Component analysis of Xylaria sp. L1 sporocarps after solid-state fermentation by okara and its safety evaluation in mice and rats

Abstract To explore the chemical components and nutrient components of Xylaria sp. L1 sporocarps, component analysis was characterized using UHPLC-LTQ-Orbitrap-MS/MS. Then, the acute toxicity and the subacute toxicity were conducted, respectively. A total of 38 compounds were detected and quantified. Meanwhile, Xylaria sp. L1 sporocarps had higher quantities of macronutrients, primarily Zn, which was remarkably higher than the human recommended daily values (p < 0.001). Importantly, no evidence of toxicity was observed in the mice after acute exposure to Xylaria sp. L1 sporocarps. In subacute toxicity studies, there were no significant differences in the body and organ weights. In the blood chemistry analysis, no significant changes occurred. Pathologically, neither histopathological changes nor gross abnormalities were observed. Thus, our study highlights the potential for using Xylaria sp. L1 sporocarps as novel food with Zn supplementation for humans. Graphical Abstract


Introduction
Termites play a key role in the global organic carbon cycling process by feeding on decaying biomass (Rogers et al. 2005). As known, fungi termitomyces are the mushroom species cultivated by macrotermitine termites (Ju and Hsieh 2007). Termitomyces can produce compounds such as polysaccharides, serine protease and fatty acid amides with possible antimicrobial, antioxidant and immune-modulating properties. Xylaria have received attention with their ability to synthesize novel and bioactive secondary metabolites. As known, Xylaria contains a wide range of many important enzymes and bioactive compounds, and it is a high value medicinal fungus for its anti-inflammatory (Chang et al. 2017), anti-tumour (Masahiko et al. 2011), antibacterial (Wu et al. 2014), anti-malarial (Ko et al. 2011) and neuroprotective activities (Pan et al. 2017). Meanwhile, some novel chemical compounds, such as geldanamycin analogues, glycosylated macrotermycins and macrolactams, were constantly produced by Xylaria sp. (Lei et al. 2018;Wang et al. 2020;Zheng et al. 2018). Although wide usages of Xylaria sp. showed that it has the potential to be developed into a nutraceutical, few studies have provided a comprehensive description of the chemical profile of Xylaria sp. (Chen et al. 2019).
Our initial study of termite-associated fungal and bacteria led to the discovery of a special fungus Xylaria sp. L1, which was derived from Guangzhou, China by our lab. Based on the ITS gene sequences, the isolated fungi Xylaria sp. L1 showed closed relatedness with Xylaria escharoidea. Furthermore, the whole genome was 36.61 Mb, and the whole genome shotgun project has been deposited at GenBank accession JALIDW000000000. One voucher specimen has been deposited in the herbarium of Hefei University of Technology under the accession number L1-SY. Using Xylaria sp. L1 to pre-treat okara biomass, fruiting bodies were collected. Despite the fruiting bodies of fungi include a source of organic nutrients, such as digestible protein, starch, fibre and certain vitamins, as well as minerals and antioxidants (Pereira et al. 2011), few studies on the toxicological characteristics and physiological functions of Xylaria sp. have been reported until now. In this study, UHPLC-LTQ-Orbitrap-MS/MS was used to explore the chemical components and nutrient components of the sclerotial powder of Xylaria sp. L1 cultivar. Then, the safety evaluation of Xylaria sp. L1 was performed through acute and subacute toxicity tests. By comparing the haematological, biochemical and histological parameters, the range of tolerable doses of Xylaria sp. L1 sporocarps on experimental rats was analyzed.

Chemical composition of Xylaria sp. L1 sporocarps
The chemical components of Xylaria sp. L1 sporocarps were conducted by UHPLC-LTQ-Orbitrap-MS/MS. Then, phytochemicals were identified by retention time, exact mass, characteristic fragmentation, online databases and the literature. As shown in Figure S1 and Table S1, a total of 38 chemical constituents were identified from the Xylaria sp. L1 sporocarps, including 6 terpenoids, 4 phenylpropanoids, 14 alkaloids, and 10 long-chain polyunsaturated fatty acids and 4 sterol compounds (as shown in the supplementary material Figure S2-S6). As known, Xylaria are often found in the fungus combs abandoned by termites, and some species of Xylaria exhibit potent anti-inflammatory, antioxidant and anti-tumour activities. It has been reported that the fungus terpenoids and sterol compounds have antibacterial, antiviral and antioxidant activities (Zhang et al. 2013). The natural-derived alkaloids including huperzine A and galanthamine, which have been discovered and explored for their potential in the management of AD (Sahoo et al. 2018). In our current study, daidzein and genistein were identified from the sporocarps of Xylaria sp. L1, and the most possible reason was that okara was used as a nutrient-rich culture medium.

Nutritional composition of Xylaria sp. L1
The proximate compositions of Xylaria sp. L1 sporocarp powder are presented in Table S2. Total carbohydrate, crude protein and crude fibre were the dominant compounds while the crude fat content was low, suggesting that the Xylaria sp. L1 sporocarps was low in fat but a rich source of protein. It is widely known that protein is responsible for multiple functions, including building cells and tissue, as well as making hormones and anti-bodies. Hence, it is important for us to get enough protein in our diet. Compared with shrimp or beef, Xylaria sp. L1 is an affordable high protein food. Thus, it could be used as a dietary supplement in the food industry. As shown in Table S3, the total amount of 15 kinds of free amino acids in the Xylaria sp. L1 sporocarps was 5404.19 mg/100g, which was much higher than that in other edible fungi (Kala c 2008). Additionally, the content of essential amino acids (EAA) and flavour amino acids (FAA) in the Xylaria sp. L1 sporocarps were 1822.63 mg/100g and 1227.65 mg/100g, respectively. Of them, Glu was the highest among the 15 detected amino acids. Meanwhile, the sweet amino acid (SAA) contents were 1363.33 mg/ 100 g, and Ribeiro et al. (2008) also reported that SAA increased the umami taste of mushrooms to a large extent. Likewise, the Xylaria sp. L1 sporocarps in this study showed a high hydrophobic amino acids (HAA) content with about 30%, and HAA was known to play an important role in antioxidant activity (Zhuang et al. 2009). Based on these advantages, Xylaria sp. L1 sporocarps provided us a supplementation for essential amino acids.
On the other hand, several minerals were also high in the sporocarps of Xylaria sp. L1. As shown in Table S4, the contents of Ca, Mg, Na and K were 17.85 mg/ 100g, 136.62 mg/100g, 312.41 mg/100g and 2606.96 mg/100g, respectively. These contents were equivalent to 1.7%, 42.69%, 22.83 and 55.47% of AI or RDA, respectively. These results indicated that the Xylaria sp. L1 was a good source of Mg and K. The lower content of Na is beneficial for nutrition, especially for patients with hypertension (Nakalembe et al. 2015). Importantly, Xylaria sp. L1 sporocarps had higher quantities of Zn, which is equivalent to 535.13% of AI or RDA. Zinc plays a huge role in metabolic processes, including immune function, protein synthesis, DNA biosynthesis and cell division (Heyneman 1996). These results indicated that Xylaria sp. L1 sporocarps has the potential as novel food with Zn supplementation for humans.

Acute toxicity and subacute toxicity
In the acute toxicity test, no signs of general toxicity and behavioural changes were observed. In the observation period, the changes of all mice in appearance, behavior, and living habits were recorded, and no observable signs of toxicity or morbidity were discovered; see Table S5. Oral administration of the Xylaria sp. L1 sclerotial powder at various doses (500, 1000 or 2500 mg/kg/d bw) did not induce any abnormal clinical signs in SD rats. No deaths or noticeable alterations in general behaviour related to treatment toxicity were recorded during the experiment. The biochemical parameters result showed obvious decreases in the level of TG at a dose of 500 mg/kg/d bw (p < 0.05) relative to the control, and liver function indexes and other blood lipid indexes were normal, so this situation was an accident. The organ coefficients of rats treated with the sporocarp powder of Xylaria sp. L1 for 28 days are presented in Table  S6, and no significant differences were observed for the relative weights of heart, liver, spleen, kidney, pancreas and testicle (p > 0.05). No significant lesions were found in the animals in each group, and no abnormal biochemical indicators were found. As shown in Figure S8, there are no significant difference in organs (heart, liver, spleen, lungs and kidneys) in the high-dose group and the control group, which showed that Xylaria sp. L1 sporocarps have no toxicity.

Conclusion
This study aimed at assessing the chemical composition, the nutritional value and safety of the sporocarp powder of Xylaria sp. L1. In this study, 38 active compounds were identified from the sporocarp powder of Xylaria sp. L1 for the first time, which could provide a basis for its rational utilization and development. The levels of Iron, Zinc, Copper and Manganese observed in the sporocarp powder of Xylaria sp. L1 were higher than the human recommended daily values. The acute toxicity and subacute toxicity study showed that the sporocarp powder of Xylaria sp. L1 is safe and shows no distinct toxicity or side effects after an oral dose of 20 g/kg/d.

Disclosure statement
No potential conflict of interest was reported by the authors.