Browsing by Author "Van Breda, Daniel John"
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- ItemEnzymatic extraction of laminarin from brown seaweed Ecklonia maxima(Stellenbosch : Stellenbosch University., 2020-03) Van Breda, Daniel John; Goosen, N. J.; Stellenbosch University. Faculty of Engineering. Dept. of Process Engineering.ENGLISH ABSTRACT: Response surface methodology was used to investigate the enzymatic extraction of β-glucan laminarin from Ecklonia maxima, a South African kelp. A commercial cellulase (Celluclast® 1.5L, “Celluclast”) was used to hydrolyse the seaweed material in the range of 0 to 4.0% (v/dw) enzyme dosage, pH 3.0 to 6.0, and 40 to 60 °C. Samples were taken at five evenly-spaced points over six-hour hydrolysis experiments. A spectrophotometric endo-1,3-β-D-glucanase assay for laminarin was developed, and samples were quantified for this response and various others. Response surfaces were regressed for solubilised yield (including supernatant dissolved solids, supernatant mass fraction, and pellet-solids loading), and the concentrations of laminarin, reducing sugars, inorganic sulfates, total phenolics, and antioxidant capacity in the hydrolysate supernatant. Response surfaces were validated, with laminarin extraction shown to be significantly influenced by pH and temperature linear and quadratic terms, but not by enzyme dosage. Reducing sugar and inorganic sulfate concentrations, solubilised yield, supernatant mass fraction, and pellet-solids loading showed the significant effect of the linear enzyme-to-substrate term. Total phenolics and antioxidant capacity, in contrast, were significantly influenced by temperature and pH only. Comparisons to alternate carbohydrase extraction (Accellerase® 1500, “Accellerase”) showed Celluclast to perform similarly to Accellerase in all responses. Further comparison to dilute-acid thermal (“conventional”) hydrolysis (pH 1.0 and 70 °C) showed that enzymatic extraction methods were superior for the release of reducing sugars, inorganic sulfate, and solubilised yield (after 4.5 hours). Conventional extraction was shown to be superior to enzymatic extraction methods for laminarin (when measured with the developed spectrophotometric assay) and antioxidant capacity. Comparison between kelp batches (May 2018 and June 2019 harvests) showed laminarin to be present in higher amounts in May 2018. Solubilised yield, reducing sugars, and supernatant mass fraction also measured significantly higher in May 2018 while inorganic sulfates, total phenolics, and antioxidant capacity were higher in June 2019. Differences were found between the spectrophotometric results of the developed laminarin enzymatic assay and the HPLC quantification of glucose in the ethanol-precipitated polysaccharide-rich fractions of the conventional, Celluclast, and Accellerase hydrolysed samples. These differences were theorised to be caused by either the inhibition of the 1,3-β-D-endoglucanase enzyme by various bioactive components in the enzymatic extracts (polyphenols, phlorotannins, and alginate), or the contamination of the HPLC results with cellulose-derived glucose. The assays showed similar readings when samples were conventionally hydrolysed (27 to 39 mgLE·gDW-1 and 36 to 39 mgGlE·gDW-1 for spectrophotometric and HPLC respectively) but enzymatic and raw extracts measured with the developed spectrophotometric assay were lower in comparison. The laminarin content of the raw supernatant was determined as 54.7 ± 12.3 mg·gDW-1(n = 3), compared to the spectrophotometric measurement of 6.7 ± 0.1 mgLE·gDW-1(n = 3). Enzymatic extraction showed no significant changes from the readings for raw material with either analysis technique, and HPLC measurement showed conventional extraction to decrease laminarin content. None of the treatment types tested increased the yield of laminarin over that found in the raw material. Further work on additional batches of E. maxima is required to ascertain the effect of enzymatic extraction on laminarin, and an additional 1,6-β-D-glucanase should be included in the analytical enzyme for spectrophotometric laminarin measurement. Inhibition of the 1,3-β-D-glucanase enzyme should be studied, and HPLC columns capable of polysaccharide separation and quantification should be considered.