Enzymatic extraction of laminarin from brown seaweed Ecklonia maxima

Van Breda, Daniel John (2020-03)

Thesis (MEng)--Stellenbosch University, 2020.

Thesis

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.

AFRIKAANSE OPSOMMING: Respons oppervlak metodologie is gebruik om die ensimatiese ekstrahering van β-glukaan laminarien uit Ecklonia maxima, ’n Suid-Afrikaans kelp, te ondersoek. ’n Kommersiële sellulase (Celluclast® 1.5L, “Celluclast”) is gebruik om seewiermateriaal te hidroliseer in die bestek van 0 tot 4.0% (v/dw) ensiemdosering, pH 3.0 tot 6.0, en 40 to 60 °C. Steekproewe is by elke vyf gelyk-gespasieerde punte oor ses-uur hidrolise eksperimente geneem. ’n Spektrofotometriese endo-1,3-β-D-glukanase proef vir laminarien is ontwikkel en steekproewe is gekwantifiseer vir hierdie respons en verskillende ander. Respons oppervlaktes is met regressie gepas vir opgeloste opbrengs (insluitend bodrywende opgeloste vaste stowwe, bodrywende massafraksie, en pellet-vaste-stof-lading), en die konsentrasies van laminarien, vermindering van suikers, anorganiese sulfate, totale fenoliese komponente, en antioksidantkapasiteit in die hidrolisaat bodrywende stof. Respons oppervlaktes is gevalideer, waar aangetoon is dat laminarienekstrahering beduidend deur pH en temperatuur liniêre en kwadratiese terme beïnvloed word, maar nie deur ensiemdosering nie. Reduserende suikers en anorganiese sulfaatkonsentrasies, opgeloste opbrengs, bodrywende massafraksie, en pellet-vaste-stof-lading het die beduidende effek van die liniêre ensiem-tot-substraat term aangetoon. Totale fenoliese komponente en antioksidantkapasiteit, in kontras, is beduidend beïnvloed deur slegs temperatuur en pH. Vergelykings met alternatiewe koolhidrase ekstrahering (Accellerase® 1500, “Accellerase”) het aangetoon dat Celluclast soortgelyk aan Accellerase in alle response optree. Verdere vergelyking met verdunde-suur termiese (“konvensionele”) hidroliese (pH 1.0 en 70 °C) het aangetoon dat ensimatiese ekstraheringmetodes superieur is vir die vrystelling van gereduseerde suikers, anorganiese sulfaat, en opgeloste opbrengs (na 4.5 ure). Konvensionele ekstrahering is bewys om superieur tot ensimatiese ekstraheringmetodes vir laminarien (as gemeet word met die ontwikkelde spektrofotometriese toets) en antioksidantkapasiteit te wees. Vergelyking tussen kelplotte (Mei 2018- en Junie 2019-oeste) het aangetoon dat laminarien teenwoordig was in hoër hoeveelhede in Mei 2018. Opgeloste opbrengs, gereduseerde suikers, en bodrywende massafraksie is ook beduidend hoër gemeet in Mei 2018, terwyl anorganiese sulfate, totale fenoliese komponente, en antioksidantkapasiteit hoër was in Junie 2019. Verskille is gevind tussen die spektrofotometriese resultate van die ontwikkelde laminarien ensimatiese toets en die HPLC-kwantifisering van glukose in die etanol gepresipiteerde polisakkariedryke fraksies van die konvensionele, Celluclast en Accellerase gehidroliseerde steekproewe. Hierdie verskille is geteoritiseer om veroorsaak te word deur of die inhibisie van die 1,3-β-D-endoglukanase ensiem by verskeie bio-aktiewe komponente in die ensimatiese ekstrakte (polifenole, florotanniene en alginaat), of die kontaminasie van die HPLC-resultate met sellulose-afgeleide glukose. Die toetse het soortgelyke lesings aangetoon as steekproewe konvensioneel gehidroliseer is (27–39 mgLE·gDW-1 en 36–39 mgGlE·gDW-1 vir spektrofotometrie en HPLC onderskeidelik) maar ensimatiese en rou ekstrakte gemeet met die ontwikkelde spektrofotometriese toets was laer in vergelyking. Die laminarieninhoud van die rou bodrywende stof is bepaal as 54.7 ± 12.3 mg·gDW-1(n=3), in vergelyking met die spektrofotometriese mate van 6.7 ± 0.1 mgLE·gDW-1(n=3). Ensimatiese ekstrahering het geen beduidende veranderinge uit die lesings vir rou materiaal met beide analisetegnieke aangetoon nie, en HPLC-meting het gewys dat konvensionele ekstrahering laminarieninhoud verminder. Geen behandelingstipes getoets het die opbrengs van laminarien verhoog in vergelyking met die in die rou materiaal gevind nie. Verdere werk op addisionele lotte van E. maxima word benodig om die effek van ensimatiese ekstrahering op laminarien vas te stel, en ’n addisionele 1,6-β-D-glukanase ensiem moet by die analitiese ensiem vir spektrofotometriese laminarienafmeting ingesluit word. Inhibisie van die 1,3-β-D-glukanase ensiem moet bestudeer word en HPLC-kolomme geskik vir polisakkariedskeiding en kwantifisering moet oorweeg word.

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