TERMINALIA FERDINANDIANA LEAF EXTRACT AND PRODUCTS CONTAINING EXTRACT OF TERMINALIA FERDINANDIANA LEAF
20200221740 ยท 2020-07-16
Inventors
Cpc classification
A23L3/3472
HUMAN NECESSITIES
A23V2002/00
HUMAN NECESSITIES
International classification
Abstract
An extract or composition containing an extract derived from Terminalia ferdinandiana (T. ferdinandiana) leaf as an antimicrobial agent to preserve or prolong storage or shelf-life of perishable animal and/or plant based products. Optionally, the extract can include T. ferdinandiana fruit extract. The leaf extract can be a methanolic, aqueous, ethyl acetate, alcohol, chloroform or hexane extract. The composition can be an antimicrobial agent for perishable animal and/or plant based products, such as food for humans or animals. Also, a method of inhibiting or controlling growth of bacteria on a food preparation surface, on a food preparation tool or utensil, on food packaging or on an internal or external surface of a food product, the method including applying bacteria includes applying a composition containing an extract of Terminalia ferdinandiana (T. ferdinandiana ) leaf, such as by dipping or drenching.
Claims
1. A composition containing an extract derived from Terminalia ferdinandiana (T. ferdinandiana) leaf as an antimicrobial agent to preserve or prolong storage or shelf-life of perishable animal and/or plant based products.
2. The composition of claim 1, further including an extract of T. ferdinandiana fruit in addition to the extract of T. ferdinandiana leaf.
3. The composition of claim 1, wherein the T. ferdinandiana leaf extract includes one or more of a methanolic extract, aqueous extract, ethyl acetate extract, alcohol extract, chloroform extract or hexane extract.
4. The composition of claim 1, wherein the T. ferdinandiana leaf extract includes a proportion of at least one antioxidant.
5. The composition of claim 4, wherein the at least one antioxidant includes one or more of a lactic acid, an ellagic acid or a trimethyl ellagic acid.
6. The composition of claim 1, as an antimicrobial agent for use used with the perishable animal and/or plant based products being fresh, cooked or semi-cooked animal and/or plant products.
7. (canceled)
8. The composition of claim 1, wherein the animal products include marine animal based product(s).
9. The composition of claim 8, wherein the marine animal based product(s) include one or more of seafood, fish, octopus, cuttlefish, squid, jellyfish, chilled cooked or raw crustaceans, shellfish, prawn, shrimp, crab, lobster, fish, muscles, oysters.
10. A method of inhibiting growth of controlling bacteria on a food preparation surface, on a food preparation tool or utensil, on food packaging or on an internal or external surface of a food product, the method including applying bacteria includes applying a composition containing an extract of Terminalia ferdinandiana (T. ferdinandiana) leaf to the respective food preparation surface, the food preparation tool or utensil, the food packaging or to the internal or external surface of the food product.
11. The method of claim 10, wherein the applying the composition includes one or more of spraying the composition onto the respective surface or putting the respective surface into a solution containing the composition.
12. The method of claim 11, including applying the composition by dipping or drenching the food product in a solution containing the composition.
13. (canceled)
14. An extract of Terminalia ferdinandiana (T. ferdinandiana) including extract of T. ferdinandiana leaf provided as an antimicrobial agent.
15. The extract of claim 14 including at least one tannin and/or at least one flavone.
16. The extract of claim 14, including at least one tannin.
17. The extract of claim 16, including one of or a combination of two or more of, chebulic acid, corilagen, chebulinic acid and chebulagic acid.
18. The extract of claim 14, including at least one flavone or flavinoid.
19. The extract of claim 14, including one or more anti-oxidants.
20. The extract of claim 19, wherein the at least one antioxidant includes an ellagic acid
21. The extract of claim 20, wherein the ellagic acid includes ellagic acid dehydrate and/or trimethyl ellagic acid.
22. The composition of claim 1, provided in a spray solution, a concentrate for subsequent dilution prior to use, a ready to use solution, a solid product for dispersal in a solution, or a solid product for inclusion in packaging or a transport container.
23. (canceled)
Description
BRIEF DESCRIPTION OF THE FIGURES
[0052] One or more embodiments or examples of the present invention will hereinafter be described with reference to the accompanying Figures, in which:
[0053]
[0054]
[0055]
[0056]
[0057] M=methanolic extract; W=aqueous extract; E=ethyl acetate extract; C=chloroform extract; H=hexane extract; Amp=ampicillin (10 g). Results are expressed as mean zones of inhibition SEM.
[0058]
[0059] In relation to the results shown in
[0060]
[0061]
DESCRIPTION OF PREFERRED EMBODIMENT(S)
[0062] T. ferdinandiana leaves were extensively dehydrated in a dehydrator. The resulting desiccated leaf material was stored at 30 C.
[0063] T. ferdinandiana fruit pulp was also extensively dehydrated in a dehydrator. The resulting desiccated fruit pulp was stored at 30 C.
[0064] The dried leaf and fruit pulp plant materials were ground into a coarse powder prior to use. A mass of 1 g of ground fruit and leaf powders was extensively extracted in 50 mL of either methanol, deionised water, ethyl acetate, chloroform or hexane or for 24 h at 4 C. with gentle shaking. The extracts were filtered through filter paper and air dried at room temperature. The aqueous extract was lyophilised by rotary evaporation in a concentrator. The resultant pellets were dissolved in 10 mL deionised water (containing 0.5% dimethyl sulfoxide DMSO) and subsequently passed through a 0.22 m filter and stored at 4 C. until used.
[0065] Antioxidant capacity: The antioxidant capacity of each sample was assessed using a modified 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging method. Ascorbic acid (0-25 g per well) was used as a reference and the absorbances were measured and recorded at 515 nm. All tests were completed alongside controls on each plate and all were performed in triplicate.
[0066] The antioxidant capacity based on DPPH free radical scavenging ability was determined for each extract and expressed as g ascorbic acid equivalents per gram of original plant material extracted.
[0067] Antibacterial screening: Environmental Shewanella strains: Shewanella putrefaciens strain 200, Shewanella baltica strain OS155, Shewanella frigidimarina strain NCIMB 400 and Shewanella loihica strain PV-4 were used. Antibacterial screening was achieved using a modified peptone/yeast extract (PYE) agar containing: 1 g/L peptone, 1.5 g/L yeast extract, 7.5 g/L NaCl, 1 g/L ammonium persulfate, 2.4 g/L HEPES buffer (pH 7.5) and 16 g/L bacteriological agar.
[0068] The S. putrefaciens and S. loihica cultures were incubated at 30 C. for 24 h. The S. baltica and S. frigidimarina cultures were incubated at 15 C. for 72 h. All stock cultures were subcultured and maintained in PYE media at 4 C.
[0069] Evaluation of antibacterial activity: Antibacterial activity screening of the T. ferdinandiana fruit and leaf extracts was assessed using a modified disc diffusion assay. Briefly, 100 L of each individual Shewanella spp. was grown separately in 20 mL of fresh nutrient broth until an approximate count of 108 cells/mL was achieved. A volume of 100 L of each bacterial suspension was spread onto nutrient agar plates and the extracts were tested for antibacterial activity using 5 mm sterilised filter paper discs. Discs were infused with 10 L of the T. ferdinandiana fruit and leaf extracts, allowed to dry and placed onto the inoculated plates. The plates were left to stand at 4 C. for 2 h before incubation.
[0070] Plates inoculated with S. putrefaciens or S. loihica cultures were incubated at 30 C. for 24 h. S. baltica or S. frigidimarina cultures were incubated at 15 C. for 72 h. The diameters of the inhibition zones were measured to the closest whole millimetre. Each assay was completed in at least triplicate. Mean values (SEM) are reported in this study. Ampicillin discs (10 g) were obtained and used as positive controls to compare antibacterial activity. Filter discs infused with 10 L of distilled water were used as a negative control.
[0071] Minimum inhibitory concentration (MIC) determination: The minimum inhibitory concentration for each extract was determined using two methods. A liquid dilution MIC assay was employed as it is generally considered the most sensitive bacterial growth inhibitory assay.
[0072] Furthermore, as microplate liquid dilution MIC assays are an often used method of quantifying bacterial growth inhibition efficacy, use of this method allows for comparisons. A solid phase agar disc diffusion assay was also used in this study as this bioassay was deemed to provide a closer representation of the environment and conditions relevant to a solid fish preservative system.
[0073] Microplate liquid dilution MIC assay: The MICs of the extracts were evaluated by standard methods. Briefly, overnight bacterial cultures were added dropwise to fresh nutrient broth and the turbidity was visually adjusted to produce a McFarlands number 1 standard culture. This was subsequently diluted 1 in 50 with nutrient broth, resulting in the MIC assay inoculum culture. A volume of 100 L sterile broth was added to all wells of a 96 well plate. Test extracts or control antibiotics (100 L) were then added to the top row of each plate and 1 in 2 serial dilutions were prepared in each column of wells by transferring 100 L from the top well to the next well in each column, etc.
[0074] A growth control (without extract) and a sterile control (without inoculum) were included on each plate. A volume of 100 L of bacterial culture inoculum was added to all wells except the sterile control wells.
[0075] Plates inoculated with S. putrefaciens or S. loihica cultures were incubated at 30 C. for 24 h. Plates inoculated with S. baltica or S. frigidimarina cultures were incubated at 15 C. for 72 h. p-Iodonitrotetrazolium violet (INT) was obtained and dissolved in sterile deionised water to prepare a 0.2 mg/mL INT solution.
[0076] A 40 L volume of this solution was added into all wells and the plates were incubated for a further 6 hours at 30 C. Following incubation, the MIC was visually determined as the lowest dose at which colour development was inhibited.
[0077] Disc diffusion MIC assay: The minimum inhibitory concentrations (MIC) of the extracts was also evaluated by disc diffusion assay. Briefly, the T. ferdinandiana fruit and leaf extracts were diluted in deionised water and tested across a range of concentrations. Discs were impregnated with 10 L of the extract dilutions, allowed to dry and placed onto inoculated plates. The assay was achieved as outlined above and graphs of the zone of inhibition versus concentration were plotted. Determination of MIC values were achieved using linear regression.
[0078] Inhibition of bacterial growth on fish fillets by T. ferdinandiana extracts: Innoculation of southern black sea bream fillets: Freshly filleted southern black sea bream (Acanthopagrus butcheri Munro) was obtained. All of the fish fillets were stored fresh at 4 C. and were purchased at 10 am on the same day as harvesting. The edges were asceptically excised and discarded from each fillet. The remainder of the fillets were excised asceptically to produce fish fillet cubes with 1 cm square ends, each with 2 surfaces (each 1 cm3) which had been exposed to atmospheric bacterial contamination. The cubes were separated into 5 groups (n=45):
[0079] (1) immersion in 1 M NaCl solution (control), (2) immersion 2000 g/mL methanolic T. ferdinandiana fruit extract in 1 M NaCl solution, (3) immersion 500 g/mL methanolic T. ferdinandiana fruit extract in 1 M NaCl solution, (4) immersion 2000 g/mL methanolic T. ferdinandiana leaf extract in 1 M NaCl solution, (5) immersion 500 g/mL methanolic T. ferdinandiana leaf extract in 1 M NaCl solution.
[0080] All test groups were immersed in the respective treatments for 6 hours. The cubes were subsequently removed from the treatments and allowed to drain asceptically. Three portions of each group were immediately sampled (day 0). The remainder of the portions for each group were stored separately in closed sterile containers at 4 C. Three further portions were sampled from each group at 5, 10 and 15 days following inoculation for growth time course studies.
[0081] Determination of colony forming units (cfu) in southern black sea bream fillets: To examine the effect of T. ferdinandiana fruit and leaf methanolic extracts on bacterial growth time in the southern black sea bream fillet, individual portions were sampled in triplicate for each treatment at 0, 5, 10 and 15 days following treatment. Each portion was individually homogenised using an overhead immersion blender and filtered through Whatman No. 54 filter paper at 4 C. Following homogenisation, 1 in 10 serial dilutions were prepared from each homegenate in a 1 M NaCl solution across the range 10-3-10-7. For enumeration of viable bacteria number, a volume of 100 L of each suspension was spread onto individual nutrient agar plates. The plates were incubated at 30 C. for 24 h and the bacterial load (colonies/mL of sample) was enumerated by direct colony counts and expressed as a % SEM of the untreated control colony counts (group 1) for each time point.
[0082] Artemia franciscana nauplii toxicity screening: Toxicity was assessed using a modified Artemia franciscana nauplii lethality assay. Briefly, 400 L of seawater containing 53 (mean 52.7, n=125, SD 11.8) A. franciscana nauplii were added to wells of a 48 well plate and immediately used in the bioassay. A volume of 400 L of the reference toxin or the diluted plant extracts were transferred to the wells and incubated at 251 C. under artificial light (1000 Lux). For each plate, a 400 L seawater negative control was run in triplicate. The wells were assessed at regular intervals and the number of dead counted. The nauplii were deemed dead if no movement of the appendages was observed within 10 seconds. After 24 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.
[0083] HPLC-MS/MS analysis: Chromatographic separations were performed using 2 L injections of sample onto an Agilent 1290 HPLC system fitted with a Zorbax Eclipse plus C18 column (2.1100 mm, 1.8 m particle size). The mobile phases consisted of (A) ultrapure water and (B) 95:5 acetonitrile/water at a flow rate of 0.7 mL/min. Both mobile phases were modified with 0.1% (v/v) glacial acetic acid for mass spectrometry analysis in positive mode and with 5 mM ammonium acetate for analysis in negative mode. The chromatographic conditions utilised for the study consisted of the first 5 min run isocratically at 5% B, a gradient of (B) from 5% to 100% was applied from 5 min to 30 min, followed by 3 min isocratically at 100%.
[0084] Mass spectrometry analysis was performed on a quadrapole time-of-flight spectrometer fitted with an electrospray ionisation source in both positive and negative mode. Data were analysed using the qualitative analysis software package.
[0085] Blanks using each of the solvent extraction systems were analysed using the Find by Molecular Feature algorithm in the software package to generate a compound list of molecules with abundances greater than 10,000 counts. This was then used as an exclusion list to eliminate background contaminant compounds from the analysis of the extracts. Each extract was then analysed using the same parameters using the Find by Molecular Feature function to generate a putative list of compounds in the extracts. Compound lists were then screened against three accurate mass databases; a database of known plant compounds of therapeutic importance generated specifically for this study (800 compounds); the Metlin metabolomics database (24,768 compounds); and the Forensic Toxicology Database by Agilent Technologies (7,509 compounds). Empirical formula for unidentified compounds was determined using the Find Formula function in the software package.
[0086] Statistical analysis: Data is expressed as the mean SEM of at least three independent experiments. One way ANOVA was used to calculate statistical significance between control and treated groups with a P value <0.01 considered to be statistically significant.
[0087] RESULTS: Liquid extraction yields and qualitative phytochemical screening: T. ferdinandiana fruit and leaf extractions (1 g) with various solvents yielded dried plant extracts ranging from 18 mg to 483 mg (fruit extracts) and 58 mg to 471 mg (leaf extracts) (Table 1).
[0088] Aqueous and methanolic extracts provided significantly greater yields of extracted material relative to the chloroform, ethyl acetate and hexane counterparts, which gave low to moderate yields. The dried extracts were resuspended in 10 mL of deionised water (containing 1% DMSO), resulting in the concentrations presented in Table 1.
TABLE-US-00001 TABLE 1 The mass of dried extracted material, the concentration after resuspension in deionised water, qualitative phytochemical screenings and antioxidant capacities of the T. ferdinandiana fruit and leaf extracts. Anti- oxidant Mass Concen- Capacity of tration of (mg Water Water Com- Dried Resus- Ascorbic Sol- Insol- Alka- Alka- Free bined Ex- pended Acid Total uble uble Cardiac loids loids Anthra- Anthra- Ex- tract Extract Equiv- Phe- Phe- Phe- Glyco- Sap- Triter- Phyto- (Mayer (Wagner Flavo- Tan- qui- qui- tract (mg) (mg/mL) alency) nolics nolics nolics sides onins penes steroids Test) Test) noids nins nones nones FM 359 35.9 660 +++ +++ +++ ++ + + + +++ ++ FW 483 48.3 264 +++ +++ +++ + +++ ++ FE 30 3 39 ++ ++ + + ++ ++ FC 62 6.2 7 + FH 18 1.8 1 LM 331 33.1 150 +++ +++ +++ +++ ++ + + + ++ +++ + + LW 471 47.1 340 +++ +++ +++ ++ +++ ++ ++ +++ + + LE 59 5.9 22 +++ +++ +++ ++ ++ LC 59 5.9 5 + LH 58 5.8 0.4 + ++ +
[0089] In Table 1, +++ indicates a large response, ++ indicates a moderate response, + indicates a minor response, indicates no response in the assay.
[0090] FM=Methanolic T. ferdinandiana fruit extract; FW=aqueous T. ferdinandiana fruit extract; FE=ethyl acetate T. ferdinandiana fruit extract; FC=chloroform T. ferdinandiana fruit extract; FH hexane T. ferdinandiana fruit extract.
[0091] LM=Methanolic T. ferdinandiana leaf extract; LW=aqueous T. ferdinandiana leaf extract; LE=ethyl acetate T. ferdinandiana leaf extract; LC=chloroform T. ferdinandiana leaf extract; LH hexane T. ferdinandiana leaf extract.
[0092] Antioxidant capacity was determined by DPPH reduction and expressed as milligrams (mg) ascorbic acid equivalence per gram (g) plant material (fruit or leaf) extracted.
[0093] Antixodidant content: Antioxidant capacity for the plant extracts (Table 1) ranged from 0.4 mg (hexane leaf extract) to a high of 660 mg ascorbic acid equivalence per gram of dried plant material extracted (methanolic fruit extract). The aqueous and methanolic extracts generally had higher antioxidant capacities than the corresponding chloroform, hexane and ethyl acetate extracts.
[0094] Growth inhibition of Shewanella spp.: To determine the ability of the T. ferdinandiana fruit and leaf extracts to inhibit Shewanella spp. growth, 10 L of each extract was screened using a disc diffusion assay. S. putrefaciens growth was inhibited by the methanolic, aqueous and ethyl acetate T. ferdinandiana fruit extracts and all of the leaf extracts (
[0095] Only the chloroform and hexane fruit extracts were devoid of S. putrefaciens growth inhibitory activity.
[0096] The methanolic and aqueous leaf extracts were particularly potent inhibitors of S. putrefaciens, each with zones of inhibition substantially >11 mm. This compared favourably with the ampicillin control (10 g) which had zones of inhibition of 8.30.6 mm. As S. putrefaciens is a main causative agent for microbial fish spoilage (at both mesophilic and psychrophilic conditions), these are particularly noteworthy results.
[0097] Whilst the methanolic, aqueous and ethyl acetate fruit extracts, as well as the chloroform and hexane leaf extracts, also inhibited S. putrefaciens growth, they were generally less potent than the corresponding methanolic extracts.
[0098] The lower efficacy of the low polarity extracts compared to the higher polarity extracts indicates that the most potent and/or most abundant growth inhibitory compounds are polar.
[0099] As seafood is generally stored using low temperature conditions, other psychrotrophic and psychrophilic Shewanella spp. have increased importance at lower temperatures.
[0100] Control of S. baltica growth, and to a lesser extent S. frigidimarina growth, become more important when seafood (e.g. fish) are stored at lower temperatures for extended periods and the contribution of S. putrefaciens decreases.
[0101] The growth of S. baltica was also susceptible to the T. ferdinandiana fruit and leaf extracts (
[0102] Consistent with the trend noted for S. putrefaciens growth inhibition, S. baltica also appeared more susceptible to the methanolic extracts than to the aqueous extract and the less polar extracts.
[0103] Furthermore, the leaf extracts were substantially more potent growth inhibitors than were the corresponding T. ferdinandiana fruit extracts.
[0104] As reported above for S. putrefaciens growth inhibition, the methanolic and aqueous leaf extracts were particularly potent inhibitors of S. baltica growth, with inhibition zones of 14.60.3 mm and 12.70.6 mm respectively.
[0105] The inhibition of S. baltica growth was particularly noteworthy as the growth of this bacterium was unaffected by ampicillin, indicating that this is an antibiotic resistant strain.
[0106] The methanolic fruit extract was also a good inhibitor of S. baltica growth (inhibition zone=9.80.4 mm). With the exception of the fruit chloroform and hexane extracts which were devoid of inhibitory activity, all other extracts were moderate inhibitors of S. baltica growth (as determined by zones of inhibition).
[0107] Growth of S. frigidimarina was also inhibited by several of the T. ferdinandiana fruit and leaf extracts (
[0108] As evident for the inhibition of the growth of S. baltica and S. frigidimarina, the methanolic extracts were generally more potent S. frigidimarina growth inhibitors than were the other corresponding solvent extracts.
[0109] The methanolic leaf extract was particularly potent, with an inhibition zone of 18.60.6 mm.
[0110] Notably, as for the other psychrotrophic bacterial species (S. baltica ), S. frigidimarina growth was also resistant to ampicillin exposure.
[0111] The aqueous leaf extract was also a potent growth inhibitor (inhibition zone =9.80.4 mm).
[0112] The fruit methanolic, aqueous and ethyl acetate extracts, as well as the leaf ethyl acetate extracts also inhibited S. frigidimarina growth, albeit with smaller zones of inhibition indicative of moderate inhibitory activity.
[0113] All chloroform and hexane extracts were completely devoid of S. frigidimarina growth inhibitory activity.
[0114] S. loihica and S. putrefaciens share similar genotypic and phenotypic characteristics and have similar optimal growth conditions.
[0115] Thus, the ability of the T. ferdinandiana fruit and leaf to inhibit S. loihica was also tested (
[0116] In contrast, an inhibition zone of 10.80.8 mm was measured for the methanolic fruit extract. Whilst the fruit aqueous, ethyl acetate and chloroform extracts, as well as the leaf hexane extracts also inhibited S. loihica growth, substantially smaller zones of inhibition were measured.
[0117] Quantification of minimum inhibitory concentration (MIC): The relative level of antimicrobial activity was further evaluated by determining the MIC values (Table 2) for each extract against the Shewanella spp. which were shown to be susceptible in the disc diffusion screening assays.
[0118] A similar trend was noted as seen for the screening assays i.e. the T. ferdinandiana leaf extracts were substantially better inhibitors of all Shewanella spp. growth than were the corresponding fruit extracts.
[0119] Furthermore, the methanolic extracts were generally the most potent growth inhibitors. The methanolic leaf extract was a particularly potent S. putrefaciens growth inhibitor, with disc diffusion (DD) and liquid dilution (LD) MIC values of 93 and 73 g/mL respectively. This is substantially more potent than the methanolic fruit extract (DD MIC 1160 g/mL; LD MIC 980 g/mL). The T. ferdinandiana methanolic leaf extract was also a potent inhibitor of S. baltica (DD MIC 104 g/mL; LD MIC 85 g/mL), S. frigidimarina (DD MIC 466 g/mL; LD MIC 391 g/mL) and S. loihica growth (DD MIC 95 g/mL; LD MIC 55 g/mL). The aqueous and ethyl acetate T. ferdinandiana leaf extracts also had low MIC values against all Shewanella spp. growth, albeit with MIC values often an order of magnitude higher than for the corresponding leaf extracts. In further contrast to the leaf extracts where the methanolic extract had the greatest potency, the ethyl acetate extract was generally the most potent of the fruit extracts.
[0120] Inhibition of of bacterial growth on southern black sea bream fillets: Whilst psychrotrophic and psychrophilic Shewanella spp. are generally acknowledged as a major cause of cold stored fish spoilage, other bacterial species would also contribute to spoilage.
[0121] Furthermore, whilst the MIC assay methods used in our study provide important information of the ability of the extracts to inhibit Shewanella spp. growth in vitro, they do not necessarily accurately portray bacterial spoilage in commercial cold stored fish. Therefore, the T. ferdinandiana fruit and leaf extracts were tested for the ability to inhibit bacterial growth in fish fillets under cold storage conditions.
[0122] As the methanolic extracts were generally the most effective growth inhibitors (Table 2), only these extracts were tested in the fish fillet spoilage assessment. T his study did not discriminate between bacterial species, instead measuring the total viable bacteria as colony forming units (cfu). Furthermore, as cold storage is the most common preservation method for fresh fish, the fish fillets used in this study were also stored at 4 C. throughout the assessment period. The results are expressed as a % cfu of the control fish fillets (no treatment, held at 4 C.) to determine the degree of improvement over cold storage alone.
[0123] All treatments with the methanolic fruit and leaf T. ferdinandiana extracts were effective at reducing the number of viable bacteria on the fillets immediately following treatment, indicating that the extracts were bactericidal at 0.5 mg/mL. Indeed, the cfu's for all treated groups were inhibited by approximately 95% compared to the control fillets.
[0124] With the exception of the 0.5 mg/mL methanolic fruit extract, all extracts remained approximately as effective following 10 days cold storage as at the start of the test, each still inhibiting bacterial growth by approximately 95% compared to the control fillets. Although the 0.5 mg/mL methanolic fruit extract was not as effective following 10 days growth, it still inhibited bacterial growth by approximately 35% compared to the control fillets.
[0125] Following 15 days cold storage, the fruit extract (both concentrations) and the 0.5 mg/mL leaf extract treatment were less effective than earlier in the trial, with bacterial growth increasing to 50-85% of the levels in the control fillets.
[0126] These values represent a significant reduction in bacterial growth and indicate that all of extracts significantly decrease spoilage for at least 15 days.
[0127] Notably, the 2 mg/mL methanolic leaf extract treatment was still very effective at inhibiting bacterial growth at 15 days. Indeed, there was still approximately 90% reduction in bacterial growth at day 15 of the trial for this treatment. Thus, treatment with 2 mg/mL methanolic T. ferdinandiana leaf extract substantially decreases bacterial spoilage at 15 days, indicating its potential for increasing the shelf life of cold stored fish.
[0128]
[0129] In
[0130] Bacterial growth for all treatment groups were measured at 5 day intervals following inoculation. Results are expressed as mean zones of inhibition SEM of 3 portions in triplicate at each time interval. * indicates results that are significantly different to the untreated control (p<0.01).
[0131] As shown in
[0132] The reference toxin was rapid in its onset of mortality, promoting nauplii death within the first 3 h of exposure, with 100% mortality evident within 5 hours (unpublished results). All of the methanolic and aqueous extracts also induced 100% mortality following 24 h exposure. Similarly, the ethyl acetate leaf extract also induced 100% mortality at 24 h exposure. All other extracts induced <50% mortality and were therefore deemed to be nontoxic.
[0133] To further quantify the effects of toxin concentration on the initiation of mortality, the extracts were serially diluted in artificial seawater to test across a range of concentrations in the Artemia nauplii bioassay. The 24 h LC50 values of the T. ferdinandiana fruit and leaf extracts towards A. franciscana are displayed in Table 2. No LC50 are reported for any chloroform or hexane extracts, nor for the fruit ethyl acetate extract as <50% mortality was seen in all tested concentrations. LC50 values substantially >1000 g/mL were determined for all of the other extracts. As extract with LC50 values >1000 g/mL towards Artemia nauplii have been defined as being nontoxic in this assay, all of the T. ferdinandiana fruit and leaf extracts were deemed to be nontoxic.
[0134] All of the methanolic and aqueous extracts also induced 100% mortality following 24 hours of exposure. Similarly, the ethyl acetate extract also induced 100% mortality at 24 hours exposure. All other extracts induced <50% mortality and were therefore deemed to be nontoxic.
[0135]
[0136] Table 2 below shows results for disc diffusion and liquid dilution MICs against S. putrefaciens, S. baltica, S. frigidimarina and S. loihica growth (g/mL) and Artemia nauplii bioassay LC50 values (g/mL) of T. ferdinandiana fruit and leaf extracts.
TABLE-US-00002 TABLE 2 MIC (g/mL) Atremia nauplii S. puirefaciens S. baltica S. frigidmarina S. loihica bioassay D.D. L.D. D.D. L.D. D.D. L.D. D.D. L.D. LC.sub.50 (g/mL) F M 1160 986 1077 991 1711 1471 1187 934 2028 F W 1869 1645 2202 2400 2774 2015 2636 1582 1980 F E 1000 860 4326 >5000 1331 1025 932 692 1655 F C 4485 >5000 F H L M 93 73 104 85 466 391 95 55 1283 L W 472 321 625 669 856 753 256 197 1375 L E 311 252 846 770 784 533 469 219 1068 L C 881 960 862 765 2562 1614 L H 841 807 1396 1160 2829 1924 D.D. = disc diffusion; L.D. = liquid dilution; Numbers indicate the mean D.D, MIC, L.D, MIC and LC.sub.50 values of triplicate determinations. indicates no inhibition; F = fruit; L = leaf; M = methanolic extract; W = aqueous extract; E = ethyl acetate extract; C = chloroform extract; H = hexane extract.
[0137] The methanolic leaf extract had the greatest antibacterial efficacy (as determined by MIC; Table 2).
[0138] Optimised HPLC-MS/MS parameters were developed and used to search for specific compound classes in the methanolic leaf extract and identify the individual components.
[0139] The resultant total compound chromatograms (TCC) for the positive ion and negative ion chromatograms are presented in
[0140] The negative ion chromatogram had significantly higher background absorbance levels than the positive ion chromatogram, due to ionisation of the reference ions in this mode, possibly masking the signal for some peaks of interest.
[0141] The positive ion chromatogram (
[0142] The metabolomics fingerprinting approach used in this study targeted two specific phytochemical classes. High tannin contents are a defining feature of Terminalia spp. and high tannin contents have been reported in T. ferdinandiana (Cock, 2015).
[0143] Furthermore, a recent study characterised a number of tannin components in T. ferdinandiana leaf and correlated them with the inhibition of the growth of several pathogenic bacteria (Courtney et al, 2014). In total, 10 tannins were putatively identified in the methanolic T. ferdinandiana leaf extract by comparison to the Metlin metabolomics, forensic toxicology (Agilent) and phytochemicals (developed in this laboratory) databases.
[0144] Chebulic acid (2.2% total peak area in+ionisation mode), chebulagic acid (1.7% total peak area inionisation mode), corilagen (7.2% total peak area inionisation mode), ellagic acid (1.0% total peak area inionisation mode) and trimethyl ellagic acid esters (1.7% total peak area in+ionisation mode), exifone (1.9% total peak area in+ionisation mode) and punicalin (2.4% total peak area inionisation mode) were present in particularly high relative abundance (as assessed by their relative % peak area). All other tannins were present in lower relative abundances.
[0145] T. ferdinandiana fruit and leaf extracts were selected for screening for the ability to block the growth of spoilage bacteria as they have potential to positively influence the shelf life of perishable food product in several ways.
[0146] A major portion of fresh food spoilage, such as meat products e.g. seafood, fish, meat etc., is the result of oxidative spoilage.
[0147] The treatment of perishable food product with preparations containing high antioxidant contents (e.g. some plant extracts) decreases lipid oxidation and thus inhibits oxidative rancidity.
[0148] T. ferdinandiana fruit and leaf extracts, individually or combined, are potent inhibitors of Shewanella spp. growth and therefore have potential as natural fish/seafood preservatives.
[0149] The T. ferdinandiana leaf extracts were particularly effective against all psychrotrophic and mesophilic Shewanella spp. and thus have potential for both fresh and cold storage fish preservation.
[0150] All tested extracts of T. ferdinandiana leaf were found to be non-toxic in Artemia fransiscana (brine shrimp) bioassay.
[0151] Furthermore, all of the T. ferdinandiana extracts were nontoxic towards Artemia nauplii and are thus safe to use as natural fish preservatives.