**1. Introduction**

*Terminalia* is the second-largest genus of the combretaceae family, with approximately 250 species growing in tropical and subtropical countries around the world [1]. More than 30 species of *Terminalia* occur in northern regions of Australia [2]. More than 50 species of *Terminalia* have found utility as ingredients in foods and beverages worldwide, as preservatives, raw material for wine and palm sugar, eaten raw and as food supplements [3]. The nutritional and therapeutic properties of *Terminalia* genus can be attributed to the presence of a wide range of phytochemicals, such as phenolic compounds, which encompasses phenolic acids, gallotannins, ellagitannins, proanthocyanidins and other flavonoids [3].

*Terminalia ferdinandiana*, popularly known as Kakadu plum, is native to Australia. Indigenous Australians (Aboriginal people) have been using this plant as a food and medicine for centuries, for example, refreshing drinks are made from fresh or dried fruits in Western Australia [2]. Fruits are traditionally used as an antiseptic, soothing balm, in colds and flu and in treating a headache [4]. A number of research outcomes have been reported on the antioxidant [5–7], antibacterial [8,9], anti-inflammatory [10], anti-apoptotic, cytoprotective and anticancer activities [11] of *T. ferdinandiana*

fruits and leaves. Phytochemical analysis has revealed that *T. ferdinandiana* fruit is a rich source of Ellagic acid and its hydrolysable tannins, ellagitannins [12]. Recently, a food safe extraction method of *T. ferdinandiana* fruits for commercial use in the food industry has been suggested [4]. Additionally, a systematic evaluation of the changes in quality and bioactivity of the fruits of *T. ferdinandiana* during processing, packaging and storage has been performed, and key chemical markers have been identified to enable standardized products to be delivered to the consumer [9].

In the last two decades, seeds and kernels from the *Terminalia* genus have been researched and reported for their nutritional properties and health-promoting activities [13–15]. To understand the relationship between the internal quality and genotype of the plant, studies of nut and kernels characteristics and composition are very common. During the industrial processing of *T. ferdinandiana* fruits, the seeds are treated as waste products and have been discarded. Recent studies on many fruit seeds or kernels have shown that they have the potential to be utilized as ingredients for value addition, they are very nutritious and could be used as alternate sources of essential minerals, fatty acids, and proteins [16–19].

To date, no reports have been published on the utilization of the by-products of *T. ferdinandiana* and there is no investigation on the chemical and nutritional composition of *T. ferdinandiana* kernels. The aim of this study was to determine the potential use of the by-product of *T. ferdinandiana* in the industry by determining proximate, mineral and fatty acid compositions to ascertain its nutritional value and potential as a source of food supplement ingredients for the food industry.

#### **2. Materials and Methods**

#### *2.1. Sample Collection and Preparation*

Fully ripe and mature fruits of *T. ferdinandiana* were collected from over 600 trees, giving a total harvest of 5000 kg, from native bush land covering an area of 20,000 km<sup>2</sup> in Northern Territory, Australia in 2015 and were authenticated by the experts in Queensland Herbarium, Brisbane Botanic Gardens Mt Coot-tha, Queensland, Australia, where botanical specimens were retained for future reference (AQ522453). Seeds were collected as the by-products after pureeing of the fruits, and were stored at −20 ◦C prior to analysis. *T. ferdinandiana* tissues are illustrated in Figure 1.

**Figure 1.** *Terminalia ferdinandiana* tissues. ( **A**) Fresh fruits; (**B**) Dried seeds; ( **C**) Kernels.

#### *2.2. Processing of Seeds*

The frozen seeds were thawed, washed and cleaned manually several times to remove the pulp residues with double distilled water. The seeds were then dried in the oven for 48 h at 40 ◦C. After drying, the seeds were individually cracked using an Engineers' vice size 125 (DAWN, Melbourne, Australia) to release the kernels from the seedcoats. The seedcoats and kernels were kept, processed and analyzed separately. The kernels were kept in air-tight containers and placed at −20 ◦C for further analysis. A flowchart depicting the processing of the seeds is illustrated in Figure 2. During the processing of fruits in the industry in a batch of 100 kg of fruits, 22 kg of seeds can be obtained as by-product. The average weight of a dry seed is 0.5 g and the moisture content is 2.8%. Average weight of a kernel is 0.04 g. The kernel is 8% of the weight of the seed. 1 kg of dry seeds can deliver 80 g of kernels.

**Figure 2.** Processing of *Terminalia ferdinandiana* seeds to release kernels.

#### *2.3. Proximate Composition Analysis*

Physicochemical analysis of the kernels of *T. ferdinandiana* was performed at an accredited laboratory (National Association of Testing Authorities (NATA), Symbio Alliance, Eight Mile Plains, Queensland, Australia). The following analyses were done according to AOAC methods: vitamin C, protein (AOAC 990.03, 992.15 & 992.15), fat (AOAC 991.36), saturated, mono-unsaturated, polyunsaturated and trans-fat (AOAC 996.06), moisture (AOAC 925.10), ash (AOAC 923.03), sodium (using ICP-AES), total sugar (AOAC 977.20) and dietary fiber (AOAC 985.29, 991.42 and 993.19). Available carbohydrate and energy were calculated using FSANZ (Food Standards Australia New Zealand) codes.

#### *2.4. Fatty Acid Analysis*

Dried kernels (ca. 1 g) were finely chopped and extracted with chloroform and methanol (2:1) followed by agitation at room temperature for one hour. The mixture was then centrifuged for 5 min at 3500 rpm and the whole process was repeated twice. The lipid extracts were mixed with boron trifluoride (BF3)-methanol reagen<sup>t</sup> (20%) and fatty acids were derivatized to fatty acid methyl esters [20]. The methyl esters of the fatty acids were dissolved in heptane and analyzed by GC-MS (Shimadzu QP2010, Shimadzu Corporation, Tokyo, Japan). The GC conditions were as follows: Restek stabilwax capillary column (30 m × 0.25 mm ID × 0.5 μm film thickness) (Restek Corporation, Bellefonte, PA, USA); oven temperature program: the column held initially at 100 ◦C after injection and the final temperature was increased to 250 ◦C, total program time was 39:00 min; injector temperature: 250 ◦C; carrier gas: Helium; linear gas velocity: 42.7 cm/s; column flow: 1.10 mL/min; split ratio: 50.00; injection volume: 1.0 μL. MS conditions were regulated as follows: ion source temperature: 200 ◦C; interface temperature: 250 ◦C; mass range: 35–500 atomic mass units. Identification of the compounds was carried out by comparison of their retention times and mass spectra with corresponding data from a standard food industry FAME Mix (Restek Corporation, Bellefonte, PA, USA). A total of 32 individual compounds were analyzed and only the detected ones were recorded along with their quantity compared with the standard.

#### *2.5. Mineral and Trace Element Analysis*

Accurately weighed 0.3 g of dried *T. ferdinandiana* kernels were taken into teflon vessels of microwave digestion system (MarsXpress, CEM, Matthews, NC, USA) and high-purity nitric acid (70% *<sup>w</sup>*/*<sup>w</sup>*, 4 mL) was added [21]. The samples were left overnight at room temperature for slow digestion gasses to evolve. The vessels were sealed and microwave-digested at increased temperature with set digestion time [22]. The digested samples were diluted and made up to 40 mL with high-purity water (Milli-Q Element system, Millipore, Bedford, MA, USA). The levels of minerals and trace elements were analyzed by inductively coupled plasma optical emission spectrometry (ICP-OES, Vista AX, Varian Australia, Mulgrave, Victoria, Australia), to measure lower levels and for greater sensitivity the analysis was carried out using ICP-MS (7500a, Agilent, Tokyo, Japan). The ICP-MS was equipped with an auto-sampler, integrated sample introduction system and a helium octopole reaction cell to remove polyatomic interferences (40Ag 35Cl on 75As). The operating conditions were as follows: radio frequency (RF) power 1350W, argon carrier gas 0.8 L/min and helium reaction cell gas flow rate 4.5 mL/min. The standard reference materials were used for the quality control and assurance and treated similarly to the samples throughout the study. The data of quality control and assurance are presented in the Supplementary Material (Table S1).

## *2.6. Statistical Analysis*

The data were calculated using Microsoft Excel 2013 (Microsoft Corporation, Redmond, WA, USA). The results are expressed as the mean of triplicate experiments unless otherwise specified.

#### **3. Results and Discussion**
