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The Production of Bacteriocins from Lactic Acid

Bacteria
THESIS APRIL 2011

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1 AUTHOR:
Myrto-Panagiota Zacharof
37 PUBLICATIONS 78 CITATIONS
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Retrieved on: 07 October 2015

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 fruits
in order to produce a variety of fermented products and beverages. Furthermore several strains of this
group produce substances of peptide structure that possesses antimicrobial activity against strains mainly
of 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 three
known bacteriocin producing strain Lactobacillus casei NCIMB 11970, Lactobacillus plantarum NCIMB
8014 and Lactobacillus lactis NCIMB 38586 were selected. The possibility of producing the bacteriocins
of these bacteria in large quantities was investigated through several techniques. Firstly, the bacteria
were 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 the
medium 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 yield
coefficient, substrate and starter inoculum concentration.
The metabolism of the bacilli was determined as homofermentative, mainly producing lactic acid In
addition carbohydrate consumption rate in the optimum pH values were measured and the constant
coefficients for substrate consumption end product inhibition effect was determined. As the optimum
conditions for growth were established, the amount of the bacteriocins produced on the optimised
medium was investigated. A simple turbidometric method based on the bacteriostatic effect of the
commercially 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 structure
substances ranging between 4 kDa to 10 kDa it was decided to develop nutrient media containing low
molecular weight nutrient sources of 4kDa and 1 kDa molecular weight which would support
simultaneously bacterial growth and high amounts of bacteriocins, in an effort to facilitate the extraction
of the bacteriocins from the nutrient broths. This was achieved through filtration of the optimised media
through series of membrane filters ranging between 30kDa to 1 kDa MWCO. Studies were conducted to
confirm 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/ml
equivalent to Nisin.
Further development of the extraction strategy of the desired substances contained the use of membrane
filters of 1 kDa and 4 kDa MWCO to separate and further concentrate them. The potency of the
concentrated bacteriocins was tested against the target strain. It was found that their activity has been
enhanced to125 IU/ml equivalent to Nisin. The substances were further separated through High
Performance Liquid Chromatography and sized using Dynamic Light Scattering method. The peptide
nature of the substances was as well confirmed. The exact phase of production of bacteriocins during the
growth of Lactobacilli was as well investigated and the substances were established as being primary
metabolites.
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 although
their practical application has to be further investigated.