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Thesis (MSc Food Sc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: Alicyclobacilli are endospore-forming, thermophilic, acidophilic bacteria (TAB) that survive the pasteurisation process and spoil acidic fruit juices through the production of the taint compound guaiacol. Guaiacol causes an undesirable odour with an unpleasant smoky, medicinal or phenolic-like taste. This thesis reports on the precursors, vanillin and vanillic acid metabolised to guaiacol by Alicyclobacillus spp. in fruit juice, the pathway of guaiacol production and the spoilage potential of contaminated fruit juices supplemented with these precursors. A high performance liquid chromatography method with UV-diode array detection (HPLC-DAD) was developed for the simultaneous detection and quantification of guaiacol and its precursors. Alicyclobacillus acidoterrestris FB2 was incubated at 45 °C for 7 d in Bacillus acidoterrestris (BAT) broth supplemented with ferulic acid, vanillin or vanillic acid. The cell concentrations were determined every 24 h and the concentration of the precursors and the production of guaiacol was determined using HPLC-DAD. The guaiacol production was also determined using the peroxidase enzyme colourimetric assay (PECA). Alicyclobacillus acidoterrestris produced guaiacol from vanillin and vanillic acid, confirming both vanillin and vanillic acid as precursors for guaiacol production by A. acidoterrestris FB2. Furthermore, a metabolic pathway directly from vanillin to guaiacol was identified in this study. However, guaiacol was not produced by A. acidoterrestris FB2 in the samples supplemented with ferulic acid and it is, therefore, not considered a direct precursor for guaiacol production by A. acidoterrestris. The spoilage potential of apple juice supplemented with either 10 mg L-1 or 100 mg L-1 vanillin or vanillic acid by A. acidoterrestris FB2 (106 cfu mL-1) was also evaluated. The production of guaiacol increased with the increase in vanillin or vanillic acid concentrations (in BAT broth and apple juice) indicating that the concentration of vanillin and vanillic acid present in fruit juice will influence the spoilage potential of the juice. Guaiacol concentrations in apple juice well above the best estimated threshold value of guaiacol for taste (0.24 – 2.00 μg L-1) and odour (0.50 - 2.32 μg L-1) was produced by A. acidoterrestris FB2 in the apple juice supplemented with 10 mg L-1 vanillin or vanillic acid. This indicates that fruit juice with a vanillin or vanillic acid concentration as low as 10 mg L-1 has the potential to spoil if the juice is contaminated with A. acidoterrestris. The concentrations of vanillin and vanillic acid in different fruit juices can be used to indicate if a specific fruit juice is susceptible to guaiacol spoilage by Alicyclobacillus spp. In the development of juice products and different blends of fruit juices, special care must be taken not to concentrate the amount of vanillin and vanillic acid present in the fruit juices. / AFRIKAANSE OPSOMMING: Alicyclobacilli is endospoor-vormende, termofiliese, asidofiliese bacterie (TAB) wat die pasteurisasie proses oorleef en suur vrugtesappe bederf met die produksie van ‘n taint komponent guaiakol. Guaiakol veroorsaak ‘n ongewensde reuk en onaangename rookagtige, medisinale of fenoliese smaak. Hierdie tesis doen verslag oor die voorloper komponente, vanillien en vanilliensuur in vrugtesappe wat gemetaboliseer word na guaiakol deur Alicyclobacillus spesies, die padweg van guaiakol produksie en die bederfbaarheid van gekontamineerde vrugtesap aangevul met hierdie voorloper komponente. ‘n Hoëprestasie vloeistof chromatografie metode met UV-deteksie (skanderend) (HPVC) is ontwikkel vir die gesamentlike deteksie en kwantifisering van guaiakol en die voorloper komponente. Alicyclobacillus acidoterrestris FB2 is geïnkubeer by 45 °C vir 7 d in Bacillus acidoterrestris (BAT) medium aangevul met feruliensuur, vanillien of vanilliensuur. Die sel konsentrasies is elke 24 h bepaal en die aangevulde komponente en die geproduseerde guaiakol is bepaal deur van HPVC gebruik te maak. Die guaiakol konsentrasies is ook bepaal deur van die peroksidase ensiem kolorimetriese bepaling (PEKB) gebruik te maak. Alicyclobacillus acidoterrestris het guaiakol geproduseer vanaf vanillien en vanilliensuur, dus is beide vanillien en vanilliensuur bevestig as voorlopers van guaiakol produksie deur A. acidoterrestris FB2. A padweg direk van vanillien na guaiakol is in hierdie studie geïdentifiseer. Guaiakol is nie geproduseer deur A. acidoterrestris FB2 in the monsters wat met feruliensuur aangevul is nie en feruliensuur is dus nie ‘n direkte voorloper van guaiakol produksie deur A. acidoterrestris. Die bederf potensiaal van appelsap aangevul met 10 mg L-1 of 100 mg L-1 vanillien of vanilliensuur deur A. acidoterrestris (106 kve mL-1) is ook geëvalueer. Die produksie van guaiakol het toegeneem met die toename in vanillien of vanilliensuur konsentrasies (in beide BAT en appelsap) wat aandui dat die konsentrasie vanillien en vanilliensuur teenwoordig in vrugtesap die bederfbaarheid van die sap sal beïvloed. Guaiakol konsentrasies in appelsap hoog bo die drumpel waardes van guaiacol vir smaak (0.24 – 2.00 μg L-1) en reuk (0.50 - 2.32 μg L-1) is geproduseer deur A. acidoterrestris FB2 in die appelsap monsters aangevul met 10 mg L-1 vanillien of vanilliensuur. Hierdie verskynsel dui aan dat vrugtesap met ‘n vanillien of vanilliensuur konsentrasies van so laag as 10 mg L-1 die potensiaal het om te bederf indien die sap gekontamineer is met A. acidoterrestris. Die konsentrasies van vanillien en vanilliensuur in verskillende vrugtesappe kan gebruik word om aan te dui of ‘n spesifieke vrugtesap ‘n hoë risiko het vir guaiakol bederf deur Alicyclobacillus spesies. Tydens die ontwikkeling van vrugtesap produkte en verskillende mengsels van vrugtesappe moet seker gemaak word dat die hoeveelhede vanillien en vanilliensuur in die sappe nie gekonsentreer word nie.

RISE Bioeconomy is working with the biorefinery concept. That is, for the company, to utilize all wood derivatives in effective processes, resulting in unique products. RISE Bioeconomy is investigating the possibilities of producing holocellulose through an oxidative delignification process of wood, using peracetic acid (PAA). The product, high-purity holocellulose, can be used for the production of for example cellulose nanofibers, a product that in recent years have increased in interest. The PAA-delignification process relies on oxidative treatment with peracetic acid, where the lignin is solubilized in water through the formation of carboxylic groups in the lignin, yielding cellulose of very low lignin content and minimal degradation of the cellulose. In addition to the holocellulose, a solution containing water, acetic acid, salts and highly oxidized lignin is obtained. The PAA-delignification has yet only been performed in lab-scale, however, the aim is to scale up the process. There has not yet been any further utilization of the oxidized lignin and it has been proposed that esterification of the carboxylic groups with an alcohol is a possible valorization route. Due to the up-scaling of the PAA-delignification, it is of large interest to find a valorization route for the lignin by-product to fulfill the biorefinery concept. This master thesis focuses on evaluating esterification of an oxidized lignin, with the aim to propose a process suitable for up-scaling. The lab trials for the PAA-delignification of wood yielded limited amounts of lignin and due to difficulties while isolating this lignin, it was not partially characterized until months into the project. Therefore, model compounds were initially used to evaluate the process setup. Compounds with muconic acid type structures are some of the expected products from PAA-delignification of wood and muconic acid was therefore used as a model compound. In addition to this, vanillic acid was used to represent the phenolic lignin compounds. Gas Chromatography/Mass Spectrometry (GC/MS) analysis of the vanillic acid esterification was shown to be a suitable method for analyzing the conversion of this reaction. Due to the difficulties with the analysis of the model compounds, no results regarding the reaction procedures were obtained. As a “proof-of-concept”, a kraft lignin was oxidized, isolated and then esterified. Two different methods of esterification were performed, with and without a molecular sieve. It was assumed that the amount of carboxylic groups would increase after oxidation and decrease after a successful esterification. The amount of carboxylic groups after each process was analyzed with Phosphorus Nuclear Magnetic Resonance (31P-NMR). For the process without molecular sieve, the 31P-NMR analysis indicated that esterification of the lignin had occurred, to a limited extent. This was confirmed by Fourier Transform Infrared Spectrometry (FTIR). The process with molecular sieve showed no indication of esterification of the lignin. The project did prove the concept of esterifying an oxidized lignin and led to several different proposals for further work in the area. Due to the very wide scope of this project and several unexpected obstacles, this project did not fully answer the research questions given.