O objetivo do trabalho foi avaliar a capacidade de cepas de bactérias ácido-láticas (BAL) em remover a aflatoxina M1 (AFM1) em solução tampão fosfato salina (TFS) e em amostras de leite. Nos ensaios com TFS, verificou-se a influência do tempo de contato (15 min. ou 24 horas) entre as células de sete cepas de BAL e AFM1, as diferenças entre a eficiência de remoção das bactérias viáveis e inviabilizadas termicamente, e a estabilidade do complexo BAL/AFM1 formado. As três cepas de BAL com maior percentual (> 33%) de remoção da AFM1 nos ensaios com TFS foram re-avaliadas utilizando-se leite UHT (ultra-high-temperature) desnatado artificialmente contaminado com AFM1. Para isso, foram utilizadas somente células inviabilizadas termicamente, verificando-se o efeito da temperatura (4ºC ou 37ºC) sobre a capacidade de remoção da toxina por 15 minutos. A remoção média da AFM1 pelas cepas de BAL em TFS variou entre 5,60±0,45 e 45,67±1,65% (n=3), sendo que as células inviáveis obtiveram percentuais de remoção de AFM1 significativamente maiores que as células viáveis, em ambos os tempos de contato analisados (15 min. ou 24 horas), não havendo diferença significativa entre os tempos. Observou-se que o complexo BAL/AFM1 obtido nos ensaios com TFS é instável, pois 40,57±4,66 a 87,37±1,82% da AFM1 retida pela bactéria foram recuperados em solução após a lavagem do complexo com TFS. As três cepas de BAL com maior percentual de remoção da AFM1 em TFS (Lactobacillus rhamnosus, Lactobacillus delbrueckii spp. bulgaricus e Bifidobacterium lactis) não apresentaram diferenças significativas nos ensaios com leite UHT a 37ºC. Somente B. lactis apresentou maior capacidade de remover a AFM1 do leite UHT a 4ºC. Os resultados demonstraram que a remoção de AFM1 empregando-se as BAL em alimentos é viável para reduzir as concentrações da toxina a níveis seguros. Entretanto, estudos adicionais são necessários a fim de investigar os mecanismos envolvidos na remoção da toxina pelas BAL com vistas à aplicação em indústrias de alimentos. / The purpose of this study was to evaluate the ability of strains of lactic acid bacteria (LAB) to remove aflatoxin M1 (AFM1) in phosphate buffer saline (PBS) and in milk samples. In the assays with PBS, the influence of contact time (15 min. or 24 hours) between the cells of seven LAB strains and AFM1 was evaluated, as well as the differences between the removal efficiency of viable and non-viable (heat-killed) bacteria, and the stability of AFM1/LAB complex produced. The three LAB strains with the highest percentage (> 33%) of AFM1 removal in the tests with PBS were reevaluated using UHT (ultra-high-temperature) skimmed milk spiked with AFM1. For these assays, only non-viable bacterial cells were used for checking the effect of temperature (4ºC or 37ºC) on the toxin removal capacity during 15 min. The mean AFM1 removal by LAB strains in PBS ranged from 5.60±0.45 and 45.67±1.65% (n=3). Non-viable cells showed AFM1 removal percentages significantly higher than viable cells in both contact times (15 min. or 24 hours), although there were not significant differences between these contact times. The AFM1/LAB complex resulted from the tests with PBS was unstable, as 40.57±4.66 to 87.37±1.82% of AFM1 retained by the bacteria were recovered in solution after washing the complex with PBS. The three LAB strains with the highest percentage of AFM1 removal in the PBS assays (Lactobacillus rhamnosus, Lactobacillus delbrueckii spp. bulgaricus and Bifidobacterium lactis) showed no significant differences in the UHT skimmed milk assays at 37ºC. Only B. lactis had greater ability to remove AFM1 in UHT milk at 4ºC. The results demonstrated that the removal of AFM1 by using LAB in foods is viable to reduce the toxin concentrations until safe levels. However, additional studies are needed to investigate the mechanisms involved in the toxin removal by LAB aiming its application in food industries.

Ethanol produced from lignocellulosic biomass has the potential to become a promisingalternative to gasoline. In this work the simultaneous saccharification and fermentation (SSF)technology was applied for ethanol production from hardwood with focus on cell growth,ethanol production and contamination.The SSF was performed at PH 5.5 and 35°C for different suspended solid concentrations(8%, 10% and 12%) of pretreated birch slurry which contained 16 % total suspended solids.Two different hexose fermenting yeast strain (Ethanol Red) and pentose fermenting yeaststrain were used.Quantifying the concentration of chemical components and metabolites in the fermentationmedium demonstrated that glucose and xylose are the major fermentable sugars in the slurry.The higher load of slurry (12%) represents a higher content of carbohydrates and potentiallyhigher end concentration of ethanol. Moreover, more lactic acid is produced with the lowerload of slurry (8 % or 10 %), presumably due to a result of a less inhibitory environment forbacterial growth. In this context, acetic acid sticks out as the most important inhibitor withconcentrations of 15.2 and 12.5 and 9.7 g/l respectively in the 12 %, 10 % and 8 % (ofsuspended solids) trials. Using pentose fermenting yeast may lead to higher ethanolproduction, lower xylose uptake and lower lactic acid formation. Cell viability and cellvitality determination from fermentation media in all the trails represented a sharplydecreasing trend during the fermentation for both Ethanol Red yeast strain and the pentosefermenting strain yeast strain apparently due to cell decomposition. / Program: MSc in Resource Recovery - Industrial Biotechnology

Depending on the previous research on LDHA gene silencing in Rhizopus oryzae CCUG 28959 through introduction of siRNA, a integral vector was constructed by inserting two copies of LDHA gene (by PCR cloning) in a fashion that it can express hpRNA in the transformed fungi, which will trigger the post transcriptional degradation of targeted mRNA through RNA degradation pathway which is known to be quelling in fungi.The vector was successfully designed with the LDHA gene, transformed in to the host organism, and also transferred to its progeny. This helps in maintaining stability of the transformed cell lines. This created vector will be advantageous at this point when compared to the use of siRNA for gene silencing, which is not a stable way. In the future, this vector can be used for down regulating other genes of interest in R. oryzae and can also be used for studying its effect on other metabolic pathways.In this study, Hygromycin resistance to the R. oryzae CCUG 28959 was shown at levels up to 1000 μg/ml, which has not been reported previously. / Program: MSc in Resource Recovery - Industrial Biotechnology

Lactic acid based thermoset were synthesised by reacting lactic acid with glycerol andfunctionalizing lactic acid branches by methacrylic anhydride. Resins with different chainlength were prepared and their thermo mechanical properties were examined through DMAanalysis and their molecular structures were analyzed by NMR method and their viscositywere investigated through rheometry analysis and three monomers were selected as the bestchain length. Degree of reaction in different reaction times was evaluated by a modifiedtitration method and bulk preparation of resin was performed by optimal process condition.DSC analysis was conducted in order to evaluate curing behaviour of resin with benzoylperoxide as cross-linking initiator. TGA analysis was performed to check thermo stability ofthe resin. Bio composites by viscose unidirectional and bidirectional knitted fabrics and alsonon woven viscose fiber with different fiber loads were prepared by ordinary hand layupimpregnation followed by compress moulding and their mechanical and thermo mechanicalproperties were characterized by tensile, flexural, charpy and DMA analysis and optimumfiber loads were identified for each fiber type. Ageing properties of prepared composites wereexamined by placing samples in climate chamber to simulate long time ageing and ageingexperiment was followed by tensile and flexural test to evaluate mechanical properties afterageing simulation. Composite`s swelling properties for water and some other solvents wereinvestigated and also their chemical resistance were evaluated by immersing them in 1M HCland KOH. The resin was also compared with a commercial oil based thermoset by preparingglass fiber reinforced composites and also effect of adding styrene to the resin were evaluated.Results of this work demonstrated that the novel synthesised have very high mechanical andthermo mechanical properties surpassing commercial oil based poly esters but ageingbehaviour is not very good however adding styrene can improve ageing properties. Also theresin is compatible with cellulosic natural fibers and forms strong composites. / Program: Masterutbildning i energi- och material

RNA silencing with direct delivery of siRNA has been used to suppress ldhA gene expression in filamentous fungus Rhizopus oryzae. Here, for the first time we show that, introducing small interfering RNA which consequently forms silencing complexes can alter the gene expression and we report a significant reduction of lactic acid production for isolates containing short (25 nt) synthetic siRNA. In all samples lactic acid production was reduced comparing with wild types. The average concentration of lactic acid production by Rhizopus oryzae during batch fermentation process where glucose has been used as a sole carbon source, diminished from 2.06 g/l in wild types to 0.36 g/l in knockdown samples which signify 5.7 times decrease. Interestingly, the average concentration of ethanol production was increased from 0.38 g/l in wild types to 0.45 g/l in knockdown samples. In some samples we were able to report even a 10 fold decrease in lactic acid production. Since R.oryzae is capable to assimilate a wide range of carbohydrates hydrolysed from lignocellulosic material in order to produce many economically valuable bulk material such as ethanol, these results suggest that RNA silencing is a useful method for industrial biotechnology to be applied in fungus Rhizopus oryzae in order to trigger the metabolism and gene expression toward a desired product.

Orientador: Jonas Contiero / Banca: Clóvis Parazzi / Banca: Luiz Carlos Basso / Banca: Pedro de Oliva Neto / Banca: Cíntia Duarte de Freitas Milagre / Resumo: O objetivo deste trabalho foi isolar micro-organismos produtores de D-(-) e L-(+) ácido lático, os quais são utilizados na síntese de polímeros empregados na produção de diversos materiais resistentes e biodegradáveis, além de otimizar a produção de ácido lático, a partir da utilização de diversos resíduos agro-industriais. Os micro-organismos mais promissores para produção de L-(+) ácido lático foram os isolados de Keffir (Ke6, Ke11, Ke8 e Ke24) e o Lactobacillus rhamnosus B103, já para a produção de D-(-) ácido lático, os mais promissores foram os isolados de iogurte (Y15C e Y15A) e o Leuconostoc mesenteroides B512. Pode-se afirmar que os micro-organismos selecionados apresentaram grande potencial para utilização na indústria de biopolímeros e indústria de alimentos. O soro de queijo e a manipueira foram os melhores resíduos para produção de L-(+) ácido lático por Lactobacillus rhamnosus B103. Quando se utilizou 160 g/L de lactose de soro de queijo, 60 mL/L de água de maceração de milho (AMM), 2 mL/L de Tween 80 e 0,10 g/L de MnSO4, observou-se alta produção de L- (+) ácido lático (142 g/L) e baixo residual de lactose (3,2 g/L). Para a otimização com manipueira, foi obtido 41,58 g/L de L-(+) ácido lático, a partir de 50 g/L de açúcar redutor total (ART), 65,40 mL/L de AMM e 1,27 mL/L de Tween 80. Nas otimizações com Leuconostoc mesenteroides B512 foi observado produção de 60,20 g/L de D-(-) ácido lático, utilizando 116,90 g/L de ART de caldo de cana e 44,25 g/L de autolisado de levedura. Nas otimizações com L. plantarum Lmism6 observou-se uma produção de 63,40 g/L de ácido lático, 0,40 g/L de ART residual e alta conversão de substrato (99,40%), quando se utilizou 70 g/L de ART de melaço, 30,00 mL/L de AMM, 2 g/L de K2HPO4 e 1 mL/L de Tween 80 / Abstract: The aim of this study was to isolate D-(-) and L-(+) lactic acid producers micro-organisms, which are used in the synthesis of polymers used in the production of many resistant and biodegradable materials and optimize the lactic acid production, from agro-industrial residues. The most promising micro-organisms for L-(+) lactic acid production were Lactobacillus rhamnosus B103, as well as, the isolated from Keffir (Ke6, Ke11, Ke8 Ke24) and the most promising D-(-) lactic acid producers were strains of yogurt (Y15C and Y15A) and Leuconostoc mesenteroides B512. Cheese whey and cassava wastewater (CW) were the best residues for L-(+) lactic acid production by Lactobacillus rhamnosus B103. Using 160 g/L of lactose from whey, 60 mL/L of CSL, 2 mL/L of Tween 80 and 0.10 g/L of MnSO4, there was higher production of L-(+) lactic acid (142 g/L) and low lactose residual (3.20 g/L). For optimizations with CW, it was obtained 41.58 g/L of L-(+) lactic acid from 50 g/L of reducing sugar, 65.40 mL/L and 1.27 mL of corn steep liquor (CSL) and Tween 80 respectively. Leuconostoc mesenteroides B512 produced 60.20 g/L of D-(-) lactic acid, using 116.90 g/L of sugarcane juice and 44.25 g/L of yeast autolysate. L. plantarum Lmism6 produced 63.40 g/L of lactic acid, with less residual reducing sugar (0.41 g/L) and higher substrate conversion (99.41%), by using 70 g/L of sugar reducing from molasses, 30 mL/L of CSL, 2 g/L of K2HPO4, and 1 mL/L of Tween 80 / Doutor

A tecnologia da microencapsulação apresenta várias aplicações na indústria de alimentos. Sabendo-se que diferentes fatores intrínsecos e extrínsecos dos alimentos podem influenciar a produção e atividade antimicrobiana das bacteriocinas produzidas pelas bactérias láticas, este estudo teve como principal objetivo avaliar a funcionalidade da encapsulação de bactérias láticas (BAL) bacteriocinogênicas em alginato de cálcio no controle de Listeria monocytogenes em salame experimentalmente contaminado. Para atingir este objetivo, foram isoladas novas cepas de BAL a partir de salame, que foram identificadas e caracterizadas quanto às propriedades das bacteriocinas produzidas, avaliando-se a influência do processo de encapsulação na produção de bacteriocinas. Foram isoladas quatro cepas produtoras de bacteriocinas, identificadas como Lactobacillus sakei (uma cepa), Lactobacillus curvatus (duas cepas) e Lactobacillus plantarum (uma cepa), nomeadas MBSa1, MBSa2, MBSa3 e MBSa4, respectivamente. As bacteriocinas produzidas pelas quatro cepas foram termoestáveis e com exceção da cepa MBSa2, sensíveis a pH acima de 8. Todas inibiram todas as cepas de Listeria monocytogenes testadas e várias espécies de BAL, mas foram inativas contra bactérias Gram negativas. As bacteriocinas foram purificadas por cromatografia de troca iônica seguida de cromatografia de interação hidrofóbica sequencial e cromatografia de fase reversa, observando-se que L. sakei MBSa1 produz um peptídeo de 4303 Da, com uma sequência parcial de aminoacidos idêntica à sequência presente em sakacina A. As cepas MBSa2 e MBSa3 produzem dois peptídeos ativos cada, idênticos nas duas cepas, um de 4457 Da e outro de 4360 Da, que apresentam sequências parciais idênticas às presentes na sakacina P e na sakacina X, respectivamente. Aparentemente, a cepa L. plantarum MBSa4 produz uma bacteriocina composta por duas sub-unidades. O DNA genômico da cepa L. sakei MBSa1 contém os genes da sakacina A e curvacina A, enquanto o DNA da cepa L. plantarum MBSa4 foi positivo para o gene da plantaricina W. A cepa L. curvatus MBSa2 foi encapsulada em alginato de cálcio e testada quanto à produção de bacteriocinas in vitro, observando-se que o processo de encapsulação não influenciou a produção de bacteriocina. Quando testada in situ, ou seja, no salame experimentalmente contaminado com Listeria monocytogenes, não foi observada ação anti-Listeria por L. curvatus MBSa2 encapsulado e não encapsulado, durante o 30 dias de fabricação do salame. / The microencapsulation technology has several applications in the food industry. Knowing that different intrinsic and extrinsic factors can influence production and antimicrobial activity of bacteriocins produced by lactic acid bacteria in foods, this study aimed at evaluating the functionality of the encapsulation of bacteriocinogenic lactic acid bacteria (LAB) in calcium alginate in the control of Listeria monocytogenes in experimentally contaminated salami. To achieve this goal, new strains of LAB were isolated from salami, identified and characterized for the properties of the produced bacteriocins, evaluating the influence of the encapsulation process in the bacteriocins production. Four bacteriocin producing strains were isolated and identified as Lactobacillus sakei (one strain), Lactobacillus curvatus (two strains) and Lactobacillus plantarum (one strain), named MBSa1, MBSa2, MBSa3 and MBSa4 respectively. The bacteriocins produced by the four strains were thermostable and with the exception of strain MBSa2, sensitive to pH above 8. All inhibited all tested Listeria monocytogenes strains and various species of LAB but were inactive against Gram-negative bacteria. The bacteriocins were purified by cation-exchange followed by sequential hydrophobic-interaction and reversed-phase chromatography, indicating that L. sakei MBSa1 produces a peptide of 4303 Da, with a partial amino acid sequence identical to the sequence present in sakacin A. L. curvatus MBSa2 and MBSa3 produce two active peptides, identical in the two strains, one of 4457 Da and the other of 4360 Da, with partial aminoacid sequences identical to those present in sakacin X and sakacin P, respectively. Apparently, L. plantarum MBSa4 produces a bacteriocin composed of two subunits. Genomic DNA of L. sakei MBSa1indicated that this strain contains genes for sakacin A and curvacin A, while the DNA of L. plantarum MBSa4 was positive for the plantaricin W gene. The strain L. curvatus MBSa2 was encapsulated in calcium alginate and tested for bacteriocin production in vitro, observing that the encapsulation process did not affect the production of bacteriocin. When tested in situ, i.e. in the salami experimentally contaminated with L. monocytogenes was not observed anti-Listeria<i/> action by L. curvatus MBSa2 encapsulated and non-encapsulated during the 30 day manufacture of salami.

Probiotics are viable nonpathogenic microbes that positively affect host health. Probiotics inhibit infection, activate immunity, and promote mucosal-barrier development. Many microbes have probiotic activity. Nonetheless, the selection of stable strains and their specific mechanism(s) of action are not fully elucidated. Bacteria from ‘Amabere amaruranu’ cultured milk from Kenya were isolated and identified by PCR sequence analysis of the 16S rRNA gene. Isolates were examined for stability to acid and bile, antimicrobial activity, mucin production, and degradation and sensitivity to antibiotics, hence their potential for probiotics. Lactobacillus isolates were acid unstable, bile-stable, nonmucinolytic, and presented antibacterial activity. L. rhamnosus cell fractions increased MUC4 and MUC3 expression in colon cells. Bacillus isolates were acid and bile stable, nonmucinolytic and lacked antimicrobial activity. In conclusion, Lactobacillus isolates that were nonmucinolytic, stable in bile, demonstrated antibacterial activity, sensitive to antibiotics, and stimulated increase MUC4 and MUC3 levels in colon cells could be potential probiotics.

A challenge study to validate a 5 log10 CFU/g reduction of non-O157 Shiga-toxin producing Escherichia coli (STEC) in dry fermented sausage (DFS) was performed. A 4.49 ± 0.474 log10 CFU/g was achieved over two trials. The results indicated that the process was not effective in reducing the pathogen to the level required of most pathogens by the USDA. Lactobacillus salivarius L28 (L28) was screened in vitro for the ability to inhibit STEC utilizing the paper disk diffusion method. This strain is a known bacteriocin producer. The results revealed that L28 would be a good candidate for use as a protective culture as large zones of inhibition were noted against the STEC. No zones of inhibition were noted against the commercial starter culture; therefore, it would not adversely impact the quality of the DFS. The supplementary L28 strain was added to a commercial starter culture to provide an additional hurdle in the protection against STEC. The sausage trial showed the additional strain did not offer a significant difference in reduction of the pathogen (p > 0.05). Further study will be required before L28 could be considered for use as a bioprotective culture.

Probiotics are marketed throughout the world to promote the health of the consumer by improving the microorganisms that normally occur in the intestinal tract (Tannock, 1997). It has also been suggested that probiotics can prevent pathogen infections by adhering to the intestinal mucosa (Lee, Lim, Teng, Ouwehand, Tuomola, & Salminen, 2000). While probiotics can be delivered to the infected areas in multiple fashions, microencapsulation is a newer form of delivering probiotics straight to the infected area. A whey protein microcapsule is thought to protect the probiotics from stomach acid and delivers the treatment to the affected area. To ensure this microencapsulation treatment is affective, the microcapsules will be stained and imaged to see if the microcapsules are constructed in a way which is consistent with the theory: a whey protein microcapsule surrounding bacteria and fat droplets. Through these experiments, it was shown that the microcapsule was not constructed as previously thought. Instead of a thin layer of protein surrounding the bacteria, it more closely resembled a solid ball of protein with bacteria and fat trapped inside. The bacteria are able to survive stomach like conditions (0.1M HCl for 8 hours) due to other forms of microencapsulation.