Submerged fermentation conditions and bioactivity profiling of mycelial extract of the Philippine <i>Oudemansiella canarii</i>

Factors	Treatments	Biomass yield (mg DW/100 mL)
Culture media	25% Mango purée (MP)	140.00 ± 14.51^b
	Coconut water (CW)	180.00 ± 8.16^a
	Combination (MP + CW)	136.67 ± 12.47^b
pH	pH 4	57.33 ± 3.40^c
	pH 5	63.33 ± 6.55^c
	pH 6	180.67 ± 2.87^a
	pH 7	189.00 ± 5.72^a
	pH 8	93.67 ± 7.41^b
	pH 9	77.33 ± 9.03^bc
Temperature	10 °C	0.00 ± 0.00^c
	20 °C	174.33 ± 12.68^b
	30 °C	223.67 ± 6.94^a
Agitation	0 rpm (static)	226.67 ± 8.02^c
	100 rpm	1385.67 ± 27.93^b
	150 rpm	1847.00 ± 80.07^a
Means with the same letters of superscript in each factor are not significantly different at 5% level of significance. Values in boldface indicate the highest biomass yield within each factor.

Table 1.

Mycelial biomass yield of O. canarii as influenced by liquid culture media, pH, temperature, and agitation.

Assay	Extract	% Inhibition
α-glucosidase	O. canarii (10 µg/mL)	1.42 ± 6.63
	Acarbose (10 µg/mL)	88.76 ± 0.68
	DMSO (33,000 µg/mL)	0.00 ± 0.00
Acetylcholinesterase	O. canarii (10 µg/mL)	−4.35 ± 3.09
	Galantamine (10 µg/mL)	98.03 ± 0.04
	Methanol (10 µg/mL)	0.00 ± 0.00
Cyclooxygenase-1 (COX-1)	O. canarii (10 µg/mL)	26.37 ± 16.92*^a
	Indomethacin (2,862 µg/mL)	93.98 ± 1.85
	DMSO (10 µg/mL)	0.00 ± 0.00
Cyclooxygenase-2 (COX-2)	O. canarii (10 µg/mL)	9.80 ± 20.00*
	Indomethacin (2,862 µg/mL)	91.86 ± 4.40
	DMSO (10 µg/mL)	0.00 ± 0.00
DPPH	O. canarii (1,000 µg/mL)	51.56 ± 2.02
	Ascorbic acid	86.88 ± 4.09
	DPPH + ethanol	0.00 ± 0.00
Values are expressed as mean ± SD. Asterisk (*) denotes a significant difference(p < 0.05) of the mushroom sample compared with negative control. Superscript (a) denotes a significant difference (p < 0.05) of the mushroom sample compared with the positive control.

Table 2.

Inhibitory activities of O. canarii mycelial ethanolic extract against α-glucosidase, acetylcholinesterase, cyclooxygenase, and DPPH radical scavenging assay.

Extract (µg/mL)	Mortality (%)		LC₅₀ value**	Hatchability (%)
Extract (µg/mL)	24 hpte*	48 hpte	LC₅₀ value**	Hatchability (%)
100,000	100.00 ± 0.00^a	100.00 ± 0.00^a	3.39 µg/mL 95% CI: [1.25, 8.56]	0.00 ± 0.00^b
10,000	100.00 ± 0.00^a	100.00 ± 0.00^a		0.00 ± 0.00^b
1,000	50.00 ± 25.00^b	100.00 ± 0.00^a		0.00 ± 0.00^b
100	33.33 ± 14.43^b	91.67 ± 14.43^a		8.33 ± 14.43^b
10	25.00 ± 25.00^bc	83.33 ± 14.43^a		16.67 ± 14.43^b
1	8.33 ± 14.43^bc	25.00 ± 25.00^b		41.67 ± 38.19^a
0.1	0.00 ± 0.00^c	0.00 ± 0.00^b		91.67 ± 14.43^a
Embryo water	0.00 ± 0.00^c	0.00 ± 0.00^b		100.00 ± 0.00^a
95% ethanol	100.00 ± 0.00^a	100.00 ± 0.00^a		0.00 ± 0.00^b
Each value represents the mean ± SD of triplicate tests (r = 3, n = 4). In each column, means with the same letter of superscript are not significantly different from each other at 5% level of significance using Tukey's HSD. * hpte = hours post-treatment exposure. ** LC₅₀ value was computed using probit analysis based on the mortality at 48 hpte and was presented with 95% confidence interval: [lower bound, upper bound].

Table 3.

Mortality and hatchability rates of zebrafish embryos after 24 and 48 h of exposure to the different concentrations of mycelial extract of O. canarii.

Treatments	Heartbeat rate (/min)	NH	TM	HM	PE	YD	DG
HD (0.34 µg/mL)	117.00 ± 6.93^a	−	−	−	−	−	+
MD (0.17 µg/mL)	117.00 ± 6.00^a	−	−	−	−	−	+
LD (0.08 µg/mL)	120.00 ± 3.46^a	−	−	−	−	−	−
Embryo water	123.00 ± 3.46^a	−	−	−	−	−	−
HD = high dose, MD = medium dose, LD = low dose (based on LC₅₀ value). Each value of the heartbeat rate represents the mean ± SD of duplicate tests (r = 2, n = 3). In each column, means with the same letter of superscript are not significantly different from each other at 5% level of significance using Tukey's HSD. (+) and (−) indicate the presence and absence of teratogenic effects of the different treatments, respectively. NH, no heartbeat; TM, tail malformation; HM, head malformation; PE, pericardial edema; YD, yolk deformity; DG, delayed growth.

Table 4.

Teratogenic effects and heartbeat rate at pharyngula phase zebrafish embryos exposed to different concentrations of mycelial extract of O. canarii at 72 h of exposure.

Extract (µg/mL)	Mortality (%)	LC₅₀ value*
100,000	100.00 ± 0.00^a	17.87 µg/mL 95% CI: [1.61, 182.84]
10,000	100.00 ± 0.00^a
1,000	100.00 ± 0.00^a
100	46.67 ± 15.28^b
10	33.33 ± 11.55^b
1	20.00 ± 10.00^b
0.1	15.00 ± 13.23^b
Saline solution	0.00 ± 0.00^b
95% ethanol	100.00 ± 0.00^a
Each value represents the mean ± SD of triplicate tests (r = 3, n = 10). In each column, means with the same letter of superscript are not significantly different from each other at the 5% level of significance using Tukey's HSD. * LC₅₀ value was computed using probit analysis based on the mortality at 12 hpte and was presented with 95% confidence interval: [lower bound, upper bound].

Table 5.

Mortality of A. salina nauplii after 12 h of exposure to the different concentrations of mycelial extract of O. canarii.