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Western SydneyUniversityResearch Proposal: Nutritional profiles andcomparisons of 4 native Carpobrotus species underdi↵erent growth conditionsTobias PalmerMay 2020AbstractThe following research proposal aims to assess the suitability of 4 Australiannative, edible halophytes from the genus Carpobrotus as an alternative food cropfor human consumption under di↵ering soil and salinity conditions.Species of Carpobrotus have traditionally been consumed as a vegetable byAfrican and Australian indigenous groups due to their nutritional value, naturalabundance and supposed medicinal value, the research proposal assesses the currentstate of nutritional knowledge available, and propose a design to test the qualities ofthe native species over a range of nutritional factors to build up a complete profile.Signature:Tobias Palmer Date: 29/05/20201Contents1 Background 12 Literature Review 13 Methodology 33.1 Materials. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33.2 Method. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33.3 Statistical analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 Gantt Chart 55 Proposed Budget 66 Results 67 Conclusion 71 BackgroundThe predicted e↵ects of climate change are likely to decrease the amount of land useableby traditional commercial agriculture. Specifically, increases in desertification, higher soilsalinity, lower water availability and overall poorer soil quality are likely e↵ects whichwould largely limit what kinds of crops that can be grown in many areas currently usedfor food production. This presents a need to research alternate crops which can grow inthese environments while still providing edible and nutritious foodstu↵ (Wyk, 2011)Halophytes from the family Carpobrotus are a traditional food source of indigenousgroups from South Africa and Australia, they are an edible succulent whose leaves andfruits are thought to have medicinal and highly nutritious properties (Pirie et al., 2013).2 Literature ReviewAt the time of writing there is no complete nutritional profile in place for any of the 4native species of Carpobrotus in Australia (C. Rosii, C.Glaucescens, C. Modestus andC. Virescens), however there has been some research done on Caprobrotus Edulis, SouthAfrica’s native Caprobrotus. Research by Rocha et al. (2017) was able to produce anutritional profile for C.Edulis shown below in figure 1.In their discussion, they compare C.Edulis to many kinds of commonly grown vegetables,with the main areas of similarity being moisture content, crude fat/crude protein and fibrecontent. They noted that the Polyunsaturated fat content of C.Edulis was significant at1Figure 1: Nutritional profile of C.edulis from Rocha et al. (2017)32.5% making it a source of dietary PUFA’s. The Authors also note that the ash contentis particularly high, and hypothesised this as being likely due to the halophytes beingcollected from a coastal setting in Portugal, and therefore accumulating large quantitiesof Na+ and Cl- , as well as other minerals during coastal exposure.Further analysis of mineral content of C.Edulis resulted in figure 2.Figure 2: Mineral analysis of C. edulis (Rocha et al., 2017)The Authors noted high values for Ca, Fe and Mn, further illustrating C.Edulis’s nutrientvalue as a source for biologically available minerals. Although the heavy metals reportedin the nutrition profile were either too low to be detected (Pb, Ni and Cd) or well belowsafe levels for ingestion, the authors do note that halophytes do bioaccumulate heavymetals if grown in contaminated soils. Typically this accumulation occurs in undergroundorgans but needs to be taken into account if grown in a commercial setting.Based on our literature review there is evidence of halophytes from the Carpobrotus2species being a valuable alternative crop based on nutritional profiles created for Caprobrotus Edulis (Rocha et al., 2017) (Varone et al., 2017), as well as micronutrient researchdone on Carpobrotus Rossii (Pirie et al., 2014) (Pirie et al., 2013).The following research proposal will aim to create a nutritional profile of all 4 nativespecies of Carpobrotus as well as C. Edulis, in order to determine which one would bemost suitable for adoption as an alternate crop. The 5 di↵erent species will also becompared across di↵erent growing conditions of salinity, and soil quality as these arefactors which are important when determining the viability of the species for human useunder non ideal conditions.3 Methodology3.1 Materials.Plant specimens for the 5 species of Carpobrotus (C. Edulis, C. Rosii, C.Glaucescens, C.Modestus and C. Virescens) are to be sourced through the University for Western Sydneyfrom trusted sources, so as to ensure that the plant samples are true representatives oftheir species.The proposed experiment requires 6 di↵erent conditions for plant growth with 6-10 plantsspecimens used by each species to create an average. For a total of 180-300 plant specimens. All plant specimens are grown in commercial 15cm diameter plastic pots with freedrainage holes at bottom. At the start of the experiment, plant specimen circumference,leaf number and average leaf size is recorded.3.2 Method.The plant samples are to be all grown under the same lighting/water schedules. Specifically the plants are grown in greenhouse conditions of high light exposure ( recordedusing Bureau of meteorology info to give approximations) with adequate ventilation toensure the inhibition of pests and fungal development. Care is taken to ensure that allplants receive the same amount of light as each other during the day.The watering schedule for the plants is automated with a drip irrigation system. Eachplant specimen receives 30 ml of water every alternate day for the duration of the 4 monthgrowing period.For each of the 5 species of Carpobrotus, there are 6 growth conditions, in order todetermine which condition results in the highest yield/nutritional value as well as whichspecies of Carpobrotus perform better under which conditions of growth.The 6 conditions are:1. Good soil/Ideal Salinity2. Poor soil/Ideal salinity3. Good soil/Low salinity34. Poor soil/Low salinity5. Good Soil/high salinity6. Poor soil/High SalinityPlants in the Good soil conditions were planted in standard commercial grade pottingmix (Hortico All Purpose Potting Mix). plants in the Poor condition were planted in amix of 50% commercial grade potting mix and beach sand.Salinity conditions were determined using the results obtained in the experiments by Pirieet al. (2013) and Varone et al. (2017), Ideal salinity was 100mM, Low salinity 0mM andHigh salinity 200mM (aprox, look up the numbers)At the end of the 3 month growth period the plant specimens circumference, leaf numberand average leaf size is recorded and the change in values to original is determined.For the nutritional profile, the plant specimens are dried at 40C for 3 days then powderedand stored at -20C, following the same method in Rocha et al. (2017)The following nutritional properties are to be determined via di↵ering methodologies.Moisture content.Moisture content is determined at the time of sample preparation, through calculatingthe di↵erence in weight of the plant samples before the 3 day oven drying and at theconclusion. Since the drying is at a low temperature but a long duration, organic volatilityis almost non existant, and the loss of matter can be determined to be almost exclusivelywater loss.Protein content.The Dumas method is used to determine the protein content of the samples. Rocha et al.(2017) used the Macro Kjeldahl method, but since there are 100’s of samples to analyse,an automatable process like the Dumas method will be used instead.Fat content.Crude fat content will be determined through solvent extraction, specifically the Blighand Dyer method. It was decided than specific fat analysis, i.e. the lipid types in thesamples is beyond the scope of this experiment, so not necessary.Carbohydrate content.Total carbohydrate content for each sample is determined through the di↵erence equation.Total carbohydrates = Sample weight (post dried) – total protein –total fat – total ash.Ash/Mineral content.Ash content is to be determined through dry ashing which is combustion at 500C in afurnace, to remove organics form the sample, leaving only the inorganic minerals.Mineral content is to be determined through atomic absorption spectroscopy, 300mg ofthe dried sample is mixed with 6ml of HNO3 (65%), 1 ml of HClO4 and 1 ml of H2O2prior to analysis, in accordance with the methodology set out in Rocha et al. (2017). the8 essential biological minerals are being tested for.Other organic compounds.4High Performance Liquid Chromatography is to be used to test for the content of thefollowing organic compounds as they are of a nutritional interest being vitamins beneficialto human diet: Retinal, retinol, beta carotene, Thaimin, Riboflavin, niacin, Pantothenicacid, pyridoxine, biotin, folic acid, hydroxocobalamin, ascorbic acid, ergocalciferol, tocopherols, tocotrienols and phytomenadione.In all cases, 10mg of sample is prepared with 10ml of ultrapure water and analysedthrough the HPLC. The retention times for each compound are compared to obtainedcalibration standard curves in order to determine a concentration.3.3 Statistical analysis.For each of the 6 growth conditions, a one way ANOVA will be carried out using ‘R’statistical software, comparing the means of the 5 Carpobrotus species for the following variables. Change to plant specimen circumference, change to average leaf number,change to average leaf size, Moisture content, Protein content, Fat content, Carbohydratecontent, Ash content, Specific mineral content, Specific Vitamin content.Where an ANOVA indicates a statistically significant di↵erence in means, Follow upstudent’s t tests are to be carried out in ‘R’ software to determine where this significancelies.4 Gantt ChartFigure 3 shows the proposed timetable for the research project, anticipated to startJanuary 2021.Figure 3: 7 month research window for proposed research project55 Proposed BudgetFigure 4 shows the proposed budget for the reseach proposal.Figure 4: 7 month research window for proposed research project6 ResultsThe purpose of this research project is to determine which native Carpobrotus speciesperform the best as nutritional crops under di↵erent growth conditions.3 of the Carpobrotus species have a similar coastal distribution (C. rossi, C. Glaucescens,C. virescens) although in di↵erent states of Australia. Since they share similarities inhabitat, we would assume that these 3 would have the most similarity in terms of nutritional potential (Pirie et al., 2013).The species C. Modestus has a coastal and inland distribution, which is dissimilar tothe other native species which are found mostly along the coast/coastal bushlands. Thisdi↵erence in distribution may represent a di↵erence in the plants physiology, which maysubsequently lead to di↵erences in the nutritional properties of the plant (Pirie et al.,2014).Since C. Edulis is not native to Australia, and the soil/growth conditions of its nativeSouth Africa is likely di↵erent to That of Australia, we would expect to see some di↵er-6ences in nutritional properties when comparing it to the other Carpobrotus species (Wyk,2011).7 ConclusionAssessing the suitability of the native Australian species of Carpobrotus would representa contribution to the growing trend of research into alternate crops, native edibles andfood security that is emerging as a vital field of research for a future likely to be severelya↵ected by the impact of global climate change (Wyk, 2011).This study represents a preliminary investigation into some basic growth conditions andtheir e↵ect on the Carpobrotus species which have the potential to be an importantcommercial crop in the future.ReferencesPirie, A., Parsons, D., Renggli, J., Narkowicz, C., Jacobson, G. A., and Shabala, S. (2013).Modulation of flavonoid and tannin production of carpobrotus rossii by environmentalconditions. Environmental and Experimental Botany, 87:19 – 31.Pirie, A. D., Davies, N. W., Ahuja, K. D., Adams, M. J., Shing, C. M., Narkowicz, C.,Jacobson, G. A., and Geraghty, D. P. (2014). Hypolipidaemic e↵ect of crude extractfrom carpobrotus rossii (pigface) in healthy rats. Food and Chemical Toxicology, 66:134– 139.Rocha, M., Rodrigues, M., Pereira, C., Pereira, H., [da Silva], M., da Rosa Neng, N.,Nogueira, J., Varela, J., Barreira, L., and Cust´odio, L. (2017). Biochemical profileand in vitro neuroprotective properties of carpobrotus edulis l., a medicinal and ediblehalophyte native to the coast of south africa. South African Journal of Botany, 111:222– 231.Varone, L., Catoni, R., Bonito, A., Gini, E., and Gratani, L. (2017). Photochemicalperformance of carpobrotus edulis in response to various substrate salt concentrations.South African Journal of Botany, 111:258 – 266.Wyk, B. V. (2011). The potential of south african plants in the development of new foodand beverage products. South African Journal of Botany, 77(4):857 – 868. Specialissue on Economic Botany.7

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