Attachment 2
To MPR #4
Project 2010-707
Optimisation of natural antimicrobial
extract formulations for inhibiting
bacterial growth on cooked, chilled
prawns
Prepared by
Yasmina Sultanbawa, Carl Paulo,
Andrew Cusack & Margaret Currie
February 2012
CRC Project 2010-707 MPR 4
Attachment 2
Optimising of natural antimicrobial extract formulations
1. Introduction
Kakadu plum powder and organic acids were selected as the most potent
antimicrobial blend in comparison to commercially available natural products in
inhibiting bacteria and hence extending the storage life of cooked chilled prawns.
Optimisation of blends of kakadu plum with lactic acid blends should overcome any
issue of flavour residue in prawns and reduce the cost of formulations.
The objective of the current research is to screen specific spoilage organisms (SSO)
previously isolated from cooked prawns during refrigerated storage against different
combinations of lactic acid and kakadu plum. The most effective formulations that
provide inhibition against the SSOs will be trialled on freshly cooked chilled prawns
to determine storage life.
2. Material and Methods
2.1.
In-vitro screening of natural antimicrobials
Bacterial strains and growth conditions
Two reference organisms were used in the assay, Staphylococcus aureus strain 6571
(NCTC - National Collection of Type Cultures, Health Protection Agency Centre for
Infection, London, UK) and Esherichia coli strain 9001 (NCTC). The test organisms
were grown for 24 h at 300C in Tryptone Soya Yeast Extract Broth (TSYEB) (Oxoid CM
129B, Basingstoke, UK), 30 g/L and yeast extract (Oxoid CM19), 6 g/L. The inoculum
was quantified by measuring the optical density (absorbance) at 540nm and adjusted
to 0.5 absorbance with TSYEB. Specific Spoilage organisms (SSOs) were isolated from
cooked chilled prawns according to the method described in Milestone Report 2
Attachment 3. The following SSOs were used for antimicrobial screening assays:
Shewanella putrefaciens 22A, Shewanella baltica 51.4, Pseudomonas spp 1034.2,
Psychrobacter faecalis 75.2 and Exiguobacterium oxidotolerans AE3 98.3. These SSOs
were grown for 24 h at 25°C in TSYEB.
Combinations of Kakadu plum and lactic acid
Commercial freeze dried Kakadu plum (Terminalia ferdinandiana) powder from
Coradji (NSW, Australia) and commercial grade lactic acid from Hela Spice Australia
(Victoria, Australia) were used. Kakadu plum powder was combined with lactic acid
according to a Central Composite Rotatable Design (CCRD) and the percentage
inhibition determined. The Kakadu plum and lactic acid were used at different
application levels ranging from 0.008 - 0.6% and 0.45 - 1.6% respectively in the 13
runs of the two-factor five-level CCRD. A total of 3 x 13 runs were generated for the
combination of Kakadu plum and lactic acid using MINITAB Release 12 Statistical
Software (State College, PA, USA). Details of the blends for kakadu plum and lactic
acid are given in Table 1 and 2.
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CRC Project 2010-707 MPR 4
Attachment 2
Optimising of natural antimicrobial extract formulations
The combinations listed in Table 1 were screened on two separate occasions against
reference strains S. aureus strain 6571 and E. coli strain 9001 to determine
inhibition.
Table 1 - Combinations of Kakadu plum and lactic acid in 96 well plates screened against
reference strains.
Runs
1-1
1-2
1-3
1-4
1-5
1-6
1-7
1-8
1-9
1-10
1-11
1-12
1-13
2-1
2-2
2-3
2-4
2-5
2-6
2-7
2-8
2-9
2-10
2-11
2-12
2-13
Kakadu plum (%)
0.300
0.100
0.300
0.500
0.300
0.500
0.300
0.300
0.300
0.017
0.100
0.583
0.300
0.15
0.15
0.15
0.29
0.25
0.05
0.15
0.15
0.15
0.0085
0.25
0.05
0.15
Lactic Acid (%)
1.250
1.000
1.250
1.000
0.896
1.500
1.250
1.600
1.250
1.250
1.500
1.250
1.250
0.448
0.625
0.625
0.625
0.750
0.750
0.800
0.625
0.625
0.625
0.500
0.500
0.625
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CRC Project 2010-707 MPR 4
Attachment 2
Optimising of natural antimicrobial extract formulations
Combinations in Table 2 were tested against the reference strains and isolated
Specific Spoilage Organisms.
Table 2 - Combinations of Kakadu plum and lactic acid in 96 well plates screened against
reference strains and SSO.
Runs
1
2
3
4
5
6
7
8
9
10
11
12
13
2.2.
Kakadu plum %
0.075
0.075
0.075
0.100
0.100
0.075
0.100
0.075
0.050
0.040
0.050
0.075
0.075
Lactic Acid %
0.474
0.563
0.563
0.563
0.625
0.563
0.500
0.563
0.625
0.563
0.500
0.651
0.563
Microplate assay procedure
Microtiter plate assay was adopted from (Sultanbawa et al., 2009). Flat bottom 96
well sterile microtiter plates with lids to prevent cross contamination (Sarstedt,
Nϋmbrecht, Germany) were used for the study. Each of the CCRD combinations of
Kakadu plum and lactic acid given in Table 1 were run on a 96 well microtiter plate.
Each 300 µL well contained 50 µL inoculum, 50 µL of the Kakadu plum and lactic acid
combinations and 100 µL Nutrient broth (NB, CM0001, Oxoid, Basingstoke, UK).
Negative or sterility control wells contained 200 µL of NB. Positive control wells
contained 50 µL inoculum and 150 µL of NB.
Optical density (OD) was determined in a spectrophotometer Infinite M200 Tecan
(Grödig, Austria) at 620 nm. Optical Density was determined prior to incubation, (T0).
Plates were placed in an incubator at 25°C and incubated for 22 h. Solutions in plates
were mixed using a plate shaker, Titertek, Flow laboratories (Germany) to avoid
clumping prior to reading the OD in the spectrophotometer after 46 h (T46).
2.3.
Analysis of results
Calculations for percent inhibition were based on (Sultanbawa et al., 2009):
Percent Inhibition = (1 - (OD test well/OD of corresponding positive control well)) x
100
Percent Inhibition = (1 - (T46 - T0) / (C46 - C0)) x 100
The OD for each replicate at time zero (positive control (C0) and test well (T0)) was
subtracted from the OD for each replicate at 46 h (positive control (C46) and test well
(T46)). The Minimum Inhibitory Concentration - MIC100 is the lowest concentration of
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CRC Project 2010-707 MPR 4
Attachment 2
Optimising of natural antimicrobial extract formulations
natural extract that results in 100% inhibition of growth. For reference strains the
Percent Inhibition was determined according to the method given in milestone
report (MPR3 Attachment 2).
3. Results and Discussion
Table 3 display the inhibition rate of the various combinations of kakadu plum and
lactic acid assessed. Through in-vivo screenings a threshold level for the synergistic
relationship between kakadu plum and lactic acid has been identified.
Table 3 - Inhibition of reference cultures with combinations of kakadu plum and lactic acid.
Runs
1-1
1-2
1-3
1-4
1-5
1-6
1-7
1-8
1-9
1-10
1-11
1-12
1-13
2-1
2-2
2-3
2-4
2-5
2-6
2-7
2-8
2-9
2-10
2-11
2-12
2-13
Kakadu plum
(%)
0.30
0.10
0.30
0.50
0.30
0.50
0.30
0.30
0.30
0.017
0.10
0.583
0.30
0.15
0.15
0.15
0.29
0.25
0.05
0.15
0.15
0.15
0.009
0.25
0.05
0.15
Lactic Acid
(%)
1.25
1.00
1.25
1.00
0.896
1.50
1.25
1.60
1.25
1.25
1.50
1.25
1.25
0.448
0.625
0.625
0.625
0.75
0.75
0.80
0.625
0.625
0.625
0.50
0.50
0.625
E. coli strain 9001
% Inhibition
100
100
100
100
100
100
100
100
100
100
100
100
100
80
100
100
100
100
100
100
100
100
90
80
90
100
S. aureus strain 6571
% Inhibition
100
100
100
100
100
100
100
100
100
100
100
100
100
100
95
100
95
100
100
100
95
100
95
90
85
100
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CRC Project 2010-707 MPR 4
Attachment 2
Optimising of natural antimicrobial extract formulations
Incorporating 0.474% lactic acid into 0.075% kakadu plum showed no effective
inhibition for bacterial reference strains nor SSOs. However when the concentration
of lactic acid was increased slightly to 0.563% almost total inhibition is achieved
(Table 4).
50
100
100
100
100
100
100
100
100
90
100
100
100
100
100
100
100
100
100
100
100
90
100
100
100
50
100
95
100
100
100
95
100
100
100
100
100
100
40
100
100
100
100
100
100
100
100
100
100
100
100
75
100
100
95
95
100
100
100
100
90
100
95
100
Exiguobacterium
oxidotolerans
AE3 98.3
Shewanella
putrefaciens 22A
0
Psychrobacter
faecalis 75.2
15
100
100
100
100
100
100
100
100
100
100
100
100
S. aureus strain
6571
E. coli strain
9001
Lactic Acid (%)
0.474
0.563
0.563
0.563
0.625
0.563
0.500
0.563
0.625
0.563
0.500
0.651
0.563
Psuedomonas
1034.2
0.075
0.075
0.075
0.100
0.100
0.075
0.100
0.075
0.050
0.040
0.050
0.075
0.075
Shewanella
baltica 51.4
1
2
3
4
5
6
7
8
9
10
11
12
13
Kakadu plum (%)
Runs
Table 4 - Inhibition of reference cultures and SSOs with combination of kakadu plum and
lactic acid.
0
95
95
95
95
95
95
95
95
90
95
95
95
Using these results a predictive model was created to determine the synergistic
effects of kakadu plum with lactic acid.
Based on predictive modelling a set of combinations for kakadu plum and lactic acid
have been identified that demonstrated optimum inhibition in-vitro (Table 5).
Table 5 - Selected combinations of kakadu plum and lactic acid for further trials.
Combination
1
2
3
4
5
Kakadu plum (%)
0.005
0.010
0.015
0.020
0.025
Lactic Acid (%)
0.750
0.750
0.625
0.625
0.625
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CRC Project 2010-707 MPR 4
Attachment 2
Optimising of natural antimicrobial extract formulations
With further consultation with industry it was established that the high price for
freeze dried kakadu plum could possibly be a major hurdle for successful commercial
implementation. A much cheaper alternative was proposed, frozen whole fruit pulp.
Future trials at concentrations listed in Table 5 will be done on whole fruit pulp as
opposed to freeze dried powder.
4. Conclusion
The main advantages of pursuing kakadu plum as an antimicrobial solution to extend
shelf life of cooked chilled prawns are:
(1) it provides a marketing edge in promoting Australian fresh cooked chilled prawns
as a clean, green product, and
(2) being a natural additive an E number does not need to be stated in labels. This
meets the needs of today’s consumer who prefer fresh, wholesome foods.
The challenges for future trials would be to reduce the required concentration due
to its high costs whilst still maintaining its antimicrobial efficacy. Another cost
reduction option available would be the substitution of freeze dried powder with the
lower cost whole fruit puree. Further investigations will be done to study the
antimicrobial efficacy of puree against reference strains of bacteria and SSOs and
also its antimicrobial and sensorial effects on cooked chilled prawns.
5. References
SULTANBAWA, Y., CUSACK, A., CURRIE, M. & DAVIS, C. (2009) An innovative
microplate assay to facilitate the detection of antimicrobial activity in plant
extracts. Journal of Rapid Methods & Automation in Microbiology, 17, 519-534.
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