Value creation of copra meal mannan into functional manno-oligosaccharides (β-MOS) using the mannanase Bacillus man B (BlMan26B)

Abstract

Agricultural wastes rich in β-mannan are an important environmental problem in tropical and sub-tropical countries. This research aims at dealing with this and investigates the valorization of mannan-rich copra meal from virgin coconut oil manufacturing into mannan-oligosaccharides (β-MOS) by enzymatic hydrolysis using β-mannanase from Bacillus licheniformis (BlMan26B). Lab-scale process, involving pre-treatment and bioconversion steps, were conducted and evaluated. Lyophilized β-MOS was analyzed and its biological activities were assessed. The size of oligosaccharides obtained ranged from dimers to hexamers with 36.7% conversion yields. The prebiotic effects of β-MOS were demonstrated in comparison with commercial inulin and fructo-oligosaccharides (FOS). In vitro toxicity assays of β -MOS on human dermal fibroblasts and monocytes showed no cytotoxic effect. Interestingly, β-MOS at concentrations ranging from 10 to 200 µg/mL also demonstrated potent anti-inflammatory activity against LPS-induced inflammation of human macrophage THP-1 in a dose-dependent manner. However, at high dose, β-MOS could also stimulate inflammation. Therefore, further investigation must be conducted to ensure its efficacy and safe use in the future. These results indicate that β-MOS have the potential to be used as valued-added health-promoting nutraceutical or feed additive after additional in-depth studies. These finding should be applicable for other agricultural wastes rich in mannan as well.

Keywords: Bacillus licheniformis β-mannanase; Defatted-copra meal; Functional; Manno-oligosaccharides; β-MOS.

Fig. 1

Optimization of bioconversion reaction and analysis of β-MOS products. (a) TLC analysis of products from various reaction conditions of substrate and enzyme at various time points (0, 1, 2, 3, 6, 9, 12, 16, 20, and 24 h). (b) HPAEC-PAD analysis of MOS composition at optimal reaction conditions. Galactomanno-oligosaccharides and manno-oligosaccharides standards were used for comparison and product quantitation. The corresponding retention times observed were 11.67 min for mannobiose, 14.65 min for mannotriose, 19.35 min for mannotretraose, and 36.42 min for mannohexaose. O-GMM3, 6¹-α-D-Galactosyl-mannotriose; O-GM3, 6¹-α-D-Galactosyl-mannobiose plus mannotriose.

Fig. 2

Promotion of growth of probiotics and potentially pathogenic bacteria by β-MOS and other oligosaccharides. Growth of the probiotic bacteria B. longum (a), L. acidophilus (b), L. amylovorus (c), L. delbrueckii (d), L. gasseri (e), S. salivarius (f) and S. thermophilus (g) and pathogenic bacteria E. coli (h) and S. enterica (i) was monitored when cultured in media containing fructooligosaccharides (FOS, squares), galactooligosaccharides (GOS, diamonds), inulin (asterisks) and β-MOS (circles). The data are given as mean ± SD (n = 3).

Fig. 3

Viability of HDF and THP-1 cells in the presence of β-MOS. β-MOS were used at 62.5–2000 µg/mL and incubated for 24 h on HDF cells (a) and at 10–2000 µg/mL for THP-1 cells (b). Puromycin (c) was used at 0.5–8 µg/mL as a control. Cell viability was tested by the resazurin assay and the MTT assay for HDF cells and THP-1 cells, respectively. The absorbance of the untreated cells was used as a reference, representing 100% viability. Each bar represents the average value of triplicates and the error bar represents the standard deviation of the mean. Statistical analysis versus untreated was performed using One-way ANOVA, Bonferroni’s multiple comparisons test: *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 4

Anti-inflammatory effects of β-MOS. The anti-inflammation was measured using VitD3-differentiated THP-1 cells. The cells were pretreated with β-MOS at concentrations ranging from 10 to 1000 µg/mL, while 0.2 µg/mL dexamethasone was used as a control. LPS was used to induce the inflammation of differentiated THP-1 cells. Each bar represents the average value of quadruplicates and the error bar represents the standard deviation of the mean. Statistical analysis was performed using One-way ANOVA, Bonferroni’s multiple comparisons test: *P < 0.05, **P < 0.01, ***P < 0.001. .

Fig. 5

Schematic diagram for the production of β-MOS using enzyme technology. (A) Pretreatment. The copra meal sample was heated in a hot air oven at 60 °C for 12 h. For the defatting process, copra meal was boiled for 2 h in twice the volume of DI water, and then kept in 4 °C for solidification and removal of the remaining coconut oil. This process was repeated three times. After that the sample was dried again in a hot air oven for 12 h at 60 °C, before suspending in 1 L of n-hexane with stirring overnight. Then the n-hexane was removed by vacuum filtration and the defatted copra meal (DCM) was dried at 60 °C for 12 h. Then the DCM was ground, sieved through 0.2 mm mesh, and kept in 4 °C until used. (B) Bioconversion. Copra meal was suspended in DI water and β-mannanase was added to the reaction at 500 U per 2.6 g mannan. The reaction mixture was incubated for 24 h at 40 °C, 250 rpm. The soluble β-MOS product in the supernatant was collected after filtration, centrifugation, and kept at −20 °C overnight before lyophilization. Pictures of copra meal waste from the virgin coconut oil company, de-fatted copra meal after pre-treatment, and lyophilized MOS from this study are shown.

Fig. 6

A summary of this research. The bioconversion of defatted copra meal from virgin coconut oil manufacturing into functional manno-oligosaccharides (β-MOS) by enzymatic hydrolysis using β-mannanase from Bacillus licheniformis (BlManB) was investigated in this study. The size of oligosaccharides, analyzed by TLC and HPLC, obtained ranged from dimers to hexamers with 36.7% conversion yields. The in vitro growth promoting effects of β-MOS were demonstrated on various probiotic bacteria compared to other oligosaccharides. In vitro toxicity assays of β-MOS on human dermal fibroblasts and monocytes showed no cytotoxic effect. For the first time, β-MOS at a concentration ranging from 10 to 200 µg/mL also demonstrated potent anti-inflammatory activity against LPS-induced inflammation of human macrophage THP-1, in a dose-dependent manner.