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Orbit Biotech, India

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Samenvatting eindwerk 1 2014-2015: Partial Characterization, Genomic DNA amplification and optimization of plantaricin production from isolated Lactobacillus plantarum (LP1 and LP2)
Probiotics are the live microorganisms that have beneficial effect on human health. It reduces the growth of harmful bacteria in the gut. Probiotics have become increasingly popular during the last two decades as a result of the continuously expanding scientific evidence pointing to their beneficial effects on human health. As a result, they have been combined with various products within the food industry.
The purpose of this rapport is to study the characteristics and antimicrobial activity of probiotic attributes of Lactobacillus plantarum (LP1 and LP2). The pathogens which the antimicrobial activity that were investigated are E.coli, S.epidermidis, K. pneumonia, Micrococcus, C. albicans, B. subtilis, V. cholera, S. aureus, E. faelcalis and S. typhi.
Probiotics that are consumed by humans has to survive the intestinal tract. The viability of these cells after consumption remains obscure as the bacteria are also influenced by unpreferred physiological condition of the gastrointestinal tract. So both strains are checked for attributes such as heat stability, acid tolerance and bile tolerance. A bacterial strain is identified as a probiotic when all of previous criteria are fulfilled to increase the viability rate of probiotics in the human body.
Lactobacillus plantarum was found to produce plantaricin whose antimicrobial activity was analyzed by agar well diffusion assay against the pathogens. The cell free supernatant decrease growth of the pathogens. This proves that the antimicrobial activity is due to the production of bacteriocin. The minimum inhibitory concentration of the plantaricin produced by LP1 and LP2 was investigated against the pathogen by the Mueller Hinton Broth dilution method to determine the lowest concentration of a particular antibiotic needed to kill the bacteria.
Samenvatting eindwerk 2 2014-2015: Partial characterization, genomic DNA amplification and optimization of reuterin production from isolated Lactobacillus reuteri (LR1 and LR2)
Research on probiotics is very important as probiotics have many opportunities in the future. These may be the antibiotics of the 21st century. This is obviously a great step forward because some pathogens already created a resistance to the currently available antibiotics. This is just one advantage of the various benefits of probiotics. Probiotics also helps the immune system to induce an optimal response. It is also relevant that the safety of probiotics must be ensured. Many experiments and projects are working on this matter and several times the safety has been confirmed. This project investigated whether Lactobacillus reuteri possesses the qualities needed to be a good probiotic. Firstly, the stock culture was checked to confirm that it is indeed Lactobacillus reuteri. The genus was confirmed by a Gram stain and various biochemical tests. Afterwards the bacteria are tested to check if it can survive in the body, otherwise it would be useless to continue working on these bacteria. There are four different tests: thermal death point (TDP), thermal death time (TDT), acid tolerance and bile tolerance. By carrying out these experiments, we showed that Lactobacillus reuteri is able to survive in the human body. Further, there will be worked with reuterin, this is a bacteriocin of the species Lactobacillus reuteri. Here, the species first have to be confirmed. This was done by carrying out a PCR reaction with specific primers for the species reuteri. After the DNA was extracted from a sample of Lactobacillus reuteri, PCR amplification was performed. In gel electrophoresis bands were obtained so the species are confirmed. Here, bands were obtained so the species are confirmed. Finally, it was determined whether L. reuteri can inhibit pathogens. For this, a bacteriocin is needed, L. reuteri has several bacteriocins but in this project we will work with reuterin. The inhibitory effect of reuterin was tested against ten different strains of pathogens; S. typhi, B. subtilis, V. cholerae, E. faecalis, S. aureus, S. epidermidis, K. pneumoniae, C . albicans, E. coli and Micrococcus.  This is determined on the basis of antimicrobial plates. The pathogen is spread on a plate with wells. These wells are filled with reuterin and when reuterin inhibits the pathogen, there will be a clear formed zone around the wells. On each plate with pathogen there were clear zones, hereby could be concluded that Lactobacillus reuteri can inhibit the pathogens listed. At last, the minimal inhibition concentration (MIC) of reuterin to inhibit the pathogens was determined.


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