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Pharmacognosy Communications Volume 4 | Issue 4 | Oct–Dec 2014
PHCOG J
Research Article Tasmannia stipitata as a Functional Food/Natural Preservative: Antimicrobial Activity and Toxicity C. Harta, P. Ilankoa, J. Sirdaartaa,b, P. Rayana,b, P.A. McDonnella and I. E. Cocka,b* a b
School of Natural Sciences, Nathan Campus, Griffith University, 170 Kessels Rd, Nathan, Queensland 4111, Australia Environmental Futures Research Institute, Nathan Campus, Griffith University, 170 Kessels Rd, Nathan, Queensland 4111, Australia
ABSTRACT: Introduction: Tasmannia stipitata (Dorrigo pepper) is an endemic Australian plant with a history of use by indigenous Australians as a food. It is taxonomically related to Tasmania lanceolata which has documented therapeutic properties as well as uses for food flavouring. Methods: T. stipitata solvent extracts were investigated by disc diffusion assay against a panel of bacteria and fungi. Their MIC values were determined to quantify and compare their efficacies. The ability to inhibit the proliferation of Giardia duodenalis was determined by direct cell counts and by using an MTS based cell proliferation assay. Toxicity was determined using the Artemia franciscana nauplii bioassay. Results: Methanolic, aqueous and ethyl acetate T. stipitata leaf and berry extracts displayed antibacterial activity in the disc diffusion assay. The berry methanolic extract had the broadest antibacterial range, inhibiting the growth of all 22 of the 23 bacteria tested (95.7%) and 2 of the 4 fungal species (50%) tested. In comparison, 18 of the bacterial species (81.8%) and 2 of the fungal species (50%) were inhibited by at least 1 of the leaf extracts. The methanol, water and ethyl acetate extracts of both berries and leaves all had similar efficacies and ranges of microbes inhibited. Whilst broad spectrum activity was seen for these extracts, they displayed only moderate to low efficacy (as determined by the zones of inhibition and MIC analyses). All extracts were more effective at inhibiting the growth Gram-negative bacteria than Gram-positive bacteria or fungi. Furthermore, the methanol, water and ethyl acetate extracts of both berry and leaf were potent inhibitors of Giardial proliferation. All T. stipitata extracts were non-toxic in the Artemia fransiscana bioassay with LC50 values greatly in excess of 1000 µg/ml. Conclusion: The lack of toxicity of the T. stipitata extracts and their moderate broad spectrum inhibitory bioactivity against bacteria, fungi and Giardia indicates their potential as natural food preservatives and as medicinal agents in the treatment and prevention of microbial diseases. KEYWORDS: Winteraceae, Tasmannia stipitata, Dorrigo pepper, antibacterial, food spoilage, food poisoning, functional food, natural preservative.
INTRODUCTION Members of family Winteraceae have been used for a broad range of dietary and medicinal purposes by a wide variety of ethnic and cultural groupings. The best documented of these is the South American species Drimys winteri. The stem and bark of this species has been used as a stimulant and as a tonic in traditional Brazilian medicinal systems.1 They are also used for the treatment of a wide *Correspondence author: Dr. I. E. Cock School of Natural Sciences and Environmental Futures Research Institute, Nathan Campus, Griffith University, 170 Kessels Rd, Nathan, Queensland 4111, Australia E-mail:
[email protected] DOI: 10.5530/pc.2014.4.4
variety of diseases and medicinal conditions including use as an analgesic, and to treat diarrhoea, inflammation, and ulcers.1,2 This species also has widespread usage in the treatment of scurvy due to its high antioxidant content.3 Of the other Winteraceae species, several have a history of ethnobotanical usage, usually for purposes related to their polygodial contents and high levels of antioxidants. Indeed, high levels of the compound polygodial (which gives the family Winteraceae a characteristic peppery flavour) (Figure 1a) and high antioxidant contents are common characteristics of Winteraceae species. Other antioxidant molecules common to Winteraceae species include safrole (Figure 1b), gallic acid (Figure 1c), chlorogenic acid (Figure 1d), quercetin (Figure 1e), rutin (Figure 1f), lutein (Figure 1g), α-tocopherol (vitamin E) (Figure 1 h), vitamin a (Figure 1i) and folic acid (Figure 1j).
© Copyright 2014 EManuscript Publishing Services, India
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Tasmanian pepper as a functional food
Figure 1. Chemical structures of antioxidant molecules common across Winteraceae species (a) polygodial, (b) safrole, (c) gallic acid, (d) chlorogenic acid, (e) quercetin, (f) rutin, (g) lutein, (h) α-tocopherol (vitamin E), (i) vitamin a, (j) folic acid.
Epidemiological studies have shown that a diet high in antioxidants may have preventative effects against the development of degenerative diseases such as cancer,4 cardiovascular diseases,5 neural degeneration,6 diabeties and obesity.7 The antioxidant activity of many plants has been associated with their phenolic contents. Many phenolic compounds have been shown to have strong antioxidant activities and may protect cells against oxidative damage by directly scavenging free radicals.8 Phenolic compounds may also interact directly with receptors or with enzymes involved in cellular signal transduction.9 Common classes of plant phenolic compounds include flavonoids, tannins and anthrocyanins. The medicinal potential of plants with high antioxidant contents has been receiving much recent attention10,11 and reports have linked antioxidant levels and redox management with anticancer activity12 There has been recent interest in the medicinal and functional food properties of Australian Winteraceae species due to recent reports of their high antioxidant contents.11 Tasmannia lanceolata (Tasmanian pepper, pepper berry) in particular has attracted attention due to reported antioxi34
dant contents of over 120 µmol/g fruit (approximately 3 times the antioxidant content of blueberries, which themselves are considered to have high antioxidant contents). The same study also reported T. lanceolata to have even greater leaf antioxidant contents (more than 4 fold higher than those reported for blueberries). Interestingly, ascorbic acid (which makes a significant contribution to the antioxidant content of many fruits) was reported to be below the threshold of detection in this study and therefore would not contribute significantly to the high antioxidant content of T. lanceolata. T. lanceolata leaves have also been reported to have phenolic antioxidant contents up to 4 times higher than in basil leaves (Ocimum basilicum),13 higher levels than determined for peppermint leaves14 and similar levels to the phenolic antioxidant contents of maple, silver birch and spruce leaves.15 The phenolic contents of T. lanceolata berries are also high, although these levels are significantly lower (less than 20%) than the leaf phenolic antioxidant levels. The contents are similar to those reported for those reported for Piper nigrum (black pepper) and Lycium barbarum (Chinese Barbary Wolfberry fruit),14 but approximately half the level of black sesame and peach kernel.15
Tasmanian pepper as a functional food
Further interest has focussed on the antimicrobial properties of high antioxidant foods and thus on their potential as natural preservatives.16-19 A recent study reported potent, broad spectrum antibacterial and anti-fungal properties for T. lanceolata extracts.20 That study not only indicated the therapeutic potential of T. lanceolata against a range of infectious diseases, but also looked at its potential as a natural preservative by specifically studying the growth inhibition of a panel of microbial species specifically associated with food spoilage and food poisoning. Due to greater consumer awareness and the negative perceptions of artificial preservatives, consumers are increasingly avoiding foods containing preservatives of chemical origin. Natural antimicrobial alternatives are increasingly being sought to increase the shelf life and safety of processed foods. Plant extracts and oils are candidates for antimicrobial agents that would be more acceptable to consumers due to their natural origin and consumer perception of safety. In addition, many plants have well established antimicrobial activity and several plant species have already been identified for their potential as natural preservatives.18-23 Thus, high antioxidant foods such as T. lanceolata have potential uses as natural preservatives, and as functional foods in the prevention and treatment of food borne diseases. Whilst T. lanceolata has received much recent interest, studies into other closely related Australian species are lacking. Tasmannia stipitata (Dorrigo pepper, northern pepperbush) is a shrub which is endemic to the rainforests and temperate woodlands of northern New South Wales and southern Queensland regions of Australia. It is a medium shrub that varies between 2-5 m in height. Individual plants are dioecious, with male and female flowers on separate plants. The aromatic leaves are lanceolate to narrowly elliptical in shape (8-13 cm in length). Fleshy dark blue-black 2 lobed berries (5-8 mm wide) develop in autumn. The berries and the leaves have similar culinary uses and tastes to T. lanceolata and are also considered to have high antioxidant and polygodial contents and many of the same therapeutic properties. Despite its ethnobotanical usage and taxonomic relationship to T. lanceolata, there is a lack of rigorous scientific studies into the therapeutic properties of T. stipitata. The current study was undertaken to test T. stipitata leaf and berry extracts for the ability to inhibit microbial growth/contamination against a variety of bacteria involved in food spoilage and/or food poisoning. Through examining the antibacterial capability of the T. stipitata extracts, we aim to assess their potential as additives to foods to retard spoilage and to potentially reduce food poisoning in processed foods.
MATERIALS AND METHODS Plant Source and Extraction
T. stipitata semi-dry berries and dried leaves were purchased from A Taste of the Bush, Australia. The berries were thoroughly dried in a Sunbeam food dehydrator and subsequently the dried plant materials were stored at -30oC. Prior to use, the plant materials were thawed and freshly ground to a coarse powder. Individual 1 g quantities of the ground leaves and berries were weighed into individual tubes and 50 ml of methanol, deionised water or ethyl acetate were added. All solvents were obtained from Ajax and were AR grade. The ground berries and leaves were individually extracted in each solvent for 24 hours at 4 oC with gentle shaking. The extracts were filtered through filter paper (Whatman No. 54) under vacuum, followed by drying by rotary evaporation in an Eppendorf concentrator 5301. The resultant dry extract was weighed and redissolved in 10 ml deionised water. Qualitative Phytochemical Studies
Phytochemical analysis of the T. stipitata extracts for the presence of saponins, phenolic compounds, flavonoids, polysteroids, triterpenoids, cardiac glycosides, anthraquinones, tannins and alkaloids was conducted by previously described assays.24–26 Antibacterial Screening Test Microorganisms
All media was supplied by Oxoid Ltd. Reference strains of Acinetobacter bayleii (ATCC33304), Klebsiella pneumoniae (ATCC31488), Proteus mirabilis (ATCC21721), Proteus vulgaris (ATCC21719) and Pseudomonas aeruginosa (ATCC39324) were purchased from American Tissue Culture Collection, USA. All other microbial strains were obtained from Tarita Morais, Griffith University. Stock cultures of Acinetobacter baylyi, Aeromonas hydrophila, Alcaligenes feacalis, Bacillus cereus, Citrobacter freundii, Enterobacter aerogenes, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeuroginosa, Pseudomonas fluorescens, Salmonella newport, Serratia marcescens, Shigella sonnei, Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus pyogenes were subcultured and maintained in nutrient broth at 4oC. Aspergillus niger, Candida albicans, Penicillium chrysogenum and Saccharomyces cerevisiae were maintained in Sabouraud media at 4oC. Evaluation of Antimicrobial Activity
Antimicrobial activity of all plant extracts was determined using a modified disc diffusion assay.27–30 Briefly, 100 µl of the test bacteria were grown in 10 ml of fresh nutrient broth media until they reached a count of approxi35
Tasmanian pepper as a functional food
mately 108 cells/ml. An amount of 100 µl of bacterial suspension was spread onto nutrient agar plates. For fungal species, 100 µl of the test species was grown in 10 ml of fresh Sabouraud media until they reached a count of approximately 106 cells/ml. A volume of 100 µl of bacterial suspension was spread onto Sabouraud agar plates. The extracts were tested for antibacterial activity using 5 mm sterilised filter paper discs. Discs were impregnated with 10 µl of the test sample, allowed to dry and placed onto inoculated plates. The plates were allowed to stand at 4oC for 2 hours before incubation with the test microbial agents. Plates inoculated with the bacterial species Alcaligenes feacalis, Aeromonas hydrophilia, Bacillus cereus, Citrobacter freundii, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeuroginosa, Pseudomonas fluorescens and Serratia marcescens and the fungal species Candida albicans, Penicillium crygogenum and Saccharomyces cerevisiae were incubated at 30oC for 24 hours, then the diameters of the inhibition zones were measured in millimetres. Plates inoculated with Acinetobacter baylyi, Enterobacter aerogenes, Enterococcus faecalis, Escherichia coli, Salmonella newport, Shigella sonnei, Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus pyogenes were incubated at 37oC for 24 hours, then the diameters of the inhibition zones were measured. Plates inoculated with Aspergillus niger were incubated at 25oC for 48 hours, then the diameters of the inhibition zones were measured. All measurements were to the closest whole millimetre. Each antimicrobial assay was performed in at least triplicate. Mean values (± SEM) are reported in this study. Standard discs of ampicillin (2 µg) and nystatin (100 µg) were obtained from Oxoid Ltd. and served as positive controls for antibacterial and antifungal activity respectively. Filter discs impregnated with 10 µl of distilled water were used as a negative control. Minimum Inhibitory Concentration (MIC) Determination
The minimum inhibitory concentration (MIC) of the extracts were determined as previously described.31,32 Briefly, the plant extracts were diluted in deionised water and tested across a range of concentrations. Discs were impregnated with 10 µl of the test dilutions, allowed to dry and placed onto inoculated plates. The assay was performed as outlined above and graphs of the zone of inhibition versus concentration were plotted for each extract. Linear regression was used to calculate the MIC values. Inhibitory Bioactivity Against Giardia duodenalis trophozoites Parasite Culture
The Giardia duodenalis S-2 (sheep strain 2) trophozoite strain used in this study were previously supplied by 36
Professor Andre Buret, University of Calgary, Canada. G. duodenalis tropozoites were maintained and subcultured anaerobically at 37oC in TYI-S-33 growth media supplemented with 1% bovine bile (Sigma), 10% Serum Supreme (Cambrex Bioproducts) and 200 IU/ml penicillin/200 µg/ml streptomycin (Invitrogen, USA). Confluent mid log phase cultures were passaged every 2 days by chilling the cultures on ice for a minimum of 10 min, followed by vortexing to dislodge the adherent trophozoites from the walls of the culture vessel. Fresh culture media (5 ml) was seeded with approximately 1 x 105 trophozoites for each passage. Evaluation of Anti-Giardial Activity by CellTiter Bioassay
Anti-Giardial activity of the extracts was assessed as in previous studies.33 Briefly, aliquots of the trophozoite suspension (70 µl) containing approximately 1 x 105 trophozoites were added to the wells of a 96 well plate. A volume of 30 µl of the test extracts or the vehicle solvent or culture media (for the negative controls) was added to individual wells and the plates were incubated anaerobically at 37oC for 12 hours in a humidified anaerobic atmosphere. A volume of 20 µl of CellTiter 96® AQueous One Solution Cell Proliferation Assay Reagent (Promega) was subsequently added to each well and the plates were incubated for a further 3 hours. Absorbances were recorded at 490 nm using a Molecular Devices, Spectra Max M3 plate reader. All tests were performed in at least triplicate and triplicate controls were included on each plate. The anti-proliferative activity of each test was calculated as a percentage of the negative control using the following formula: Giardial growth (% untreated control) = (Act/Acc) x 100 Act is the corrected absorbance for the test extract (calculated by subtracting the absorbance of the test extract in media without cells from the extract/cell/test combination) and Acc is the corrected untreated control (calculated by subtracting the absorbance of the untreated control in media without cells from the untreated/cell/media combination). Evaluation of Anti-Giardial Activity by Direct Parasite Enumeration
Anti-Giardial activity of the extracts was also assessed by direct enumeration of parasite numbers in the presence or absence of extracts. For each test, aliquots of the trophozoite suspension (70 µl) containing approximately 1 x 105 trophozoites were added to the wells of a 96 well plate. A volume of 30 µl of the test extracts or the vehicle solvent or culture media (for the negative controls) was
Tasmanian pepper as a functional food
added to individual wells and the plates were incubated anaerobically at 37oC for 8 hours in a humidified anaerobic atmosphere. Following the 8 h incubation, all tubes were placed on ice for a minimum of 10 min, followed by vortexing to dislodge the adherent trophozoites from the walls of the culture vessel. The suspensions were mounted onto a Neubauer haemocytometer (Weber, UK) and the total trophozoites per ml were determined. The anti-proliferative activity of the test extracts was determined and expressed as a % of the untreated control trophozoites per ml.
well plate and immediately used for bioassay. A volume of 400 µl of diluted plant extracts or the reference toxin were transferred to the wells and incubated at 25 ± 1oC under artificial light (1000 Lux). A negative control (400 µl seawater) was run in triplicate for each plate. All treatments were performed in at least triplicate. The wells were checked at regular intervals and the number of dead counted. The nauplii were considered dead if no movement of the appendages was observed within 10 seconds. After 72 h all nauplii were sacrificed and counted to determine the total% mortality per well. The LC50 with 95% confidence limits for each treatment was calculated using probit analysis.
Determination of IC50 Values Against Giardial trophozoites
For IC50 determinations, the plant extracts were tested by both methods across a range of concentrations. The assays were performed as outlined above and graphs of the zone of inhibition versus concentration were plotted for each extract. Linear regression was used to calculate the IC50 values.
Statistical Analysis
Toxicity Screening Reference Toxin for Toxicity Screening
Liquid Extraction Yields and Qualitative Phytochemical Screening
Data are expressed as the mean ± SEM of at least three independent experiments.
RESULTS
Potassium dichromate (K2Cr2O7) (AR grade, Chem-Supply, Australia) was prepared as a 1.6 mg/ml solution in distilled water and was serially diluted in artificial seawater for use in the Artemia franciscana nauplii bioassay.
Extraction of 1 g of dried T. stipitata berry and leaf with various solvents yielded dried plant extracts ranging from 114 mg (leaf ethyl acetate extract) to 293 mg (leaf water extract) (Table 1). Deionised water and methanol generally gave relatively high yields of dried extracted material, whilst ethyl acetate extracted lower masses for both the berries and leaves. The dried extracts were resuspended in 10 ml of deionised water resulting in the extract concentrations shown in Table 1.
Artemia franciscana Nauplii Toxicity Screening
Toxicity was tested using a modified Artemia franciscana nauplii lethality assay.34–37 Briefly, 400 µl of seawater containing approximately 43 (mean 43.2, n = 155, SD 14.5) A. franciscana nauplii were added to wells of a 48
Total Phenolics
Water Soluble
Water Insoluble
Cardiac Glycosides
Saponins
Triterpenes
Polysteroids
Alkaloids (Meyer test)
Alkaloids (Wagners test)
Flavanoids
Tannins
Free Anthraquinones
Combined Anthraquinones
Methanol
279
27.9
+++
+++
++
-
+++
+
-
-
-
+++
-
-
-
Water
207
20.7
+++
+++
+++
-
++
+
-
-
-
+++
-
-
-
Ethyl Acetate
170
17
+
+
+
-
+
++
-
-
-
++
-
-
-
Methanol
232
23.2
+++
+++
++
-
+++
+
-
-
-
+++
-
-
-
Water
293
29.3
+++
+++
+++
-
++
+
-
-
-
+++
-
-
-
Ethyl Acetate
114
11.4
+
+
+
-
-
+
-
-
-
++
-
-
-
Extract
Resuspended Extract Concentration (mg/ml)
Leaf
Berry
Mass of Dried Extract (mg)
Table 1: The mass of dried extracted material, the concentration after resuspension in deionised water and qualitative phytochemical screenings of T. stipitata berry and leaf extractions.
+++ indicates a large response; ++ indicates a moderate response; + indicates a minor response; - indicates no response in the assay.
37
Tasmanian pepper as a functional food
Antimicrobial Activity
To determine the antimicrobial activity of the crude plant extracts, aliquots (10 µl) of each extract were tested in the disc diffusion assay against a panel of bacteria and fungi associated with food spoilage and food poisoning. Gramnegative bacterial growth was inhibited by a broad range of the tested plant extracts (Figure 2). Indeed, the growth of all of the Gram-negative species was inhibited by at least 1 of the T. stipitata extracts. The berry ethyl acetate extract displayed the broadest antibiotic specificity, inhibFigure 2. Antibacterial activity of (a) T. stipitata berry and (b) T. stipitata leaf extracts against Gram-negative bacteria measured as zones of inhibition (mm). BM = T. stipitata berry methanolic extract; BW = T. stipitata berry water extract; BE = T. stipitata berry ethyl acetate extract leaf; LM = T. stipitata leaf methanolic extract; LW = T. stipitata leaf water extract; LE = T. stipitata leaf ethyl acetate extract leaf; 1 = A. baylii (clinical isolate); 2 = A. baylii (ATCC33304); 3 = A. faecalis; 4 = A. hydrophilia; 5 = C. freundii; 6 = E. aerogenes; 7 = E. coli; 8 = K. pneumoniae (clinical isolate); 9 = K. pneumoniae (ATCC31488); 10 = P. mirabilis (clinical isolate); 11 = P. mirabilis (ATCC21721); 12 = P. vulgaris (ATCC21719); 13 = P. aeruginosa (clinical isolate); 14 = P. aeruginosa ATCC39324); 15 = P. fluorescens; 16 = S. newport; 17 = S. marcenscens; 18 = S. sonnei; Amp = ampicillin (2 µg) control. Results are expressed as mean zones of inhibition ± SEM.
Qualitative phytochemical studies (Table 1) showed that methanol and water extracted the widest range of phytochemicals. Both showed high levels of phenolics (both water soluble and insoluble phenolics) and flavonoids, as well as moderate to high levels of saponins. The ethyl acetate extracts had low to moderate levels of phenolics, triterpenes and flavonoids. Low levels of saponins were also reported for the berry ethyl acetate extract. Neither tannins nor alkaloids were detected in any of the extracts tested.
38
Tasmanian pepper as a functional food
were generally not as effective as the berry extracts, with 3 bacterial strains (E. aerogenes, P. aeruginosa (clinical isolate) and P. aeruginosa reference strain (ATCC39324)) growing in the presence of all leaf extracts. Gram-positive bacterial growth was also inhibited by both berry and leaf extracts, although the range of bacteria was more limited than for Gram-negative bacteria (Figure 3). Four of the 5 Gram-positive bacterial species (80%) were Figure 3. Antibacterial activity of (a) T. stipitata berry and (b) T. stipitata leaf extracts against Gram-positive bacteria measured as zones of inhibition (mm). BM = T. stipitata berry methanolic extract; BW = T. stipitata berry water extract; BE = T. stipitata berry ethyl acetate extract leaf; LM = T. stipitata leaf methanolic extract; LW = T. stipitata leaf water extract; LE = T. stipitata leaf ethyl acetate extract leaf; Amp = ampicillin (2 µg) control. Results are expressed as mean zones of inhibition ± SEM.
iting the growth all of the Gram-negative bacteria tested, although the measured zones of inhibition against most were relatively low (below 8 mm for all bacterial species). The most potent growth inhibition was seen for the berry ethyl acetate extracts against S. sonnei (inhibition zone of 9.6 ± 0.6 mm). Indeed, the berry ethyl acetate extract was generally more effective than the berry methanol and water extracts against the majority of bacteria that it inhibited, despite extracting much less material. The leaf extracts
39
Tasmanian pepper as a functional food
that whilst the T. stipitata extracts inhibited the growth of a broad panel of both Gram-positive and Gram-negative bacteria, the relatively small zones of inhibition indicate only low to moderate efficacy. Fungal growth was less susceptible to the T. stipitata extracts than was bacterial growth (as determined by zones of inhibition) (Figure 4). A. niger was the most resistant, growing in the presence of all of the extracts tested. Figure 4. Inhibitory activity of (a) T. stipitata berry and (b) T. stipitata leaf extracts measured as zones of inhibition (mm) against fungal species. BM = T. stipitata berry methanolic extract; BW = T. stipitata berry water extract; BE = T. stipitata berry ethyl acetate extract leaf; LM = T. stipitata leaf methanolic extract; LW = T. stipitata leaf water extract; LE = T. stipitata leaf ethyl acetate extract leaf; Nys = nystatin (100 µg) control. Results are expressed as mean zones of inhibition ± SEM.
inhibited by at least 1 berry extract compared to 3 bacteria (60%) inhibited by at least 1 leaf extract. S. aureus and S. epidermidis were the most susceptible of the Gram-positive bacteria, being inhibited by the methanol, water and ethyl acetate of both the berries and leaves. In contrast, S. pyogenes growth was not inhibited by any of the T. stipitata extracts. The berry was more versatile than the leaf at inhibiting Gram-positive bacterial growth, as determined by the number of susceptible bacteria. It is noteworthy
40
Tasmanian pepper as a functional food
This species was a particularly resistant strain, also growing in the presence of ampicillin (unpublished results). P. chrysogenum was also resistant to the T. stipitata extracts, although its growth was not inhibited by the ampicillin control (unpublished results). In contrast, C. albicans and S. cerevisiae were inhibited by 6 (60%) and 5 (50%) of the 10 plant extracts respectively.
inhibiting microbial growth at low to moderate concentrations, with MIC values against the susceptible bacterial and fungal species generally less than 2000 µg/ml (< 20 µg impregnated in the disc), indicating the potential of these extracts in controlling food spoilage and inhibiting food poisoning. The MIC values determined against P. mirabilis were particularly interesting, with values as low as 87 µg/ml (0.9 µg impregnated into the disc) for the berry water extract. Similarly low P. mirabilis MIC values were also seen for several other extracts. Interestingly, whilst the inhibition zone studies indicated low antifungal efficacy, lower MIC values (generally