Adequate intake of probiotics helps in maintaining the homeostasis of the gut microbiota, which is of great significance for human intestinal health. However, probiotics are vulnerable to stress and necessitate stringent storage conditions in industrial settings, which pose challenges to the vitality maintenance of probiotics in transportation and shelf-life. Therefore, it is essential to develop the preparation process capable of protecting the high vitality of probiotics. Drying beneficial for sustaining probiotic vitality is often used to maintain the strain stability. Among the drying methods, freeze-drying is widely used. However, during the freeze-drying process, probiotics encounter environmental stress conditions, which lead to cellular damage and even death. Therefore, the freeze-drying protectants that can significantly improve probiotic survival rate have become a research hotspot in the industrial application of probiotics. This article systematically summarizes both the commonly used and the newly discovered protectants for freeze-drying of probiotics and elucidates their mechanisms of action. It provides an overview of the factors affecting cell viability post freeze-drying while outlining the prospective research directions of freeze-drying protectants. This review is expected to furnish theoretical substantiation for the development of freeze-dried probiotics with high vitality.

[Objective] We investigated the diversity, composition, and functions of soil bacterial communities of wildGlycyrrhiza uralensis Fisch., a typical halophyte and medicinal plant, in the habitats with different degrees of salinization. The study can help to reveal the linkage between soil salinity and microbiome related to the growth, development, and quality formation ofG.uralensis, being essential for improving the quality of cultivatedG.uralensis. [Methods] Soil samples were collected from six main habitats of wildG.uralensis. High-throughput sequencing was employed to compare the diversity, composition, and functions of soil bacterial communities among the habitats of un-salinization (US), light salinization (LS), moderate salinization (MS), and heavy salinization (HS) and to excavate the dominant bacteria in different habitats. [Results] The soil bacterial richness and diversity of wildG.uralensis were higher in LS and MS habitats than in US and HS habitats and were the lowest in the HS group. The principal component analysis (PCA) revealed differences in the composition and functions of soil bacterial communities between groups with different degrees of salinization (P < 0.05). The redundancy analysis (RDA) showed that total salinity (TS) was an important factor influencing the composition and functions of the soil bacterial community in the native habitat. The dominant bacterial genera in the US and LS groups were the same, all of which were plant-beneficial bacteria, includingNocardioides,Streptomyces, andMarmoricola. The significantly dominant genera in the MS group included both the beneficial bacteria unidentified_Acidobacteria and the halophilic bacteriaHalomonas andMarinobacter. The soil bacteria in the HS group were dominated by salinophilic or salinity-tolerant bacteria, includingHalomonas,Marinobacter,Truepera,Alifodinibius,Salinimicrobium, andSalegentibacter. The PICRUSt prediction results underlined the potential of soil bacteria in interactions with plants in US, LS, and MS groups, suggesting that beneficial bacteria in the three habitats influenced the growth, development, and quality formation of wildG.uralensis. Moreover, the prediction results emphasized that the soil bacteria endowed the HS group with the potential of self-repairing for adaptation to highly saline environments and improving the salt tolerance of wildG.uralensis. This result suggested that halophilic and salt-tolerant bacteria in the HS habitat played a key role in the salt tolerance of wildG.uralensis. The MS habitat had both kinds of bacteria, being worthy of attention. [Conclusion] The soil bacterial diversity and richness of wildG.uralensis were remarkably higher in LS and MS habitats than in US and HS habitats. The composition and functions of soil bacterial communities in US and LS habitats were similar and differed significantly from those of the HS habitat, and the MS habitat had characteristics of both the above.

The conventional rescue system of newcastle disease virus (NDV) contains a cDNA clone plasmid and three helper plasmids expressing NDV: nucleocapsid protein (NP), phosphoprotein (P) and polymerase protein (L), respectively. These four plasmids have to be transfected into the same host cell simultaneously to complete the assembly of the virus, which is relatively inefficient. [Objective] To improve the rescue efficiency of NDV, this study aims to establish a two-plasmid rescue system. [Methods] The expression cassettes of NP, P, and L genes were constructed and sequentially cloned into the eukaryotic expression vector pCI to generate a single-helper plasmid, named pCI-NPL, capable of co-expressing NP, P, and L proteins. During this process, the genomic cDNA of the NDV LaSota strain was incorporated downstream of the CMV promoter in the pCI expression vector. Simultaneously, the reporter gene EGFP was inserted into the genome between the P and M genes, accompanied by the introduction of a hammerhead ribozyme sequence at the 5′ end and a hepatitis delta virus ribozyme sequence at the 3′ end. This culminated in the development of the full-genome transcriptional plasmid termed pCI-LaSota-EGFP. The two plasmids pCI-LaSota-EGFP and pCI-NPL were co-transfected as a two-plasmid system into BHK-21 cells to rescue the recombinant virus rLaSota-EGFP. The biological characteristics of the virus were then examined. [Results] RT-PCR, fluorescence microscopy, Western blotting, and growth characterization confirmed that rLaSota-EGFP was correctly constructed and expressed the foreign gene. The rescued recombinant virus rLaSota-EGFP had similar biological characteristics to wild-type (WT) LaSota. [Conclusion] A novel two-plasmid rescue system for NDV based on the CMV promoter was successfully established, laying a foundation for the efficient rescue of recombinant NDV and other paramyxoviruses.

Equine coronavirus (ECoV) is an emerging virus attacking the gastrointestinal tract in horses, and the infected adult horses mainly present fever, abdominal pain, and diarrhea. In 1975, ECoV infection first appeared in the United States, and since then it has been prevalent in many countries and regions. Only one recombinant strain of ECoV was isolated from the small intestine of a donkey experiencing diarrhea in Shandong Province, China. [Objective] Understanding the genetic composition, genetic relationship, and biological characteristics of ECoV strains in China can lay a foundation for unveiling the epidemic status and genetic evolution trend of ECoV and provide materials for the research and development of products for the prevention and control of ECoV. [Methods] RT-PCR was employed to detect the stool samples from a horse experiencing diarrhea in Huangpi District, Wuhan City, Hubei Province. The virus was isolated from the positive samples and verified by indirect immunofluorescence assay (IFA) with monoclonal antibodies targeting the S1 protein of ECoV. Based on the whole genome sequencing results of the isolate, the phylogenetic analysis and sequence alignments of the whole genome, N gene, and NS2 gene were performed. [Results] An ECoV strain was successfully isolated and named ECoV-JL. Transmission electron microscopy (TEM) showed that the isolated virus particles were spheroidal and had a capsule membrane and a typical spiroid structure of coronaviruses. The tissue culture infectious dose 50% (TCID₅₀) of ECoV-JL reached a peak of 10^6.16 TCID₅₀/mL 72 h post infection in HRT-18 cells. ECoV-JL strains could infect three human cell lines: HRT-18 (human ileocecal cancer cells), Caco-2 (human colorectal adenocarcinoma cells), and Huh7 (human liver cancer cells). The genome sequence of ECoV-JL and the ECoV genome sequences in GenBank showed the similarity within the range of 97.9%–99.0%. ECoV-JL was in a separate branch of the phylogenetic tree and far related to other strains, which indicated that ECoV-JL might be derived from recombination mutations. The NS2 gene presented more mutations, and the difference in NS2 gene was the main reason for the poor homology between ECoV-JL and other strains. [Conclusion] We isolated and identified an ECoV strain from the stool samples of horses with diarrhea and named it ECOV-JL. The study about the biological characteristics and phylogenetic relationship of this strain reflected the characteristics of the epidemic strains in Hubei Province, providing a clue for the epidemic status and evolution trend of ECoV in China.

Atopic dermatitis (AD) is a highly prevalent allergic skin disease characterised by recurrent attacks and severe itching. The pathogenesis of AD involves a variety of factors including genetic susceptibility, epidermal barrier dysfunction, microbiome dysbiosis, immune imbalance, and the environment, while the available therapeutic drugs have severe side effects and limited efficacy. Studies have demonstrated that gut microbiota, particularly probiotics, play a role in AD. Probiotics can alleviate AD symptoms by inhibiting pathogens, enhancing barriers, improving the intestinal environment, and balancing the Th1/Th2 immune response, among other mechanisms. In this review, we summarized the skin and intestinal microecological characteristics of AD patients and systematically elucidated the mechanisms of probiotics in alleviating AD from the pathogenesis and influencing factors of AD, aiming to provide theoretical support for probiotics in the treatment of AD and related allergic skin diseases.

Hepatitis E virus (HEV) is a major zoonotic pathogen that causes acute viral hepatitis worldwide. HEV has high genetic diversity, and the incidence of various genotypes or subtypes is strongly correlated with host species, geographic location, and prevention and control methods. HEV strains HEV-3, comprising 3a–3i subtypes, were present in Europe and America, whereas HEV-3 and HEV-4 were prevalent in Asia. The epidemic strains in China have evolved from HEV-1 to HEV-4. Recent studies have shown that gene recombination, amino acid mutations, and synonymous codon usage patterns are part of the mechanisms underlying HEV evolution. In particular, amino acid mutations are the main driving force for the continued prevalence of the virus. This paper reviews the classification, global epidemic characteristics, and evolutionary mechanism of HEV, aiming to provide a reference for the prevention and control of hepatitis E and vaccine development.

The roles of gut microbiota and its metabolites in the pathogenesis and rehabilitation of neurodegenerative diseases, gastrointestinal diseases, and musculoskeletal systemic diseases in the elderly are receiving increasing attention. Gut microbiota and its metabolites can regulate the functions of the cranial nervous system and the musculoskeletal system through various pathways, involving the immune, endocrine, and nervous systems. Conversely, the gut, brain, and musculoskeletal system can act on the intestinal system via inflammatory, metabolic, and mitochondrial pathways to regulate the gut microbiota. Accordingly, bidirectional signaling mechanisms are formedvia the gut-brain, gut-muscle, and gut-brain-muscle axes, which affect the organism health. This review summarizes that gut microbiota establishes gut-brain-muscle interconnections mainly through metabolites, intestinal permeability, and immune-neural pathways, providing new ideas for improving the brain neuroplasticity and muscle health.

[Objective] To investigate the community structure, diversity, and plant-growth-promoting functions of endophytic bacteria in the protocorms ofEpidendrum sp. and mine the core bacterial groups associated with the seed germination. [Methods] We collected five samples including three types of protocorms (germinated on MS1 medium, pine bark, and rotten oak leaves) and two types of substrates (pine bark and rotten oak leaves). High-throughput sequencing of the 16S rRNA gene was employed to compare the diversity and community composition of bacterial communities under different germination conditions. The conventional method was used to isolate the endophytic bacteria from the protocorms germinated on pine bark and rotten oak leaves (i.e., from symbiotic seed germination), and the growth-promoting potential of the isolates was evaluated. [Results] A total of 2 735 operational taxonomic units (OTUs) were obtained from the five samples, belonging to 876 genera, 453 families of 41 phyla, among whichProteobacteria andActinobacteria were the dominant phyla. The results of principal coordinates analysis showed that there were differences in the bacterial community structure between the protocorms ofEpidendrum sp. and the substrates, and the community structure of endophytic bacteria in the protocorms germinated on the MS1 medium was closest to that on pine bark. Functional prediction indicated that the endophytic bacteria in the protocorms germinated on rotten oak leaves had higher nitrogen-fixing ability than that in other types of protocorms. Nineteen isolates were obtained from protocorms geminated on pine bark and rotten oak leaves, belonging to 16 species of 12 genera.Tumebacillus flagellatus,Bradyrhizobium cenepequi, andWeizmannia ginsengihumi were the common species in the protocorms germinated on pine bark and rotten oak leaves.Pseudomonas koreensis andW.ginsengihumi had the potential of solubilizing phosphorus and producing indole-3-acetic acid (IAA) and siderophores. [Conclusion] TheEpidendrum sp. protocorms germinated in different environments harbor rich endophytic bacteria. The endophytic bacteria isolated from the protocorms from symbiotic seed germination had plant growth-promoting functions, such as fixing nitrogen, solubilizing phosphorus, and producing IAA and siderophores. This study provides a scientific basis for mining the microbial resources related to seed germination of orchids and studying the interactions between orchids and microorganisms.

[Objective] Prolidase is an enzyme that can hydrolyze proline or hydroxyproline residues from the C-terminal dipeptides (Xaa-Pro). A putative prolidase-encoding gene was identified in the genome ofPyrococcus yayanosii CH1 isolated from the deep sea. In this study, we characterized the enzymatic properties ofPyprol encoded byPYCH_07700in vitro, aiming to find a new prolidase. [Methods] Pyprol was heterologously expressed in the hyperthermophilic archaeonThermococcus kodakarensis TS559. The dipeptide Met-Pro was used as a substrate to test the prolidase activity of the purified recombinant protein. [Results] Pyprol showed the best performance at 100 ℃ and pH 6.0.Pyprol binding to Co²⁺ exhibited the maximum activity, and the optimal metal ion concentration was 1.2 mmol/L.Pyprol had catalytic activity in a wider pH range and can tolerate higher concentrations of metal ions than the prolidasePfprol fromP.furiosus.Pyprol was a piezotolerant protein with an optimal hydrostatic pressure of 40 MPa. It exhibited enhanced activities at 40, 70, and 100 ℃ under 40 MPa, compared with at the atmospheric pressure. [Conclusion] Pyprol is a novel thermostable and piezotolerant prolidase ofP.yayanosii CH1, which is an obligate piezophilic hyperthermophilic archaeon strain isolated from a deep-sea hydrothermal vent.

[Background] Bacteria ofEnterobacteriaceae are the main pathogens of foodborne and clinical infections, posing a threat to human and animal health. The numerical identification method using API 20E as the "gold standard" is one of the main identification methods forEnterobacteriaceae. However, the existing numerical identification methods have problems such as laborious sample addition, low accuracy, and high prices. [Objective] To develop a semi-automatic, high-precision, and low-cost biochemical kit for identifying the bacteria ofEnterobacteriaceae based on the numerical identification method. [Methods] On the basis of the theoretical model and supporting software of the numerical identification system forEnterobacteriaceae established by our team, we designed and optimized 24 biochemical matrix trace formulations. Furthermore, a semi-automatic freeze-drying identification strip was developed. Using the commercialized numerical identification strip API 20E, MALDI-TOF MS, and 16S rRNA gene sequencing as the controls, we evaluated the performance of the developed biochemical kit that integrated semi-automatic biochemical identification strips and online analysis software. [Results] The biochemical spectra of 458 strains ofEnterobacteriaceae were obtained, with the overall identification accuracy of 98.5% at the genus level and 96.5% at the species level. The kit only needed twice sample addition to obtain the identification results, with the price only 4.46% that of API 20E. The shelf life of the product was 7 days, and the biochemical experiments had repeatability. [Conclusion] We develop a semi-automatic biochemical kit for identification ofEnterobacteriaceae based on the numerical identification method, which has simple operation, a low cost, and high accuracy. This study provides technical support for the identification ofEnterobacteriaceae and the development of biochemical products for numerical identification of other families and genera of bacteria.