Research on High throughput Detection Method of Lingzhi Ergothionein
Ergothioneine (EGT), also known as 2-thio-Lhistidine trimethyl inner salt, is a natural amino acid that is abundant in many animals and plants. It is only synthesized in some microorganisms (actinomycetes, streptomyces), fungi, and certain cyanobacteria, and cannot be synthesized by animal organisms themselves. The human body can only ingest EGT from food and accumulate high concentrations of EGT in various cells and tissues through the highly specific organic cation transporter novel type-1 (OCTN1). EGT is considered a strong antioxidant, and Servillo et al. explored its antioxidant mechanism, believing that it has a unique redox mechanism, and proposed a unique antioxidant effect of EGT in cells. The standard oxidation-reduction potential of EGT is -0.06V, while the potential of other thiols is generally between -0.32V and -0.2V. Therefore, EGT is more stable than other antioxidants in physiological pH environments and is less prone to spontaneous oxidation. Research has shown that EGT has multiple important physiological functions, such as anti-inflammatory effects; Cell protection function achieved by protecting DNA; The antidepressant function achieved by promoting neuronal differentiation and neurogenesis; Eye protection function achieved by resisting oxidative stress; Cardiovascular and cerebrovascular protection function achieved by interrupting the expression of adhesion molecules related to atherosclerosis; EGT has a wide range of application prospects in industries such as medicine, food, and cosmetics due to its preventive and therapeutic effects on neurodegenerative diseases achieved by inhibiting toxicity, and its inherent safety.

The production methods of EGT include chemical synthesis, edible mushroom extraction, and microbial fermentation. Due to the high cost and complex process of the first two, deep fermentation of edible mushroom hyphae is currently the mainstream mode of EGT industrial production. However, natural strains have low EGT production, and it is necessary to cultivate high-yield industrial strains of EGT through breeding techniques. The methods of cultivating industrial edible mushroom strains include mutagenesis breeding, hybridization breeding, genetic engineering breeding, and protoplast fusion breeding. However, each breeding method requires multiple large-scale EGT content tests. It should be noted that the significance of EGT content testing in the breeding process is more to compare the content between samples. At present, the detection methods for EGT include high-performance liquid chromatography, high-performance capillary electrophoresis, and thin-layer electrophoresis. The most widely used method is high-performance liquid chromatography, which can accurately detect the content of EGT from complex material systems. However, high-performance liquid chromatography has its obvious shortcomings, that is, it needs to explore different detection conditions for different samples. Moreover, due to the late peak time of EGT and the expensive standard samples, the screening of high-yield samples from a large number of samples in breeding can lead to long detection time and high cost, which greatly hinders the development and application research of EGT. Therefore, it is urgent to establish a high-throughput rapid detection method that can accurately determine the concentration of EGT between samples.
Due to its strong antioxidant properties and moderate reducibility to divalent iron ions (neither affected by contact with oxygen in the air nor reduced due to weak reducibility), EGT can react with divalent iron ions to form colorless chelates, while thiocyanate ions react with divalent iron ions to form red complexes. Based on these characteristics, iron thiocyanate was selected as the reducing agent in this study, and ergotamine standard was used to react with iron thiocyanate to establish a high-throughput detection system for ergotamine. At the same time, the high-throughput detection system was validated by varying EGT yields of different Ganoderma lucidum strains. The results of this study will provide new methods and ideas for the screening of high-yield EGT microorganisms and the breeding of new high-yield EGT strains.

Based on the strong reducing physicochemical properties of ergothionein, before constructing the EGT thiocyanate iron system, experiments were conducted to construct alkaline potassium permanganate system, bromocresol green system, blue bottle methylene blue system, guaiacol system, and ferric chloride EGT system. The results showed that the alkaline potassium permanganate system failed due to low stability; The color development of bromocresol green system failed due to pH dependence; In the blue bottle methylene blue system, due to the strong reducibility of the reduced state methylene blue, it is also easily oxidized to methylene blue when in contact with oxygen in the air, resulting in the failure of the experiment without any phenomenon; The guaiacol system also failed due to strong reducibility and interference from oxygen contact; The ferric chloride system failed due to low color rendering. The EGT iron thiocyanate system can be used for the study of high-throughput detection methods for EGT.

During the construction of the EGT thiocyanate iron detection system, it was found that residual phenolic substances in the extraction solution would affect the color reaction of the system and reduce its sensitivity when the tested sample was extracted using the ordinary EGT crude extraction method, namely alcohol precipitation nitrogen blowing constant volume extraction. Therefore, after nitrogen blowing constant volume extraction, a chloroform phenol removal step needs to be added to the crude extraction solution.
Ganoderma lucidum is a traditional and precious medicinal fungus in China, containing various bioactive substances and physiological functions. Its ergothionein content is relatively high among many edible fungi, but as a commercially produced strain, its yield is still low. Therefore, cultivating a new strain of Ganoderma lucidum with high ergothionein production is of great significance and practical application value. Based on the above reasons, our laboratory has obtained a batch of pseudo fusion products through protoplast fusion regeneration, isolation and identification. The Ganoderma lucidum fusion products used in the validation process of this method are all derived from this source.
The method established in this study is based on the physical and chemical properties of EGT itself, and the detection system is sensitive and stable. It is suitable for rapid comparison of large quantities of samples in the breeding process of EGT producing industrial fermentation strains. In theory, only simple sample EGT extraction optimization is needed to achieve high-throughput rapid detection of EGT content in the vast majority of strains, breaking the limitations of original EGT research and providing new methods and ideas for the overall development and application research of EGT.