weight by 134.35%.Therefore,the application of biochar and wood vinegar in saline-alkali soil could improve the soil phosphorus availability,increase the weight of rice grains,thereby realizing the resource utilization of agricultural waste and the sustainable development of agriculture.

Response Surface Methodology as an Approach for Optimization of Vinegar Fermentation Conditions Using Three Different Thermotolerant Acetic Acid Bacteria

This study aimed to investigate optimal fermentation conditions of biological acetic acid fermentation for vinegar production. Optimization was performed on 3 acetic acid bacteria strains namely VMA1, VMA7 and VMAO using Response Surface Methodology (RSM). A Box-Behnken-Design (BBD) was achieved with three different independent process parameters involving: fermentation temperature, original alcohol concentration and original acetic acid concentration and one dependent variable (acetic acid yield). The results showed that the mathematical models describe correctly the relationship between responses and factors (F values of the models (p R2 (coefficient of correlation) respectively 0.96, 0.94, 0.98, and adjusted R2 0.95, 0.92, 0.98). The maximum acidity was obtained respectively at fermentation temperatures, original alcohol concentrations and original acetic acid concentrations ranging from [37.5°C - 45°C], [16% - 20% (v/v)], [1.5% - 2% (w/v)] for VMA1, [40°C - 45°C], [14.5% - 20% (v/v)], [1.7% - 2% (w/v)] for VMA7 and [42°C - 45°C], [17% - 20% (v/v)], [1.5% - 2% (w/v)] for VMAO. The use of these acetic strains in the production of vinegar may seriously lead to a decrease or even an ablation of the costs related to the cooling of bioreactors especially in warm and hot countries, in the context of global warming.

Effect of Mongolian Vinegar Soaked Licorice on Liver Fibrosis Induced by Carbon Tetrachloride Combined with High-fat Diet in Rats

[Objectives]To determine the improvement effect of vinegar soaked licorice on liver fibrosis induced by carbon tetrachloride(CCl_(4))combined with high-fat diet in rats.[Methods]Subcutaneous injection of 40%-60%CCl_(4)olive oil solution(0.3 mL/100 g)combined with a high-fat diet was used for 5 weeks to establish the rat model with liver fibrosis.After the modeling,the rats were divided into a low dose(0.8 g/kg),a medium dose(2.5 g/kg),a high dose(5 g/kg)group,a colchicine(1.5 mg/kg)positive group,and a vinegar group(2 mL/kg).The serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)levels in the rats were measured automatically.The serum hyaluronic acid(HA)was detected by radioimmunoassay,and the serum laminin(LN)and procollagen type III peptide(PIIIP)levels were measured by enzyme-linked immunoassay.Liver histopathological morphological changes were observed by HE and Masson staining,and expressions of cytochrome CYP2E1 and transcription factor Nrf2 were detected by immunohistochemistry.[Results]The rat liver fibrosis model was established successfully at the 6~(th)week.Compared with the model group,the levels of ALT,AST,HA,LA,PIIIP,CYP2E1 and Nrf2 of all the examined indexes in the dosing group were decreased(P<0.05).As shown in the pictures of liver pathological tissue sections,the liver fibrosis was significantly alleviated in the positive group and the 3 administration groups.[Conclusions]Vinegar soaked licorice can significantly improve the liver fibrosis induced by carbon tetrachloride combined with high-fat diet in rats,and the effect of the high-dose group was similar to that of the positive group.

Effects of Different Sterilization Conditions on Active Components and Flavor of Apple Vinegar

[Objectives]To explore the effects of different sterilization conditions on nutrition and flavor of apple vinegar.[Methods]Five kinds of high temperature short time(HTST)sterilization conditions were selected to treat apple vinegar,and the volatile aroma components and the content of active components in apple vinegar before and after sterilization were analyzed.[Results]Compared with the control,the contents of total acid and malic acid in the samples after sterilization changed little,but the contents of citric acid increased significantly(P<0.01),and the contents of total phenols,ascorbic acid and total flavonoids decreased.Ethyl acetate,isopentyl acetate,ethyl caprylate,phenethyl acetate,1-pentanol,phenylethyl alcohol,acetic acid,and sec-butyl ether were the characteristic aroma components which contributed to the flavor of apple vinegar.As sterilization temperature increased,the content of esters decreased,while the content of acids,alcohols and aldehydes increased.The contents of nutrition,active components and volatile aroma components in apple vinegar under 100℃and 30 s sterilization conditions had little change compared with other sterilization conditions,so 100℃and 30 s were the optimal sterilization conditions.[Conclusions]Under different sterilization conditions,the content of flavor components in apple vinegar will change greatly,which will affect the quality of apple vinegar.

Optimization on Fermentation Conditions of Orange Vinegar by Box-behnken Design

The optimal process for orange vinegar was investigated in order to explore the effect of process parameters and obtain the superior process parameters.The process for orange vinegar was optimized using single-factor methodology and Box-behnken methodology, and the effect of process parameters was analyzed in the light of response surface plots. Results were as follows. The optimal process of alcohol fermentation was as follows： original sugar degree of 16%, yeast inoculum size of 5% and p H value of 3.5 through single-factor experiment. The optimal fermentation process of acetic acid optimized by response surface methodology was as follows： inoculum size of 8.52%, original alcohol content of 6.0% and p H value of 3.3, in the optimal condition the yield of acetic acid was 53.11 g/L. Single factor action and the interaction of two factors were presented on the yield of acetic acid.The effects of inoculum size and p H were extremely significant, and that of original alcohol content significant. The interaction of inoculum size and original alcohol content was non-significant, that of inoculum size and p H also non-significant. On contrast, original alcohol content and p H had significant interaction.

Process Optimization of Acetic Acid Fermentation of Hawthorn Vinegar

[Objective] Fermentation process of hawthorn vinegar was investigated in order to explore the effect of process andobtain superior process of hawthorn vine- gar. [Method] Fermentation process of hawthorn vinegar was firstly optimized using Box-Behnken methodologyof 3-factor and 3-level, and the effect of process was an- alyzed. Secondly, it was also finished to build secondary multivariate regression model related to yield of acetic acid. [Results] The optimal fermentation process ob- tained by response surface methodology was as follows： inoculum size of 10%, or- iginal alcohol content of 7.0% and temperature of 30 ^（3. In optimal condition, yield of acetic acid was 51.76 g/L which was close to predicted value （51.77 g/L）. In the model, interaction of inoculum size and original alcohol content was extremely signif- icant. In contrast, interaction of others was non-significant. [Conclusion] Response surface methodology is advisable to be used for process optimization of hawthorn vinegar.

Influence of Brewing Method on Quality of Apple Vinegar

[Objective] The paper was to compare the quality of apple vinegars prepared by different methods and screen an optimum brewing method for apple vinegar production. [Methed] The quality of apple vinegar brewed by three traditional brewing methods and a new method developed by our previous work was analyzed. Three traditional methods were solid state fermentation （SSF）, liquid state fermentation （LSF） and immobilized microorganism fermentation （IMF）, and the new method was multi-microorganisms co-immobilization technology（MMCT）, which used co-immobilized beads of ethanol-producing yeast, aroma-improving yeast and lactic acid bacteria （with a ratio of 6：3：1） for alcoholic fermentation and then used immobilized acetic acid bacteria for vinegar fermentation. [Result] The general quality of apple vinegar brewed by MMCT was superior to the others. Its total acidity reached to 3.845 g/100 ml, unvolatile acidity was about 0.600 g/100 ml, amino-nitrogen was higher than 0.510 g/100 ml, and the composing of flavor compounds was almost similar to that of SSF brewed apple vinegar. [Conclusion] The MMCT method was proven to be the optimum one for high quality apple vinegar brewing and might be widely used in the future.

Preliminary study of application effect of bamboo vinegar on vegetable growth

Tiffs paper reports the effects of moso bamboo （Phyllostachys pubescense） vinegar, with different diluents, on the growth of lettuce, cole and cucumber based on field tests. The results show that moso bamboo vinegar with 500-800 times dilution had good effect on the growth of tested vegetables. The harvest of vegetables increased from 18.8%-20.2% compared with a control. The height and the weight of the average single vegetable tested also increased. The main components of moso bamboo vinegar were analyzed by GC-MS and the positive effect on the growth was, in the first instance, attributed to a synergistic effect of trace amounts of the main components of bamboo vinegar.

Fermentation characteristics of vinegar residue and some natural materials

Solid state fermentation is an attractive process to produce cellulase economically due to its lower financial investment and lower operating costs. Generally available natural materials in our study included vinegar residue, wheat bran, rice bran and wheat straw. Cellulase production was carried out by solid state fermentation using these materials as the substrate of Trichoderma koningii. The ingredients of natural materials, associated with the effects of water content, time and nitrogen sources on cellulase synthesis were studied. A comparatively high cellulase activity （〉4 IU·g^-1 SDM） was obtained in the fermentation batch. The optimum culture time of vinegar residue, wheat bran and wheat straw were 72 hours, but that office bran was 60 hours. Total water contents of wheat bran, rice bran and wheat straw should not exceed 50% and that of vinegar residue should not be more than 60%. It was also shown that nitrogen salts contributed much to fermentation. （NH4）3PO4 and urea achieved good results in promoting enzyme activity.

Effect of Wood Vinegar on Seed Germination and Water Implantation of Corn

To analyze the effect of wood vinegar on seed germination and seedling growth, the seeds of corn are dipped in wood vinegar of different densities. The results showed that significant effects were found through all the treatments on the seed germination rate, the seed germinating energy and the germinating index. The regress CUBICS curves were developed to describe the relation. The reasonable parameters range was obtained. At the same time, the corn was cultivated by wood vinegar of different densities, aiming to study the effect of wood vinegar on biomass. It showed that all treatments had obvious effects on the seedling length and dry weight aboveground, the chlorophyll and dry weight underground were not included. The research results could be used to direct the wood vinegar deeply refining process and product development.

Effect of esophagus and stomach cancer-preventing vinegar on N-nitrosoproline formation in human body

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Upgrading the Fermentation Process of Zhejiang Rosy Vinegar by Purebred Microorganisms

Purebred microorganisms were employed to upgrade the fermentation process of Zhejiang rosy vinegar. The fermentation cycle was greatly shorten from 5 months to 72 d. The transformation rate of raw materials was increased from 1:4.5 in the traditional fermentation to 1:5 or more in the upgraded fermentation. The content of organic acids in the traditional vinegar (TRV), the upgraded vinegar (UPV) and the submerged fermentation vinegar (SFV) were also investigated by HPLC. No significant difference was found regarding the proportion of phenylethanol to the total volatile components in UPV (7.47% ± 0.00324%) and TRV (7.23% ± 0.00329%), but it was significantly higher than that in SFV (2.26% ± 0.00143%). This study provides deep insight into upgrading the fermentation process of Zhejiang rosy vinegar by purebred microorganisms.

Optimization of Acetic Acid Fermentation Process of Apple Cider Vinegar

This paper studied the fermentation rules of apple cider vinegar from fruit juice,to provide a theoretical guidance for the production of apple cider vinegar.Using Fuji apples as raw materials,the process parameters(fermentation temperature,fermentation time,stirring speed,and inoculation amount)of apple cider vinegar fermentation were optimized through single-factor experiments and response surface analysis.The results indicated that the fermentation temperature had no significant effect on the total acid content of apple cider vinegar fermentation,the fermentation time had an extremely significant effect on the total acid content of apple cider vinegar fermentation,and the stirring speed and inoculation amount had a significant effect on the total acid content of apple cider vinegar fermentation.Through process optimization,the optimal process parameters for apple cider vinegar fermentation are fermentation temperature of 33℃,fermentation time of 39 h,stirring speed of 1500 r/min,and acetic acid bacteria inoculation amount of 7%.Under such conditions,the total acid content of fermented apple cider vinegar is 62.22 g/L,very close to the predicted value of the model,indicating that the process parameters of acetic acid fermentation obtained by response surface methodology(RSM)optimization are reliable and can be used for actual production prediction.

Antidiabetic and antioxidant activities of Nypa fruticans Wurmb. vinegar sample from Malaysia

Objective: To study the antidiabetic and antioxidant activities of nipa palm vinegar(NPV) used in traditional Malay medicine for treating diabetes. Methods: NPV was extracted using liquid-liquid extraction method and the obtained samples were subjected to antidiabetic studies using normal and streptozotocin-induced diabetic rat models whereas antidoxidant activities were investigated via in vitro antioxidant tests namely 2,2-diphenyl-1-picrylhydrazyl and 2,2'-azinobis-3-ethylbenzothiozoline-6-sulfonic acid free radicals scavenging activities and the reducing power assay. Results: Single administration of NPV and its extracts were not effective in both normal and diabetic rats. In intraperitoneal glucose tolerance test, NPV and its aqueous extract showed significant blood glucose lowering effect. In the sub-acute study, compared with the diabetic control, aqueous extract of NPV showed the most notable blood glucose lowering effect(56.6%) and a significant improvement in serum insulin levels(79.8%, P<0.05). To assess NPV's antioxidant activity, three in vitro antioxidant tests were employed: 2,2-diphenyl-1-picryhydrazyl and 2,2'-azinobis-3-ethylbenzothiozoline-6-sulfonic acid free radical-scavenging assays, and the reducing power assay. Ethyl acetate extract had the greatest antioxidant potential and content of phenolic and flavonoid compounds. A linear positive correlation between the antioxidant parameters was observed. Chemical profiling analysis of aqueous extract of NPV revealed the presence of acetic acid(35.25%), the main active constituent which significantly contributed to the observed antidiabetic activity. Conclusions: Aqueous extract of NPV possesses antihyperglycaemic activities comparable to the metformin, while the ethyl acetate extract precipitated significant antioxidant effects attributable to its high phenolic content. These findings suggest that antioxidant compounds of NPV do not contribute much towards the overall observed antidiabetic effect.

Inhibitory Effect of Wood Vinegar Produced from Apricot Shell on Aspergillus fumigatus

[Objectives] This study was conducted to explore the antifungal activity of wood vinegar produced from apricot shell. [Methods]The minimal inhibitory concentration（ MIC） of wood vinegar against Aspergillus fumigatus was determined on agar plates. Then,the mycelial growth and the morphology of the apical vesicles of A. fumigatus cultured in the medium supplemented with 1/2 MIC wood vinegar were observed to evaluate the effect of wood vinegar on A. fumigatus.RNA-Seq was performed to explore the antifungal mechanism of wood vinegar. [Results] The wood vinegar produced from apricot shell could inhibit the growth of A. fumigatus,with a MIC of 20 μl/ml. In the medium supplemented with 1/2 MIC wood vinegar,the mycelial growth of A. fumigatus was suppressed,some apical vesicles were incompletely developed. The gene ontology（ GO） term enrichment analysis based on RNA-Seq showed that the differentially expressed genes（ DEGs）were enriched in metabolic process,cellular process and single-organism process in the category of biological processes,cell,cell part,membrane,organelle and membrane part in the category of cellular components,catalytic activity and binding in the category of molecular function. The significantly up-regulated DEGs were mostly involved in cell,cell part,organelle,organelle part and macro molecular complex in the category of cellular component,while the significantly down-regulated DEGs were mostly involved in biological process,cellular process and single-organism process in the category of biological process,catalytic activity,binding,transporter activity,and nucleic acid binding transcription factor activity in the category molecular function. Moreover,KEGG（ Kyoto Encyclopedia of Genes and Genomes） pathway analysis showed that the DEG unigenes involved in metabolism were the most,followed by those involved in genetic information processing.[Conclusions]The results indicated that the wood vinegar produced from apricot shell might inhibit the growth of A. fumigatus by interfering with the expression of multiple genes involved in multiple signaling pathways.