In vivo toxicity of bioreactor-grown biomass and exopolysaccharides from Malaysian tiger milk mushroom mycelium for potential future health applications

Natural mycelial biomass (MB) and exopolysaccharides (EPS) of Malaysian tiger milk mushroom Lignosus rhinocerus are considered high-end components due to their high commercial potential value in drug discovery. This study aims to evaluate the toxicity of the mushroom extracts’ generated in a bioreactor using the zebrafish embryo toxicity (ZFET) model assay as a new therapy for treating asthma. Both MB and EPS extracts, at concentrations 0.16–10 mg/mL, were tested for ZFET and early development effects on Zebrafish Embryos (ZE) during 24–120 h post-fertilisation (HPF). Findings revealed that MB was deemed safe with an LC50 of 0.77 mg/mL; the EPS were non-toxic (LC50 of 0.41 mg/mL). Neither MB nor EPS delayed hatching nor teratogenic defects in the treated ZE at a 2.5 mg/mL dose. There were no significant changes in the ZE heart rate after treatments with MB (130 beats/min) and EPS (140 beats/min), compared to that of normal ZE (120–180 beats/min). Mixing both natural compounds MB and EPS did not affect toxicity using ZFET testing; thus, intimating their safe future use as therapeutic interventions. This represents the first study to have used the ZFET assay on MB and EPS extracts of L. rhinocerus for future health applications.


Results
Zebrafish embryo survival rate after MB and EPS exposure. The survival rate of zebrafish embryos following MB and EPS exposure was studied between 0 and 120 h at MB and EPS extract concentrations of 0.16-10 mg/mL. The study period included larvae as the zebrafish embryos hatch typically 48 to 72 h postfertilisation (HPF). The survival rate of untreated embryos, between 0 and 120 HPF, was 100% (Fig. 1a). At 48 HPF, the survival percentage for embryos treated with MB fell to 85% and 60% at > 5 mg/mL and 10 mg/mL, respectively. At 72 HPF, the survival rate declined to 80%, 65%, and 10% at < 2.5 mg/mL, 5 mg/mL, and > 10 mg/ mL, respectively. At concentrations > 1.25 mg/mL, the survival rate at 96 HPF was 20%, and after 120 HPF; it was observed that no embryos survived at concentrations > 1.25 mg/mL (Fig. 1a). The survival rate of embryos www.nature.com/scientificreports/ (prior to hatching) and larvae (post-hatching) treated with EPS (0.110 mg/mL) during the five days is shown in Fig. 1b. Between 0 and 120 h of HPF, untreated embryos (control) exhibited a 100% survival rate. After 72 h of HPF exposure, the survival rate declined to 90%, 85%, and 50% at a concentration of 0.63 mg/mL, 1.25 mg/mL, and 5 mg/mL, respectively. At 96 HPF, the survival rate declined to 75% at concentrations < 0.63 mg/mL and 30% at concentrations > 1.25 mg/mL. At 120 HPF, survival rates at concentrations 0.63 mg/mL declined to 30%, while survival rates at concentrations > 1.25 mg/mL were 0%, with no surviving embryos. Overall, the results suggest that MB and EPS extracts delay hatching at doses < 1.25 mg/mL.

Zebrafish embryos mortality after MB and EPS exposure. Overall, MB and EPS extracts had dose-
and time-dependent fatal effects. Figure 2 shows a high survival rate (90%) of zebrafish embryos at concentrations of MB and EPS extracts < 1.25 mg/mL. Both MB and EPS extracts had a low survival rate at high concentrations (> 1.25 mg/mL), and none survived after 96 HPF. As a result, the fatal concentration for 50% (LC 50 value) of zebrafish embryos exposed to MB was 0.77 mg/mL, while the LC 50 value of the EPS extract was 0.41 mg/mL.
Zebrafish embryos hatching after MB and EPS exposure. Based on the embryo observations, increasing the mushroom extract concentrations can decrease the percentage hatchability. Figure 3a illustrates the hatching rate of zebrafish embryos treated with MB and EPS (both at 0.1610 mg/mL) at 0-120 HPF. No significant changes in the hatching rate were found when the zebrafish embryos were treated with MB extract at a 0.63 mg/mL concentration. However, at 48 HPF, the rate declined to 80% at concentrations > 1.25 mg/mL. At 72 HPF, the hatching rate was lowered to 65% at 5 mg/mL. Further reduction was observed (25% hatching rate) when treated with 10 mg/mL MB, implying a high death rate after 72 HPF. The hatching rate of EPS did not alter significantly after the treatment with 0.63 mg/mL MB. Less than 85% of the embryos hatched were observed after a 48-h treatment with EPS at > 1.25 mg/mL. However, due to a significant mortality rate at 72 HPF, zebrafish larvae treated with EPS at 10 mg/mL doses had the lowest hatching rate (30%).  www.nature.com/scientificreports/ Zebrafish embryos heart rate after MB and EPS exposure. During the development of many model species, including zebrafish, the heart is the major functioning organ 38 . Previous research has shown that the average heart rate of zebrafish embryos is 120-180 bpm, which is much closer to that of humans 39 . As shown in Fig. 4, the heart rates of zebrafish larvae at 96 HPF (4 days) for both the MB (Fig. 4a) and EPS ( Fig. 4b) treatments were 130 and 140 bpm, respectively. Both extracts exhibited no significant difference in the heart rate of zebrafish larvae at 96 HPF at lower concentrations (relative to higher doses in Fig. 3), ranging between 0.161.25 mg/mL for MB and 0.161.25 mg/mL for EPS. The heart rate of zebrafish larvae at these concentrations was not determined because both MB and EPS extracts at 2.5, 5, and 10 mg/mL demonstrated very little to no survival at 96 HPF.

Morphology of the larvae and zebrafish embryos after MB and EPS exposure.
Potential morphological abnormalities in embryos and larvae were measured from 0 to 120 HPF. There was no apparent teratogenic effect on embryos and larvae after 120 h of exposure to MB and EPS at 0.63 mg/mL and 1.25 mg/ mL, respectively (Fig. 5). These findings infer that MB and EPS have no teratogenic effects on zebrafish embryo development prior to-and post-hatching. The unaffected development of zebrafish embryos and larvae after exposure to 0.63 mg/mL MB and 1.25 mg/mL EPS are shown in Fig. 6 and Fig. 7; however, numerous defects were observed when the concentration of MB and EPS increased to 10 mg/mL ( Fig. 8 and Fig. 9). Coagulated embryos observed between 24 HPF (segmentation) and 48 HPF (pharyngula), along with the loss of yolk sac preventing hatching, were the most common abnormalities reported using MB treatments. Moreover, EPStreated zebrafish hatched at 72 HPF, where tail deformity and damaged blood cells were observed after 120 HPF, with various defects included missing fins, guts, and melanophores.

Discussion
Lignosus rhinocerus is well-known for its therapeutic values, particularly as potential treatment of respiratory diseases. Previous reports have highlighted that the sclerotia, mycelium, and exopolysaccharides of L. rhinocerus contain similar bioactive compounds to β-glucans 21 . Nowadays, β-glucans have gained appeal for several emerging applications, including biopolymers 40 and biomedicines 41 . Notable potential therapeutic properties recently uncovered of mushroom-derived β-glucans include: (a) new or complementary immunotherapies www.nature.com/scientificreports/ against Coronavirus disease (SARS-CoV-2) 18 ; (b) new therapeutic agent for mitigating diseases associated with gastrointestinal mucosal damage, such as peptic ulcers and inflammatory bowel disease 42 ; (c) anticancer drugs for lung and breast cancer 43 ; and (d) asthmatic treatment 28,44 . However, there is a substantial gap in knowledge surrounding the toxicity (if any) of these mushroom-derived bioactive compounds, particularly on MB-EPS extracts. Therefore, the use of Zebrafish trials could aid product development and implementation. Hence, this work investigated and reported on the acute toxicity of zebrafish embryos post exposure to MB and EPS derived from a rare Malaysian-origin Tiger Milk mushroom L. rhinocerus grown in a bioreactor. The ZFET approach was used to expose fertilised zebrafish embryos to quantities of L. rhinocerus extract, MB (0.16-10 mg/mL), and EPS (0.16-10 mg/mL) shown to be non-toxic. Overall, both MB and EPS at 2.5 mg/ mL concentrations did not delay embryo hatching and had a > 80% survival rate between 24 and 120 HPF. In addition, there were no significant differences in the embryo heart rate between the MB and EPS concentrations of 1.25 mg/mL. At MB and EPS doses of > 0.63 mg/mL and > 1.25 mg/mL, respectively, teratogenic effects were observed with evident zebrafish embryo defects. The test revealed that MB has a larger LC 50 value of 0.77 mg/mL than EPS, with a lower LC 50 value of 0.41 mg/mL. Although both MB and EPS extracts were obtained from L. rhinocerus mycelium, the compound composition may differ owing to the fruiting body and mycelial extraction procedures 45,46 . L. rhinocerus mycelium and culture broth demonstrated similar or increased bioactivities, including antioxidant capacities, compared to the use of fruiting bodies 45 . Moreover, EPS exhibited a lower LC 50 value than MB did due to its different mycelial extraction methodology. This is possibly related to MB being directly obtained from dried fungal mycelium, whereas EPS is derived from post series of physicochemical extractions using active fungal mycelia 21,47 . The embryo's ability to burst through the chorion (Fig. 8) and hatch after five days may be limited by morphological defects such as tail deformity. A coagulated embryo and the absence of a heartbeat are both considered deadly.
Certain medicinal mushrooms have also been tested for their toxicity on zebrafish embryos in comparison to Lignosus species. Recent research on Ganoderma lucidum exposure found that MB did not affect ZE hatching at concentrations ranging from 250 to 5000 g/mL and EPS at 3000 g/mL. Notably, neither MB nor EPS were teratogenic at concentrations < 3000 g/mL 35 . Neither EPS or endopolysaccharide (ENS) concentrations of 1 mg/ mL in G. applanatum cause embryo hatching delays. They were shown to have an 88% survival rate when tested from 24 to 120 HPF 48 . Consequently, this new ZFET data could be helpful in the identification of potential health risks associated with the MB-EPS consortia. However, more testing is merited to identify the LC 50 value of MB-EPS extract for large-scale human trials and larger animals before this innovation may be used commercially     21 found that MB production (~ 6 g/L: 30 g dry form) from a 5-L bioreactor culture supports high EPS yield, which can be produced in large quantities. When compared to dried polysaccharides, powdered MB is more applicable in the pharmaceutical industry. The latter is notable as 300 mg of dry tuber biomass from the Malaysian L. rhinocerus has been reported to potentially improve respiratory health in both in vivo and in vitro models 34 . This study therefore constitutes the first toxicity investigation of L. rhinocerus grown in a bioreactor, with the results compared with that of extracts from other Lignosus species. Table 1 shows details of four studies assessing the effect of L. rhinocerus MB on cervical cancer cells (24 mg/mL) 50 , neurite bearing cells (1.75-5.93 mg/mL) 51 , MTT assay for normal human cells (200 μg/mL) 45 , and developmental toxicity in pregnant Sprague-Dawley (SD) rats (3.4 mg/mL) 52 . Notwithstanding this, there is no published research on the toxicity of EPS. The study results are significant where the Zebrafish 3.0 toxicity model was used to evaluate and assess what was to be non-toxic mycelial biomass (0.77 mg/mL) and EPS (0.41 mg/mL) in L. rhinocerus bioreactor samples. This Zebrafish model offered evidence that the use of Malaysian bioactive mycelial biomass and polysaccharides L. rhinocerus may be safe as a new therapeutic intervention.
Furthermore, the findings from this research highlight the increasing trend towards the intensive yet sustainable exploitation of bio-based resources from food and marine ecosystems, from the emergence of the bioeconomy 11 . These bio-inspired materials may be refined and scaled up for commercial use through advances in biotechnology, as described here 49 . Notably, this emerging area will be future-proofed through accelerating digitalisation, where metadata outputs will potentially inform food for therapeutics, cosmetics, personal care products, and smart packaging, along with offering putative interventions to help mitigate the Covid-19 disease 10,51,53 .

Conclusion
In conclusion, this is the first study on the use of ZFET assay on bioreactor-grown Malaysian medicinal tiger milk mushroom L. rhinocerus MB and EPS extracts. MB (LC 50 : 0.77 mg/mL) was harmless, whereas EPS (LC 50 : 0.41 mg/mL) are practically non-toxic. The ZFET assay offers a fast, affordable, robust, and efficient early development approach to evaluating extracts from medicinal fungi for future use as asthmatic medication. Specifically, this study provides evidence of the potential of L. rhinocerus as an alternative or adjuvant to the current drugs used for the management of respiratory diseases. Additionally, for the early medication development process, zebrafish can be utilised to quickly discover potentially dangerous chemicals and prioritise compounds for additional pre-clinical and clinical testing. The adaptation of conventional instruments in conjunction with new nanotechnology discoveries will help to further increase the use of zebrafish for drug screening.  21 . The sclerotium was cultured on a potato dextrose agar (PDA) plate (Sigma-Aldrich, Dorset, UK) and incubated at 30 °C under dark conditions. The strain was stored and maintained on PDA slants at 4 °C 54 .
Culture conditions. The fungal inoculum was prepared according to Wan Mohtar et al. 55 blueprints fungal production plan, including two seed culture stages. The mycelium was cultivated for ten days under dark conditions at an initial pH of 5, 150 rpm, and 30 °C with slight adjustments for the first seed culture. Four mycelial agar squares (1 cm x 1 cm each) were cut from a ten-day-old plate culture and inoculated in a 250 mL Erlenmeyer flask using sterile scalpels (100 mL of medium). The first seed culture was then homogenised for 10 sec with a sterile Waring hand mixer to produce more hyphal tips with uniform mycelium diameters. The homogenised mycelial culture was transferred to a 500 mL shake flask (200 mL medium) as the inoculum for the second seed culture and incubated for 11 d under dark circumstances on an orbital shaker at initial pH 5 Sample preparation for the toxicity test. Dried MB and EPS were prepared at room temperature for toxicity testing. A 10 mg/mL of stock solution was prepared by dissolving dried MB and EPS in embryo media (Danio-SprintM media), which was then diluted two-fold and further in a 96-well microplate (200 µL/well) using serial dilutions to obtain seven different concentrations in the 0.16-10 mg/mL range. For a standard control, zebrafish embryos in embryo media solution were used as an untreated control sample (0 mg/mL).
Upkeep and breeding of zebrafish system. A couple of adult zebrafish were placed in a breeding tank the day before the breeding occurred to set up the system. The following day, embryos were cleansed and incubated in the embryo medium (Danio-SprintM media) for two hours. Only healthy fertilised embryos were selected for the ZFET testing; meanwhile, the dead and coagulated embryos were discarded 35 .
Zebrafish embryo toxicity (ZFET) test. Firstly, at 0 HPF, zebrafish embryos were exposed to samples (200 µL) in 96-well microplates (embryo/well) at seven different concentrations ranging from 0.16 to 10 mg/ mL. The experiments were designed with an exposure group, both treated and untreated, containing 12 embryos each. The successfully treated embryos were cultured at ambient temperature (25 °C to 28 °C) for five days. The cumulative mortality and development abnormalities of zebrafish embryos and larvae were observed and examined for every 24 HPF from 0 to 120 HPF. Data of the survival rate, hatching rate, heart rate, morphological malformations, and teratogenic defects were captured and recorded using an inverted microscope coupled with a digital camera. The heartbeats were counted using a stopwatch (three embryos/min). Lethal endpoints were defined based on coagulation and the nonappearance of a heartbeat. Developmental defects such as pericardial oedema, yolk sac oedema, non-hatched, twisted body, and twisted tail were observed and recorded. The LC 50 values were considered based on the principle of toxicity, in which > 1 mg/mL are considered relatively harmless, 0.1-1 mg/mL non-toxic, 0.01-0.1 mg/mL slightly toxic, 0.001-0.01 mg/mL moderately toxic, 0.0001-0.001 mg/ mL highly toxic, and > 0.0001 mg/mL are super toxic. www.nature.com/scientificreports/ evaluated using the same methods. The heart rates of three different animals were presented as a mean standard error of mean (SEM). A one-way analysis of variance (ANOVA) was used to determine significant differences, followed by a Dunnett's Multiple Comparison post-hoc test. differences between the means of the treated group and embryos in embryo media were set at p 0.001***, p 0.01**, p 0.05*.