Doctoral Thesis

Development of hurdle technology
using plant extracts for control offoodborne pathogens(Summary)

TABITHA MLOWOKA KAYIRA
Department of Biofunctional Science and Technology
Graduate School of Biosphere Science
Hiroshima University

September 2020

Despite many countries installing food safety intervention measures, foodborne
outbreaks including those caused by salmonella and listeria are on the rise. Use of synthetic
preservatives which is one such common approach, does not satisfy consumers because ofsafety and nutritional issues regarding food containing chemical preservatives such as sodium
benzoates and potassium sorbates. Consumers are increasingly aware of health implications
associated with consumption of food containing some synthetics or conventional preservatives
as such demand food which is safer. This has been food industries driving force towards naturalpreservatives which includes dietary herbs and spices which appeal to consumers because havealready been part of human diet since antiquity and have Generally Recognized as Safe
(GRAS) substances according to Food and Drug Administration (FDA). However, dietary
herbs and spice extracts exhibit activity at a concentration which may not be economical toapplication. Also, higher levels impact negatively on the sensory attributes of food and water.
Against this background, there is need to find ways of reducing herbs and spice extracts
effective levels in order to widen their application. This study aimed at identifying dietary
herbs and spices with antibacterial activity and then improve the activity by combining withother GRAS antimicrobials against foodborne pathogens. Outbreaks concerning vibrios are on
the rise. This study focused on control of Vibrio cholerae (V. cholerae)
Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration(MBC) were determined by agar and broth dilution assays respectively. Those extracts that
demonstrated lower (stronger activity) MIC and MBC were selected for time-kill experimentto verify killing effect and comprehend bactericidal pattern. Additionally, combinatorial study
was done with selected extracts in order to create hurdle effect.
Ethanolic extracts of lemon eucalyptus exhibited strong antibacterial activity with aMIC of 0.025 % followed by curry plant with a MIC of 0.05 % against Gram-positive bacteria
tested. Lemon eucalyptus and curry plant showed a MBC of 0.1 % against Gram-positivebacteria. MIC of lemon eucalyptus reduced from 0.025-0.0125 % in the presence of glycine
0.2 % at pH 5.5. Activity of curry plant was improved from MIC of 0.05 % to 0.0125-0.025 %with glycine 0.2 % at pH 5.5. MBC of lemon eucalyptus and curry plant was enhanced from

0.1% to 0.025-0.05 % in the presence of glycine 0.5 % at pH 5.5. Ethanolic extracts of clove,lemon eucalyptus, licorice, rosemary, sage and thyme were active against V. Cholerae with aMIC of 0.2 % except thyme (0.4 %). Activity of plant extracts was amplified throughincorporation of sodium acetate. Mixture of plant extract with sodium acetate (0.4%) at pH 7.0
reduced MICs of clove, lemon eucalyptus, rosemary, sage and thyme to 0.025 % and that oflicorice to 0.05 %. At pH 6.4, combinations were more effective reducing MICs of clove, lemon
eucalyptus, rosemary and sage to 0.0125 % with sodium acetate 0.2 %. Plant extracts andsodium acetate at pH 7.0 resulted in inhibitory activity while at pH 6.4 vibriocidal wasobserved. At pH 7.0, the mixture resulted in additive effect, nevertheless, at pH 6.4, synergy
with Fractional Inhibitory Concentration (FIC) index of 0.375-0.5 was observed. In
contaminated water, time kill curves confirmed bactericidal effect in a dose and time dependent
manner. Extracts of licorice and sage showed significant reduction in viable cell count after
exposure of 4-8 h. Within 4-8 h, extract of sage eliminated V. cholerae.
Lemon eucalyptus demonstrated most potential and broad inhibitory activity. Curry
plant exhibited specificity against Gram-positive bacteria while rosemary was more specificon V. cholerae. Plant extracts showed high potency (low MIC) when NaOAc or glycine wasincorporated. More amplified activity was established when plant extract-NaOAc/glycine
mixture was at mildly acidic pH. The obtained data was evaluated to have a combinatorial
effect which could be a potential candidate for gentle but effective ways of controlling
foodborne bacterial pathogens (hurdle technology). In contaminated water licorice and sageexerted significant anti-vibrio activity indicating a potential for use as additives wherereduction in bacterial count or preservation is desired. More importantly, is definite exposure
time established in this study to achieve complete disinfection. This may have a positive
implication in control of V. cholerae in water which serves as one of main vehicles fortransmission. Furthermore, no off odour was detected with both extracts in water after
incubation period. This suggests that sage and licorice extracts could be applied in water as
decontaminants. Further study may utilize these findings to examine activity of thecombinations in food and water.