Elsevier

Food Chemistry

Volume 334, 1 January 2021, 127428
Food Chemistry

Production of mannooligosaccharides from various mannans and evaluation of their prebiotic potential

https://doi.org/10.1016/j.foodchem.2020.127428Get rights and content

Highlights

Abstract

Aspergillus quadrilineatus endo-β-mannanase effectively degraded konjac glucomannan (66.09% w/v), copra meal (38.99% w/v) and locust bean galactomannan (20.94% w/v). High performance liquid chromatography (HPLC) analysis of KG hydrolysate indicated its mannooligosaccharides (MOS) content (656.38 mg/g) with high amounts of DP 5 oligosaccharide. Multi-scale characterization of mannan hydrolysate was done using FTIR and 13C NMR which revealed α and β form of galactose or glucose in MOS, respectively. CM and LBG hydrolysates (1 mg/mL) have shown cytotoxic effect and reduced cell viability of Caco-2 cells by 45% and 62%, respectively. MOS DP (1–4) derived from LBG supported better Lactobacilli biofilm formation as compared to KG hydrolysate containing high DP MOS (5–7). Lactobacilli effectively fermented MOS to generate acetate and propionate as main short chain fatty acids. Lactobacilli produced leucine, isoleucine and valine as branched chain amino acids when grown on LBG hydrolysate.

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.

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