Distribution of endophytic bacteria and preliminary screening of steroidal saponin producing strains in Xiangmaidong at different growth stages
Xiangmaidong, also known as Hubei maidong Liriope spicata var. prolifera Y.T. Ma, is one of the source plants of Shanmaidong in the Pharmacopoeia of the People’s Republic of China (2010 edition). Steroid saponins are its main active ingredient, and its sapogenin basic skeleton belongs to the derivative of spirostane (composed of 27 carbon atoms). According to the C25 configuration in the spirostane structure and the cyclic state of the F ring, it can be divided into the following four main types: spirostanol (C25 is S configuration), isosterostanol (C25 is R configuration), and furostanol (F ring is an open chain derivative).), modified spirostanol alcohols (F ring is a five membered tetrahydrofuran ring). Fourteen types of steroidal saponins have been isolated from Ophiopogon japonicus, with the main glycosides being diosgenin and ruscogenin, both of which are C25 (S) isomers. Among them, the content of saponins C (Ls-S3) in Ophiopogon japonicus is relatively high. Steroid saponins have multiple pharmacological activities, and except for a few that can be chemically synthesized, they are mainly obtained directly from traditional Chinese medicinal materials through chemical extraction. Xiangmaidong steroid saponins are no exception. However, due to issues such as long production cycles, continuous cropping obstacles in the field, low saponin content, high extraction costs, and significant environmental pollution, the sustainable production and supply of Xiangmaidong steroidal saponins are constrained. Therefore, it is necessary to seek new ways to prepare saponins in large quantities.

Previously, there were studies reporting the isolation of endophytic bacteria capable of producing steroidal saponins from Dianzhong and Huazhong buildings. Inspired by this, our research team attempted to isolate and discover endophytic bacterial strains that metabolize and produce steroidal saponins from Xiangmaidong. Using pure culture methods and 16SrDNA-PCRDGGE technology, we analyzed and identified the distribution of endophytic bacteria in samples (tubers or roots) of Xiangmaidong during its initial appearance, rapid expansion, and mature harvesting periods. At the same time, color reaction, TLC, and HPLC methods were used to preliminarily evaluate the saponin production ability of the obtained pure cultured strains, in order to obtain the target strains and provide theoretical support for the “factory, scale, and rapid” production of steroidal saponins.

Using traditional microbial pure cultivation methods, this study isolated 15 genera and 22 species of endophytic bacteria from the root tubers and fibrous roots of Xiangmaidong at different growth stages. Among them, there were 4 species of Bacillus and 4 species of Staphylococcus, 2 species of Pseudomonas, and the remaining 12 genera, including Salmonella, Enterobacter, Pseudoxanthomonas, Morganella, Agrobacterium, Microbacterium, Porphyromobacter, Acinetobacter, Lysinibacter, and Sphingosine. There is only one species of bacteria in the genera Sphingomonas, Sphingomonas, and Curtobacterium.

Through 16SrDNA PCR DGGE identification, this study found that the endophytic bacterial community structure of Xiangmaidong was basically the same during the initial appearance period, rapid expansion period, and mature harvesting period of its tubers, with little difference in strain groups. Through sequence alignment, 15 genera and 21 species of bacteria were identified from the isolated sequences, including 3 species of Bacillus and 3 species of Staphylococcus, 2 species of Serratia and 2 species of Pseudomonas, and the remaining 11 genera, including Pseudomonas, Pectobacterium, Bordetella, Stenotrophomonas, Porphyromobacter, Curtobacterium, Enterobacter, Methylobacterium, nitrogen fixing. There is only one strain of Azotobacter, Microbacterium, and Pseudoclavibacter. Compared to the 22 strains of pure endophytic bacteria mentioned above, DGGE has identified 7 new genera: Serratia, Pectobacterium, Bordetella, Stenotrophomonas, Methylobacterium, Azotobacter, and Pseudomonas.

This study evaluated the saponin production ability of pure cultivable endophytic strains and preliminarily identified three strains of bacteria, Pseudoxanthomonas mexicana, Staphylococcus caprae, and Microbacterium testaceum, with significant characteristics of producing steroidal saponins. According to the HPLC detection results, these three endophytic bacteria all showed characteristic peaks at the time points of 15.98, 57.64, and 75.19 minutes, indicating that at least three saponins in their fermentation products may have the same structure, which needs further experimental verification. At present, there are two main strategies for detecting steroidal saponins: one is direct detection, and the other is indirect detection through qualitative and quantitative analysis of cleaved saponins. Scholars have used HPLC method to determine the content of dioscin in Dioscorea nipponica, arrowroot, sisal, and lily buds. Due to the lack of standard and isolated pure samples of Xiangmaidong steroidal saponins in this experiment, the prepared Xiangmaidong root steroidal saponins (mixture) were temporarily used as positive controls. By comparing whether characteristic peaks at the same time point could appear under the same detection conditions, the glycoside production characteristics of endophytic bacteria were preliminarily identified. In the future, our laboratory will further separate and purify the fermentation product saponin extract, and use mass spectrometry, nuclear magnetic resonance and other methods for more accurate characterization.

Based on the DGGE analysis of the high variable region of 16SrDNA V6 ～ V8, and combined with the screening results of pure cultured endophytic bacteria capable of producing steroidal saponins, this experiment found a phenomenon: during the rapid expansion period and mature harvesting period of Xiangmaidong’s root tubers, Mexican pseudoyellow pseudomonas P. can be detected from its root tubers The presence of mexicana was not detected in the initial stage of root tubers. It has been confirmed that the biomass and content of steroidal saponins in the root tubers of Xiangmaidong gradually increase from the initial stage and reach their peak during the rapid expansion and maturity stages of harvesting. Does this phenomenon imply that Pseudomonas aeruginosa in Mexico plays a positively correlated role in the accumulation of steroidal saponins in the tubers of Ophiopogon japonicus? This also requires further experimental verification.