Influence of blanching on the bioactive compounds of Terminalia ferdinandiana Exell fruit during storage

Changes in quality of fruits and vegetables during processing and storage might impact on the nutritional and economical value of food products. The present study aimed to evaluate the influence of blanching on the content of vitamin C and ellagic acid (EA) as the main bioactive compounds present in Kakadu plum (KP) fruits (Terminalia ferdinandiana) during storage at 40oC to mimic typical temperature when wild harvested. Changes in the profile of fatty acids, malondialdehyde (MDA) production, as a biomarker for lipid peroxidation, and antioxidant properties of KP fruits were evaluated. The results revealed that vitamin C decreased between 25 and 52% over the storage period. Statistically significant differences in the concentration of vitamin C were associated with temperature (p < 0.05) and blanching (p < 0.05), whereas no significant differences in EA during storage were observed. DPPH radical scavenging capacity and total phenolic content of both blanched and control samples decreased by 80% and 35%, respectively, at the end of the storage period compared to day 0. In addition, the change in DPPH activity is significantly correlated (Pearson R2 = 0.829, p ˂ 0.01) with the breakdown of ellagitannins. Furthermore, KP fruit demonstrated excellent antioxidative properties by reducing MDA production. It was concluded that blanching causes significant vitamin C loss whereas neither blanching nor long-term storage at elevated temperature affect the EA content. The results also indicate that the antioxidant compounds present in the KP fruits provided considerable protection against fatty acid oxidation during storage.


Introduction
Terminalia ferdinandiana Exell fruit, commonly known as Kakadu Plum (KP), fruit extracts demonstrated several health advantages including in vitro anti-inflammatory [1], antioxidant [2], anticancer [3], and antibacterial [4,5] activities. A relatively high content of specific minerals, vitamins and sugars as well as ETs and EA [2,6] have also been reported in this fruit. As a results KP fruits and derived non-reactive unsaturated fatty acids to secondary oxidation products [18]. Particularly malondialdehyde (MDA) is one of the most abundant aldehydes generated during autoxidative degradation of PUFA [17].
Several methods of food processing have been evaluated to preserve the quality attributes of plant-based food products [19,20]. Blanching is the oldest and simplest technique that has been applied to maintain food quality before drying, frying and canning, and to inactivate quality-deteriorating enzymes [21]. In addition, blanching, normally carried out by boiling water immersion, steam and radiations [12,22], has shown advantages in stabilizing nutritional quality [21,23].
Water blanching is the most commercially popular method [21]. The treatment time and temperature of the blanching process depend on the required degree of enzyme inactivation (mostly 90% inactivation) and amount of heat transferred to the samples [23]. For instance, Kim and coworkers [23] blanched Samnamul (Aruncus dioicus var kamtschaticus) in hot water at various temperatures (80, 90 and 98 °C from 15 s to 10 min) and have reported that polyphenol oxidase and peroxidase activities were maximally inhibited by blanching for 30s at 98 °C. Menon et al. [24] and Xanthakis et al. [19] reported the "optimal" polyphenol oxidase and ascorbate oxidase inactivation in cocoa beans and frozen mangoes, respectively, when water blanched at 90 °C for 5 min.
Therefore, the present study was carried out to investigate the influence of water blanching on the vitamin C and ellagic acid (EA) contents, main bioactive compounds in KP fruits and derived products, during storage of KP fruits wild harvested from Kimberley, Western Australia. Furthermore, oxidative rancidity and changes in the fatty acid content were determined to examine if the antioxidant compounds in KP fruit provide oxidative stability during storage.

Plant materials and storage experiment
The KP fruits were wild harvested in January 2020 from Bidyadanga (located 180 km south of Broome, Kimberley, Western Australia). The samples were transported to the laboratory under refrigerated conditions and then immediately stored at -80 °C for further analysis. A preliminary blanching study was conducted with total of 60 randomised fruits (20 fruits per blanching temperatures of 80 °C, 90 and 100 °C) for 5 min using a pre-heating water bath (TW20, John Morris Scientific LTD, Germany) in order to select the optimum blanching temperature. The 5 min duration was based on previous studies [19,24] while 45 days storage was time length selected due to the fact that free radicals could cause damage to lipids with increased storage time [25].
A total of 800 fruits were randomly selected for the storage study, in which 400 fruits were subjected to blanching treatment at 90 °C for 5 min as given below (Fig. 1). The remaining fruits were control samples (no heat treatment). Both the blanched and control fruit samples were immediately frozen at -80 °C and freeze-dried under vacuum at -50 o C for 72 h, using a Scanvac Coolsafe superior freeze drier (Labgear, Brisbane, QLD, Australia). Half of the freezedried fruits with seeds (n = 200) in each sample group (control or blanched) were finely ground into a homogenous powder using a Retsch MM301 cryomill (Retsch GmbH, Haan, Germany). Approximately, 20-30 whole fruits and 20-25 g of freeze-dried powder from each sample group were packed in individual vacuum pouches (150 mm x 200 mm x 70 μm; Length x Height x Thickness) (Pac Plus, QLD, Australia). The pouches were sealed and the air in the pouch was completely removed using a vacuum seal C 500 Multivac Chamber machine (Multivac Sepp Haggenmüller GmbH & Co. KG, Germany). Figure 1 illustrates the flowchart for processing the Kakadu plum fruit for the storage trial and the experimental design.
The pouches containing either the whole fruit or fruit powder samples were stored at 40 °C for 45 days. An elevated storage temperature at 40 °C was used to mimic the hot climate conditions of Northern Australia. Duplicate samples were collected at time zero before packaging (Day 0), on the 27th day (Day 27) and 45th day (Day 45) for further analysis. A total of 105 samples were collected in duplicate including heat treatment (control and blanching), powder and whole fruit, different storage times (Day 0, 27th & 45th ). Once sampled, the whole fruit stored in pouches were finely ground with the seeds using ball milling as with the powder samples prior to the determination of vitamin C, EA, TPC, DPPH radical scavenging capacity, and Thiobarbituric Acid Reactive Substances' (TBARS).

Total phenolic content (TPC) and 2,2-diphenyl-1picrylhydrazyl (DPPH) radical scavenging capacity
Approximately 100 mg (in triplicate) of samples was mixed with 5 ml aqueous acidified ethanol (80% ethanol, 19.8% water, 0.2% HCl) (AAE), vortexed and sonicated for 10 min. The mixture was centrifuged (3900 rpm, 5 min at 20 °C; Eppendorf Centrifuge 5810 R, Hamburg Germany) and the supernatant was collected. The extraction was repeated three times and the supernatants were combined and subjected to the TPC assay using the Folin-Ciocalteu reagent and DPPH radical scavenging capacity assay according to Moore & Yu [26] as described previously [27] without modification.

Analysis of ellagic acid (EA) and vitamin C
The extraction and analysis of EA and vitamin C were carried out using UHPLC-PDA as described previously [27] without modification.
Where, Abs is absorbance.

Fatty acid analysis
The lipids were extracted in a process according to Srivarathan et al. [32] with slight modifications. Briefly, approximately 200 mg of the powdered sample (in triplicate) were mixed with methanol in glass test tubes and vortexed for 5 min. The tubes were sonicated for 15 min at room temperature (RT). Chloroform (CHCl 3 ) and Milli Q water were added to the tubes at the ratio of 2:1:1 (methanol, chloroform, water; v/v/v), followed by centrifugation at 800 rpm for 5 min at RT (Eppendorf Centrifuge 5804, Eppendorf, Hamburg, Germany) and the lower layer was collected.
The sample in the tubes was re-extracted with chloroform and methanol (1:1, v/v), centrifuged (800 rpm for 5 min) and the supernatant was collected and combined with the previous collected solution. Finally, the combined solution was evaporated at 45 °C under nitrogen flow using a Ratek dry heat blocker (DBH20D, Ratek Instruments Pty Ltd., Melbourne, VIC, Australia) and reconstituted in chloroform (10 mg/mL) before derivatization to fatty acid methyl esters (FAME) as previously described by Srivarathan et al. [32]. FAME analysis was conducted using A Thermo ISQ-7000™ Single quadrupole GC-MS system (Thermo Scientific, Brisbane, QLD, Australia) equipped with an Agilent DB-23 fused silica capillary column (60 m × 0.25 mm diameter; i.d, 0.15 μm film thickness; Agilent Technologies, Santa Clara, CA, USA). A Supelco 37-component FAME mix standard (Sigma-Aldrich, NSW, Australia) was used for compound identification and quantification.

Statistical analysis
Data are expressed as mean ± SEM (standard error of mean). Differences between the samples were determined using a one-way analysis of variance (ANOVA) followed by Tukey post hoc test using IBM SPSS Statistics 25 (SPSS Inc. Chicago, IL, USA). Further correlations were obtained by Pearson correlation coefficient in bivariate correlations. p < 0.05 was considered as significant different.
fruit at 90 o C was selected as the heat treatment condition for the present study. Vitamin C content was reduced by approximately 25-52% over the storage period of 45 days (Table 1). Statistically significant (p < 0.05) effect of blanching treatment was observed on the vitamin C loss, accelerated by the storage condition. Similarly, Sommano et al. [33] found a higher loss of vitamin C in KP fruit product caused by heating and high temperature and longer holding times. The almost twofold higher vitamin C loss (50%) in the blanched samples compared to the control samples (25% loss) might be due to the plant tissue disruption during thermal treatment, which enabled the fruit to be exposed to oxidative reactions during storage at high temperature (40 o C) [12,19,21,34]. In agreement with our findings, Ullah and co-workers [35] also reported a reduction of vitamin C in Azadirachta indica leaves by 59 and 72% after storage for 1 and 2 months at 50 o C, respectively. Similar trends in the reduction of vitamin C were also observed in strawberry fruit stored at high temperatures (40-45 0 C) compared with fruit stored at refrigerated conditions (4-6 0 C) [36].
The L-ascorbic acid (LAA) content of the studied KP fruit at the beginning of storage varied from 6 to 8% dry weight of whole fruits which is lower than that reported in previous studies of 18% [2]. However, it is still considerably higher than the LAA content found in common dietary sources of vitamin C such as orange, grapefruit, lemon, lime, kaffir lime and musk lime [34].

Vitamin C
Results of a preliminary blanching experiment displayed in Fig. 2 showed that KP fruit samples blanched at 90 o C retained significantly (p˂0.05) more vitamin C (7103.3 ± 56.8 mg/100 g DW) than that at 80 o C (5489.4 ± 211.8 mg/100 g DW) and 100 o C (5365.9 ± 42.7 mg/100 g DW). Therefore, blanching KP    the control samples. The DPPH radical scavenging capacity decreased significantly of almost 80% at the end of the storage period, whereas the TPC decreased only about 35% between the first and the last day of storage. A positive correlation (Pearson R 2 = 0.829, p˂0.01) between the decrease in DPPH radical scavenging capacity and ETs was observed. ETs can prevent free radical damage via the hydrogen donation process [46] which might contribute to the variation of TPC and DPPH values during storage ( Table 3). Previously reported studies [47,48]
Previous studies have indicated that the unsaturated fatty acids (UFA) are easily oxidized in many fruits during storage [17,49]. Although the UFA constitute more than 65% of

Ellagic acid (EA) content
The change in EA (both free and total) during storage is shown in Table 2. The difference in FEA between the three sampling times (day 0, day 27 and day 45) as well as the sample pre-treatment (blanched vs. control) was not significant (p > 0.05). A similar result of relative EA stability have been reported in previous studies [6,37] which might be due to either the chemical thermostability of EA (melting point of 450 °C and a boiling point of 796.5 °C) [38] or the ability of endogenous ascorbic acid on preventing the oxidation of EA [6]. However, a significant (p˂0.05) difference in TEA between day 0 and the final sampling day (day 45) were observed ( Table 2). This might be caused by ETs degradation/ polymerization/structural changes presumably linked to the storage temperature (40 o C) or involvement in scavenging free radicals [39,40]. Yu et al. [41] also found that punicalagin released from pomegranate leaf infusion, one of the main ETs in KP fruit [27], underwent degradation after storing at 20-25 o C for one day.
Furthermore, the samples analysed in this study were vacuum sealed prior to storage. Vacuum packaging has been successfully used to prevent quality loss associated with moisture absorption and reactive oxygen metabolism during storage [42,43]. This is probably the reason for the high amount of TEA (1800 to 2100 mg/100 g DW; Table 2) retained in the sample, comparable with previous frozen storage studies [6,37,44] and slow viamin C degradation because of the stress protective effect of vacuum packaging. In addition, the vitamin C, EA and ETs in KP fruits are located predominantly in the fruit flesh [2,45] and therefore comparable to the previous results reported for the KP lyophilized fruit flesh [2,6].

Antioxidant capacity
Significant (p < 0.05) differences in TPC and DPPH radical scavenging capacity between the storage days are shown in Table 3. However, sample pre-treatment did not affect the antioxidant capacity as both TPC and DPPH values were statistically not different (p > 0.05) between the blanched and 214.6 ± 6.1 bA Values are expressed as mean ± SEM, n = 6. DW: dry weight of the fruits including the stone, TPC: total phenolic contents, DPPH: 1,1-diphenyl-2-picrylhydrazyl, mg GAE/g DW: milligram gallic acid equivalent per gram dry weight, µmol TE/g DW: micromole Trolox equivalent per gram dry weight, Different capital letters in the same row indicate significant difference; Different small letters in the same column show significant difference might also be responsible for quenching free radicals and breaking the auto-oxidative chain reaction initiated during storage [52][53][54][55].
Akter and collaborators [8] reported the fatty acid content of KP fruit kernels and found linoleic acid as the major constituent, followed by oleic acid, palmitic acid and stearic acid. These results agree with the findings in the present study. Results of this study are also in accordance with previous reports regarding the fatty acid profile of the seeds of different Terminalia species [56][57][58][59] and commonly reported for nuts, seeds and fruits of wild edible plants [56,59]. However, differences in the fatty acid composition between the blanched and control samples were negligible, suggesting that the blanching process did not have any significant effect on the fatty acid composition.
The ratio of PUFA/SFA ≥ 0.4 is commonly used as an "health index" for food or diet rich in fatty acids [60,61] since PUFA can help to protect type 2 diabetes [62], stroke [63], and cardiovascular diseases [64]. The PUFA/ SFA ratio of each analysed KP fruits sample is ~ 2 ( Table 4). This is similar to the value reported for G. dura (1.89), C. tetragonoloba (1.71) and L. albus (1.97) [65], suggesting the potential use of KP fruit as alternative source of PUFA. Table 5 shows the change in TBARS of the samples during storage. In the process of lipid oxidation, the quantity of MDA is expected to increase, followed by the reaction with two molecules of TBA via acid catalysed reaction [29], which in turn produces TBARS. However, the results shown the total fatty acids in each sample (Table 4), the fatty acid content of KP did not significantly (p > 0.05) change over the storage period. These results are similar to those reported for extra virgin olive oil after 36 months of storage at 15, 22 and 37 °C [50]. It has been demonstrated that storing vegetable oil mixed with gallic acid, ellagic acid and quercetin at 50 °C decreased the oxidation process [51]. Therefore, the antioxidant compounds in KP fruit, especially ETs, EA and vitamin C, are most likely responsible for the observed "antioxidant defence" during storage. These antioxidants Table 4 Effect of blanching and storage on the fatty acid profile as determined by FAME analysis, at the beginning (day 0) and the end (day 45) of the storage trial Common Name   Values are expressed as mean ± SEM, n = 6. DW: dry weight of the fruits including the stone; a, b indicates significant differences between the samples at the beginning of the storage; x, y represents significant differences between the samples at the end of the storage trial; *, ** represents significant difference in each sample at the beginning (Day 0) and end of the storage (Day 45); the mean difference on each storage day is determined using One-Way ANOVA followed by Tukey's Post Hoc analysis; difference in the fatty acid content of sample between days is determined using Chi-Square tests published version of the manuscript.

TBARS
Funding This research was funded by the Cooperative Research Centre for Developing Northern Australia (CRCNA) and the support of its investment partners the Western Australian, Northern Territory and Queensland Governments and the Australian Research Council (ARC) Industrial Transformation Training Centre for Uniquely Australian Foods (Grant number: IC180100045).

Open Access funding enabled and organized by CAUL and its Member Institutions
Data Availability The data are available from the corresponding author on reasonable request.

Declarations
Ethics approval Not applicable.
Informed consent Not applicable.

Conflict of interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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in Table 6 indicate a significant (p˂0.05) reduction (~ 50%) in TBARS in the samples collected at day 45 vs. day 0. This further substantiated that the antioxidants present in KP fruit participated in preventing lipid oxidation because the degradation of ETs and vitamin C has been strongly correlated with decrease in TBARS on the 45th day (TEA Pearson R 2 = 0.809, p˂0.01; TVC Pearson R 2 = 0.897, p˂0.01). Similarly, Maqsood and Benjakul [66] reported preventive effects of tannic acid on lipid oxidation using the TBARS assay. Natural (poly) phenolic extracts [67] and fat diet containing EA [68] also reduced MDA production evidenced by a decrease in TBARS value.
Given the fact that MDA can be harmful for many physiological processes [69], the present results are important regarding the potential use of KP fruit (blanched or untreated) to prevent lipid oxidation.

Conclusion
This study, for the first time, revealed that blanching significantly reduced the vitamin C content of KP fruits stored at 40 o C for 45 days. However, the blanching treatment did not have a significant effect on the antioxidant capacity. Neither the storage duration nor blanching has showed any effect on the FEA and fatty acid contents. These results suggest that KP fruits, stored as whole dried fruit, by Indigenous communities at a relatively high temperature of up to 40 °C in remote areas of Australia, retained a high content of EA, fatty acids and vitamin C as well as a strong antioxidant capacity.
Acknowledgements The authors would like to acknowledge the Traditional Owners of the lands on which the Terminalia ferdinandiana was harvested and respect the knowledge and experience the Traditional Owners hold regarding the care, harvest and use of these plants.  Control powder 1.4 ± 0.05 aC 1.0 ± 0.04 aB 0.63 ± 0.04 bA Control whole fruits 1.48 ± 0.09 aB 0.9 ± 0.02 aA 0.77 ± 0.004 cA Values are expressed as mean ± SEM, n = 6. DW: dry weight of the fruit including the stone, TBA: thiobarbituric acid, MDA: malondialdehyde, TBARS: thiobarbituric acid-reactive substances, Different capital letters in the same row show significant difference; Different small letters in the same column show significant difference