Figure 3. Substrate bags colonized by G. lucidum 7 days after inoculation

Fibre content analysis

Samples were analysed and calculations performed for neutral detergent fibre (NDF), acid detergent fibre (ADF) and acid detergent lignin (ADL) using the ANKOM 220 Fibre Analyzer (ANKOM Technology Corporation, NY, USA) according to the Van Soest and co-workers (1991) method [12]. NDFom-NDF was not assayed with a heat stable amylase and was expressed exclusive of residual ash. ADFom-ADF expressed exclusive of residual ash. Lignin (sa)-Lignin was determined by solubilization of cellulose with sulphuric acid.
The ADF content of samples was calculated as follows:
ADF (g/kg DM) = Sample weight after boiling (g DM) – Ash weight (g DM)/ ADF (g/kg DM) / Weight of sample (g DM) × 1 000
The NDF content of samples was calculated as follows:
NDF (g/kg DM) = Sample weight after boiling (g DM) – Ash weight (g DM) / Weight of sample (g DM) × 1 000
The ADL content of samples was calculated as follows:
ADL (g/kg DM) = Sample weight (g DM) – Ash weight (g DM) / Weight of sample (g DM) × 1 000

Crude Protein

Crude protein (CP) was determined for the raw substrate and the SSF product in triplicate at each selected stage (time 0 and time 18 weeks). The nitrogen (N) content of NDF residues was analysed by combustion assay [13]. One-gram (1 g) samples were weighed accurately to determine the CP content by inserting it into a Leco Nitrogen analyser and the total N content determined on combustion in oxygen [14]. A factor of 6.25 was used to convert the N content of the samples to CP content [13].

Gross Energy determination

One gram (1 g) of the samples were analysed in triplicate to determine the GE content according to the procedures described by the AOAC [15].

Factorial design and data analysis

Data processing and analyses were performed with R version 4.0.2 [16] within R Studio version 1.2.5042 [17]. Data were imported using the ‘gsheet‘ package [18]. Data exploration, wrangling and visualisation were conducted using the ‘tidyverse‘ package [19]. Analysis of variance (ANOVA; α = 0.05) of the data were performed using base R functions. ANOVA was applied to determine if mushroom species (Ganoderma lucidum, Ganoderma #1 and Pleurotus ostreatus ) and grind (sieve sizes of 2 to 5 cm, 1 to 2 cm and < 1 cm, for grind 3, 2 and 1, respectively) affected dependent variables (ADF, ADL and NDF) from the first and last sampling period, 0 to 18 weeks. Means were separated using the Least Significant Difference (LSD) test function from the ‘agricolae‘ package [19]. A linear regression analysis was applied to identify if any associations existed between the volume of the dependent variables over the 18-week sampling period. Pearson correlation coefficient was applied in the scatter plots and plotted with the ‘ggpubr‘ package [21] to determine the strength of the associations presented.

Results

Crude Protein

Crude protein (CP) determination of the various substrates was performed after 18 weeks of SSF (Figure 4). The undigested control yielded mean CP concentrations (g/kg DM) of 39.98 (±3.31), Ganoderma lucidum55.58 (±1.13), Pleurotus ostreatus 61.48 (±1.46) andGanoderma #1 isolate yielded 53.25 (±0.38) as shown in Table 1.
Table 1: Crude protein (CP) concentrations (g/kg DM) of mushroom treatments after 18 weeks solid-state fermentation of S: undigested Acacia mellifera control, M1: Pleurotus ostreatus substrate, M2: Ganoderma lucidum substrate, M3:Ganoderma #1 substrate.