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DNA barcoding is a valuable tool for fish species identification by food regulators, however, it does not perform well when multiple species are present within the same food product. PCR cloning has high potential to be used in combination with DNA barcoding to overcome this challenge. The objective of this study was to examine the use of PCR cloning combined with DNA barcoding to identify fish in a mixed-species product that cannot be identified with standard DNA barcoding. A total of 15 fish ball mixtures were prepared with known amounts of Nile tilapia (Oreochromis niloticus), Pacific cod (Gadus macrocephalus), and walleye pollock (Gadus chalcogrammus). The fish balls underwent DNA extraction in triplicate, followed by DNA barcoding across the full barcode (655 bp) and SH-E mini-barcode (226 bp) of the cytochrome c oxidase subunit 1 (CO1) region. Samples that did not pass sequencing according to regulatory standards were further analyzed with PCR cloning. Full barcoding enabled identification of at least one species in 80% of the fish ball mixtures compared to 51% for minibarcoding. The results of PCR cloning with samples that did not pass DNA barcoding showed identification success rates of 61% for clones (54 of 90) that underwent full barcoding and 51% for clones (111 of 220) that underwent mini-barcoding. All fish balls made of just one species tested positive for that species (i.e., tilapia, cod, or pollock).. The combination of standard full barcoding and PCR cloning enabled identification of Nile tilapia in all 12 mixed-species fish balls and Pacific cod in 6 of 12 (50%) of mixed-species fish balls. In comparison, the combination of standard mini-barcoding and PCR cloning enabled identification of Nile tilapia in all 12 mixed-species fish balls and Pacific cod in 9 of 12 (75%) of mixed-species fish balls. Overall, the results of this study show that PCR cloning may be an effective method to identify certain fish in mixed-species products when standard DNA barcoding fails. However, additional research is needed to understand the limitations associated with primer bias.

Rhizopus oryzae is a filamentous fungi which produces lactic acid and ethanol in fermentations. R. oryzae has numerous advantages for use industrial production of L-(+)-lactic acid but the yield of lactic acid produced on the basis of carbon consumed is low. Metabolic flux analysis of R. oryzae has shown that most of the pyruvate produced at the end of the glycolysis is channelled to ethanol, acetyl-CoA and oxaloacetate production. This study aimed to answer some questions addressed on the regulation of pyruvate branch point in R. oryzae and for this purpose biochemical characterisation of the enzymes acting at this branch point and cloning the genes coding for these enzymes have been done. Pyruvate decarboxylase was purified and characterised for the first time from R. oryzae. The purified enzyme has a Hill coefficient of 1.84 and the Km of the enzyme is 8.6 mM for pyruvate at pH 6.5. The enzyme is inhibited at pyruvate concentrations higher than 30 mM. The optimum pH for enzyme activity shows a broad range from 5.7 and 7.2. The monomer molecular weight was estimated as 59&plusmn / 2 kDa by SDS-PAGE analysis. Pyruvate decarboxylase (pdcA and pdcB) and lactate dehydrogenase (ldhA and ldhB) genes of R. oryzae have been cloned by PCR-cloning approach and the filamentous fungi Aspergillus niger was transformed with these genes. The A. niger transformed with either of the ldh genes of R. oryzae showed enhanced production of lactic acid compared to wild type. Citric acid production was also increased in these transformants while no gluconate production was observed Cloning of hexokinase gene from R. oryzae using degenerate primers was studied by the use of GenomeWalker kit (Clontech). The results of this study were evaluated by using some bioinformatics tools depending on the unassembled clone sequences of R. oryzae genome.