- Title
- Bioactivity evaluation of manno-oligosaccharides produced from spent coffee grounds using a Bacillus sp. derived endo-1,4-β-mannanase
- Creator
- Magengelele, Mihle
- Subject
- Uncatalogued
- Date Issued
- 2022-10-14
- Date
- 2022-10-14
- Type
- Academic theses
- Type
- Master's theses
- Type
- text
- Identifier
- http://hdl.handle.net/10962/365233
- Identifier
- vital:65719
- Description
- Coffee is one of the most popular beverages produced worldwide; however, its processing results in the generation of spent coffee grounds (SCGs). SCG as an agro-industrial waste which leads to adverse environmental effects, such as carbon dioxide and methane production, when disposed of in landfills. SCGs contain high levels of polysaccharides such as mannan, specifically galactomannan; thus, the utilisation of this waste is an important subject. Recently, there has been a growing interest in the production of nutraceutical mannooligosaccharides (MOS) through the enzymatic hydrolysis of mannans. MOS have been reported to exhibit various bioactive properties, including prebiotic effects, the ability to inhibit pathogens and antioxidant activity. In this study, a Bacillus sp. derived endo-1,4-β-mannanase, Man26A, was used for the production of MOS from model mannan substrates; ivory nut mannan (INM), locust bean gum (LBG) and guar gum (GG). After incubation, Man26A exhibited saccharification yields of 30.18, 36.86 and 34.93% for INM, LBG and GG, respectively. Kinetic studies showed that Man26A had a high binding affinity and catalytic efficiency for LBG (Km = 10.8 mg/mL and kcat/Km = 8.8 min-1 mg-1mL) than INM (Km = 28.9 mg/mL and kcat/Km = 3.8 min-1 mg-1mL) and GG (Km = 50.2 mg/mL and kcat/Km = 2.6 min-1 mg-1mL). The hydrolysis products from these model mannan substrates were quantitatively and qualitatively analysed using high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC), respectively. INM hydrolysis resulted in the production of mannose (M1) - mannotriose (M3), while LBG hydrolysis resulted in the generation of M1 - M2 (mannobiose) and mannopentaose (M5) - mannohexaose (M6) as the dominant sugars. On the other hand, GG hydrolysis mainly produced M5 - M6, and some oligosaccharides with a degree of polymerisation (DP > 6). Putative galactosyl-MOS; GM2 and GM3, were also observed in the HPLC chromatograms of both LBG and GG hydrolysates. The MOS produced from these model mannan substrates were stable over a broad pH range of 2 - 10. Furthermore, MOS produced by enzyme hydrolysis showed antioxidant properties, with MOS obtained from INM showing higher antioxidant activity than those from LBG and GG. A mannan-rich agro-processing waste, SCG, was pretreated using NaOH and hydrolysed using Man26A under the optimised conditions obtained from the model mannan hydrolysis studies for MOS generation. Structural analysis studies performed using Fourier-transform infrared spectroscopy (FT-IR) and thermogravimetric analysis (TGA) confirmed the structure of untreated and pretreated SCGs, and some chemical differences were observed in the untreated and pretreated SCGs. TGA analysis specifically showed that pretreated SCG was more resistant to temperature induced decomposition than untreated SCG. The removal of lignin during the pretreatment of SCG was observed by TGA, whereas the decomposition of lignin was only observed in untreated SCG. Using FT-IR, α-linked D-galactopyranose units (812 cm-1) and β-linked D-mannopyranose units (817 cm-1) were observed in both untreated and pretreated SCGs, confirming the galactomannan presence. MOS were successfully produced from the hydrolysis of NaOH pretreated SCG by Man26A, where M2 (1.04 mg/mL) and M3 (1.20 mg/mL) were the main products. The effect of bile salts, α-amylase, trypsin and hydrochloric acid on SCG-MOS was investigated, and they did not degrade SCG-MOS. The effect of SCG MOS on the in vitro survival of beneficial bacteria was investigated. SCG-MOS enhanced the growth of Lactobacillus bulgaricus, Bacillus subtilis and Streptococcus thermophilus, and led to the production of short chain fatty acids (SCFAs). The growth of beneficial bacteria in the presence of SCG-MOS was 2-fold higher than in the presence of the glucose control and sugar-free control. Bacterial SCFAs production was more in carbon source containing broth than sugar-free broth. In terms of autoaggregation influence, L. bulgaricus, B. subtilis and S. thermophilus grown in the presence of SCG-MOS showed aggregation percentages of 18.21, 20.98 and 17.99%, respectively. The formation of biofilms by these bacterial cells in the presence of SCG-MOS were approximately 2-fold higher than the values obtained in the positive mannose control and sugar-free control. Utilisation of SCG-MOS activated putative mannan degrading genes in beneficial bacteria, resulting in the production of mannan degrading enzymes, such as β-mannanase, β-mannosidase and α-galactosidase. In conclusion, this study demonstrated that enzymatic hydrolysis of SCG using Man26A resulted in the production of M2 and M3 as the predominant MOS. These MOS have prebiotic effects, which may be essential for the improvement of animal and human health. The MOS possibly act in the digestive tracts of mammals by enhancing the production of beneficial secondary metabolites, such as SCFAs, and enhancing autoaggregation and biofilm formation of beneficial bacteria, which may likely lead to competitive exclusion of pathogenic bacteria in the host’s digestive tract.
- Description
- Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Format
- computer
- Format
- online resource
- Format
- application/pdf
- Format
- 1 online resource (127 pages)
- Format
- Publisher
- Rhodes University
- Publisher
- Faculty of Science, Biochemistry and Microbiology
- Language
- English
- Rights
- Magengelele, Mihle
- Rights
- Use of this resource is governed by the terms and conditions of the Creative Commons "Attribution-NonCommercial-ShareAlike" License (http://creativecommons.org/licenses/by-nc-sa/2.0/)
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RU Department of Biochemistry and Microbiology (2015-)
| Thumbnail | File | Description | Size | Format | |||
|---|---|---|---|---|---|---|---|
| View Details Download | SOURCE1 | MANGENGELELE-MSC-TR22-168-EMB.pdf | 2 MB | Adobe Acrobat PDF | View Details Download |