During the fermentation process, the color of DLF changed from green to dark brown（Figure 1）accompanied by a sweet smell. The microstructure was clearly different between fermented DLF (FDLF) and unfermented DLF (UDLF) (Figure 1). An irregularly shaped and rough surface was mainly observed in FDLF, whereas the microstructure of DLF was more regular and the surface smooth. This change of microstructure may increase the surface area of the substrate and facilitate the full reaction between enzyme and substrate.
Effect of solid-state fermentation on proteins and amino acids
The protein content is the most important parameter that determines the overall quality of animal feed products. It is well known that the protein content can vary depending on the microorganism used and their carbon and nutrient accessibility, as well as the cultivation conditions, such as carbon and nutrient sources, water content and pH. The analysis showed that the average concentration of CP in the raw flour was 28.42% (Table 1), close to the figure reported by Teixeira et al.  but lower than the values reported by Moyo and Masika , most likely due to differences in the growth stage and planting conditions . SSF significantly increased the CP concentrations from 28.42% to 40.98% (Table 1). The enrichment of CP could be the result of increased fungal biomass, suggesting that the treated substrate could act as a good protein source for livestock. However, it could also be due to the concentration effect caused by the aggravation of dry matter loss.
To analyze the influence of fermentation on the DLF protein profile, SDS-PAGE was performed. SSF affected the characteristics of proteins in DLF. The molecular weight of the main protein fractions in the unfermented DLF was 55 kDa (Figure 2). The maximal degradation of large proteins in the FDLF was almost complete after 24 h of fermentation. This was likely because highly active proteases were able to decompose the large proteins secreted by the microorganisms during fermentation . This reduction of protein sizes is important to increase the digestibility of protein. However, from the third day of fermentation, the color of the protein bands gradually deepened and bands appeared at around 33-45 kDa and 60-140 kDa, similar to the results reported by Zuo et al. . This could be due to the production of single-celled proteins in the late fermentation period. This phenomenon indicated that fermentation not only degraded the macromolecular proteins in DLF but also increased the abundance of other proteins with high molecular weight.
Drumstick leaves contain all essential amino acids, but the content varies greatly depending on growth environments, cultivation mode, tree age, leaf maturity and other conditions [34, 35]. Evaluation of the amino acid profile confirmed significant differences between the fermented substrate and raw flour (Table 2). The levels of most amino acids increased, whereas glutamic acid and lysine decreased. The alterations in amino acid profiles may vary depending on the microorganism used . The inoculated microorganisms may use glutamic acid and lysine for metabolic activity, resulting in a reduction in their concentrations in the fermented substrate compared to those in raw flour. After fermentation, the total amino acid content of DLF was 24.88%, which was significantly higher than that of the unfermented substrate. This may be due to enzymatic hydrolysis of large proteins into small amino acids or the microbial synthesis of some amino acids. The concentration of essential amino acids and non-essential amino acids in the fermented substrate also increased, by 1.81% and 1.76%, respectively. Animals not only have dietary requirements for essential amino acids but also need nutritionally nonessential amino acids to achieve maximum growth and production performance. Therefore, increasing non-essential amino acids in animal feeds is beneficial. It has been reported that peptides are more rapidly utilized than proteins and amino acids . The small peptide concentration in our study increased from 5.72% to 12.03% after fermentation. The increased peptide content may positively affect the bioactivity of DLF because they may contribute to antioxidative and metal-chelating activities .
Effect of solid-state fermentation on the chemical composition
The results regarding the chemical composition of the fermented substrate and raw flour are shown in Table 1. SSF increased the content of crude ash, neutral washing fiber and lignin. In addition, the CF concentration of the fermented substrate was decreased markedly, by 70.07%, compared to the control. There were also clear reductions in total sugars and reducing sugars from 18.49% and 12.38% to 5.34% and 1.69%, respectively (Table 1). Meanwhile, the ether extract (fat) concentration decreased by 30.94% compared with that before fermentation. This suggests that the microbial fermentation process consumed carbohydrates and fats, especially small molecules of sugars. At the same time, SSF resulted in an increase in the mineral content, probably due to the metabolic activity of the microorganisms or dry matter loss.
Effect of solid-state fermentation on total flavonoid, phenolic content and antioxidant capacity
Antioxidants are free radical scavengers that can protect the body from free radicals that can cause a variety of diseases, including ischemia, asthma, anemia, dementia and arthritis. Phenols and flavonoids are considered to be some of the safest natural antioxidants, and fermentation technology is an effective way to increase the concentration of these compounds. In this study, the concentration of total phenols and flavones reached the maximum value on the first day of fermentation. Afterwards, the content of total phenols and flavonoids decreased as the fermentation continued (Figure 3). Dey et al.  proposed that the reason for the increased content was that various extracellular enzymes secreted by microorganisms destroy the intact cell wall structure of plants, releasing flavonoids from within cells and phenols bound to the cell walls during fermentation. At the same time, microorganisms can produce some phenols through secondary metabolism. However, prolonged fermentation may lead to the diffusion and oxidation of phenolic substances.
It is crucial to evaluate the antioxidant potential of extracts using more than one method due to the different mechanisms of antioxidant activity. In the present study, the antioxidant activity of fermented substrates was measured using ABTS+, DPPH free radical scavenging methods and FRAP. The antioxidant activity showed a trend of first increasing and then decreasing with increasing fermentation time (Figure 3), similar to results obtained with okra seeds during fermentation by Adetuyi and Ibrahim . The antioxidant activity of DLF after fermentation was slightly lower than that before fermentation. Hossain et al.  suggested that this might be because of a too long fermentation time and reduced content of phenols and other substances. Many studies have shown that the antioxidant capacity of plants is directly related to the content of phenolic compounds and flavones. In this study, Pearson’s correlation analysis showed that flavonoids were only slightly positively correlated with DPPH radical scavenging rate and total antioxidant capacity, whereas total phenols were highly positively correlated with the three antioxidant indexes tested (Table 3), indicating that the total phenols and flavonoid concentrations of FDLF were closely related to antioxidant activity. However, total phenols and flavonoids are not the only factors affecting antioxidant activity. Small peptides and amino acids, such as leucine, methionine, tyrosine, histidine, and tryptophan, can also make the fermented sample more reductive. In addition, determination of phenolic substances with the Folin-Ciocalteu reagent may be influenced by a variety of non-phenolic compounds, such as reducing sugars, aromatic amino acids and citric acids, which often do not have free radical scavenging capacity. In general, the synergistic effect between phenols and other components in the solid fermentation of DLF may be the main reason for this phenomenon.
Effect of solid-state fermentation on anti-nutritional factors
Tannins, phytic acid and glucosinolates are the main anti-nutrients in DLF [41-43]. Tannins can precipitate proteins, amino acids, alkaloids and other organic molecules in aqueous solution, which hinder the absorption of some nutrients due to complexation. Further, the bitter taste of tannins may affect the palatability of feed. High concentrations of tannins have an adverse effect on animal productivity and digestibility. Phytic acid is an organic acid that cannot be digested by animals with a single stomach. Therefore, phytic acid is eventually expelled in an animal’s feces, which may be degraded by aquatic microorganisms after entering water, releasing phosphorus and resulting in serious eutrophication of water . Although glucosinolates are non-toxic, they may decompose into glucose, isothiocyanate, nitrile, thiocarcinate and other toxic compounds through endogenous myrosinase. Their main anti-nutritional effects include reducing the palatability of feed, inducing iodine deficiency and damaging liver and kidney function. They are also more harmful to non-ruminants than ruminants . Nevertheless, phytase, tannase and other enzymes can degrade these substances. Aspergillus is considered to be the main source of these enzymes. In this study, the phytic acid and tannin concentrations of DFL were 18.31 mg/g and 14.60 mg/g, respectively, which are close to the values of 22.3 mg/g and 16.3 mg/g reported by Stevens et al. . After fermentation, the levels of tannins, phytic acid and glucosinolates were drastically decreased (Table 1). This might have been caused by the secretion of tannase, phytase and other biological enzymes, which are able to break down tannins, phytic acid and glucosinolates.