Pigeon pea [Cajanus Cajan (L.) Millsp.] is an important edible legume, and its seeds are widely consumed in developing countries due to the high nutritional values (Gai et al., 2022). Currently, pigeon pea is the sixth most important food legume in the world regarding cultivation area and production (Gai et al., 2021). Moreover, the leaves, stems, and flowers of pigeon pea are rich in bioactive flavonoids and stilbenes, which are commonly used as traditional folk medicines for the treatment of wounds, dysentery, measles, jaundice, etc. (Tan et al., 2022; Wu et al., 2009). In China, pigeon pea leaves are highly appreciated in the pharmaceutical industry because they can be processed into the drug “Tongluo Shengmu Capsule” for the treatment of ischemic necrosis of the femoral head (Gai et al., 2020). As by-products of edible seeds and medicinal leaves, pigeon pea roots (PPR) are often abandoned in large quantities in agricultural and pharmaceutical production. In most practices, farmers dispose of PPR through burning (Zhang et al., 2013), which can result in both resource wastage and environmental pollution. In fact, there are still some bioactive ingredients in PPR, among which genistin and genistein are considered to be two main compounds (Liu et al., 2010). As typical isoflavones, genistin and genistein are phytoestrogens associated with versatile biological properties including antitumor, antioxidant, anti-inflammatory, antiviral, antibacterial, anticancer, tyrosine kinase inhibition, etc. (Feng et al., 2015; Gai et al., 2021; Wang et al., 2019). However, genistein is reported to possess better intestinal absorption potential, bioavailability, and antioxidant activity as compared with its glucoside precursor genistin (Jin et al., 2013; Zhang et al., 2013), and has been widely used as active pharmaceutical ingredient, food additive, and dietary supplement (Liu et al., 2010; Wang et al., 2019; Zhang et al., 2012). Thus, the deglycosylation of genistin to genistein in PPR can increase the availability of more valuable extracts from this abandoned resource.

In comparison with acidic/alkaline hydrolysis, biotransformation by the aid of biocatalysts such as enzymes or microorganisms possesses advantages of mild reaction conditions, simple operation, low cost, environmental friendliness, and high stereoselectivity and regioselectivity (Ekiz, Duman, & Bedir, 2018; Jiang, Li, & Fan, 2021; Liu, Zhou, Cui, Wang, & Wang, 2021), which is the method preferentially recommended to obtain bioactive aglycones through the deglycosylation of glycosides (Borges, Borges, Bonato, Said, & Pupo, 2009; Singhvi & Zinjarde, 2020). It was reported that the immobilized β-glucosidase could effectively convert genistin to genistein in extracts of PPR (Zhang et al., 2013). Although the utilization of immobilized enzymes could reduce the cost, the hydrolysis reaction was carried out in the solution of crude extracts and the operational conditions were demanding. The application of microorganisms for transformation does not require the step of enzyme purification. Moreover, the natural regeneration cofactors in microorganisms enable enzyme production more economically and continuously, which can fulfill the demands of industrial production through large-scale fermentation (Choudhary, Gupta, Dhar, & Kaul, 2021; dos Santos & Silva, 2019). Jin et al. (2013) reported that the fermentation of PPR by edible Aspergillus oryzae and Monacus anka could achieve the transformation of genistin to genistein by the aid of β-glucosidase secreted by microorganisms. However, this work mainly focused on the deglycosylation of genistin to genistein in PPR, ignoring the fact that flavonoid glycosides are mainly distributed in the vacuoles of plant cells or cross-linked to cellulose in cell walls through hydrogen bonds (Fu et al., 2008), which introduces difficulty for the deglycosylation of them to valuable aglycones. It is known that cellulase can hydrolyze and degrade cellulose in plant cell walls, which is beneficial for releasing the intracellular glycosides and conjugated glycosides (Wilkins, Widmer, Grohmann, & Cameron, 2007; Zhang et al., 2012). Hence, searching for the novel microorganisms that can produce cellulase and β-glucosidase for PPR fermentation is expected to obtain high yield of genistein.

Endophytic fungi have drawn considerable attention due to their ability to produce various extracellular enzymes, making them potential sources of novel biocatalysts for modifying natural products (Borges et al., 2009; Ekiz, Yılmaz, Yusufoglu, Kırmızıbayrak, & Bedir, 2019; Wang et al., 2020; Xiang et al., 2018). In addition, endophytic fungi may secrete hydrolysis enzymes that can break the cell wall barrier to access the host plant and obtain nutrients, which is attributed to their particular living environment and the long-term coexistence with hosts (An et al., 2022; Xiao et al., 2022). Moreover, some endophytic fungi were found to able to produce β-glucosidase that enabled the deglycosylation of low-activity glucoside precursors to high-activity aglycones (Chiu et al., 2020; Singhvi & Zinjarde, 2020). In view of this, the versatility of endophytic fungi offers the possibility to screen novel microorganisms with abilities of cell wall hydrolysis and glucoside deglycosylation. It is worth mentioning that our research team has isolated an endophytic fungus (J-21) from pigeon pea that is associated with the aforementioned potentiality.

In recent years, an emerging application is the utilization of solid-state fermentation as a bioprocess tool to increase the contents of nutraceutical compounds in agrifood/agro-industrial by-products by enhancing their extractability, thus adding value to these by-products (Cano y Postigo, Jacobo-Velázquez, Guajardo-Flores, Garcia Amezquita, & García-Cayuela, 2021; Paz-Arteaga et al., 2023; Vilas-Franquesa, Casertano, Tresserra-Rimbau, Vallverdú-Queralt, & Torres-León, 2023). Solid-state fermentation enables fungi to utilize the solid agrifood/agro-industrial by-products as the substrate (source of carbon), and can increase the extractability of nutraceuticals by destroying plant cell walls through fungal colonization, as well as the liberation of bound nutraceuticals due to the action of hydrolytic enzymes produced by fungi (Cano y Postigo et al., 2021). Semi-solid-state fermentation is proposed based on solid-state fermentation with the addition of a small amount of free liquid to dried solid matrices, which can promote the fungal nutrient uptake and enable growth in a shorter fermentation period (Gupta & Jana, 2019). Both solid-state fermentation and semi-solid-state fermentation are economical and environmentally friendly techniques associated with the outstanding advantage of lower energy cost and less wastewater production without compromising high product yields (Paz-Arteaga et al., 2023). The present study focused on the application of semi-solid-state fermentation of PPR to enhance the extractability of genistein using the novel endophytic fungus J-21. Based on the economical characteristics of semi-solid-state fermentation, it was reasonable to assume that this study would offer a cost-effective alternative for obtaining valuable genistein from agrifood by-products PPR.

In this work, the morphological identification and phylogenetic analysis of endophytic fungus J-21 were initially performed, followed by determining activities of cellulase and β-glucosidase. Afterwards, yields of genistein in PPR fermented by endophytic fungus J-21 using different strategies were compared, and the fermentation parameters of the optimal strategy were systematically optimized. Subsequently, the surface morphology of PPR before and after fermentation was observed by scanning electron microscope (SEM). Also, the antioxidant and antibacterial activities of extracts before and after fermentation were evaluated. To our knowledge, this is the first study to simultaneously utilize activities of cellulases and β-glucosidase of endophytic fungus to obtain high yield of genistein from agrifood by-products PPR.