Optimisation of the enzymatic hydrolysis of rainbow trout processing by-products to manufacture liquid fertiliser

Date
2017-12
Journal Title
Journal ISSN
Volume Title
Publisher
Stellenbosch : Stellenbosch University
Abstract
ENGLISH ABSTRACT: Fish processing by-products are a potential source of proteins for a variety of applications, amongst them fertilisers. Since these by-products are otherwise dumped in landfills, resulting in environmental contamination, their recovery presents an economic opportunity to derive value from these otherwise discarded materials. There exist two main technologies for fish fertiliser production; fish emulsion and fish protein hydrolysates (FPH). Fish fertilisers contain both macro (nitrogen, phosphorous, potassium, calcium and magnesium) and micro (iron, manganese, zinc, copper, molybdenum, and boron) nutrients, with the ability to provide nutrients to both the plants and the soil micro-organisms, thereby building a healthy soil profile. Depending on the characteristics, fish fertilisers can be applied both as soil additives and as foliar spray. South Africa produced 1800 metric tonnes of rainbow trout in 2008, of which a significant amount ends up as by-products after processing. Such a supply of rainbow trout by-products is substantial to sustain the protein recovery processes at a commercial scale. This work is an investigation into the production of a liquid fertiliser, suitable both as a foliar and soil application, using rainbow trout processing by-products (heads) as raw material. The work was divided into three stages. The first stage involved determining the optimum hydrolysis conditions for two pre-selected proteolytic enzymes; SEBPro XL, an exoprotease and SEBDigest F59P, an endoprotease. To determine optimal conditions, three independent variables namely temperature, pH and E/S ratio were optimised at five levels (−α, −1, 0, +1, and +α) by using response surface methodology, central composite design. The degree of hydrolysis, which is the extent of the hydrolysis process, was the dependent variable. The DH was measured and monitored according to the modified spectrophotometric o-phthaldialdehyde (OPA) method. Optimal hydrolysis conditions for optimum DH were found to be a temperature of 60oC for both enzymes. The optimum pH for SEBPro XL was 6.9 whilst for SEBDigest F59P, the optimum pH was 7.6. Within the employed experimental domain, the optimum temperatures and pH were determined at an enzyme to substrate ratio (E/S) of 0.05% and 0.67% for SEBDigest F59P and SEBPro XL respectively. The E/S ratios were based on the protein content of the by-product fish heads. The second stage involved hydrolysing with the two enzymes at their optimum conditions for three hours. The main purpose of this stage was to maximise the DH using the two enzymes and their combinations. With three combinations of the two enzymes being designed, a total of five enzyme treatments were considered for the hydrolysis experiments. These five enzyme treatments were employed in the hydrolysis process and their fish protein hydrolysates (FPH) were compared by way of DH, amino acid (AA) content, free amino acid (FAA) content, macro and micro mineral content of the fertiliser, as well as the heavy metals. The results showed that using SEBPro XL alone results in higher levels of total AA and a higher proportion of FAA in the FPH compared to all other treatments. Its DH was also higher than that of all other treatments. All enzyme treatments produced FPH of within limits heavy metals as per the requirements of the fertiliser regulations and significant amounts of macro and micro nutrients. In the third and final stage of the investigation, SEBPro XL was further employed at varying E/S ratios to maximise on the DH and shorten the reaction time. The E/S ratios ranged from 1% to 5%. The reactions were run for four hours each. At the end of hydrolysis, the DH was 38% for a 1% ratio, 43.1% for 2% ratio, 50.9% for 3% ratio, 58.2% for 4% ratio and 60.2% for a 5% E/S ratio. Therefore depending on the desired DH, a choice can be made for an enzyme concentration and time of hydrolysis. This work established the optimum temperature and pH for hydrolysing rainbow trout processing by-products heads with two enzymes, SEBPro XL and SEBDigest F59P. It also demonstrated the possibility of producing foliar fertiliser from rainbow trout processing by-products. The fertiliser’s nutrient content lies within the legislative constraints. By manipulation of the reaction conditions, temperature, pH and enzyme concentration, as well as reaction time, its quality may be pre- determined.
AFRIKAANSE OPSOMMING: Verwerkte by-produkte van vis is 'n potensiële bron van proteïene vir 'n verskeidenheid van toepassings, onder andere soos kunsmis. Sedert hierdie by-produkte in stortingsterreine gestort word, kan dit lei tot omgewing besoedeling waar hierdie herstel ʼn ekonomiese geleentheid verteenwoordig om waarde uit hierdie weggooi materiaal af te lei. Daar bestaan twee hoofsaaklike tegnologieë vir die kunsmis produksie van vis nl. vis emulsie en vis proteïen hidrolisate (FPH). Vis kunsmis bevat beide makro- (stikstof, fosfor, kalium, kalsium en magnesium) en mikro- (yster, mangaan, sink, koper, molibdeen, en boor) voedingstowwe, wat die vermoë het om voedingstowwe aan beide plante en die grond mikro-organismes te voorsien asook om ’n gesonde grondprofiel te bou. Afhangende van die karaktereienskappe kan vis kunsmis toegepas word beide grondbymiddels en as blaar besproeiing. Suid-Afrika het 1800 metrieke ton reënboog forel in 2008 geproduseer waarvan 'n beduidende hoeveelheid beland het as by-produkte na die verwerking daar van. So aansienlike aanbieding van reënboog forel by-produkte word gebruik om die proteïen herstel prosesse, teen 'n kommersiële skaal in stand te hou. Hierdie werk dien as 'n ondersoek in die vervaardiging van 'n vloeibare kunsmis tipe wat geskik sal wees vir beide blaarbespuiting en grond toediening wat met die hulp van reënboog forel verwerkte by-produkte (koppe) as rou materiaal, sal dien. Die werk word in drie fases gedeel. Die eerste fase is betrokke by die bepaling van die optimum hidroliese voorwaardes vir twee geselekteerde ensieme SEBPro XL ('n exoprotease) en SEBDigest F59P ('n endoprotease). Om optimale omstandighede te bereken, sal drie onafhanklike veranderlikes nl. die temperatuur, pH en ensiemkonsentrasie optimiseer word teen vyf vlakke (-α, -1, 0, 1, en + α), met behulp van die reaksie oppervlak metode, sentrale saamgestelde ontwerp. Die graad van hidrolise, was die afhanklike by die omvang van die hidroliese proses. Die DH is gemeet en gemonitor volgens die aangepaste spektrofotometries o-phthaldialdehyde (OPA). Die optimale hidroliese omstandighede vir die optimale DH was gevind teen 'n temperatuur van 60oC vir beide ensieme. Die optimale pH vir SEBPro XL was 6,9 terwyl SEBDigest F59P se optimale pH was 7.6 was. Binne die perke van die eksperimentele domein, is die optimaal temperature en pH bepaal by 'n ensiem die substrate verhouding (E/S) van 0,05%, 0,67% vir SEBDigest F59P en SEBPro XL onderskeidelik te bepaal. Die E/S verhoudings was gebaseer op die proteïeninhoud van die by- produk viskoppe. Die tweede fase is betrokke met die hidrolisering van twee ensieme by hul optimale vir twee ure lank. Die hoofdoel van hierdie fase was om die DH te maksimeer deur gebruik te maak van twee ensieme en hul kombinasies. Met drie kombinasies van die twee ensieme wat ontwerp is, was 'n totaal van vyf ensieme behandelings in aanmerking gekom vir die hidroliese eksperimente. Hierdie vyf ensiem behandelings was in diens gestel in die hidroliese proses en hul vis proteïen hidrolisate (FPH) was vergelyk deur middel van DH, aminosuur (AA) inhoud, bevry aminosuur (FAA) inhoud, makro en mikro minerale inhoud van die kunsmis sowel as die swaar metale. In vergelyking met die ander behandelinge, het die resultate getoon dat die allenige gebruik van die SEBPro XL hoër vlakke van totale AA getoon het asook 'n hoër proporsie van FAA in FPH. Sy DH was ook hoër as dié van al die ander behandelings. Alle ensiem behandelings produseer FPH binne die perke van swaar metale soos uiteengesit deur die kunsmis regulasies vereistes asook die aansienlike hoeveelhede van makro en mikro voedingstowwe. In die derde en finale fase van die ondersoek, is SEBPro XL verder in diens gestel teen wisselende E/S verhoudings om die DH te maksimeer en die reaksie tyd te verkort. Die E/S verhoudinge wissel van 1% tot 5%. Die reaksies is gehardloop vir vier ure elk. Aan die einde van hidrolise, was die DH 38% vir 'n 1% verhouding, 43.1% vir ʼn 2% verhouding, 50.9% vir ʼn 3% verhouding, 58.2% vir ʼn 4% verhouding en laastens 60.2% vir ʼn 5% verhouding. Afhangende van die gewenste DH, kan 'n keuse gemaak word vir 'n ensiem konsentrasie en tyd van hidrolise. Hierdie werk het die optimale temperatuur en pH vir die hidrolisering van verwerkte reënboog forel by-produkte gevestig deur middel van twee ensieme nl. SEBPro XL en SEBDigest F59P. Dit het ook die moontlikheid om blaar voedingstowwe te vervaardig van reënboog forel by-produkte, gedemonstreer. Die kunsmis se voedingstowwe lê binne die wetgewende beperkings. Deur die reaksie kondisies, temperatuur, pH en ensiem konsentrasie asook reaksie tyd te manipuleer, kan die kwaliteit vooraf bepaal word.
Description
Thesis (MEng)--Stellenbosch University, 2017.
Keywords
Rainbow trout industry, Hydrolytic enzymes, Foliar fertilization, Fishery processing, Agricultural wastes
Citation