Bioactivity evaluation of manno-oligosaccharides produced from spent coffee grounds using a Bacillus sp. derived endo-1,4-β-mannanase
- Authors: Magengelele, Mihle
- Date: 2022-10-14
- Subjects: Mannosidase beta , Oligosaccharides , Prebiotics , Probiotics , Coffee waste , Coffee grounds
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/365233 , 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. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
- Authors: Magengelele, Mihle
- Date: 2022-10-14
- Subjects: Mannosidase beta , Oligosaccharides , Prebiotics , Probiotics , Coffee waste , Coffee grounds
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/365233 , 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. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
The use of probiotics in the diet of farmed South African abalone Haliotis midae L
- Authors: Maliza, Siyabonga
- Date: 2015
- Subjects: Haliotis midae -- South Africa , Abalones -- South Africa , Haliotis midae -- Feeding and feeds , Haliotis midae -- Effect of chemicals on , Haliotis midae -- Growth , Haliotis midae -- Immunology , Probiotics
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5382 , http://hdl.handle.net/10962/d1018178
- Description: Physiological stress in farmed abalone can lead to immunosuppression and increase the susceptibility to bacterial, viral and parasitic disease, often followed by mortality. Thus, handling and poor water quality can reduce farm production efficiency. Probiotics in aquaculture have been effective in a wide range of species in enhancing immunity, survival, improving feed utilisation and growth. Three putative probionts identified as a result of in vitro screening had been beneficial to laboratory-reared abalone in a previous study. The aim of this study was to produce an abalone feed that contains a suite of probionts that may promote abalone growth and health under farming conditions. The objectives were to compare growth and physiological responses (i.e., haemocyte and phagocytosis counts) of abalone fed a commercial feed (Abfeed®S 34, Marifeed, Hermanus) supplemented with probiotics (i.e., the probiotic diet) to abalone fed the commercial feed without probiotic supplementation as a control treatment in a factorial design with handling method as an independent variable. This experiment was conducted at HIK Abalone Farm (Pty Ltd) for a period of eight months with initial weight and length 36.1 ± 0.05 g and 58.6 ± 0.06 mm abalone-1. Another experiment was carried out at Roman Bay Sea Farm (Pty) Ltd with initial weight and length 34.7 ± 0.17 g and 62.3 ± 0.18 mm abalone-1, but this experiment included one factor only, i.e. the presence and absence of the probionts in the feed. At HIK there was no significant interaction between diet and handling on average length and weight gain month-1 after four (p=0.81 and p=0.32) and eight (p=0.51 and p=0.53) months, respectively. Average length (additional handling = 73.9 ± 0.52 mm, normal farm handling = 75.8 ± 0.57 mm) and weight gain (mean: additional handling = 68.5 ± 1.20 g, normal farm handling = 74.3 ± 1.86 g) increased significantly in animals that were handled under normal farm procedure and were either fed probiotic or control diet after eight months (p=0.03 and p=0.02, respectively). There was no iii difference in length gain or weight gain of abalone fed the probiotic diet and those fed the control diet (ANOVA: F(1,16)=0.04, p=0.84; F(1,16)=0.14, p=0.71, respectively). After four months phagocytotic count was significantly different between dietary treatments with mean values of 74.50 ± 10.52 and 63.52 ± 14.52 % phagocytosis count per sample for the probionts and control treatment, respectively (p=0.04), there was no difference after eight months at HIK Abalone Farm. There was no effect of stressor application (p=0.14) and no interaction between dietary treatment and stressor application for this variable i.e., phagocytosis count (p=0.61). There was no difference in feed conversion ratio between treatments with values ranging from 2.9 to 3.8. At Roman Bay Sea farm, there was no significant difference in mean length gain between abalone fed the probiotic and control diet after eight months (repeated measures ANOVA: F(4,28)=16.54. Mean weight gain of abalone fed the probiotic diet was significantly greater than those fed the control diet after eight months (repeated measures ANOVA: F(4,28)=39.82, p(0.00001). There was no significant difference in haemocyte counts between animals fed either probiotic or control diet after four and eight months at Roman Bay Sea farm (p>0.05).
- Full Text:
- Date Issued: 2015
- Authors: Maliza, Siyabonga
- Date: 2015
- Subjects: Haliotis midae -- South Africa , Abalones -- South Africa , Haliotis midae -- Feeding and feeds , Haliotis midae -- Effect of chemicals on , Haliotis midae -- Growth , Haliotis midae -- Immunology , Probiotics
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5382 , http://hdl.handle.net/10962/d1018178
- Description: Physiological stress in farmed abalone can lead to immunosuppression and increase the susceptibility to bacterial, viral and parasitic disease, often followed by mortality. Thus, handling and poor water quality can reduce farm production efficiency. Probiotics in aquaculture have been effective in a wide range of species in enhancing immunity, survival, improving feed utilisation and growth. Three putative probionts identified as a result of in vitro screening had been beneficial to laboratory-reared abalone in a previous study. The aim of this study was to produce an abalone feed that contains a suite of probionts that may promote abalone growth and health under farming conditions. The objectives were to compare growth and physiological responses (i.e., haemocyte and phagocytosis counts) of abalone fed a commercial feed (Abfeed®S 34, Marifeed, Hermanus) supplemented with probiotics (i.e., the probiotic diet) to abalone fed the commercial feed without probiotic supplementation as a control treatment in a factorial design with handling method as an independent variable. This experiment was conducted at HIK Abalone Farm (Pty Ltd) for a period of eight months with initial weight and length 36.1 ± 0.05 g and 58.6 ± 0.06 mm abalone-1. Another experiment was carried out at Roman Bay Sea Farm (Pty) Ltd with initial weight and length 34.7 ± 0.17 g and 62.3 ± 0.18 mm abalone-1, but this experiment included one factor only, i.e. the presence and absence of the probionts in the feed. At HIK there was no significant interaction between diet and handling on average length and weight gain month-1 after four (p=0.81 and p=0.32) and eight (p=0.51 and p=0.53) months, respectively. Average length (additional handling = 73.9 ± 0.52 mm, normal farm handling = 75.8 ± 0.57 mm) and weight gain (mean: additional handling = 68.5 ± 1.20 g, normal farm handling = 74.3 ± 1.86 g) increased significantly in animals that were handled under normal farm procedure and were either fed probiotic or control diet after eight months (p=0.03 and p=0.02, respectively). There was no iii difference in length gain or weight gain of abalone fed the probiotic diet and those fed the control diet (ANOVA: F(1,16)=0.04, p=0.84; F(1,16)=0.14, p=0.71, respectively). After four months phagocytotic count was significantly different between dietary treatments with mean values of 74.50 ± 10.52 and 63.52 ± 14.52 % phagocytosis count per sample for the probionts and control treatment, respectively (p=0.04), there was no difference after eight months at HIK Abalone Farm. There was no effect of stressor application (p=0.14) and no interaction between dietary treatment and stressor application for this variable i.e., phagocytosis count (p=0.61). There was no difference in feed conversion ratio between treatments with values ranging from 2.9 to 3.8. At Roman Bay Sea farm, there was no significant difference in mean length gain between abalone fed the probiotic and control diet after eight months (repeated measures ANOVA: F(4,28)=16.54. Mean weight gain of abalone fed the probiotic diet was significantly greater than those fed the control diet after eight months (repeated measures ANOVA: F(4,28)=39.82, p(0.00001). There was no significant difference in haemocyte counts between animals fed either probiotic or control diet after four and eight months at Roman Bay Sea farm (p>0.05).
- Full Text:
- Date Issued: 2015
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