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.