3.1 pH, degree of protein hydrolysis, and proximate composition
The HE and CP canola meals were fermented withA. niger NRRL 334 andA. oryzae NRRL 5590 over a 72-h period. After 48 h of fermentation, significant growth of hyphae was observed, and spores started to form on the surface after 72 h of fermentation as shown in Figure 1. For pH upon fermentation (Table 1a), significant differences (p <0.001) between meal types (CP vs. HE) and among fermentation time groups were observed. The pH for all fermented samples increased with fermentation time, with the CP meals showing changes after 48 h while changes occurred earlier (24 h) for HE meals. The HE meals had an overall higher pH (~8.2) than CP meals (~7.6) at the end of the fermentation period (72 h), regardless of the strains used. In contrast to the result of the present study, Olukomaiya et al. (2020) found a reduction in the pH value from 6.26 to 6.13 after SSF for 7 days using A. ficuum , A. sojae , and co-culture Lactobacillus salivarius on autoclaved, solvent-extracted canola meal. The authors described this to be caused by an increase in organic acid content during SSF, especially when usingL. salivarius . Aljubori et al. (2017) also reported relatively stable or decreased pH values when using L. salivarius . The differences in results may be explained by the variations in canola meals, fungi strains, and fermentation conditions used in these studies in comparison to the present study. In addition, the organic acids might have been used as nutrients by A. niger and A. oryzaeduring SSF, leading to increases in pH.
The protein hydrolysis, measured as the degree of hydrolysis (DH), increased throughout the fermentation period (Table 1b). Meal type, strain used, fermentation time, and their interactions were all significant factors (p <0.001) affecting DH. The DH of the meals increased from zero (0 h) to 15-30% (24 h), then to ~40% (48 h), and finally to 45-50% after 72 h. For the first two days (0-48 h), the DH increased quite rapidly due to the large surface area that was exposed to oxygen, which helped with hyphae growth. The proteins were most likely hydrolyzed partially by the proteases synthesized by fungal cells, resulting in increased DH with the release of free amino acids. The relatively compact nature of the lower layers of the meal significantly lowered the speed of growth as fungi could not penetrate easily to the bottom of the solid substrate, hence the lower rate of DH increase at 72 h. The increase in DH was slower for HE meals (15.8-23.4%) as compared to CP meals (28.3-30.1%) on the first day (24 h) of fermentation, with the strain A. oryzae being less efficient at protein hydrolysis, particularly in the HE meal. At the end of the fermentation cycle, the DH was similar for CP meals and the HE meal fermented by A. oryzae (45.1-46.4%), while that of the A. niger HE meal was higher (52.3%).
The changes to the proximate composition were similar for CP and HE meals fermented with both strains. For protein, lipid and ash contents, significant impacts (p <0.001) from meal type and fermentation time were observed. As shown in Table 2a, the crude protein level increased from ~34 to ~37% (a 9-11% increase) after 72 h of fermentation in the CP meals, regardless of the strain used. The protein levels were higher in the HE meals, ranging from 39.7-41.5%, and remained relatively constant upon fermentation (though still increased by A. niger ). In contrast to protein, the crude lipid levels decreased from ~12.5 to ~9% for CP meals, whereas the contents in the HE meals decreased from ~2.5 to ~1% (Table 2b). There was a small increase in the ash content for CP meals from ~6 to ~7% after 72 h of fermentation, while that of HE meals increased from ~8 to ~10% (Table 2c). Compared to HE meals, CP meals had a lower initial crude protein content due to the higher proportion of oil; this trend remained even after a 72-h period of SSF. The high levels of residual oil in the CP meals can be inhibitory to fungal growth and lead to similar results found by Simon et al. (2017). They introduced and compared several strains (Aureobasidium pullulans NRRL-58522 3.0,Aureobasidium pullulans NRRL-Y-2311-1, Trichoderma reeseiNRRL 3653, Fusarium venenatum NRRL 26139, Paecilomyces variotti NRRL 1115, Rhizopus microspores var. oligosporusNRRL 2710, Neurospora crassa NRRL 2332, Mucor circinelloides , and Pichia kudriavzeii ) as SSF cultures on HE and CP meals and concluded that fungal strains preferred HE meals due to the lower level of residual oil.
The increase in the levels of crude protein and ash is attributed to the effect of concentration. The carbohydrates, including sucrose and fibre, acted as carbon sources for the fungi to grow, and carbon dioxide (CO2) was produced as a result, concentrating the remaining compounds (protein and ash). Wang et al. (2012) reported a 27% increase in the crude protein content using composite strains with Candida tropicalis CICIM Y0079(T). The results agreed with those of Plaipetch and Yakupitiyage (2014), who found a 9% increase in the crude protein content in Saccharomyces cerevisiaeyeast-fermented canola meal. Simon et al. (2017) reported an increase in crude protein from 42 to 44-50% (d.b.) in fermented HE meal and a ~14% increase in fermented CP meal, depending on the strains. The increases in the nitrogen content of 11.6, 23.1, 34.2, 47.6, and 65.4% after 1, 2, 5, 8 and 10 days of incubation, respectively, were reported by Pal Vig and Walia (2001).