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The Production of Bacteriocins from Lactic Acid Bacteria

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  Abstract Abstract  Lactobacilli are a group of Lactic Acid Bacteria widely used in the dairy industry nowadays. These bacteria are used as natural acidifiers for the inoculation of bulk quantities of milk, vegetables and fruitsin order to produce a variety of fermented products and beverages. Furthermore several strains of thisgroup produce substances of peptide structure that possesses antimicrobial activity against strains mainlyof the same genera, called bacteriocins. The only commercially available bacteriocin is Nisin produced by  Lactococcus lactis . As such, the need of investigating the production of such substances from other strains of LAB to develop a technique for their production on a large scale emerged. Initially threeknown bacteriocin producing strain  Lactobacillus casei NCIMB 11970, Lactobacillus plantarum NCIMB8014 and  Lactobacillus lactis NCIMB 38586  were selected. The possibility of producing the bacteriocinsof these bacteria in large quantities was investigated through several techniques. Firstly, the bacteriawere grown into simple batch cultures without pH control where the physicochemical needs for their optimum growth were determined. Through the determination of the optimum nutritional conditions for the propagation of the bacilli an optimised medium for growth occurred. The growth efficiency on themedium was tested on a 2L STR reactor operated batchwise with continuous pH control. The optimum pH conditions for the growth of the coccus were determined as well as parameters such as cellular yieldcoefficient, substrate and starter inoculum concentration.The metabolism of the bacilli was determined as homofermentative, mainly producing lactic acid Inaddition carbohydrate consumption rate in the optimum pH values were measured and the constantcoefficients for substrate consumption end product inhibition effect was determined. As the optimumconditions for growth were established, the amount of the bacteriocins produced on the optimisedmedium was investigated. A simple turbidometric method based on the bacteriostatic effect of thecommercially available Nisin on the growth of the selected target strain  L.delbruckii subsp. lactis  NCIMB 8117 was established. The assay developed was used in order to certify the exact amount of  bacteriocins produced by the  Lactobacilli .It was then confirmed that the chosen  Lactobacilli are indeed producing bacteriocins and the need for developing a successful separation and extraction strategy occurred. As bacteriocins are peptide structuresubstances ranging between 4 kDa to 10 kDa it was decided to develop nutrient media containing lowmolecular weight nutrient sources of 4kDa and 1 kDa molecular weight which would supportsimultaneously bacterial growth and high amounts of bacteriocins, in an effort to facilitate the extractionof the bacteriocins from the nutrient broths. This was achieved through filtration of the optimised mediathrough series of membrane filters ranging between 30kDa to 1 kDa MWCO. Studies were conducted toconfirm that these media can equally support bacteriocin production to the unfiltered media. The selected  Lactobacilli were found to produce an amount of bacteriocins ranging between 90 IU/ml top 100 IU/mlequivalent to Nisin.Further development of the extraction strategy of the desired substances contained the use of membranefilters of 1 kDa and 4 kDa MWCO to separate and further concentrate them. The potency of theconcentrated bacteriocins was tested against the target strain. It was found that their activity has beenenhanced to125 IU/ml equivalent to nisin. The substances were further separated through HighPerformance Liquid Chromatography and sized using Dynamic Light Scattering method. The peptidenature of the substances was as well confirmed. The exact phase of production of bacteriocins during thegrowth of   Lactobacilli was as well investigated and the substances were established as being primarymetabolites.The stability of the potency of the produced bacteriocins was investigated through numerous techniques.The produced bacteriocins were proven to be bacteriostatic against strains of the same genera althoughtheir practical application has to be further investigated.
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