Ismail Akyol

Biogenic amines formation in Streptococcus thermophilus isolated from home-made natural yogurt

Twelve different biogenic amines formation in 58 isolates of Streptococcus thermophilus from home-made natural yogurt were investigated in histidine (HDB) and lysine decarboxylase broth (LDB). All S. thermophilus isolates had an ability to produce twelve different biogenic amines in HDB and LDB. Most of the S. thermophilus isolates formed low amounts of histamine (1-50 mg/L) from histidine. Apart from one isolate, S. thermophilus produced tyramine at low (47 isolates) and medium (10 isolates) levels. The amount of each specific biogenic amine produced by S. thermophilus was generally lower than 100 mg L(-1). Also, the presence of hdcA gene was investigated using PCR technique and relation between gene and histamine production was conducted in S. thermophilus isolates. This study showed that most of the S. thermophilus isolates have the ability to form biogenic amines, especially histamine, and tyramine, which is an important consideration when selecting strains as starter cultures.

The function of lactic acid bacteria and brine solutions on biogenic amine formation by foodborne pathogens in trout fillets.

The influences of lactic acid bacteria and brine solutions on the biogenic amine formation by Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Enterococus faecalis, Pseudomonas aeruginosa, Listeria monocytogenes, Aeromonas hydrophila and Salmonella paratyphi A in fermented trout fillets were investigated. Fish fillets were divided into four groups, group 1 without any lactic acid bacteria inoculation, group 2 and group 3 with different salt concentration inoculated with lactic acid bacteria and food-borne pathogens, and group 4 inoculated with lactic acid bacteria and food-borne pathogens without a salt solution. The histamine content in trout fillets in group 4 was found to be more than 10mg/100g, while the other groups contained less than 7.5mg/100g. The highest tyramine production was found for group 1 and group 3, ranging from 3 to 18mg/100g. Lactic acid bacteria did not seem to play an important role on biogenic amine production by food borne pathogens, while adding brine solution on fillets has inhibitory effects on some of the biogenic amines.

Expression of fungal cellulase gene in Lactococcus lactis to construct novel recombinant silage inoculants.

The facultative anaerobic bacterium Lactococcus lactis has been used as a host for expression of a gene isolated from the anaerobic rumen fungus Neocallimastix sp. The coding region of the cellulase gene was obtained from the fungus with the aid of polymerase chain reaction amplification. The gene was then transformed into pCT vector system and the constructed recombinant plasmid was introduced into two L. lactis strains (IL403 and MG1363) by electroporation. The gene encoding the fungal originated cellulase was expressed in both strains successfully although the expression level was relatively lower in comparison with the original enzyme activity. Genetically modified L. lactis strains were used as silage inoculants for pre-biodegradation of the plant biomass during ensiling. That treatment resulted in a notable reduction of the acid detergent fiber (ADF) and neutral detergent fiber (NDF) contents of the plant biomass used as silage material. Inoculation with recombinant strain IL1043 resulted in 4.8 and 9.7 % decrease in NDF and ADF contents, respectively while the inoculation of silage with strain MG1363 decreased the ADF content by >5 %.

Carboxymethylcellulase production by the anaerobic rumen fungusNeocallimastix sp. GMLF7

Anaerobic fungi have highly active fibrolytic enzymes and these enzymes are attractive for scientific research. We isolated a ruminal fungus of the genusNeocallimastix sp., named GMLF7, which could survive on a variety of cellulosic material such as carboxymethylcellulose (CMC), fibrous cellulose, avicel and wheat straw. Carboxymethylcellulase production was investigated with the above carbon sources and high CMCase activity was obtained with CMC (73.75 U/ml), fibrous cellulose (72.68 U/ml) and avicel (70.03 U/ml). While growth temperature of the microorganism was 39°C, for CMCase activity optimal temperature was 50°C and optimal pH 6.5. Enzyme reached maximum activity in 60–180 min at 50°C. Substrate concentration also affected the enzyme activity which was increased more than 2 fold with 40 mg/ml CMC. Activity was measured in presence of various divalent ions and reagents, and Co2+ and DTT has a positive effect on the enzyme activity.

Redirection of Pyruvate Pathway of Lactic Acid Bacteria to Improve Cheese Quality

Metabolic engineering in Lactic acid bacteria (LAB) has focused on changing of pyruvate metabolism to increase production of desired flavor compounds. A constructed mutant strain should contain no foreign DNA and antibiotic resistance genes. Therefore, food grade lactate dehydrogenase (ldh d) and diacetyl reductase (dar d) mutant strains were created using two plasmid system in this study. Metabolic end products (pyruvate, lactate, formate and acetoin) of these strains in glucose medium and in cheese were determined using HPLC. Created mutant and wild type strains were used as a starter culture in cheese. Compared to the wild type strain, different levels of metabolites were observed in cheese during three weeks of ripening. The ldh d strains produced less lactate but high acetoin as a result of gene deletion. Deletion of dar gene decreased the production of acetoin. The dar deficient strains have low diacetyl reductase activity and are able to reduce significant amounts of acetoin but not terminate it completely. Genetic modification made the shift from homolactic to mixed acid fermentation, but the desired compound production hardly improved. The basis of these results and techniques are promising for the further studies.

Cloning of a xylanase gene xyn2A from rumen fungus Neocallimastix sp. GMLF2 in Escherichia coli and its partial characterization

Anaerobic fungi belonging to the family Neocallimastigaceae are native inhabitants in the rumen of the most herbivores, such as cattle, sheep and goats. A member of this unique group, Neocallimastix sp. GMLF2 was isolated from cattle feces and screened for its xylanase encoding gene using polymerase chain reaction. The gene coding for a xylanase (xyn2A) was cloned in Escherichia coli and expression was monitored. To determine the enzyme activity, assays were conducted for both fungal xylanase and cloned xylanase (Xyl2A) for supernatant and cell-associated activities. Optimum pH and temperature of the enzyme were found to be 6.5 and 50°C, respectively. The enzyme was stable at 40°C and 50°C for 20 min but lost most of its activity when temperature reached 60°C for 5-min incubation time. Rumen fungal xylanase was mainly released to the supernatant of culture, while cloned xylanase activity was found as cell-associated. Multiple alignment of the amino acid sequences of Xyl2A with published xylanases from various organisms suggested that Xyl2A belongs to glycoside hydrolase family 11.

Regulation of flpA , flpB and rcfA Promoters in Lactococcus lactis

E. coli fumarate nitrate reductase (FNR) binds to conserved FNR sites to regulate transcription under anaerobic condition. L. lactis subsp. cremoris MG1363 strain contains two FNR-like proteins (FlpA and B) encoded by flpA and flpB genes and the rcfA gene–encoded RcfA in L. lactis subsp. lactis IL1403 strain. Potential FNR-binding sites were located upstream of these genes. The flpA promoter is expressed in MG1363 anaerobically and aerobically. The flpB and rcfA promoters have typical class II FNR-dependent promoters and are activated anaerobically in MG1363 and IL1403, respectively. Despite their strong homology, the Flp and RcfA proteins cannot substitute for each other and control these promoters in the heterologous strains. The flpA and flpB promoters require FlpA and FlpB for activation in the MG1363 background. This was confirmed by expressing FlpB under nisin control in flp mutants and monitoring flpA promoter expression. In flpB– backgrounds, both FlpA and FlpB were required for flpA promoter expression. FlpB could not complement for the lack of FlpA protein in flpA− backgrounds.

Molecular study on cloned endoglucanase gene from rumen bacterium.

An endoglucanase gene was subcloned from anaerobic rumen bacterium Ruminococcus flavefaciens strain 17. To express endoglucanase gene in Escherichia coli and Streptococcus bovis JB1, an endoglucanase gene fragment was inserted into pVA838-based shuttle vectors. Removal of endoglucanase gene promoter and expression of endoglucanase by promoter of S. bovis JB1 α-amylase gene (pACMCS) was also achieved. Survival of constructs pVACMCI, pTACMC and pACMCS, which carry endoglucanase gene, and stability of endoglucanase gene in S. bovis JB1, were observed. Maximal endoglucanase activities from S. bovis JB1/pVACMCI were 2- to 3-fold higher than from E. coli/pVACMCI. Specific cell activity of E. coli/pACMCS was found to be approximately 2- to -3 fold higher than the both E. coli/pVACMCI and E. coli/pTACMC. Specific cell activity of S. bovis JB1/pACMCS was also found to be approximately 2-fold higher than the both S. bovis/pVACMCI and S. bovis JB1/pTACMC.

Cloning and Overexpression of the als, pflA, and adhB Genes in Streptococcus thermophilus and Their Effects on Metabolite Formation.

Streptococcusthermophilus is a lactic acid bacterium and used as starter culture in the dairy industry, mainly in the manufacture of yoghurt, with Lactobacillus delbrueckii subsp. bulgaricus. It produces lactic acid as a major fermentation end product and some carbonyl compounds through sugar metabolism. The level of metabolites could be improved using molecular biotechnology. The genes of als, encoding α-acetolactate synthase (Als), the pflA, encoding pyruvate-formate lyase activating enzyme (PflA), and the adhB which encodes alcohol dehydrogenase (AdhB) of S. thermophilus NCFB2393 strain were amplified by polymerase chain reaction and separately cloned into the overexpression vector pNZ276 under the control of the lacA promoter. The strains were transformed individually with the constructed plasmids. Their abilities to generate important metabolites such as pyruvate, lactate, formate, acetaldehyde, acetoin, ethanol, and 2,3-butanediol in LM17 medium were analyzed using high-performance liquid chromatography. High level of 2,3-butanediol was obtained by overexpressing the als gene. The level of formate increased slightly by overexpressing the pflA gene. The overexpression of the adhB gene, on the other hand, resulted in a significant increase in the ethanol level.

Regulation of the acid induciblercfB promoter inLactococcus lactis subsp.lactis

Although Lactococcus is one of the most extensively studied lactic acid bacteria, little information is available on the regulation of the catabolite receptor protein (CRP) and acid induced gene expression. ThercfB gene, encoding RcfB protein which has unknown function in Lactococcus lactis IL1403, was analysed. Promoter region contains consensus CRP binding site and acid inducible sequence at upstream of start site. A 402 bprcfB promoter was fused to upstream of b-galactosidase gene by cloning into promoterless pAK80 plasmid. Promoter activity corresponding to b-galactosidase was measured from Lactococcus lactis IL1403 transformant grown in medium M17 containing different carbon sources. We have found that thercfB promoter expression was highly induced by acidity and carbohydrate sources used in medium and growth phase of the culture.