The purpose of this investigation was to examine the change in pulmonary ventilation (V_E), ventilatory equivalent of oxygen (VE_O_2) and lactic acid (LA) in relation to oxygen uptake (V_O_2) as predictors of the lactate threshold (LT). Eight healthy male (21.9 ± 3.0 years) subjects conducted three incremental-load tests. In each test the initial work rate consisted of 4 minutes of unloaded pedaling ("0" load) followed by incremental-load work of 360 Kgm • min^-1 at 60 rpm for trial I and trial II, while during trial III the work rate consisted of 540 Kgm • min^-1 of incremental-load work at 90 rpm. Work load was increased every third minute until the subject reached voluntary exhaustion. Blood samples from a forearm vein were collected during trial II (60 rpm) and trial III (90 rpm) and analyzed for lactic acid. In our subjects the measured (x̄ ± SD) lowest VE_O_2 for O_2 in relation to V_O_2 for trial I of 22.9 ± 1.9 occurred at a V_O_2 of 1.27 ± 0.8 L • min^-1. For trial II the VE_O_2 of 22.4 ± 1.3 occurred at a V_O_2 of 1.30 ± 0.09 L • min^-1, while for trial III a VE_O_2 of 24.4 ± 2.5 occurred at a V_O_2 of 1.84 ± 0.15 L • min^-1. The lowest VE_O_2 and onset of LA accumulation as calculated from individual exponential equations relating V_E to V_O_2 yielded V_O_2 values at 1.77 ± 0.18 L • min^-1 and 1.74 ± 0.25 L • min^-1 for trial II, and 1.83 ± 0.19 L • min^-1 and 2.02 ± 0.53 during trial III. Utilizing ln[LA]-ln V_O_2 equations, the LT occurred at a V_O_2 of 1.30 ± 0.07 L • min^-1 during trial II and 1.85 ± 0.12 L • min^-1 during trial III. It was concluded that during the 60 rpm test that the lactate threshold was best predicted by measured lowest VE_O_2 and the plot of the In[LA] to In V_O_2 relationship. The methods used in this study provide a valid and reliable estimate of the lactate threshold and support the use of measured lowest VE_O_2 , a respiratory gas exchange measure, as an indirect measure of the lactate threshold.

Lic.-avh. Stockholm : Karolinska institutet, 2005.

Thesis (MSc (Wine Biotechnology))--University of Stellenbosch, 2007. / Bacteriocins are ribosomally synthesized antimicrobial peptides produced by bacteria and are active against other bacteria, either in the same species (narrow spectrum) or across genera (broad spectrum). The application of bacteriocins during the vinification process might help to prevent the production of undesired compounds by inhibiting the indigenous bacterial microflora and allowing malolactic fermentation to be conducted by a selected bacterial strain. Furthermore, the use of bacteriocins might allow reducing the total sulphur dioxide amount in wine. The purpose of this study was the selection of lactic acid bacteria (LAB) belonging to the genera Oenococcus, Lactobacillus and Pediococcus with the ability to produce bacteriocins, with respective biological activity against undesired indigenous wine LAB and the capability to complete malolactic fermentation. The first objective of this study was the screening of LAB isolated from South African red wines for the production of bacteriocins. Only 27 strains out of 330 wine isolates, belonging to the species Lb. plantarum, Lb. paracasei, Lb. hilgardii and O. oeni, showed activity towards various wine-related and non wine-related indicator strains with the colony-overlay method. It is the first time that bacteriocin activity is reported in O. oeni. The second objective was the detection and identification of known structural bacteriocin genes of Lb. plantarum wine strains. Furthermore, the web server BAGEL was used to in silico analyse putative bacteriocin-encoding genes in the genome of O. oeni and primers were designed to amplify four possible bacteriocin-encoding genes. A PCR-based screening revealed the presence of the plantaricin encoding genes plnA, plnEF, plnJ and plnK in five selected Lb. plantarum strains. Moreover, PCR analysis rendered positive results with all four chosen putative bacteriocin-encoding genes in the eight tested O. oeni strains with antimicrobial activity. The latter genes of O. oeni were heterologously expressed in different Escherichia coli host strains, but no antimicrobial activity could be detected. The third objective of this study was the transformation and expression of the heterologous bacteriocin genes nisin A and pediocin PA-1 in two selected Lb. plantarum strains. To enhance their antimicrobial activity a plasmid containing the nisin A gene was successfully cloned into the two strains. Indeed, an enhanced antimicrobial activity could be detected, but the transformed plasmid was not stable. The fourth objective in this project was the evaluation of bacteriocin production in liquid media. A co-culture experiment with a plantaricin producing Lb. plantarum strain and an Enterococcus faecalis strain as indicator was performed. A complete inhibition of cell growth of Ent. faecalis was observed within 72 hours. The last objective was the evaluation of the impacts of phenolic compounds on the activity of nisin and pediocin. The short term influence of two phenolic acids, two flavan-3-ols, grape tannins and oak tannins on the activity of nisin and pediocin PA-1 was investigated. No influence on the activity was detected. Furthermore, synergistic effects on bacterial growth inhibition were observed. This study confirms the potential use of either bacteriocin additives or bacteriocin-producing LAB in order to control the bacterial microflora during the vinification process.

<p>Pivski trop čini približno 85% od ukupnih sporednih proizvoda proizvodnje piva, i dostupan je po veoma niskim cenama tokom čitave godine. Pivski trop ima veliku perspektivu za primenu u biotehnologiji i proizvodnji visoko vrednih proizvoda. Jedna od veoma ekolo&scaron;ki i ekonomski isplativih alternativa je upotreba pivskog tropa u proizvodnji mlečne kiseline, jer se poslednjih par decenija uočava intenzivan rast potražnje za mlečnom kiselinom. Mlečna kiselina je najvažnija hidroksikarbonska kiselina &scaron;iroko rasprostranjena u prirodi, sa velikom primenom u prehrambenoj, farmaceutskoj, tekstilnoj i hemijskoj industriji i industriji prerade kože.<br />Cilj istraživanja ove doktorske disertacije je ispitivanje primene pivskog tropa u proizvodnji mlečne kiseline. Prvo je izvr&scaron;ena optimizacija enzimske hidrolize pivskog tropa u cilju dobijanja &scaron;to je moguće veće koncentacije redukujućih &scaron;ećera neophodne za mlečno-kiselu fermentaciju. Hidrolizat pivskog tropa je dobijen enzimskom hidrolizom dodatkom komercijalnih enzima za razgradnju skroba i celuloze. Parametri čiji je uticaj na efikasnost enzimske hidrolize ispitanu su: pH vrednost, temperatura hidrolize i količina dodatih enzima. Nakon &scaron;to su određeni najbolji uslovi razgranje pivskog tropa, dobijeni postupak hidrolize je primenjen u proizvodnji hidrolizata pivskog tropa koji je kori&scaron;ćen u mlečno-kiselim fermentacijama.<br />Nakon toga je ispitana mlečno-kisela fermentacija sa dva proizvodna mikoorganizma. Kao proizvodni mikroorganizmi u mlečno-kiselim fermentacijama primenjena su dva soja bakterija mlečne kiseline: Lactobacillus fermentum PL-1 i Lactobacillus rhamnosus ATCC 7469. Ispitan je uticaj dodatka različitih koncentracija ekstrakta kvasca (0,5-5,0%) uz korekciju pH vrednosti tokom fermentacije sa dodatkom kalcijum-karbonata. U zavisnosti od udela L-(+)- i D-(-)-mlečne kiseline koje nastaju tokom fermentacije izabran je proizvodni mikroorganizam koji proizvodi vi&scaron;e L-(+)-mlečne kiseline.<br />U daljim ispitivanjima je ispitan uticaj korekcije pH pomoću natrijum-hidroksida kao i dodatak različitih koncentracija ekstrakta kvasca (0,5-5,0%) i redukujućih &scaron;ećera (2,7; 5,4 i 8,1%) u hidrolizatu pivskog tropa na mlečno-kiselu fermentaciju pomoću odabranog soja bakterija mlečne kiseline. Na osnovu dobijenih rezultata izabrana je najbolja koncentracija redukujućih &scaron;ećera i ekstrakta kvasca koji će se koristiti u daljim istraživanjima.<br />Takođe je ispitana i mogućnost zamene skupog ekstrakta kvasca i glukoze sa obnovljivim sirovinama, kao &scaron;to su pivski kvasac, džibra i bistra džibra.<br />Ispitan je uticaj dodatka različitih koncentracija pivskog kvasca (0,5-5,0%), džibre (5-20%) i bistre džibre (5-50%) pre fermentacije kao i dodatak bistre džibre u dolivnoj fermentaciji, na mlečno-kiselu fermentaciju hidrolizata pivskog tropa.<br />Ispitan je i dolivni postupak fermentacije hidrolizata pivskog tropa dodatkom glukoze, glukoze i ekstrakta kvasca i sladovine. Takođe je ispitana mogućnost izvođenja vi&scaron;e uzastopnih fermentacija sa imobilisanim ćelijama odabranog soja bakterija mlečne kiseline u kalcijum-alginatu.<br />Na osnovu eksperimentalnih rezultata zaključujeno je da je dodatak kalcijum-karbonata imao pozitivan uticaj na proizvodnju mlečne kiseline sa L. fermentum i L. rhamnosus. Sa dodatkom kalcijum-karbonata povećali su se utro&scaron;ak redukujućih &scaron;ećera, koncentracija i prinos mlečne kiseline i vijabilnost ćelija L. fermentum i L. rhamnosus. Ekstrakt kvasca i kalcijum-karbonat su imali značajan uticaj na proizvodnju mlečne kiseline sa L. fermentum i L. rhamnosus. U fermentacijama sa L. fermentum najveći prinos ukupne mlečne kiseline (44%) je postignut sa dodatkom 5,0% ekstrakta kvasca i 2,0% kalcijum-karbonata. U fermentacijama sa L. rhamnosus najveći prinos ukupne mlečne kiseline (98%) i L-(+)-mlečne kiseline (96%) je ostvaren u fermentaciji sa dodatkom 2,0% ekstrakta kvasca i 2,0% kalcijum-karbonata. Na osnovu rezultata odlučeno je da se u daljim ispitivanjima mlečno-kisele fermentacije hidrolizata pivskog tropa kao proizvodni mikoorganizam koristi L. rhamnosus.<br />Primenom natrijum-hidroksida za korekciju pH je skratila fermentaciju za 48 sati a ostvareno je i značajno povećanje zapreminske produktivnosti L-(+)-mlečne kiseline (za 200%, povećanje sa 0,21 na 0,63 g/l&middot;h-1). Korekcija pH u svim daljim istraživanjima je vr&scaron;ena sa dodatkom natrijum-hidroksida.<br />U mlečno-kiselim fermentacijama sa različitim početnim koncentracijama redukujućih &scaron;ećera (2,7; 5,4 i 8,1%) i sa dodatkom različitih koncentracija ekstrakta kvasca (0,5-5,0%), najveći prinos L-(+)-mlečne kiseline i zapreminska produktivnost od 91,29% i 1,69 g/l&middot;h-1, kao i vijabilnost ćelija L. rhamnosus od 9,7&middot;109 CFU/ml ostvareni su u fermentaciji sa početniom koncentracijom redukujućih &scaron;ećera od 5,4% i dodatkom 5,0% ekstrakta kvasca.<br />Na osnovu ostvarenih rezultata u istraživanjima sa dodatkom džibre i dodacima tokom fermentacije kao i u fermentacijama sa imobilisanim ćelijama je kori&scaron;ćen hidrolizat pivskog tropa sa početnom koncentracijom redukujućih &scaron;ećera od 5,4%.<br />U mlečno kiseloj fermentaciji sa dodatkom pivskog kvasca najveći prinos L-(+)-mlečne kiseline (89,01%) i zapreminska produktivnost (0,89 g/l&middot;h-1) L-(+)-mlečne kiseline su ostvareni u fermentaciji sa dodatkom 5,0% pivskog kvasca i korekcijom početne koncentracije redukujućih &scaron;ećera na 5,0%. Na osnovu rezultata utvrđeno je da se može izvr&scaron;iti delimična ili potpuna zamena ekstrakta kvasca pivskim kvascem uz značajno smanjenje cene podloge za mlečno-kiselu fermentaciju, bez značajnog smanjenja efikasnosti mlečno-kisele fermentacije.<br />U mlečno-kiseloj fermentaciji sa dodatkom džibre i bistre džibre najveć koncetracija, prinos i zapreminska produktivnost L-(+)-mlečne kiseline od 31,03 g/l, 86,15% i 0,93 g/l&middot;h-1, ostvareni su u fermentaciji sa dodatkom 50% bistre džibre. Najvi&scaron;a koncentracija, prinos i zapreminska produktivnost L-(+)-mlečne kiseline ostvareni u dolivnoj fermentaciji sa dodatkom glukoze i bistre džibre tokom mlečno-kisele fermentacije su iznosili su 48,02 g/l, 87,82% i 0,96 g/l&middot;h-1.<br />U fermentacijama sa dodatkom nutritijenata tokom mlečno-kisele fermentacije najveća vrednost koncetracije, prinosa i zapreminske produktivnosti L-(+)-mlečne kiseline od 116,08 g/l, 93,32% i 2,04 g/L&middot;h-1, su ostvarene u fermentaciji sa dodatkom glukoze i ekstrakta kvasca tokom fermentacije. Na osnovu rezultata utvrđeno je da se dolivni postupak fermentacije može koristiti u cilju povećanja efikasnosti mlečno-kisele fermentacije.<br />Izvr&scaron;ena je imobilizacija ćelija L. rhamnosus u kalcijum-alginatu uz izuzetno visoku vijabilnost (1010 CFU/ml). Imobilisane ćelije L. rhamnosus su uspe&scaron;no kori&scaron;ćene u tri mlečno-kisele fermentacije. Prinos L-(+)-mlečne kiseline i zapreminska produktivnost su u sve tri fermentacije bili izuzetno visoki, pri čemu su najveći prinos L-(+)-mlečne kiseline i zapreminska produktivnost od 95,2% i 1,76 g/l&middot;h-1, ostvareni u drugoj fermentaciji. Upotrebom imobilisanih ćelija L. rhamnosus je osim povećanja prinosa i zapreminske produktivnosti L-(+)-mlečne kiseline skraćena fermentacija za 12 sati u poređenju sa &scaron;aržnim fermentacijama.</p> / <p>Brewers spent grain represents (BSG) about 85% of the total by-products from brewing process and is available at low price during the whole year. Due to its chemical composition BSG has great potential use in biotechnology and production of high-value products. One of very eco-friendly and economical alternative uses of BSG is in production of lactic acid (LA), since in the last few decades the demand for the LA has significantly risen, mostly because of development of biodegradable lactic polymers, which are eco-friendly and nontoxic.<br />Lactic acid is the most important hydrocarboxylic acid with an asymmetrical carbon atom, widely distributed in nature, and it has shown great potential in fields of food, pharmaceutical, textile, leather and chemical industries.<br />The aim of this doctoral thesis was to investigate the application of BSG in lactic acid production. First, the optimization of enzymatic hydrolysis of BSG was conducted, with the goal to achieve high reducing sugar concentrations, as much as possible, that are necessary on LA fermentation. BSG hydrolysis was conducted by usage of commercial enzymes for degradation of starch and cellulose. Effect of pH value, temperature and enzyme dosage on BSG hydrolysis efficiency was investigated. After the best conditions for BSG hydrolysis were determined, the optimized procedure for BSG hydrolysis was used for the production of BSG hydrolysate that will be used in LA fermentations.<br />After optimization of BSG hydrolysis, LA fermentation by two LA producing microorganisms was investigated. The strains investigated were two LA bacteria strains: Lactobacillus fermentum PL-1 and Lactobacillus rhamnosus ATCC 7469. The effect of yeast extract (0.5; 1.0; 2.0; 3.0; 4.0, and 5.0%) addition in BSG hydrolysate, with the correction of pH value during LA fermentation by the addition of calcium-carbonate, on LA fermentation was investigated. Based on the results achieved for L-(+)- and D-(-)-LA ratio the LAB strains that produced more L-(+)-LA was chosen for further research.<br />In further research the effect of pH correction (with addition of NaOH), yeast extract (0.5, 1.0, 2.0, 3.0, 4.0, and 5.0%) addition and reducing sugar concentration (2.7; 5.4 and 8.1%) in BSG hydrolysate on LA fermentation was investigated. Based on the results achieved the best yeast extract and reducing sugars concentrations was determined and used in further analysis or research. Also the possible replacement of expensive yeast extract and glucose with cheap alternatives, like brewer`s spent grain and stillage was investigated. The effect of brewer`s spent grain (0.5; 1.0; 2.0; 3.0; 4.0, and 5.0%), whole stillage (5, 10, 15 i 20%) and thin stillage (5, 10, 15, 20, 30, 40, 50%) addition before fermentation as well as thin stillage addition in fed-batch fermentation in BSG hydrolysate on LA fermentation were investigated.<br />Also fed-batch fermentation procedure (addition of glucose, glucose and yeast extract and wort during fermentation) was investigated. The possible application of cells immobilized in Ca-alginate for LA fermentation of BSG hydrolysate was also investigated.<br />Based on the results it was concluded that BSG can be successfully utilized as a raw material in production of LA, after optimization of hydrolysis and addition of nitrogen source.<br />According to the results of chemical composition before and after optimized hydrolysis 78.6% of total cellulose was hydrolyzed.<br />Addition of calcium-carbonate had positive effect on LA production by L. fermentum i L. rhamnosus. With the addition of calcium-carbonate reducing sugar utilization, LA yield and concentration and cell viability (both L. fermentum i L. rhamnosus) increased. Addition of calcium-carbonate and yeast extract had a positive effect on LA fermentation by L. fermentum and L. rhamnosus. In LA fermentation by L. fermentum the highest LA yield (44%) was achieved with addition of 5.0% of yeast extract and 2.0% of calcium-carbonate. In L. rhamnosus fermentations the highest total LA yield (98%) and L-(+)-LA yield (96%) was reached when 2.0% of yeast extract and 2.0% of calcium-carbonate were added.<br />Based on the results achieved it was concluded that BSG hydrolysate, with the addition of yeast extract, is a good fermentation media for LA fermentation with L. rhamnosus, and it was decided that L. rhamnosus will be used in further research of LA fermentation on BSG hydrolysate.<br />Addition of NaOH instead of calcium-carbonate for the pH correction shortened the fermentation time by 48 h and increased the L-(+)-LA volumetric productivity (by 200%, from 0.21 to 0.63 g/L&middot;h-1). Based on this results pH correction in further experiments was done by addition of NaOH.<br />In LA fermentation with different reducing sugar (2.7, 5.4 and 8.1%) and yeast extract concentrations (0.5-5.0%), the highest L-(+)-LA yield and volumetric productivity of 91.29%, and 1.69 g/L&middot;h-1, respectively, as well as L. rhamnosus cell viability (9.67 log CFU/mL), were achieved with the reducing sugar content of 5.4% and yeast extract content of 5.0%.<br />Based on this results in further experiment with the addition of stillage, in fed-batch fermentation and fermentation with immobilized cell BSG hydrolysate with 5.4% of reducing sugars and 5.0% yeast extract was used.<br />In fermentation with the addition of brewer&rsquo;s spent yeast the highest L-(+)-LA yield (89.01%) and volumetric productivity (0.89 g/L&middot;h-1) were achieved in the fermentation of BSG hydrolysate with 5.0% of reducing sugar and 5.0% of brewer&rsquo;s yeast. Based on the results achieved it was concluded that yeast extract can be partial or complete replaced by brewer&rsquo;s spent yeast with significant decrease of media cost, without the decrease in LA fermentation efficiency.<br />In fermentation with the addition of thin stillage the highest L-(+)-LA concentration, yield, and volumetric productivity of 31.03 g/L, 86.15%, and 0.93 g/L&middot;h-1, respectively, was obtained in fermentation with the addition of 50% of thin stillage. The highest L-(+)-LA concentration, yield, and volumetric productivity achieved in fed-batch fermentation with the addition of glucose and thin stillage during fermentation, were 48,02 g/L, 87,82% i 0,96 g/L&middot;h-1.<br />In fed-batch fermentation the highest L-(+)-LA concentration, yield, and volumetric productivity of 116.08 g/L, 93.32%, and, 2.04 g/L h-1, respectively, were achieved in fermentation with glucose and yeast extract addition during fermentation. The results showerd that fed-batch fermentation could be used to increase L-(+)-LA fermentation efficiency<br />Immobilization of L. rhamnosus cells with high viability (1010 CFU/mL) in Ca-alginate was conducted. Immobilized cells we successfully utilized in three repeated batch fermentation. L-(+)-LA yield and volumetric productivity were very high in all three batch fermentation, with the highest results achieved (95.20% and 1.76 g/L&middot;h-1, respectively) in second fermentation. Application of immobilized L. rhamnosus cells increased L-(+)-LA yield and volumetric productivity and shortened the fermentation time for 12 h in comparison with batch fermentation.</p>

Abstract Bone tissue engineering has become a rapidly expanding research area because it offers a promising new approach for bone repair and regeneration. Compared to traditional autograft and allograft procedures, bone tissue engineering techniques based on the use of scaffolding materials in combination with autogenous stem cells can eliminate problems of donor site morbidity associated with the harvest of bone tissue, and its short supply. Clearly, the choices of material as well as a scaffold design that enhance bone regeneration are major challenges in the tissue engineering approach. Fibers in the micro-range in combination with textile-based technologies are consider as potential routes for the production of complex scaffolds since they can be used to generate a wide range of morphological structures and geometrically varied structures with high precision. Therefore in this thesis the specific objects were to: (i) develop a biocompatible composite fiber from poly(lactic acid) (PLA) and hydroxyapatite (HA) by melt spinning, (ii) design a 3D textile scaffold utilizing weaving and (iii) evaluate the scaffolds’ performance as a bone substitute material in vitro. In the present study PLA/HA composite fibers were successfully produced, and found to possess sufficient mechanical strength even at high loading concentrations (i.e. 20wt %), to be useful in a textile process. In addition, the material was shown to be biocompatible and the presence of HA in the PLA composite significantly enhanced the initial cell attachment. In a 3D woven scaffold, bone marrow derived human mesenchymal stem cells (hMSCs) differentiated into osteoblasts and mineralized bone formation in vitro was observed through-the-thickness of the scaffold. Taken together, these results indicate the potential feasibility of PLA/HA composite fiber in a 3D woven scaffold for use as a bone substitute material in tissue engineering applications. / Tiivistelmä Luupuutosten korvaaminen kudosteknologisesti on kehittynyt nopeasti ja tutkimustulokset tarjoavat lupaavia mahdollisuuksia tuottaa uutta luuta luupuutosalueelle. Perinteisiin potilaan omasta luusta tehtyihin luusiirteisiin ja pankkiluusiirteisiin verrattuna potilaan omat kantasolut voivat vähentää ongelmia, joita ovat siirremateriaalin rajallinen saatavuus ja vieraan kudoksen aiheuttamat reaktiot. On tärkeä etsiä hyviä materiaaleja, joista voidaan valmistaa sellaisia kolmiulotteisia (3D) rakenteita, joilla tehostetaan luun paranemista ja uuden luun muodostumista. Kutomalla tuotetut tukirakenteet mahdollistavat kantasolusiirteille kolmiulotteisuuden, jota voidaan säädellä monipuolisesti ja tarkasti. Tämän väitöstutkimuksen tarkoituksena oli: (i) kehittää bioyhteensopiva kuitu maitohappopolymeeristä poly lactic acid (PLA) ja hydroksiapatiitista (HA) kuituekstruusiolla, (ii) suunnitella ja kutoa tästä kuidusta 3D tekstiilirakenne, ja (iii) tutkia sen toimivuus ja ominaisuudet luunmuodostusta tukevana materiaalina soluviljelyolosuhteissa. Tämä tutkimus osoittaa, että PLA kuitua voidaan seostaa hydroksiapatiitilla, ja PLA/HA kuidut ovat mekaanisesti kestäviä sisältäessään jopa 20 painoprosenttia hydroksiapatiittia. Siten kuidut ovat tekstiilin valmistuksessa käyttökelpoisia. Lisäksi materiaali osoittautui bioyhteensopivaksi, ja hydroksiapatiitti paransi solujen tarttumista PLA kuituun viljelyn alkuvaiheessa. Ihmisen luuytimestä peräisin olevat sidekudoksen kantasolut (hMSCs) erilaistuivat soluviljelmässä luuta muodostaviksi soluiksi eli osteoblasteiksi, ja tuottivat mineralisoitunutta luun väliainetta kautta koko kudotun tukirakenteen. Johtopäätöksenä on, että PLA/HA yhdistelmäkuitua voidaan kutoa kolmiulotteiseksi tukirakenteeksi, ja sitä on mahdollista käyttää apuna korvattaessa luupuutoksia kudosteknologian keinoin.

Doctor of Philosophy / Department of Grain Science and Industry / X. Susan Sun / Biobased polymers derived from renewable resources are increasingly important due to acute concerns about the environmental issues and limited petroleum resources. Poly(lactic acid) (PLA) is such a polymer that has shown great potential to produce biodegradable plastics. However, low glass transition temperature (Tg), low thermal stability, slow biodegradation rate, and high cost limit its broad applications. This dissertation seeks to overcome these limitations by reinforcing PLA with inorganic nanoparticles and low-cost agricultural residues. We first synthesized PLA nanocomposites by in situ melt polycondensation of L-lactic acid and surface-hydroxylized nanoparticles (MgO nanocrystals and TiO2 nanowires) and investigated the structure-property relationships. PLA grafted nanoparticles (PLA-g-MgO, PLA-g-TiO2) were isolated from the bulk nanocomposites via repeated dispersion/centrifugation processes. The covalent grafting of PLA chains onto nanoparticle surface was confirmed by Fourier transform infrared spectroscopy and thermalgravimetric analysis (TGA). Transmission electron microscopy and differential scanning calorimetry (DSC) results also sustained the presence of the third phase. Morphological images showed uniform dispersion of nanoparticles in the PLA matrix and demonstrated a strong interfacial interaction between them. Calculation based on TGA revealed that more than 42.5% PLA was successfully grafted into PLA-g-MgO and more than 30% was grafted into PLA-g-TiO2. Those grafted PLA chains exhibited significantly increased thermal stability. The Tg of PLA-g-TiO2 was improved by 7 °C compared with that of pure PLA. We also reinforced PLA with low-value agricultural residues, including wood flour (WF), soy flour (SF), and distillers dried grains with solubles (DDGS) by thermal blending. Tensile measurements and morphological images indicated that methylene diphenyl diisocyanate (MDI) was an effective coupling agent for PLA/WF and PLA/DDGS systems. MDI compatibilized PLA/WF and PLA/DDGS composites showed comparable tensile strength and elongation at break as pure PLA, with obviously increased Young’s modulus. Increased crystallinity was observed for PLA composites with SF and DDGS. Such PLA composites have similar or superior properties compared with pure PLA, especially at a lower cost and higher biodegradation rate than pure PLA. The results from this study are promising. These novel PLA thermoplastic composites with enhanced properties have potential for many applications, such as packaging materials, textiles, appliance components, autoparts, and medical implants.

Doctor of Philosophy / Department of Grain Science and Industry / Praveen V. Vadlani / Lactic acid is an important platform chemical that has long history and wide applications in food, polymer, pharmaceutics and cosmetic industries. Lactic acid has two optical isomers; namely D-lactic acid and L-lactic acid. Racemic mixture of lactic acid are usually used as preservatives and ingredients in solvents, or as precursors for different chemicals. Currently there is an increasing demand of optical pure lactic acid as a feedstock for the production of poly-lactic acid (PLA). PLA is a biodegradable, biocompatible and environmental friendly alternative to plastics derived from petroleum based chemicals. Optically pure D or L-lactic acid is used for the synthesis of poly D or L- lactic acid (PDLA, PLLA). Blend of PDLA with PLLA results in a heat-resistant stereocomplex PLA with excellent properties. As a consequence, large quantity of cost effective D-lactic acid is required to meet the demand of stereocomplex PLA. Lignocellulosic biomass is a promising feedstock for lactic acid production because of its availability, sustainability and cost effectiveness compared to refined sugars and cereal grain-based sugars. Commercial use of lignocellulosic biomass for economic production of lactic acid requires microorganisms that are capable of using all sugars derived from lignocellulosic biomass. Therefore, the objectives of this study were: 1) to produce high level of optically pure D-lactic acid from lignocellulosic biomass-derived sugars using a homofermentative strain L. delbrueckii via simultaneous saccharification and fermentation (SSF); 2) to develop a co-culture fermentation system to produce lactic acid from both pentose and hexose sugars derived from lignocellulosic biomass; 3) to produce D-lactic acid by genetically engineered L. plantarum NCIMB 8826 ∆ldhL1 and its derivatives; 4) to construct recombinant L. plantarum by introduction of a plasmid (pLEM415-xylAB) used for xylose assimilation and evaluate its ability to produce D-lactic acid from biomass sugars; and 5) to perform metabolic flux analysis of carbon flow in Lactobacillus strains used in our study. Our results showed that D-lactic acid yield from alkali-treated corn stover by L. delbrueckii and L. plantarum NCIMB 8826 ∆ldhL1 via SSF were 0.50 g g[superscript]-1 and 0.53 g g[superscript]-1 respectively; however, these two D-lactic acid producing strains cannot use xylose from hemicellulose. Complete sugar utilization was achieved by co-cultivation of L. plantarum ATCC 21028 and L. brevis ATCC 367, and lactic acid yield increased to 0.78 g g[superscript]-1 from alkali-treated corn stover, but this co-cultivation system produced racemic mixture of D and L lactic acid. Simultaneous utilization of hexose and pentose sugars derived from biomass was achieved by introduction of two plasmids pCU-PxylAB and pLEM415-xylAB carrying xylose assimilation genes into L. plantarum NCIMB 8826 ∆ldhL1, respectively; the resulting recombinant strains ∆ldhL1-pCU-PxylAB and ∆ldhL1-pLEM415-xylAB used xylose and glucose simultaneously and produced high yield of optically pure D-lactic acid. Metabolic flux analysis verified the pathways used in these Lactobacillus strains and provided critical information to judiciously select the desired Lactobacillus strain to produce lactic acid catering to the composition of raw material and the optical purity requirement. This innovative study demonstrated strategies for low-cost biotechnological production of tailor-made lactic acid from specific lignocellulosic biomass, and thereby provides a foundational manufacturing route for a flexible and sustainable biorefinery to cater to the fuel and chemical industry.

This project investigates the moisture barrier properties of bio-based polymers and ways of improving them. The first section addresses the effect of crystallinity on the water permeability of poly(lactic acid) (PLA). The second section investigates PLA/talc composites and PLA/ montmorillonite nanocomposites. The third section is focused on a new polymer, polybutylene succinate (PBS), and its nanocomposites with montmorillonite. In the first section, the water vapour transmission rates (WVTR) through samples of polylactic acid of different crystallinities have been measured. Three different grades of commercial PLA were used with different ratios of L-lactide and D-lactide to give a range of crystallinities from 0 to 50%. Sheets of PLA were prepared by melt compounding followed by compression moulding and annealing at different temperatures and for different times to give the range of crystallinities required. Crystallinity was measured by differential scanning calorimetry (DSC) and the morphology of the samples was observed under crossed polars in a transmitted light microscope. Water vapour transmission rates through the films were measured at 38°C and at a relative humidity of 90%. It was found that the measured values of WVTR decreased linearly with increasing crystallinity of the PLA from 0 to 50%. The results are discussed in terms of the effect of crystallinity on solubility and shown to fit the tortuous path model. The model was also successfully used to explain published data on water permeability of polyethylene terephthalate. In the second section, a series of PLA/talc composites and PLA/ montmorillonite nanocomposites were prepared by melt compounding followed by compression moulding. The morphologies of the composites were investigated using transmission electron microscopy (TEM) and wide-angle X-ray diffraction (WAXD) and it was found that the fillers were well dispersed in the polymer matrix. The average aspect ratio of the compounded talc was found to be 8, and that of the nanoclay was found to be 50. Water vapour transmission rates (WVTR) through the films were measured at 38°C and at a relative humidity of 90%. It was found that the measured values of WVTR decreased with increasing filler content and the results gave good agreement with predictions from the Nielsen tortuous path model. In the third section, PBS/ montmorillonite nanocomposites were prepared by melt compounding followed by compression moulding. The melting and crystallisation behaviour of the pure PBS samples were investigated using differential scanning calorimetry (DSC) and cross polarised optical microscopy. A slight decrease of the degree of crystallinity was found in PBS containing 5% nanoclay. The morphology of the composites was investigated using transmission electron microscopy (TEM) and wide-angle X-ray diffraction (WAXD) and it was confirmed that that composite structures were intercalated. Water vapour transmission rates (WVTR) through the PBS sheets were measured using a MOCON Permatran-W®398. The measured values of WVTR decreased with increasing nanoclay content. However, the experimental values were all higher than the values predicted by the Nielsen tortuosity model. This result shows that in the case of PBS, which is a highly crystalline polymer, the nanoclay is not as well dispersed and is not as effective in reducing water vapour permeability as in the case of PLA.

Thesis (MSc)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: Thirty lactic acid bacteria were isolated from the intestinal tract of Drosophila simulans Stuvervant and nine lactic acid bacteria from Merlot grapes collected from the same winery in the Stellenbosch region, South Africa. The isolates were grouped according to morphological, biochemical and physiological characteristics. Isolates selected from each group were identified to species level by PCR with species-specific primers, PCR-based DGGE and 16S rDNA sequencing. The majority of isolates from the intestinal tract of Drosophila simulans Stuvervant belonged to the species Lactobacillus plantarum, but Lactobacillus paracasei, Lactobacillus sanfranciscensis, Leuconostoc mesenteroides subsp. mesenteroides, Lactococcus lactis subsp. lactis, Enterococcus faecalis and Pediococcus pentosaceus were also identified. As far as we could determine, this is the first report on the isolation of L. paracasei, L. sanfranciscensis, L. mesenteroides subsp. mesenteroides, L. lactis subsp. lactis, E. faecalis and P. pentosaceus from vinegar flies. Lactobacillus plantarum has previously been isolated from Merlot grapes. The genotypic relatedness among isolates of L. plantarum isolated from the intestinal tract of vinegar flies and from Merlot grapes were determined by RAPD-PCR. The isolates were grouped into four genotypically well-separated clusters. Thirteen isolates from grape must and five from flies yielded identical RAPD-PCR banding patterns and grouped into one cluster, suggesting that they are descendants from the same strain. This suggests that L. plantarum has the ability to use vinegar flies as a vector. / AFRIKAANSE OPSOMMING: Dertig melksuurbakterieë is vanuit die dermkanaal van Drosophila simulans Stuvervant geïsoleer en nege melksuurbakterieë vanuit Merlot-druiwe. Die druiwe is afkomstig van dieselfde wynkelder in die Stellenbosch-area van Suid-Afrika. Die isolate is volgens morfologiese, biochemiese en fisiologiese eienskappe gegroepeer. Verteenwoordigende isolate vanuit die fenotipiese groepe is tot spesievlak met behulp van lukraak ge-amplifiseerde polimorfe-DNA (RAPD) polimerase ketting-reaksie (PKR), PKR met spesie-spesifieke inleiers, PKR-gebaseerde denaturerende gradient-jel elektroforese (DGGE) en 16S rDNA sekwensering geïdentifiseer. Die meerderheid isolate uit die ingewande van Drosophila simulans Stuvervant is as Lactobacillus plantarum geklassifiseer. Stamme van Lactobacillus paracasei, Lactobacillus sanfranciscensis, Leuconostoc mesenteroides subsp. mesenteroides, Lactococcus lactis subsp. lactis, Enterococcus faecalis en Pediococcus pentosaceus is ook geïdentifiseer. Sover bekend, is dit die eerste keer dat L. paracasei, L. sanfranciscensis, L. mesenteroides subsp. mesenteroides, L. lactis subsp. lactis, E. faecalis en P. pentosaceus uit asynvlieë geïsoleer is. Lactobacillus plantarum is voorheen uit Merlot-druiwe geïsoleer. Die genotipiese ooreenkoms tussen die stamme van L. plantarum wat uit die asynvlieë en Merlot-druiwe geïsoleer is, is deur middel van RAPD-PKR bepaal. Hiervolgens is die stamme in vier genotipies goed-gedefinieerde groepe geplaas. Dertien isolate vanuit druiwemos en vyf vanuit asynvlieë het identiese RAPD-PKR bandpatrone vertoon en het in een groep gesorteer. Hierdie resultate dui daarop dat die stamme heel moontlik uit een voorouer ontstaan het en dat asynvlieë heel moontlik as vektor vir L. plantarum dien.

Thesis (MSc) -- University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Lactic acid bacteria play an essential role in the majority of fermented foods by producing organoleptic compounds and increasing the shelf life. The best-studied antimicrobial compounds are bacteriocins, i.e. ribosomally synthesized peptides. Most of these peptides have a narrow spectrum of activity and are usually only active against bacteria from the same ecological niche. The fact that all bacteriocins are degraded by proteolytic enzymes enlarges their potential use as natural food preservatives. The ideal would be to replace or reduce chemical preservatives such as sulfur dioxide, nitrates and nitrites. Bacteriocins are classified into four groups according to their structural and functional characteristics. Plantaricin 423, produced by Lactobacillus plantarum 423, is heat stable, plasmid encoded, relatively small (3.5 kDa) and is classified as a class Iia bacteriocin. The peptide is active from pH 1.0 to 10.0 and inhibits Gram-positive bacteria, including Lactobacillus spp., Leuconostoc spp., Oenococcus oeni, Pediococcus spp., Enterococcus spp., Propionibacterium spp. and pathogens such as Bacillus cereus, Clostridium spp. and Listeria monocytogenes. Production of bacteriocins may occur constitutively or may be regulated by a cell-density dependent system called quorum sensing. Plantaricin 423 is produced throughout logarithmic growth, with no apparent change in production levels when the producer strain is cultured in the presence of plantaricin 423 or Listeria innocua and Lactobacillus sakei. This led us to believe that plantaricin 423 may be produced constitutively. A reporter system was constructed which consisted of the plantaricin 423 promoter, P423, fused to the luxAB genes and cloned into a shuttle vector, pTRKH2. The newly constructed plasmid, pTAB4, was transformed to a bacteriocin-negative mutant of L. plantarum (423 B} Despite several repeats, no luciferase activity was recorded and no RNA homologous to the luxAB genes was detected. The region necessary for expression of plantaricin 423 may be located stream-up of the -80 region homologous to the -80 and -40 conserved repeats of regulated class II bacteriocins. Inclusion of the latter region in the reporter construct may result in the successful expression of luxAB. / AFRIKAANSE OPSOMMING: Melksuurbakteriee speel 'n belangrike rol in die meeste gefermenteerde voedselsoorte deur die produksie van organoleptiese komponente en die verlenging van rakleeftyd. Van aile antimikrobiese komponente is bakteriosiene (ribosomaal gesintetiseerde peptiede) die beste bestudeer. Hierdie peptiede het gewoonlik 'n nou spektrum van antimikrobiese werking en is meestal aktief teen bakteriee in dieselfde ekologiese nis. Die feit dat bakteriosiene deur proteolitiese ensieme in die spysverteringskanaal vernietig word, verhoog die potensiele gebruik van bakteriosiene as preserveermiddels. Die ideaal sal wees om die konsentrasie van chemiese preserveermiddels soos swaweldioksied, nitrate en nitriete te verlaag of rnoontlik te vervang met bakteriosiene. Bakteriosiene word in vier groepe op grond van hul strukturele en funksionele karaktereienskappe geklassifiseer. Plantarisien 423, geproduseer deur Lactobacillus plantarum 423, is hitte-stabiel, word deur 'n plasmied gekodeer, is relatief klein (3.5 kDa) en sorteer onder die klas Iia bakteriosiene. Die peptied is aktief oor 'n wye pH-reeks (pH 1.0-10.0) en inhibeer Gram-positiewe bakteriee, insluitend Lactobacillus spp., Leuconostoc spp., Oenococcus oeni, Pediococcus spp., Enterococcus spp., Propionibacterium spp. en patogene soos Bacillus cereus, Clostridium spp. en Listeria monocytogenes. Produksie van bakteriosiene kan konstitutief plaasvind of kan gereguleer word deur 'n seldigtheids- afhanklike sisteem naamlik "quorum sensing". Plantarisien 423 word regdeur logaritmiese groei geproduseer, met geen verandering in produksievlakke wanneer die produserende stam in die teenwoordigheid van plantarisien 423 of Listeria innocua en Lactobacillus sakei gekweek word nie. Dit het gelei tot die hipotese dat plantarisien 423 moontlik konstitutief geproduseer word. 'n Verklikkersisteem bestaande uit 'n fusie van die plantarisien 423 promoter, P423, aan die luxAB gene is gekonstrueer en in die pendelplasmied pTRKH2 gekloneer. Die nuutgekonstrueerde plasmied, pTAB4, is na 'n bakteriosien-negatiewe mutant van L. plantarum (stam 423 B-) getransfonneer. Ten spyte van etlike herhalings kon geen lusiferase-aktiwiteit opgespoor word nie en kon ook geen homologie in die RNA met die luxAB gene opgespoor word nie. Dit is moontlik dat die area nodig vir uitdrukking van plantarisien 423 verder stroom-op van die -80 area, homoloog aan die -80 en -40 gekonserveerde herhalings van reguleerbare klas II bakteriosiene, gesetel is. Insluiting van laasgenoemde area in die verklikker-konstruk mag lei tot die suksesvolle uitdrukking van luxAB.