THE PRODUCTION OF BACTERIOCINS FROM LACTIC ACID BACTERIA M.P.Zacharof ¹and R.W. Lovitt²...
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THE PRODUCTION OF BACTERIOCINS FROM LACTIC ACID BACTERIA M.P.Zacharof ¹and R.W. Lovitt² Multidisciplinary Nanotechnology Centre, Swansea University, Swansea, SA2 8PP, UK¹ School of Engineering, Multidisciplinary Nanotechnology Centre, Swansea University, Swansea, SA2 8PP, UK² Introduction Lactic acid Bacteria produce antimicrobial activity metabolites majorly active against other bacteria of the same genre. They are called bacteriocins. They could be used as natural anticontaminants on their own or synergistically with other factors. [2] Nisin though is the only commercially available bacteriocin. This emerges the need to further exploit these metabolites. Implications on their production though include the following. ● They are growth related metabolites so they are influenced from factors that negatively affect microbial growth (end product inhibition due to lactate, glucose exhaustion due to fastidious growth, microbial contamination)[1] ● They are extracellular metabolites of peptide nature though sensitive to conditions that may denaturise their peptide nature ● Their extraction becomes difficult as they may form micelles or clumps with the nitrogen sources already in the medium.[3] References 1. Beasley, S. S.; Saris, P. E. J., Nisin-producing Lactococcus lactis strains isolated from human milk. Journal of Applied and Environmental Microbiology 2004, 70, 5051-5053. 2. Benuerroum N.; Sandine, W. E., Inhibitory action of nisin against Listeria monocytogenes. Journal of Dairy Science 1998, 71, 3237-3245. 3. Bernadeau M. Vernoux, J. P., ,Henri-Dubernet, S., Gueguen M.,, The Lactobacillus genus. International Journal of Food Microbiology 2007, 41, 103-125. Aims and Objectives ● The previously referred hurdles are taken into consideration and a suitable strategy is developed ● Three known bacteriocin producing strains were selected, Lactobacilli L.plantarum NCIMB 8014, L.casei NCIMB 11970 and L.lactis NCIMB 8586 ● A common optimised growth medium was developed including optimum physicochemical conditions ● A low molecular weight medium was developed to facilitate their extraction ● As extraction method filtration is selected and as purification method high precision chromatography was selected ● A quantification method was developed based on the antimicrobial effect Nisin has. ● The quantification method will include a carefully selected target strain and a growth medium that ensured optimum growth of the target strain will be developed ● Studies was conducted on the potency and efficiency of the bacteriocins against the target strain in relation to various parameters Conclusions and Future Recommendations Lactobacilli do indeed produce primary metabolites of antimicrobial activity that could be exploited commercially. A novel growth strategy for their in vitro production was developed. A novel quantification method of their amount was developed. A cost effective , easy to implement and handle separation , concentration and purification method was developed Their potency was tested against various factors. A mathematical model could be developed forecasting the production of bacteriocins. The bacteriocins could be tested in a wider area of target strains. Several other nutrient sources could be tested. Results and Discussion An optimized medium was developed which fully supported the growth off Lactobacilli. LMWM were developed through serial filtration of the optimised medium by a 30 kDa, 4 kDa and 1kDa membrane filters. Having proved that the media supported Lactobacilli growth, bacteriocin production had to be investigated. A quantification assay was developed. The assay was developed on the growth rates of the target strain L.delbruckii occurring under different concentrations of Nisin induced in the medium. The assay had a limit value of 200 IU/ml of Nisin or of Nisin-like activity antimicrobial compound. Beyond this value no growth occurs. The Lactobacilli were tested for bacteriocin production both through measurement of protein content and through testing the supernatant for antimicrobial activity As extraction method filtration was selected. The supernatants deriving from the growth of the selected Lactobacilli on the develop media ,were initially filtrated through a 4kDa MWCO membrane filter and the retentates deriving were further through a 1kDa filter. These retentates were tested against the target strain. They were also sized and they protein nature was certified through trypsinisation. The potency of the bacteriocins was tested under numerous conditions that could possibly deactivate them. These condition include heat treatment, storage capacity, filtration and dilution. It was proven that bacteriocins are relatively stable molecules that should be commercially exploited. y = -0.0019x + 0.3453 R 2 = 0.9758 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0 50 100 150 200 N isin IU /m l μ m ax (h-1) μ m ax (h-1)of L.delbruckiiundera range ofNisin Concentrations betw een 0-200 IU /m Linearfitting is used 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 0 2 4 6 8 10 Tim e (h) B io m a s s (g / 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 0 2 4 6 8 10 Tim e (h) B io m a ss (g Figure: Figure: N orm algrow th of L .delbruckii ( )and on N orm alG row th of L .delbruckii ( )on perm R etentate (unf/d m ed)( ), retentate (4 kD a m ed)( ) R etentate (unf/d m ed)( ), retentate (4 kD A nd retentate (1 kD a m ed) L .casei A nd retentate (1 kD a m ed) L .plantarum 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 0 2 4 6 8 10 12 Tim e (h) B io m a s s (g Figure:N orm algrow th of L .delbruckii ( )R etentate (unf/d m ed)( ), retentate (4 kD a m ed)( ) and retentate (1 kD a m ed ) L .lactis As separation method HPLC was selected. The 1kDa concentrated samples of the fermented broth of selected Lactobacilli. The method was proven successful clearly showing peaks. 0 20 40 60 80 100 120 0 20 40 60 80 100 120 Tim e (h) B a c terio cin am o u n t (IU 0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 Tim e (h) B acterio cin am o un t (IU Figure. Figure Stability ofpotency ofL .casei Bacteriocin up to 96h Stability ofpotency ofL .plantarum B acteriocin up Treated w ith H eat(80 °C )(◊) Treated w ith H eat(80 °C )(◊) Treated w ith M icrofiltration (□) Treated w ith M icrofiltration (□) & w ithoutany treatm ent(∆ ) & w ithoutany treatm ent(∆ ) 0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 Tim e (h) B a cterio cin am o u n t (IU FigureStability ofpotency ofL .casei B acteriocin up to 96h Treated w ith H eat(80 °C )(◊)Treated w ith M icrofiltration (□)& w ithoutany treatm ent(∆ ) Figure 2 Diagram of mode off action of nisin against the pathogenic strains Figure 1 Molecular Structure of the bacteriocin nisin Figure 3 Dose response model for bacteriocin activity Figure 4 Bacteriocin production on all the media categori Table 1.Summary table of the HPLC separation results for the bacteriocins 5 6 7 8 9 10
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Transcript of THE PRODUCTION OF BACTERIOCINS FROM LACTIC ACID BACTERIA M.P.Zacharof ¹and R.W. Lovitt²...
THE PRODUCTION OF BACTERIOCINS FROM LACTIC ACID BACTERIA
M.P.Zacharof ¹and R.W. Lovitt²Multidisciplinary Nanotechnology Centre, Swansea University, Swansea, SA2 8PP, UK¹
School of Engineering, Multidisciplinary Nanotechnology Centre, Swansea University, Swansea, SA2 8PP, UK²
IntroductionLactic acid Bacteria produce antimicrobial activity metabolites majorly active against other bacteria of the same genre. They are called bacteriocins. They could be used as natural anticontaminants on their own or synergistically with other factors. [2] Nisin though is the only commercially available bacteriocin. This emerges the need to further exploit these metabolites. Implications on their production though include the following. ●They are growth related metabolites so they are influenced from factors
that negatively affect microbial growth (end product inhibition due to lactate, glucose exhaustion due to fastidious growth, microbial contamination)[1]
●They are extracellular metabolites of peptide nature though sensitive to conditions that may denaturise their peptide nature
●Their extraction becomes difficult as they may form micelles or clumps with the nitrogen sources already in the medium.[3]
References
1. Beasley, S. S.; Saris, P. E. J., Nisin-producing Lactococcus lactis strains isolated from human milk. Journal of Applied and Environmental Microbiology 2004, 70, 5051-5053.
2. Benuerroum N.; Sandine, W. E., Inhibitory action of nisin against Listeria monocytogenes. Journal of Dairy Science 1998, 71, 3237-3245.
3. Bernadeau M. Vernoux, J. P., ,Henri-Dubernet, S., Gueguen M.,, The Lactobacillus genus. International Journal of Food Microbiology 2007, 41, 103-125.
Aims and Objectives●The previously referred hurdles are taken into consideration and a suitable
strategy is developed●Three known bacteriocin producing strains were selected, Lactobacilli
L.plantarum NCIMB 8014, L.casei NCIMB 11970 and L.lactis NCIMB 8586 ●A common optimised growth medium was developed including optimum
physicochemical conditions●A low molecular weight medium was developed to facilitate their extraction●As extraction method filtration is selected and as purification method high
precision chromatography was selected●A quantification method was developed based on the antimicrobial effect
Nisin has.●The quantification method will include a carefully selected target strain and a
growth medium that ensured optimum growth of the target strain will be developed
●Studies was conducted on the potency and efficiency of the bacteriocins against the target strain in relation to various parameters
Conclusions and Future RecommendationsLactobacilli do indeed produce primary metabolites of antimicrobial activity that could be exploited commercially. A novel growth strategy for their in vitro production was developed. A novel quantification method of their amount was developed. A cost effective , easy to implement and handle separation , concentration and purification method was developedTheir potency was tested against various factors. A mathematical model could be developed forecasting the production of bacteriocins. The bacteriocins could be tested in a wider area of target strains. Several other nutrient sources could be tested.
Results and DiscussionAn optimized medium was developed which fully supported the growth off Lactobacilli. LMWM were developed through serial filtration of the optimised medium by a 30 kDa, 4 kDa and 1kDa membrane filters. Having proved that the media supported Lactobacilli growth, bacteriocin production had to be investigated. A quantification assay was developed. The assay was developed on the growth rates of the target strain L.delbruckii occurring under different concentrations of Nisin induced in the medium. The assay had a limit value of 200 IU/ml of Nisin or of Nisin-like activity antimicrobial compound. Beyond this value no growth occurs.The Lactobacilli were tested for bacteriocin production both through measurement of protein content and through testing the supernatant for antimicrobial activity
As extraction method filtration was selected. The supernatants deriving from the growth of the selected Lactobacilli on the develop media ,were initially filtrated through a 4kDa MWCO membrane filter and the retentates deriving were further through a 1kDa filter. These retentates were tested against the target strain. They were also sized and they protein nature was certified through trypsinisation. The potency of the bacteriocins was tested under numerous conditions that could possibly deactivate them. These condition include heat treatment, storage capacity, filtration and dilution. It was proven that bacteriocins are relatively stable molecules that should be commercially exploited.
y = -0.0019x + 0.3453
R2 = 0.9758
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0 50 100 150 200Nisin IU/ml
μ m
ax (h
-1)
μ max (h-1) of L.delbruckii under a
range of Nisin Concentrations
between 0-200 IU/mLinear fitting is used
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
0 2 4 6 8 10Time (h)
Bio
ma
ss
(g
/L)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
0 2 4 6 8 10
Time (h)
Bio
mass (
g/L
)
Figure: Figure:
Normal growth of L.delbruckii () and on Normal Growth of L.delbruckii () on permeate ()
Retentate (unf/d med) (), retentate (4 kDa med) () Retentate (unf/d med) (), retentate (4 kDa med) ()
And retentate (1 kDa med) L.casei And retentate (1 kDa med) L.plantarum
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
0 2 4 6 8 10 12
Time (h)
Bio
ma
ss
(g
/L)
Figure: Normal growth of L.delbruckii () Retentate (unf/d med) (), retentate (4 kDa med) () and retentate (1 kDa med) L.lactis
As separation method HPLC was selected. The 1kDa concentrated samples of the fermented broth of selected Lactobacilli. The method was proven successful clearly showing peaks.
0
20
40
60
80
100
120
0 20 40 60 80 100 120
Time (h)
Bacte
rio
cin
am
ou
nt
(IU
/ml)
0
20
40
60
80
100
120
140
0 20 40 60 80 100 120
Time (h)
Bacte
rio
cin
am
ou
nt
(IU
/ml)
Figure. Figure
Stability of potency of L.casei Bacteriocin up to 96h Stability of potency of L.plantarum Bacteriocin up to 96h
Treated with Heat (80 °C) (◊) Treated with Heat (80 °C) (◊)
Treated with Microfiltration (□) Treated with Microfiltration (□)
&without any treatment (∆) &without any treatment (∆)
0
20
40
60
80
100
120
140
0 20 40 60 80 100 120Time (h)
Bacte
rio
cin
am
ou
nt
(IU
/ml)
Figure Stability of potency of L.casei Bacteriocin up to 96h Treated with Heat (80 °C) (◊) Treated with Microfiltration (□) & without any treatment (∆)
Figure 2 Diagram of mode off action of nisin against the pathogenic strains
Figure 1 Molecular Structure of the bacteriocin nisin
Figure 3 Dose response model for bacteriocin activity Figure 4 Bacteriocin production on all the media categories
Table 1.Summary table of the HPLC separation results for the bacteriocins
5 6
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8 9
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