Wenjiao Xue

The use of biocatalysis technology to manufacture rare natural products can solve the contradiction between the market demand for rare natural products in large health industry fields and the protection and sustainable development of wildlife resources. However, the currently available research on fungal endophytes, which are great potential resources for glycoside hydrolase biocatalysts, is still insufficient. In this study, endophytic fungi from Epimedium brevicornum Maxim. were isolated in the Qinling Mountains, identified and tested for their potential to biotransform epimedium extracts into minor epimedium flavonoids. A total of 84 representative morphotype strains were isolated and identified via ITS rDNA sequence analyses and were grouped into 32 taxa. The Shannon‒Wiener index (H’, 3.089) indicated that E. brevicornum Maxim. harboured abundant fungal resources. Ten strains showed strong β-glucosidase activity and exhibited the ability to biotransform major epimedium flavonoids into deglycosylated minor epimedium flavonoids, such as baohuoside I and icaritin, via various glycoside-hydrolysing pathways. Among these strains, strains 8509 and F8889, which were initially characterized as Aspergillus ochraceus and A. protuberus, have the potential for further development in the biotransformation of epimedium extracts into minor epimedium flavonoids because of their excellent biosafety, enzyme activity, and enzymatic characteristics. The enzyme activity of the crude enzyme obtained by freeze-drying from the F8509 fermentation broth supernatant reached 78.24 ± 2.48 U/g. Further research revealed that major glycosylated flavonoids from 100 g/L epimedium extracts were bio-transformed completely into minor deglycosylated flavonoids in 90 min after the addition of 1 g/L crude enzyme. In addition, the liquid phase separation conditions were optimized, and ethyl alcohol and water were ultimately used as the mobile phase for efficient separation of the conversion products at equal flow degrees. This study not only identified a series of candidates for the biotransformation of minor epimedium flavonoids but also provided an efficient purification method. More importantly, this study also demonstrated the important value of endophytes in the biotransformation of rare natural products.

The genus Weissella, commonly found in fermented foods, is a significant group of lactic acid bacteria (LAB) with potential probiotic properties. Several Weissella strains have been proposed as probiotics due to their biotechnological capabilities. However, a few strains may exhibit opportunistic pathogenic behavior, which restricts the widespread use of all Weissella strains in food applications. This study sought to expand our understanding of the biotechnological capabilities of Weissella spp. by examining the safety and functional characteristics of strains isolated from spontaneous fermentation. In this investigation, nine Weissella strains were evaluated for their safety and probiotic potential. The safety assessment revealed that the antibiotic resistance profiles of strains 16‐2, 38‐3, 69‐3, 91‐3, 91‐5, 104‐4, and 106‐5 were comparable or superior to the reference strain LGG. Hemolytic activity and ammonia production were also evaluated, but no positive results were observed. Further probiotic experiments demonstrated that strain 91‐5 exhibited superior performance in several areas, including survival rates in simulated gastrointestinal fluids, cell surface properties (hydrophobicity and adhesion to Caco‐2 cells), ABTS+ scavenging ability, antimicrobial activity, and cholesterol assimilation in vitro. Additionally, strain 104‐4 produced an exopolysaccharide (EPS) yield of 35.11 g/L after 48 h of culture in MRS‐sucrose (60 g/L) medium, surpassing most previously reported values. These findings suggest that strains 91‐5 and 104‐4 show promise as potential probiotic candidates for the development of new functional food supplements. Furthermore, this research expands the theoretical basis for considering Weissella strains as novel probiotics.

To assess the probiotic potential of strain 68-1 with rutin conversion capabilities, isolated from Chinese traditional Jiangshui, a complete genomic analysis and in vitro tests were conducted. The Oxford Nanopore Technologies (ONT, Oxford, UK)–Illumina (San Diego, CA, USA) hybrid sequencing platform was used for whole genome sequencing and the results showed that strain 68-1 had a chromosome sequence of 3,482,151 bp, with 46.53% GC content and five plasmids with a sequence length ranging from 2009 bp to 48,711 bp. Strain 68-1 was identified as Lactiplantibacillus pentosus based on the whole genome sequence. A total of 133 genes encoding for carbohydrate-active enzymes (CAZymes) were identified and genes that may be involved in rutin conversion were found in the L. pentosus 68-1 genome. L. pentosus 68-1 showed excellent tolerance to gastrointestinal juice and adhesion properties, and corresponding genes were identified. In addition, L. pentosus 68-1 exhibited strong antibacterial and antifungal activity, where organic acids may play a crucial role in its antagonistic ability. Moreover, the gene cluster for plantaricin_EF production was detected. No high virulence factor was found in the L. pentosus 68-1 genome and no hemolytic effect was observed. In addition, L. pentosus 68-1 showed resistance to ampicillin, gentamycin, and kanamycin, and the genomic analysis indicated that horizontal ARG transfer should not be possible. The results show that L. pentosus 68-1 could be developed as a novel probiotic candidate to improve rutin bioavailability in the food and feed industry.

Ginsenoside CK, a kind of rare ginsenoside transformed from protopanaxadiol saponins extracted from the genus Panax, has been proven to possess favorable bioactivities such as anti-inflammatory, anti-cancer, anti-diabetes, and hepatoprotective effects. The current study is targeted to determine the effect of ginsenoside CK on hepatitis induced by concanavalin A (Con A). Mice were treated with different dosages of ginsenoside CK for 7 days, and Con A (15 mg/kg) was intravenously injected to induce autoimmune hepatitis (AIH) after the last administration. The results demonstrated that pretreatment with ginsenoside CK (40 mg/kg) could obviously ameliorate the increase in serum indicators related to liver function such as AST, ALT, and ALP, and hepatic lesions induced by Con A. Meanwhile, ginsenoside CK suppressed hepatocyte apoptosis, which was observed in pathological data, and immunoblotting results showed that the expression of Bax, Bcl-2, and other proteins was regulated by CK. Furthermore, the release of inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and IL-6 in mice with AIH were lowered by the administration of 40 mg/kg of ginsenoside CK. Furthermore, ginsenoside CK elevated the gene expression of Nrf2 and Sirt1 and augmented downstream target genes such as HO-1. In addition, a significant inhibition effect of the TLR4/NF-κB signal was observed in 40 mg/kg CK-pretreated mice compared with the model group. To sum up, the results indicated that ginsenoside CK has a notable hepatoprotective effect against AIH by activating Sirt1/Nrf2 and suppressing the TLR4/NF-κB signaling pathway.

In this study, we aimed to explore the potential targets and functional mechanisms of Rk1 combined with Rg5 (Rk1+Rg5) against type II diabetes mellitus (T2DM). Network pharmacology and molecular docking were used to predict and verify the targets and signaling pathways of Rk1+Rg5 against T2DM. The results were further confirmed by a db/db mouse model and a model using PA-induced L6 cells. According to network pharmacology, a total of 250 core targets of Rk1+Rg5 towards T2DM were identified; the insulin resistance signaling pathways were enriched by KEGG. Results of molecular docking indicated good binding affinity of Rk1 and Rg5 to Akt1. In vivo and in vitro studies further showed that Rk1+Rg5 is an inhibitor of skeletal muscle insulin resistance. The results showed that Rk1+Rg5 significantly improved the hyperglycemic state of db/db mice, alleviated dyslipidemia, and promoted skeletal muscle glucose uptake. This phenomenon was closely related to the alleviation of the insulin resistance in skeletal muscles. Finally, the combination activated the Akt signaling pathway and promoted GLUT4 translocation to the cell membrane for glucose uptake. Altogether, our findings, for the first time, demonstrate that the combination of Rk1 and Rg5 could be beneficial for anti-T2DM, possibly involving ameliorated insulin resistance.

Plant growth-promoting bacteria (PGPB) have been considered promising biological agents to increase crop yields for years. However, the successful application of PGPB for biocontrol of sharp eyespot in wheat has been limited, partly by the lack of knowledge of the ecological/environmental factors affecting the colonization, prevalence, and activity of beneficial bacteria on the crop. In this study, an endophytic bacterium XN08 with antagonistic activity against Rhizoctonia cerealis (wheat sharp eyespot pathogenic fungus), isolated from healthy wheat plants, was identified as Burkholderia ambifaria according to the sequence analysis of 16S rRNA. The antibiotic synthesis gene amplification and ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) analyses were used to characterize the secondary metabolites. The results showed that the known powerful antifungal compound named pyrrolnitrin was produced by the strain XN08. In addition, B. ambifaria XN08 also showed the capacity for phosphate solubilization, indole-3-acetic acid (IAA), protease, and siderophore production in vitro. In the pot experiments, a derivate strain carrying the green fluorescent protein (GFP) gene was used to observe its colonization in wheat plants. The results showed that GFP-tagged B. ambifaria could colonize wheat tissues effectively. This significant colonization was accompanied by an enhancement of wheat plants' growth and an induction of immune resistance for wheat seedlings, which was revealed by the higher activities of polyphenol oxidase (PPO), peroxidase (POD), and phenylalanine ammonia-lyase (PAL). As far as we know, this is the first report describing the colonization traits of B. ambifaria in wheat plants. In addition, our results indicated that B. ambifaria XN08 might serve as a new effective biocontrol agent against wheat sharp eyespot disease caused by R. cerealis.

To obtain novel fungi with potent β-glucosidase for minor ginsenoside production, Panax bipinnatifidus var. bipinnatifidus, which is a traditional medicinal plant containing various ginsenosides, was first employed to isolate endophytic fungi in this study. A total of 93 representative morphotype strains were isolated and identified according to ITS rDNA sequence analyses, and they were grouped into three phyla (Ascomycota, Basidiomycota, and Mucoromycota), five classes (Dothideomycetes, Sordariomycetes, Eurotiomycetes, Agaricomycetes, and Mucoromycetes), and 24 genera. Plectosphaerella (RA, 19.35%) was the most abundant genus, followed by Paraphoma (RA, 11.83%) and Fusarium (RA, 9.70%). The species richness index (S, 34) and the Shannon–Wiener index (H’, 3.004) indicated that P. bipinnatifidus harbored abundant fungal resources. A total of 26 endophytic fungal ethyl acetate extracts exhibited inhibitory activities against at least one pathogenic bacterium or fungus. In total, 11 strains showed strong β-glucosidase activities and also presented with the ability of ginsenoside biotransformation with varied glycoside-hydrolyzing pathways. Excitingly, three genera, namely, Ilyonectria, Sarocladium, and Lecanicillium, and all 11 taxa were first found to have the ability to transform ginsenosides in our study. The results indicated that P. bipinnatifidus could be a new fungi resource with potential novel natural compounds with antimicrobial activity and potent β-glucosidase for varied minor ginsenoside production.

The objective of this study was to assess in vitro probiotic potential of Lactobacillus strains derived from artisanal fermented vegetables in Shaanxi, China. In total, 74 acid-producing Gram-positive strains with rod-shaped under the microscope were isolated from 16 samples of spontaneously fermented vegetables. Out of 74 strains, 26 showed high survival rate under low pH and high bile salts conditions and were subjected to molecular identification by 16S rRNA gene sequencing analysis. The results showed that 15 isolates belonged to Lactobacillus plantarum, 9 isolates belonged to Lactobacillus brevis, and the 2 remaining strains belonged to Weissella viridescens. The 24 Lactobacillus strains were investigated for their survival rate to transit simulated gastrointestinal tract, cell surface hydrophobicity, auto-aggregation, co-aggregation with pathogen, adhesion to Caco-2, antimicrobial activity, antibiotics susceptibility, radical scavenging ability, α-glucosidase inhibition, and the cholesterol assimilation. The results showed that the probiotic characteristics were strain-dependent, and several strains exhibited great probiotic potential with specific health benefits, which indicated that they might be excellent candidates for production of functional foods. Interestingly, it was first found that L. plantarum generally had higher antibacterial activities, α-glucosidase inhibition ability, and antibiotics susceptibility compared to L. brevis in this study. The results indicated that Lactobacillus strains isolated from fermented vegetables in Shaanxi, China, could be exploited as a promising novel probiotic source.

The plant Chloranthus japonicus Sieb is known for its anticancer properties and mainly distributed in China, Japan, and Korea. In this study, we firstly investigated the diversity and antimicrobial activity of the culturable endophytic fungi from C. japonicus. A total of 332 fungal colonies were successfully isolated from 555 tissue segments of the medicinal plant C. japonicus collected from Qinling Mountains, China. One hundred and thirty representative morphotype strains were identified according to ITS rDNA sequence analyses and were grouped into three phyla (Ascomycota, Basidiomycota, Mucoromycota), five classes (Dothideomycetes, Sordariomycetes, Eurotiomycetes, Agaricomycetes, Mucoromycetes), and at least 30 genera. Colletotrichum (RA, 60.54%) was the most abundant genus, followed by Aspergillus (RA, 11.75%) and Diaporthe (RA, 9.34%). The Species Richness Index (S, 56) and the Shannon-Wiener Index (H′, 2.7076) indicated that C. japonicus harbored abundant fungal resources. Thirteen out of 130 endophytic fungal ethyl acetate extracts exhibited inhibitory activities against at least one pathogenic bacterium or fungus. Among of these, F8158, which was identified as Trichoderma cf. harzianum, exhibited good antagonistic capacities (the percent inhibition of mycelial growth ranged from 47.72~88.18) for different pathogens and has a potential application in biological control. In addition, it is noteworthy that the strain F8157 (Thanatephorus cucumeris, an opportunistic pathogen) showed antibacterial and antifungal activity, which is reported firstly in this study, and should be investigated further. Taken together, these results indicated that the endophytic fungi from C. japonicus may be of potential interest in screening bio-control agents and discovering of new bioactive compounds.

Endophytic fungi have been emerged as fruitful resources for producing structurally fascinating and biologically active secondary metabolites. However, endophytic fungi from medicinal plants of Qinling Mountains–the most important natural climatic boundary between the subtropical and warm temperate zones of China with an astonishingly high level of biodiversity–have rarely been explored as potential sources of novel fungal species and active secondary metabolites. In this study, a total of 371 fungal colonies were successfully isolated from 510 tissue segments of the medicinal Tupistra chinensis Baker collected from Qinling Mountains, China. Roots of T. chinensis Baker are used as a folk medicine to ameliorate pharyngitis and treat rheumatic diseases. A total of 100 representative morphotype strains were identified according to ITS rDNA sequence analyses and were grouped into three phyla (Ascomycota, Basidiomycota, Mucoromycota), seven classes (Dothideomycetes, Sordariomycetes, Eurotiomycetes, Microbotryomycetes, Agaricomycetes, Leotiomycetes, Mortierellomycetes), and at least 35 genera. The genera of Collectotrichum (IF, 29.92%), Fusarium (IF, 8.36%), Aspergillus (IF, 8.09%), and Dactylonectria (IF, 5.39%) were most frequently isolated from the tissues of T. chinensis Baker. The Species Richness Index (S, 65) and the Shannon-Wiener Index (H′, 3.7914) indicated that T. chinensis Baker harbored abundant fungal resources. Moreover, five isolates were potential new taxa because of low similarity of ITS sequences ranged from 95.09%∼96.61%. Fifteen out of 100 endophytic fungal ethyl acetate extracts exhibited inhibitory activities against at least one pathogenic bacterium or fungus. Two important lead compounds produced by two stains (F8047 and F8075) with high antimicrobial activities were identified using high performance liquid chromatography (HPLC) and ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry (UPLC–QTOF MS) analyses. In addition, it was noteworthy that the strain F8001, which may be a potential new species, showed antimicrobial activity and should be investigated further. Overall, these results indicated that the endophytic fungi from T. chinensis Baker could be exploited as a novel source of bioactive compounds.