The impact of saving and credit cooperatives on food security in the West Amhara Region of Ethiopia

In rural Ethiopia, among other things, lack of adequate financial service is considered as the basic problem to alleviate rural poverty and to solve the problem of food insecurity. Commercial banks are restricted to urban centres. Providing rural financial service through RUSACCO to the poor has been proposed as a tool for economic development and for achieving food security. Evidence from research in this regard has been so far scanty, especially in rural Ethiopia. The aims of this study are to analyze the determinants of membership, to identify socioeconomic and demographic factors that influence members’ participation in RUSACCOs and to quantify the impact of RUSACCOs on member households’ food security. The study was conducted in two purposely selected woredas in the Amhara region one from food insecure (Lay Gayint woreda) and the other from food secure (Dejen woreda). Six RUSACCOs were selected randomly from these two woredas. Both qualitative and quantitative data were collected. Key informant interviews, focus group discussions and survey techniques were used to collect primary data. Collected data was then analyzed using mixed methods depending on the nature of data. For quantitative data analysis appropriate statistical models were used. The study result reveals that the number of members in each RUSACCO is very small. However, the majority of non-member respondents are willing to join RUSACCO. Lack of information about the benefits of RUSACCO membership is the main problem why many rural poor do not join RUSACCOs. Members participate in different aspects of the cooperatives, starting from attending general assembly up to board membership. They also participate actively in saving and borrowing activities of RUSACCO. The majority of the respondents believe the RUSACCO is a vital instrument in combating food insecurity. The empirical findings indicate that gender, marital status, occupation, educational level, participation in local leadership and participation in other income generation means determine the decision of rural poor to join a RUSACCO or not. The amount of saving is determined by household head occupation, farming experience and income level. While age of household head, primary occupation, farming experience, date of membership, annual total consumption expenditure, amount of saving and participation in other income generation activities influence members’ amount of borrowing by RUSACCO members. Finally, the study confirms that RUSACCO participation improves household food security. RUSACCO membership has made positive impact on household total consumption expenditure and food expenditure.

Lactobacillus rossiae, a vitamin B12 producer, represents a metabolically versatile species within the genus Lactobacillus

Lactobacillus rossiae is an obligately hetero-fermentative lactic acid bacterium, which can be isolated from a broad range of environments including sourdoughs, vegetables, fermented meat and flour, as well as the gastrointestinal tract of both humans and animals. In order to unravel distinctive genomic features of this particular species and investigate the phylogenetic positioning within the genus Lactobacillus, comparative genomics and phylogenomic approaches, followed by functional analyses were performed on L. rossiae DSM 15814(T), showing how this type strain not only occupies an independent phylogenetic branch, but also possesses genomic features underscoring its biotechnological potential. This strain in fact represents one of a small number of bacteria known to encode a complete de novo biosynthetic pathway of vitamin B-12 (in addition to other B vitamins such as folate and riboflavin). In addition, it possesses the capacity to utilize an extensive set of carbon sources, a characteristic that may contribute to environmental adaptation, perhaps enabling the strain's ability to populate different niches.

Biodiscovery of natural products from microbes associated with Irish coastal sponges

Marine sponges have been an abundant source of new metabolites in recent years. The symbiotic association between the bacteria and the sponge has enabled scientists to access the bacterial diversity present within the bacterial/sponge ecosystem. This study has focussed on accessing the bacterial diversity in two Irish coastal marine sponges, namely Amphilectus fucorum and Eurypon major. A novel species from the genus Aquimarina has been isolated from the sponge Amphilectus fucorum. The study has also resulted in the identification of an α–Proteobacteria, Pseudovibrio sp. as a potential producer of antibiotics. Thus a targeted based approach to specifically cultivate Pseudovibrio sp. may prove useful for the development of new metabolites from this particular genus. Bacterial isolates from the marine sponge Haliclona simulans were screened for anti–fungal activity and one isolate namely Streptomyces sp. SM8 displayed activity against all five fungal strains tested. The strain was also tested for anti–bacterial activity and it showed activity against both against B. subtilis and P. aeruginosa. Hence a combinatorial approach involving both biochemical and genomic approaches were employed in an attempt to identify the bioactive compounds with these activities which were being produced by this strain. Culture broths from Streptomyces sp. SM8 were extracted and purified by various techniques such as reverse–phase HPLC, MPLC and ash chromatography. Anti–bacterial activity was observed in a fraction which contained a hydroxylated saturated fatty acid and also another compound with a m/z 227 but further structural elucidation of these compounds proved unsuccessful. The anti–fungal fractions from SM8 were shown to contain antimycin–like compounds, with some of these compounds having different retention times from that of an antimycin standard. A high–throughput assay was developed to screen for novel calcineurin inhibitors using yeast as a model system and three putative bacterial extracts were found to be positive using this screen. One of these extracts from SM8 was subsequently analysed using NMR and the calcineurin inhibition activity was con rmed to belong to a butenolide type compound. A H. simulans metagenomic library was also screened using the novel calcineurin inhibitor high–throughput assay system and eight clones displaying putative calcineurin inhibitory activity were detected. The clone which displayed the best inhibitory activity was subsequently sequenced and following the use of other genetic based approaches it became clear that the inhibition was being caused by a hypothetical protein with similarity to a hypothetical Na+/Ca2+ exchanger protein. The Streptomyces sp. SM8 genome was sequenced from a fragment library using Roche 454 pyrosequencing technology to identify potential secondary metabolism clusters. The draft genome was annotated by IMG/ER using the Prodigal pipeline. The Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession AMPN00000000. The genome contains genes which appear to encode for several polyketide synthases (PKS), non–ribosomal peptide synthetases (NRPS), terpene and siderophore biosynthesis and ribosomal peptides. Transcriptional analyses led to the identification of three hybrid clusters of which one is predicted to be involved in the synthesis of antimycin, while the functions of the others are as yet unknown. Two NRPS clusters were also identified, of which one may be involved in gramicidin biosynthesis and the function of the other is unknown. A Streptomyces sp. SM8 NRPS antC gene knockout was constructed and extracts from the strain were shown to possess a mild anti–fungal activity when compared to the SM8 wild–type. Subsequent LCMS analysis of antC mutant extracts confirmed the absence of the antimycin in the extract proving that the observed anti–fungal activity may involve metabolite(s) other than antimycin. Anti–bacterial activity in the antC gene knockout strain against P. aeruginosa was reduced when compared to the SM8 wild–type indicating that antimycin may be contributing to the observed anti–bacterial activity in addition to the metabolite(s) already identified during the chemical analyses. This is the first report of antimycins exhibiting anti–bacterial activity against P. aeruginosa. One of the hybrid clusters potentially involved in secondary metabolism in SM8 that displayed high and consistent levels of gene–expression in RNA studies was analysed in an attempt to identify the metabolite being produced by the pathway. A number of unusual features were observed following bioinformatics analysis of the gene sequence of the cluster, including a formylation domain within the NRPS cluster which may add a formyl group to the growing chain. Another unusual feature is the lack of AT domains on two of the PKS modules. Other unusual features observed in this cluster is the lack of a KR domain in module 3 of the cluster and an aminotransferase domain in module 4 for which no clear role has been hypothesised.

Studies on factors relating to the development of defects in commercial cheese

Defects in commercial cheese result in a downgrading of the final cheese and a consequential economic loss to the cheese producer. Developments of defects in cheese are often not fully understood and therefore not controllable by the producer. This research investigated the underlying factors in the development of split and secondary fermentation defect and of pinking defects in commercial Irish cheeses. Split defect in Swiss-type cheese is a common defect associated with eye formation and manifests as slits and cracks visible in the cut cheese loaf (Reinbold, 1972; Daly et al., 2010). No consensus exists as to the definitive causes of the defect and possible factors which may contribute to the defect were reviewed. Models were derived to describe the relationship between moisture, pH, and salt levels and the distance from sample location to the closest external block surface during cheese ripening. Significant gradients within the cheese blocks were observed for all measured parameters in cheeses at 7 day post/after manufacture. No significant pH gradient was found within the blocks on exit from hot-room ripening and at three months post exit from the hot-room. Moisture content reached equilibrium within the blocks between exit from hot-room and 3 months after exit from hot-room while salt and salt-to-moisture levels had not reached equilibrium within the cheese blocks even at three months after exit from hot-room ripening. A characterisation of Swiss-type cheeses produced from a seasonal milk supply was undertaken. Cheeses were sampled on two days per month of the production year, at three different times during the manufacturing day, at internal and external regions of the cheese block and at four ripening time points (7 days post manufacture, post hot-room, 14 days post hot-room and 3 months in a cold room after exit from hot-room). Compositional, biochemical and microbial indices were determined, and the results were analysed as a splitplot with a factorial arrangement of treatments (season, time of day, area) on the main plot and ripening time on the sub-plot. Season (and interactions) had a significant effect on pH and salt-in-moisture levels (SM), mean viable counts of L. helveticus, propionic acid and non-starter lactic acid bacteria, levels of primary and secondary proteolysis and cheese firmness. Levels of proteolysis increased significantly during hot-room ripening but also during cold room storage, signifying continued development of cheese ripening during cold storage (> 8°C). Rheological parameters (e.g. springiness and cohesiveness) were significantly affected by interactions between ripening and location within cheese blocks. Time of day of manufacture significantly affected mean cheese calcium levels at 7 days post manufacture and mean levels of arginine and mean viable counts of NSLAB. Cheeses produced during the middle of the production day had the best grading scores and were more consistent compared to cheeses produced early or late during day of manufacture. Cheeses with low levels of S/M and low values of resilience were associated with poor grades at 7 days post manufacture. Chesses which had high elastic index values and low values of springiness in the external areas after exit from hot-room ripening also obtained good commercial grades. Development of a pink colour defect is an intermittent defect in ripened cheese which may or may not contain an added colourant, e.g., annatto. Factors associated with the defect were reviewed. Attempts at extraction and identification of the pink discolouration were unsuccessful. The pink colour partitioned with the water insoluble protein fraction. No significant difference was observed between ripened control and defect cheese for oxygen levels and redox potential or for the results of elemental analysis. A possible relationship between starter activity and defect development was established in cheeses with added coulourant, as lower levels of residual galactose and lactose were observed in defective cheese compared to control cheese free of the defect. Swiss-type cheese without added colourant had significantly higher levels of arginine and significantly lower lactate levels. Flow cell cytometry indicated that levels of bacterial cell viability and metabolic state differed between control and defect cheeses (without added colourant). Pyrosequencing analysis of cheese samples with and without the defect detected the previously unreported bacteria in cheese, Deinococcus thermus (a potential carotenoid producer). Defective Swiss-type cheeses had elevated levels of Deinococcus thermus compared to control cheeses, however the direct cause of pink was not linked to this bacterium alone. Overall, research was undertaken on underlying factors associated with the development of specific defects in commercial cheese, but also characterised the dynamic changes in key microbial and physicochemical parameters during cheese ripening and storage. This will enable the development of processing technologies to enable seasonal manipulation of manufacture protocols to minimise compositional and biochemical variability and to reduce and inhibit the occurrence of specific quality defects.

Thuricin CD: a potential therapeutic targeted against Clostridium difficile-associated diarrhea (CDAD)

Clostridium difficile is mainly a nosocomial pathogen and is a significant cause of antibioticassociated diarrhea. It is also implicated in the majority of cases of pseudomembranous colitis. The main etiological agent of C. difficile-associated diarrhea (CDAD) is perturbations to the gut microbiota by broad-spectrum antibiotics. Recently, thuricin CD, a two-peptide narrow spectrum sactibiotic bacteriocin with potent activity against C. difficile has been discovered. It is produced by Bacillus thuringiensis DPC6431. The efficacy of thuricin CD against a range of C. difficile clinical isolates has been determined in the form of minimum inhibitory concentration (MIC) values and compared to metronidazole, vancomycin, ramoplanin and actagardine in this thesis. Furthermore, by assessing paired combinations of the above-mentioned antimicrobials, it was determined that ramoplanin and actagardine function in a synergistic manner against the majority of C. difficile isolates. The functions of the genes in the thuricin CD gene cluster have also been elucidated by cloning the cluster and expressing thuricin CD in a heterologous Bacillus subtilis host and are described herein. In addition, the immunity mechanisms employed by the B. thuringiensis DPC6431 producer to protect itself from the antimicrobial actions of thuricin CD have also been elucidated. It has been shown that a small immunity peptide, TrnI, is involved in thuricin CD immunity, most likely by intercepting the thuricin CD peptides and/or blocking their access to the thuricin CD receptor. This immunity peptide and also the ABC-transporter system TrnFG serve to protect the B. thuringiensis host against thuricin CD.

Toxicity of manufactured nano-ZnO to Lemna minor

The rapid development of nanotechnology has led to a rise in the large-scale production and commercial use of engineered nano-ZnO. Engineered/manufactured nano-ZnO are applied in a broad range of products such as drugs, paints, cosmetics, abrasive agents and insulators. This can result in the unintended exposure of human beings to nano-ZnO and will inevitably result in the release of nano-ZnO in to the environment. Thus, it is necessary to assess the risk of nano-ZnO to the environment. In this thesis the toxicity of nano-ZnO was analysed using the aquatic, primary producer lesser duckweed (Lemna minor), and the mechanism of toxicity was analysed. Both short-term (one week) and long-term (six weeks) toxicity of nano-ZnO (uncoated) were determined. Results show that the toxicity of nano-ZnO added to the aquatic growth medium increases with increasing concentration and that toxicity accumulates with exposure time. A study of nano-ZnO dissolution reveals that the main reason for nano-ZnO toxicity on Lemna minor is the release of Zn ions. Nano-ZnO dissolution is pH dependent, and toxicity matches the release of Zn2+. Functional coating materials are commonly added to nano-ZnO particles to improve specific industrial applications. To test if coating materials contribute to nano-ZnO toxicity on lesser duckweed, the effect of silane coupling agent (KH550) coated nano-ZnO on Lemma minor was investigated. Results show that coating can decrease the release of Zn ions, which reduces toxicity to Lemna minor, in contrast to uncoated particles. Another commonly hypothesized reason for nano-ZnO toxicity is the formation of Reactive Oxygen Species (ROS) on the particles surface. As part of this thesis, the ROS formation induced by nano-ZnO was studied. Results show that nano-ZnO catalyse ROS formation and this can negatively affect duckweed growth. In conclusion, this work has detailed potentially toxic effects of nano-ZnO on Lemna minor. This study has also provides references for future research, and informs regulatory testing for nanoparticle toxicity. Specifically, the outcomes of this study emphasize the importance of exposure time, environmental parameters and coating material when analysing NPs toxicity. Firstly, impacts of longer exposure time should be studied. Secondly, environmental parameters such as pH and medium-composition need to be considered when investigating NPs toxicity. Lastly, coating of NPs should always be considered in the context of NPs toxicity, and similar NPs with different coatings require separate toxicity tests.

Isolation and characterisation of antifungal compounds from lactic acid bacteria and their application in wheat and gluten-free bread

As part of the “free-from” trend, biopreservation for bread products has increasingly become important to prevent spoilage since artificial preservatives are more and more rejected by consumers. A literature review conducted as part of this thesis revealed that the evaluation of more suitable antifungal strains of lactic acid bacteria (LAB) is important. Moreover, increasing the knowledge about the origin of the antifungal effect is fundamental for further enhancement of biopreservation. This thesis addresses the investigation of Lactobacillus amylovorus DSM19280, Lb. brevis R2: and Lb. reuteri R29 for biopreservation using in vitro trials and in situ sourdough fermentations of quinoa, rice and wheat flours as biopreservatives in breads. Their contribution to quality and shelf life extension on bread was compared and related to their metabolic activity and substrate features. Moreover, the quantity of antifungal carboxylic acids produced during sourdough fermentation was analysed. Overall a specific profile of antifungal compounds was found in the sourdough samples which were strain and substrate dependently different. The best preservative effect in quinoa sourdough and wheat sourdough bread was achieved when Lb. amylovorus DSM19280 fermented sourdough was used. However, the concentration of the antifungal compounds found in these biopreservatives were much lower when compared with Lb. reuteri R29 as the highest producer. Nevertheless, the artificial application of the highest concentration of these antifungal compounds in chemically acidified wheat sourdough bread succeeded in a longer shelf life than achieved only by acidifying the dough. This evidences their partial contribution to the antifungal activity and their synergy. Additionally, a HRGC/MS method for the identification and quantification of the antifungal active compounds cyclo(Leu-Pro), cyclo(Pro-Pro), cyclo(Met-Pro) and cyclo(Phe-Pro) was successfully developed by using stable isotope dilutions assays with the deuterated counterparts. It was observed that the concentrations of cyclo(Leu-Pro), cyclo(Pro-Pro), and cyclo(Phe-Pro) increased only moderately in MRS-broth and wort fermentation by the activity of the selected microorganism, whereas the concentration of cyclo(Met-Pro) stayed unchanged.