Microbial metabolic interaction in fermentation ecosystem and cooperation in flavor compounds formation of Chinese cereal vinegar

Shanxi aged vinegar(SAV)is a famous cereal vinegar in China,which is produced through a solid-state fermentation where diverse microbes spontaneously and complex interactions occur.Here,combined with the metatranscriptomics,the microbial co-occurrence network was constructed,indicating that Lactobacillus,Acetobacter and Pediococcus are the most critical genera to maintain the fermentation stability.Based on an extensive collection of 264 relevant literatures,a transport network containing 2271 reactions between microorganisms and compounds was constructed,showing that glucose(84%of all species),fructose(67%)and maltose(67%)are the most frequently utilized substrates while lactic acid(64%),acetic acid(45%)are the most frequently occurring metabolites.Specifically,the metabolic influence of species pairs was calculated using a mathematical calculation model and the metabolic influence network was constructed.The topology properties analysis found that Lactobacillus was the key role with robust metabolic control of vinegar fermentation ecosystem and acetic acid and lactic acid were the main metabolites with feedback regulation in microbial metabolism of SAV.Furthermore,systematic coordination of positive and negative impacts was proved to be inevitable to form flavor compounds and maintain a natural microbial ecosystem.This study provides a new perspective for understanding microbial interactions in fermented food.

De novo transcriptome assembly of Aureobasidium melanogenum CGMCC18996 to analyze theβ-poly(L-malic acid)biosynthesis pathway under the CaCO_(3) addition

β-Poly(L-malic acid)(PMLA)is a water-soluble biopolymer used in food,medicine and other industries.To date,the biosynthesis pathway of PMLA has not been fully elucidated.In this study,we sequenced the transcriptom e of strain Aureobasidium melanogenum under 20 g/L CaCO_(3) addition.The resulting sequencing reads were assembled and annotated for the differentially expressed genes(DEGs)analysis and novel transcripts identification.The result indicated that with the CaCO_(3) addition,the tricarboxylic cycle(TCA)cycle and glyoxylate pathway were up-regulated,and it also found that a non-ribosomal peptide synthetase(NRPS)like protein was highly expressed.The DEGs analysis showed a high expression level of malate dehydrogenase(MDHC)and phosphoenolpyruvate carboxykinase(PCKA)in the CaCO_(3) group,which indicated a cytosolic malate activity.We speculated that the malate should be transported to or synthesized in the cytoplasm,which was then polymerized to PMLA by the NRPS-like protein,accompanied by the up-regulated TCA cycle providing ATP for the polymerization.Depending on the analysis,we assumed that an NRPS-like protein,the TCA cycle,and the cytosolic malate together are contributing to the PMLA biosynthesis.

Molecular basis of cross-interactions between Aβ and Tau protofibrils probed by molecular simulations

Amyloid β-protein(Aβ) and Tau, two common pathogenic proteins associated with Alzheimer’s disease(AD), cross-interact, and thus co-assemble into hybrid aggregates. However, molecular mechanism of the cross-interactions remains unclear. To explore the issue, docking and molecular dynamics(MD) simulations were coupled to study the cross-interactions between Aβ pentamer and Tau pentamer. Four stable hybrid decamer conformations including double layer, single layer, block, and part-in were obtained by protein-protein docking software HADDOCK 2.2. Then, MD simulations were used to explore the molecular mechanism of cross-interactions between Aβ pentamer and Tau pentamer. The results of MD simulations showed that the part-in structure was the most stable among all the above four representative ones. The binding energy between Aβ and Tau was about-759.77 kJ·mol-1in the part-in structure. Moreover, the part-in conformation would undergo conformational transition, which would improve its hydrophobicity and make the structure more compact. This work offers a structural understanding of cross-interactions between Aβ and Tau linked to AD.

Multi-component quantitative and feed-forward neural network for pattern classification of raw and wine-processed Corni Fructus

Background:To promote the quality evaluation,clarify the processing mechanism and distinguish origins of Corni Fructus(cornus)from different regions.Methods:This study developed a high performance liquid chromatography method for simultaneous determination of 5-hydroxymethylfurfural,2 phenolic acids and 4 iridoid glycosides and the reference fingerprint of cornus from different regions.In addition,the feedforward neural network model provided a pattern classification of sample regions.Results:The content of morroniside and loganin were the highest in all raw cornus samples ranging from 9.45μg/mg to 16.3μg/mg and 6.64μg/mg to 13.7μg/mg,respectively.The level of sweroside in raw cornus from Henan(0.83μg/mg^(-1).39μg/mg)and Zhejiang(0.64μg/mg^(-1).17μg/mg)were greater than other origins.After wine-processing,the glucose or fructose were dehydrated to increase the levels of 5-hydroxymethylfurfural.The C-4 position of-COOCH3 of hot-sensitive iridoid glycosides was hydrolyzed to generate-COOH as stable components.Polyphenol derivatives may be degraded to increase the content of phenolic acid.Subsequently,an excellent feedforward neural network model for identification of raw cornus and wine-prepared cornus was established which could distinguish the sample origins.Conclusion:This work provided a trustworthy method to evaluate the quality and distinguish the sources of cornus.Meanwhile,the clear processing mechanism provided a scientific foundation for controlling the cornus quality during wine-processing.

Lindqvist-type Polyoxometalates Act as Anti-breast Cancer Drugs via Mitophagy-induced Apoptosis

Objective Lindqvist-type polyoxometalates(POMs)exhibit potential antitumor activities.This study aimed to examine the effects of Lindqvist-type POMs against breast cancer and the underlying mechanism.Methods Using different cancer cell lines,the present study evaluated the antitumor activities of POM analogues that were modified at the body skeleton based on molybdenum-vanadium-centered negative oxygen ion polycondensations with different side strains.Cell colony formation assay,autophagy detection,mitochondrial observation,qRT-PCR,Western blotting,and animal model were used to evaluate the antitumor activities of POMs against breast cancer cells and the related mechanism.Results MO-4,a Lindqvist-type POM linking a proline at its side strain,was selected for subsequent experiments due to its low half maximal inhibitory concentration in the inhibition of proliferation of breast cancer cells.It was found that MO-4 induced the apoptosis of multiple types of breast cancer cells.Mechanistically,MO-4 activated intracellular mitophagy by elevating mitochondrial reactive oxygen species(ROS)levels and resulting in apoptosis.In vivo,breast tumor growth and distant metastasis were significantly reduced following MO-4 treatment.Conclusion Collectively,the results of the present study demonstrated that the novel Lindqvist-type POM MO-4 may exhibit potential in the treatment of breast cancer.

Purification and Characterization of an Antibacterial Protein from the Cultured Mycelia of Cordyceps sinensis

An antibacterial protein was isolated from the cultured mycelia of Cordyceps sinensis, and was designated as Cordyceps sinensis Antibacterial Protein （CSAP）. CSAP was single-chained, with an apparent molecular mass of 35 × 10^3 revealed by SDS-PAGE and a novel hydrophobic N-terminal sequence N-ALATQHGAP. The antimicrobial assays showed CSAP could inbibit the growth of Gram-positive and Gram-negative bacteria but no significant inhibition against fungi or yeasts. Further more, the antibacterial activity of CSAP was not bactericidal but bacteriostatic. It was the first time that an antibacterial protein was described in the Cordyceps species, which might involve in the chemical defense mechanism of the hosts.

Synthesis and Characterization of Carboxyl-terminated Polyethylene Glycol Functionalized Mesoporous Silica Nanoparticles

Colloidal mesoporous silica nanoparticles functionalized with carboxy-terminated polyethylene glycol(CMS-PEG-COOH) were successfully synthesized by covalently grafting dicarboxy-terminated polyethylene glycol(HOOC-PEG-COOH) on the surface of the amino functionalized CMS nanoparticles with amide bond as a cross linker. Moreover, the structural and particle properties of CMS-PEG-COOH were characterized by nuclear magnetic resonance spectroscopy(1 H-NMR), transmission electron microscopy(TEM), dynamic light scattering(DLS), nitrogen adsorption-desorption measurements, X-ray diffraction(XRD), and Fourier transform infrared spectroscopy(FT-IR). The nanomaterials presented a relatively uniform spherical shape morphology with diameters of about 120 nm,and favorable dispersibility in weak acid solution. The CMSPEG-COOH exhibited no changes in the state of amorphous, while the mesopores sizes of 5.25 nm might provide the nanomaterials with large capacity for the loading and releasing of drugs. So the results indicated that CMSPEG-COOH might be a critical nanomaterial for drug delivery system in the future.

Carbonaceous Nanofibers-titanium Dioxide Nanocomposites: Synthesis and Use as a Platform for Removal of Dye Pollutants

A mild chemistry route was developed to prepare carbonaceous nanofibers-titanium dioxide(CNF-TiO_2) nanocomposites for removal of dye pollutants. In the process of the template-directed hydrothermal carbonization(HTC), ultrathin Te nanowires were adopted as templates and glucose as the carbon source, and TiO_2 was decorated on CNF via the hydrolysis of tetrabutyltitanate in the presence of CNF in ethanol. The as-prepared materials were characterized by scanning electron microscopy(SEM), transmission electron microscopy(TEM), energy-dispersive X-ray(EDX) and X-ray diffraction(XRD). SEM and TEM observations displayed that TiO_2 nanoparticles were anchored on the CNF. EDX and XRD data confirmed that the assynthesized samples were CNF-TiO_2, and TiO_2 belonged to anatase titania. Taking advantage of combined benefits of carbonaceous nanofibers and titanium dioxide, these CNF-TiO_2 nanocomposites exhibited higher removal efficiency in a short time and showed good reusability. It was showed that over 97% of Rhodamine B could be removed in 15 min without generating the solid and liquid wastes. The removal efficiency of dyes was still over 80% after reuse in five cycles. All the results demonstrate that the as-prepared CNF-TiO_2 composites are effective materials for fast and effective removal of dye pollutants and thus can provide a new platform for dye decontamination.

Breeding of NHase hyper-producing <i>Rhodococcus ruber</i>strain LUV_30-06and verification of mutants by RAPD

A nitrile hydratase (NHase) hyper-producing Rhodococcus ruber strain LUV30-06 was bred by mutagenization on the starting strain CGMCC3090 with ultraviolet irradiation and lithium chloride. The NHase activity of the strain LUV30-06 was increased by 21.99% (3881.3 U/ml), as compared with that of R. ruber CGMCC3090 (3181.4 U/ml). The mutant strain UV30-06 has been proved genetically stable with higher NHase activity in seven successive subcultures as well as random amplified polymorphic DNA analysis (RAPD).

Chemical composition and antimicrobial activity of essential oil isolated from the cultured mycelia of Ganoderma japonicum

Objective：To explore a new natural antibiotic. Methods：The chemical composition of the essential oil from Ganodermajaponicum （G. japonicum）mycelia was analyzed by gas chromatography-mass spectrometry（GC-MS）, The antimicrobial activity of the oil was evaluated against eighteen microorganisms, including bacteria, mildew and yeast by using a disc diffusion method. Furthermore, the minimum inhibitory concentrations（MIC） and the minimum bactericidal concentrations（MBC） of the essential oil against twelve clinical pathogens were determined. Results：The main components of the oil were nerolidol, decadienal, linalool and benzyl alcohol. The antimicrobial results indicated that the oil inhibited all the tested bacterium, especially Methicillin-resistant Staphylococcus aureus （MRSA） in which the antibacterial activity exhibited a MBC of 1.03 mg/ml. Conclusion： The essential oil of G. japonicum mycelium has significant inhibitory activity. It is a potential medicinal resource that can be used as a natural antibiotic.

Preparation and Characterization of Carboxyl Functionalized Fluorescent Mesoporous Silica Nanoparticles Containing 8-Hydroxyquinolinate Zinc Complexes

Fluorescent mesoporous silica nanoparticles functionalized with carboxyl group(Znq-CMSCOOH) were successfully synthesized by in situ formation route of 8-hydroxyquinolinate zinc complexes in channels of mesoporous silica nanoparticles and post-grafting of carboxyl group on the surface. Moreover,the particle size and structural properties of Znq-CMS-COOH were characterized by transmission electron microscopy(TEM),field emission scanning electron microscopy(FE-SEM),dynamic light scattering(DLS),Fourier transform infrared spectroscopy(FT-IR),UV-vis spectrometer, fluorescence spectrometer and nitrogen adsorption-desorption measurements. The obtained results suggest that the Znq-CMS-COOH presents the uniform spherical shape with the mean diameter of about 85 nm and the obvious wormhole arrangement mesoporous. In addition, the Znq-CMS-COOH possesses green fluorescence with the emission peaks at 495 nm. So the Znq-CMS-COOH, which is beneficial to further modification and tracing, might be a great potential carrier for applying in drug delivery system in the future.

Unraveling the core functional bacteria and their succession throughout three fermentation stages of broad bean paste with chili

The entire fermentation process of traditional Chinese broad bean paste with chili comprises three individual stages:Tianbanzi,chili pei,and paste fermentation(Tianbanzi-chili pei mixture).Three stages share average 77.53%of all bacteria(89 genera),indicating that the similar environment leads to the similar bacterial communities.One,one,and three genera are exclusive to Tianbanzi,chili pei,and paste stages,respectively,due to the special physical and chemical properties for each stage.Total acidity,pH,and NaCl are important endogenous factors that promote the succession of bacterial communities.According to the dynamics of organic acids,reducing sugars,amino acids,and volatile compounds,60-,210-,and 180-day are considered the best fermentation periods for Tianbanzi,chili pei,and paste,respectively,to balance time cost and product quality.Three(Tetragenococcus,Lactobacillus,and Pseudomonas),four(Tetragenococcus,Lactobacillus,Bacillus,and Pseudomonas),and five(Tetragenococcus,Lactobacillus,Bacillus,Pseudomonas,and Pediococcus)genera are considered the core functional bacteria of Tianbanzi,chili pei,and paste fermentation,respectively.

Preparation and Crystal Structure of 15α-Hydroxyl-oleanolic Acid

The compound 15α-hydroxyl-oleanolic acid was biotransformed from oleanolic acid with Colletotrichum lini AS3.4486,and its structure was characterized by 1H NMR,13C NMR,HR-ESI-MS and single-crystal X-ray diffraction.The crystal of the title compound belongs to monoclinic,space group R3 with a=34.255（5）,b=34.255（5）,c=6.8671（14）,β=90°,Z=9,V=6978（2）3,Mr=490.70,Dc=1.051 mg/m3,μ=0.069 mm-1,F（000）=2430,R=0.0375 and wR=0.0808.The title compound is stacked into a three-dimensional network through hydrogen bonds.

Self-induced Secretion Expression of Trehalose Synthase in Bacillus subtilis WB800n

Bacillus subtilis is a non-pathogenic Gram-positive bacterium that has been widely used to produce industrially and pharmaceutically relevant proteins. Trehalose, a non reducing disaccharide used as protective agent and additive in foodstuffs and pharmaceutical products, can be prepared by trehalose synthase(TreS). The present work aims to construct a robust recombinant B. subtilis to achieve the secretory expression of TreS. In this study, the treS gene from Pseudomonas putida ATCC47054 was amplified by PCR and further cloned and expressed in B. subtilis WB800 N using pHT01 as expression vector. For avoiding the use of inducer, promoter P_(srfA) was used to replace the promoter P_(grac) in pHT01 and verify the activity of recombinant trehalose synthase. The TreS activity assay was employed to evaluate the performance of recombinant B. subtilis W800 N under different phosphate concentrations, carbon sources, carbon source concentrations, nitrogen sources and pH. The results showed that the P_(srfA) promoter had a good regulation effect under pH 8.0 condition, and the enzyme activity reached 6 000 U/L. Using the PhoD as the secretory signal peptide, TreS was effectively secreted, and the extracellular enzyme activity reached 2 100 U/L, accounting for 35% of the total enzyme activity. By optimizing the medium and fermentation conditions, the extracellular enzyme activity reached 6 900 U/L in 5 L of fermentor, and the proportion reached 48%. The pHT01-P_(srfA)-PhoD-treS secretory recombinant B. subtilis constructed in this study has great potential in trehalose synthase production.

Effect of Fermented Feed on Intestinal Microbial Community Diversity of Chicks

[Objective]The paper was to study the effects of fermented feed on structure and diversity of intestinal flora in the duodenum,jejunum,ileum and cecum of chicks,and to clarify the mechanism of fermented feed on growth and health of chicks.[Method]The 3601-day-old Hyline brown egg chicks were randomly divided into three treatment groups,six replicates each group,and were fed with basal diet,basal diet+antibiotics and basal diet+fermented feed,respectively.The structure and distribution of intestinal microbial community of egg chicks in each group were determined by Illumina MiSeq sequencing technology.[Result]The Chaol index and Shannon index indicated that the abundance and diversity of microorganisms in unfermented feed group were significantly higher than those in fermented feed group,and the abundance and diversity of flora in the duodenum,jejunum and ileum of each group were significantly higher than those in the cecum.[Conclusion]Feeding fermented feed significantly increases the abundance of Lactobacillus and reduces the number of pathogenic bacteria such as DSV and Bacieroides in the intestinal tract,which also indirectly proves that Lactobacillus inhibits the proliferation of DSV and prevents intestinal diseases.Feeding fermented feed increases the abundance of Firmicutes,significantly decreases the proportion of Bacteroidetes and Firmicutes,and promotes the growth of chicks.

Fast Determination of Vitamin B₂Based on Molecularly Imprinted Electrochemical Sensor

Under the condition of weak acidity of pH 5.2, a sensitive vitamin B2 electrochemical sensor based on molecularly imprinted nonconducting polymer of o-aminophenol by potentiostatic polymerization in the presence of template(vitamin B2) on a glassy carbon electrode was prepared, and its performance was studied. The sensor exhibited good sensitivity and selectivity to VB2. The detection limit went down to 2.3851nM, and a linear relationship between the current incremental and the concentration was found in the range of 10~120nM. And the sensor could use in detection of VB2 real sample for a long time and show good reproducibility. The average recovery rate to VB2 was 98.41%.

Preparation and Crystal Structure of 12β,15α-dihydroxyprogesterone

Progesterone（1） was biotransformed into the title compound 12β,15α-dihydroxy-progesterone（2） with Colletotrichum lini AS3.4486.The biotransformation process was monitored with HPLC.The structure of 2 was determined by 1H NMR,13C NMR,ESI-MS and single-crystal X-ray diffraction.The crystal of the compound belongs to orthorhombic,space group P212121 with a = 8.0671（9）,b = 12.3970（15）,c = 18.532（3） ,Z = 4,V = 1853.3（4）3,Dc = 1.242 mg/m3,μ = 0.084 mm-1,F（000） = 752,R = 0.0373 and wR = 0.0850.Single-crystal X-ray diffraction analysis reveals that a 1D supramolecular structure of 2 has been constructed by multiply intermolecular O（2）-H（2）…O（4） and O（3）-H（3）…O（2） H-bonding interactions.

Toxicological advances of traditional medicine in 2020

There were many researches concerning toxicology of traditional medicine and active natural products during the past 12 months.This annual toxicology review summarized different toxic analysis methods of traditional medicine,evaluated models,toxic target organs,toxic mechanisms,popular research issues and herbs in 2020.Caenorhabditis elegans came to use for the assessment of toxicity.Omics technology such as genomics,transcriptome,metabolomics and proteomics were applied extensively.2020 toxicology research demonstrated that liver,kidney and heart were the mainly toxic target organs of traditional medicine.Their toxic mechanisms included cell apoptosis,metabolic disorder,oxidative stress,inflammatory damage,liver and renal fibrosis and even inducing carcinogenesis.In addition,the safety assessment of Aconitum carmichaeli Debx.,Tripterygium wilfordii Hook.f.and Polygonum multiflorum Thunb.as well as their detoxification methods were still hot issue.Therefore,study on the toxicity mechanism of target organs,processing and extract methods,quality control and dose control,new models and methods should be used in the prevention of traditional medicine toxicology in the future.

Toxicological advances of traditional medicine in 2019

There were many researches concerning toxicology of traditional medicine(TM)and active natural products during the past 12 months.This annual toxicology review summarized target organs of TM like liver,kidney and heart.Safety medication of TM has been concerned to different kinds of people,including infants,children,pregnancy and the postnatal period.Besides rodents,zebrafish embryoes have been regarded as common models to evaluate the safety of TM.New technologies in toxicology focus on rapidly screening and identification of toxins in TM.Multispectral optoacoustic tomography imaging the precise location of TM-induced liver injury with 3D information and integrating serum exosomal microRNA and liver microRNA profiles are used to explain the mechanism of TM-induced hepatotoxicity.Taken together,study on the toxicity mechanism of other target organs,drug safety in elders,new models and methods should be paid attention to in the prevention of TM toxicology in the future.

Toxicological advances of traditional medicine in 2021

In 2021,there have been many studies on the toxicological evaluation of traditional medicine,as well as natural active ingredients.Among them,the toxicity evaluation of evodiamine,Gardenia Jasminoides J.Ellis,and anthraquinone in the liver,kidneys,heart,and other organs has become a popular topic.Their toxic mechanisms include oxidative stress,inflammatory response,apoptosis,mitochondrial damage,and disorders of lipid and amino acid metabolism.In response to the drawbacks of time-consuming,expensive,and ethical restrictions of animal models,a variety of techniques,such as 3D organoid models,metabolomics,toxicokinetics,bioprinting methods,and network toxicology methods,have been gradually employed in the toxicity evaluation of traditional medicine.This review summarizes the drug toxicity and safety assessment of traditional medicine in 2021.In the future,attention should be paid to preventing traditional medicine toxicity.