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. 2024 Jun 18;14(1):14015.
doi: 10.1038/s41598-024-63803-4.

Optimized production and characterization of endo-β-mannanase by Aspergillus niger for generation of prebiotic mannooligosaccharides from guar gum

Suresh Nath  1 , Naveen Kango  2
Affiliations

Optimized production and characterization of endo-β-mannanase by Aspergillus niger for generation of prebiotic mannooligosaccharides from guar gum

Suresh Nath et al. Sci Rep. .

Abstract

Optimized production of Aspergillus niger ATCC 26011 endo-β-mannanase (ManAn) on copra meal resulted in 2.46-fold increase (10,028 U/gds). Purified ManAn (47 kDa) showed high affinity towards guar gum (GG) as compared to konjac gum and locust bean gum with Km 2.67, 3.25 and 4.07 mg/mL, respectively. ManAn efficiently hydrolyzed GG and liberated mannooligosaccharides (MOS). Changes occurring in the rheological and compositional aspects of GG studied using Differential scanning calorimetry (DSC), Thermal gravimetric analysis (TGA) and X-ray diffraction (XRD) revealed increased thermal stability and crystallinity of the partially hydrolyzed guar gum (PHGG). Parametric optimization of the time and temperature dependent hydrolysis of GG (1% w/v) with 100 U/mL of ManAn at 60 °C and pH: 5.0 resulted in 12.126 mg/mL of mannotetraose (M4) in 5 min. Enhanced growth of probiotics Lactobacilli and production of short chain fatty acids (SCFA) that inhibited enteropathogens, confirmed the prebiotic potential of PHGG and M4.

Keywords: A. niger; Guar gum; Partially hydrolyzed guar gum; Prebiotics; Probiotics; β-mannanase.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Optimized production and characterization of A. niger ATCC 26011 endo-β-mannanase. (a) RSM showing interaction of pH and moisture content. Agro-waste copra meal was used as substrate in SSF. (b) Purification profile of ManAn using DEAE cellulose-52. (c) SDS-PAGE showing protein profile and zymogram (47 kDa and 40 kDa) (MW: mol. wt. marker, Lane 1: crude enzyme, Lane 2: Dialyzed protein precipitate, Lane 3: purified ManAn (47 kDa), Lanes 4–6: Zymogram of Lanes 1–3 (d) temperature optimum and thermal stability (e) pH optimum and pH stability (f) Lineweaver–Burk plots showing affinity of purified ManAn towards GG, KG and LBG.
Figure 2
Figure 2
Optimized production of MOS from GG by ManAn. (a) TLC showing time dependent GG hydrolysis. (b) TLC showing temperature dependent GG hydrolysis. (c) Parametrically optimized production of MOS using RSM. (d) MOS fractionation using Bio-gel P2 as visualized by FACE (S: standards and fractions 18–39). (e) Elution profile of MOS through Bio-gel P2.
Figure 3
Figure 3
HPLC showing MOS generation by ManAn from GG (a) MOS standards (b) Guar gum hydrolysate showing presence of MOS (c) Biogel-P2 fraction containing purified mannotetraose (M4).
Figure 4
Figure 4
Rheological properties of PHGG. (a) Viscometric analysis and molecular weight determination of hydrolysate using ManAn. (b) Scanning electron micrographs showing morphological alterations in guar gum upon treatment with ManAn.
Figure 5
Figure 5
Changes in thermal behavior of guar gum after hydrolysis with ManAn (a) GG and (b) PHGG (c) XRD showing the crystallinity index of GG and PHGG.
Figure 6
Figure 6
Growth of probiotic (a) L. delbrueckii NCIM 2025, (b) L. acidophilus NCIM 5306, (c) L. rhamnosus MTCC 5957 in M9-minimal medium containing PHGG, M4, FOS, glucose and mannose as sole carbon source and the effect of postbiotic metabolites on pathogenic S. aureus MTCC 96 and C. albicans MTCC 227 (1-PHGG, 2-Mannose, 3-Glucose, 4-FOS, 5-medium control, 6-M4 and 7-Guar gum control).
Figure 7
Figure 7
Characterization of postbiotics using 1H and 13C NMR spectra of the culture filtrates of the Lactobacillus spp. grown on PHGG. The peaks show the production of short chain fatty acids (SCFA).
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