Production of mannooligosaccharides from various mannans and evaluation of their prebiotic potential
Introduction
Mannans are hetero-polysaccharides chiefly composed of repeating units of mannopyranose linked by β-1,4- glycosidic bonds (Soni & Kango, 2013). Common mannans include locust bean galactomannan (LBG; mannose: galactose 4:1), Konjac glucomannan (KG; mannose: glucose 4:3) and guar galactomannan (mannose: galactose 2:1). Some agro-industrial products such as copra meal (CM; mannose: galactose 3:1), palm kernel cake (PKC) and spent coffee ground are rich source of mannans (Soni and Kango, 2013, Passos et al., 2019). Copra meal is brown color residue obtained after copra oil extraction and contains 43–45% carbohydrates, 19–20% protein, 12% crude fibre and 5% lignin along with xylose and arabinose in trace amounts (Prajapati, Suryawanshi, Agrawal, Ghosh, & Kango, 2018). Mannanases (β-mannanases EC 3.2.1.78 and β-mannosidases EC 3.2.1.25) hydrolyze mannans into various DP (DP 2–10) MOS and mannose (Jana, Suryawanshi, Prajapati, Soni, & Kango, 2018). MOS are categorized as prebiotic non-digestible short chain oligosaccharides because of their selective fermentation by gut microbes, specially Lactobacilli, Bifidobacteria etc. and beneficial short chain fatty acid (SCFA) production (Mary, Prashanth, Vasu, & Kapoor, 2019). Prebiotic MOS can enrich growth of health promoting gut microbiota in humans as well as animals by imparting anti-obesity, anti-neoplastic, anti-allergic, hypocholesterolemic and immunomodulatory effects (Garcia Diaz et al., 2018, Chauhan et al., 2012). Short DP prebiotic oligosaccharides (<4), on behalf of their excellent solubility and diffusion, confer better health promoting effects as compared to high DP oligosaccharides (>4) (Endo et al., 2016, Jana and Kango, 2020). The important characteristics of prebiotics include stability in gastric juice, resistance to gastrointestinal (GI) enzymes, non-absorption in upper GI tract or large intestine and selective utilization by probiotic gut microbiota (Aachary and Prapulla, 2010, Garcia Diaz et al., 2018). Several in vitro and in vivo studies have demonstrated utility of MOS as Lactobacilli and Bifidobacteria growth promoting carbohydrates (Jana and Kango, 2020, Mary et al., 2019, Li et al., 2019, Zheng et al., 2018, Srivastava et al., 2017, Pongsapipatana et al., 2016, Ghosh et al., 2014).
Lactobacilli are well known for their beneficial effects including help in maintaining good enteric environment by producing SCFA that suppresses the growth of pathogenic bacteria and production of branched chain amino acid (BCAA) which also has significant health promoting benefits (Gibson and Roberfroid, 1995, Kubota et al., 2008, Mutaguchi et al., 2013). Probiotics are live microorganisms that, when administered in appropriate amounts, confer several health benefits not only in the gut but also in other parts of the body such as the urogenital tract and oral cavity (Monteagudo-Mera, Rastall, Gibson, Charalampopoulos, & Chatzifragkou, 2019). Probiotics could produce different types of SCFA such as acetate, butyrate, propionate, formate etc. under in vitro and in vivo conditions by fermenting oligosaccharides. In context of health promotion, acetate has been reported as an enteropathogen inhibitor and it can also interact with central nervous system to reduce the appetite. Similarly, butyrate and propionate have been reported to induce gut hormone production in human (Rios-Covian et al., 2016).
In the present study, Aspergillus quadrilineatus RSNK-1 β-mannanase was used to hydrolyze mannan-rich defatted copra meal (DfCM), copra meal (CM), konjac glucomannan (KG) and locust bean galactomannan (LBG). Multi-scale characterization of hydrolysates was carried out using high performance liquid chromatography (HPLC), Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (NMR) spectroscopy. Cytotoxicity of MOS containing mannan hydrolysates were ascertained by studying their effect on colorectal cell line (Caco-2). Lactobacilli were used to measure prebiotic potential of mannan hydrolysates containing MOS by biofilm formation, SCFA and BCAA production.
Section snippets
Chemicals
3, 5 di-nitrosalicylic acid (DNS), potassium sodium tartrate, sodium hydroxide, locust bean gum (LBG), WST reagent, acetic acid, butyric acid, propionic acid, ammonium hydroxide and Trypsin-EDTA solution were purchased from Sigma Aldrich, USA. Mannose (M1), Mannobiose (M2), Mannotriose (M3) and Mannotetraose (M4) were purchased from Megazyme (Bray, Ireland). Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), penicillin and streptomycin were procured from Invitrogen, USA. TLC
FTIR analysis
MOS generation was confirmed by analyzing functional groups present in the mannan hydrolysate using Bruker Alpha ECO-ATR FTIR spectroscopy. Spectral analysis of each sample was performed for 32 scans in the range 500–4000 cm−1.
Nuclear magnetic resonance (NMR) analysis
13C NMR spectroscopic analysis of mannan hydrolysates was carried out on a JEOL ECX 500 NMR spectrometer. In liquid state NMR, small amount of deuterium oxide (D2O) and tetramethylsilane (TMS) were added for spin locking and internal reference, respectively (Prajapati et
Cytotoxicity assay
Caco-2 cells were cultured in glucose containing DMEM supplemented with 10% heat inactivated fetal bovine serum (FBS), non essential amino acids (100 µg/mL), penicillin (100 U/ mL) and streptomycin (100 mg/mL). Cells were passaged every week and maintained at 37°C with 5% CO2 in a humidified CO2 incubator (Nuaire, USA).
Cytotoxicity assay of hydrolysates (DfCM, CM, KG and LBG) was performed in presence of the
colon colorectal cell line (Caco-2) and cell proliferation was measured using
WST-1
Mannan hydrolysis
End products derived from the enzymatic hydrolysis of mannan containing substrates by endo-β-mannanase revealed the generation of different MOS of varying DP e.g. DfCM and LBG both showed formation of DP2, DP3 and DP4 in their hydrolysates while CM showed DP2 and DP4 as major end products. KG showed the generation of MOS containing large amounts of DP2 and DP5 oliogs alongwith some unidentified high DP MOS (Fig. 1). A. quadrilineatus RSNK-1 produced enzyme cocktail with predominant endo
Conclusions
A. quadrilineatus RSNK-1 consortia with endo-β-mannanase was utilized for galactomannan (DfCM, CM and LBG) and glucomannan (KG) hydrolysis. Multi-scale analysis (HPLC, FTIR and 13C NMR) of mannan hydrolysates revealed the generation of various DP MOS and their characteristics. LBG hydrolysate containing short DP (<4) MOS showed better inhibition of Caco-2 cells than KG hydrolysate containing high DP MOS. Lactobacilli effectively formed biofilms upon LBG and DfCM mannan hydrolysate
CRediT authorship contribution statement
Rahul Kumar Suryawanshi: Conceptualization, Methodology, Software, Validation, Formal analysis, Writing - original draft, Writing - review & editing. Naveen Kango: Conceptualization, Validation, Supervision, Writing - review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgment
Author (RKS) is thankful to the University Grants Commission (UGC), New Delhi for providing financial support as SRF (National fellowship-OBC). Authors are thankful to Sophisticated Instrument Center (SIC) and DST PURSE (II) at Dr. Harisingh Gour Vishwavidyalaya (A Central University), Sagar for providing FTIR and NMR facilities.
References (40)
- et al.
Dietary modulation of the human colonic microbiota: Introducing the concept of prebiotics
Journal of Nutrition
(1995) - et al.
Characteristics and bioactive properties of mannooligosaccharides derived from agro-waste mannans
International Journal of Biological Macromolecules
(2020) - et al.
Production optimization and characterization of mannooligosaccharide generating β-mannanase from Aspergillus oryzae
Bioresource Technology
(2018) - et al.
Structural characterization and evaluation of prebiotic activity of oil palm kernel cake mannanoligosaccharides
Food Chemistry
(2017) - et al.
Biofilm formation by Lactic acid bacteria and resistance to environmental stress
Journal of Bioscience and Bioengineering
(2008) - et al.
Thin layer chromatographic determination of organic acids for rapid identification of Bifidobacteria at genus level
Journal of Microbiological Methods
(2001) - et al.
Structural diversity and prebiotic potential of short chain β-manno-oligosaccharides generated from guar gum by endo-β-mannanase (ManB-1601)
Carbohydrate Research
(2019) - et al.
Characterisation of the oligosaccharides produced on hydrolysis of galactomannan with β-D-mannanase
Carbohydrate Research
(1983) - et al.
Structural features of spent coffee grounds water-soluble polysaccharides: Towards tailor-made microwave assisted extractions
Carbohydrate Polymers
(2019) - et al.
Molecular cloning of kman coding for mannanase from Klebsiella oxytoca KUB-CW2-3 and its hybrid mannanase characters
Enzyme and Microbial Technology
(2016)