Reduction of soybean meal non-starch polysaccharides and α-galactosides by solid-state fermentation using cellulolytic bacteria obtained from different environments

Abstract

Soybean meal (SBM) is an important protein source in animal feed. However, the levels of SBM inclusion are restricted in some animal species by the presence of antinutritional factors (ANFs), including non-starch polysaccharides (NSPs) and α-galactosides (GOSs). The aim of this study was to reduce the soybean meal NSPs and GOSs by solid-state fermentation (SSF) using a combination of cellulolytic bacteria isolated from different environments (termites, earthworms, corn silage and bovine ruminal content). To analyse the key enzymatic activities, the isolates were grown in minimal media containing NSPs extracted from SBM. The selected bacterial strains belonged to the genera Streptomyces, Cohnella and Cellulosimicrobium. SSF resulted in a reduction of nearly 24% in the total NSPs, 83% of stachyose and 69% of raffinose and an increase in the protein content. These results suggest that cellulolytic bacteria-based SSF processing facilitates SBM nutritional improvement. In addition, the use of fermented SBM in animal diets can be recommended.

Figure 1. Dendrograms displaying ITS profile cluster analyses of the cellulolytic strains from each environment: (A) garden soil, (B) earthworm, (C) corn silage, (D) termite, (E) rotten leaves and (F) bovine rumen content.

The profile distances between the cellulolytic strains were calculated based on the DICE similarity coefficient. Dendrograms were created using GelCompar II (Applied Maths) software with a position tolerance of 2%.

Figure 2. The total cellulase activity screen of the cellulolytic strains.

The activity was assessed by filter paper hydrolysis. The strains were incubated beforehand in minimal medium containing NSP extracted from soybean meal. One unit of total cellulase activity was defined as the amount in µmoles of glucose released per min/ml of bacterial culture supernatant.

Figure 3. Endo-β-xylanase activity screening in cellulolytic strains.

The activity was assessed using p-nitrophenyl-β-D-xylopyranoside as a substrate for hydrolysis. Bacteria were incubated beforehand in minimal medium containing NSP extracted from soybean meal; one unit of total xylanase activity was defined as the µmoles of p-nitrophenol released per min/ml of bacterial culture supernatant.

Figure 4. Box-and-whisker plots showing comparisons of the contents of (A) stachyose, (B) raffinose, (C) non-starch polysaccharide and (D) protein obtained after SSF between the inoculated group and the non-inoculated group.

The results are expressed in grams of ANFs or protein per 100 g of FSBM.

Figure 5. Dominance of the inoculated strains after SSF as revealed by their RFLP profiles.

The RFLP profiles from the 16S rRNA genes of the selected strains (S7, CR18 and T5) were generated using a culture-independent approach. DNA was extracted directly from FSBM (inoculated and non-Inoculated groups), and the PCR amplicons were digested with the restriction enzymes Bmt I and BtsC I. The figure shows the RFLPs of the selected strains (S7, CR18 and T5) and the RFLP profiles from five replicates of inoculated and non-inoculated SSFs. The arrows indicate the presence of the S7 and T5 strains as dominant in the fermentation process.