Incorporation of fingerroot ( Boesenbergia sp.) rhizome extract powder on chemical, microbial and sensory properties of dry - roasted macadamia ( Macadamia sp.) nut during 12 months of storage

Macadamia nut is an important crop with high economic value. It is commonly dried and roasted into an instant crisp. Due to high oil content, the dry - roasted macadamia nut is feasible to oil rancidity as well as microbial contamination. In order to improve the economic value of macadamia nuts, it is very urgent to stabilise the quality of roasted macadamia nuts during storage and distribution. Fingerroot ( Boesenbergia sp.) belonging to the Zingiberaceae family is one of the most important Vietnamese perennial medicinal plants. Its rhizome contained numerous bioactive phytoconstituents like essential oils, flavonoids, phenolics exhibiting diversified biological activities beneficial for human health. This research evaluated the possibility of incorporation of fingerroot rhizome extract powder (0 - 6%) into the dry - roasted macadamia nut to improve its chemical, microbial and sensory properties during 12 months of storage. Results showed that 4.5% of fingerroot rhizome extract powder could be supplemented into the dry - roasted macadamia nut to reduce coliform load (1.28±0.03 log CFU/g), peroxide value (0.78±0.02 meq/kg), thiobarbituric acid reactive substances (0.65±0.03 mg malonaldehyde/kg), free fatty acid (0.76±0.02% oleic acid) while enhancing overall acceptance (8.24±0.17 score). This research revealed that fingerroot rhizome extract would be a promising antimicrobial and antioxidant natural source to preserve fatty foodstuffs efficiently.


Introduction
The macadamia tree (Macadamia sp.) is widely cultivated in Central Highlands and other provinces in the Southeast region of Vietnam ( Figure 1). It is a diversified tree with a great cultivation potential, a high socio-economic efficiency with an open international market. It could be propagated in remote and mountainous areas as defending forests to improve the forest coverage proportion and the ecological balance. By planting this tree, the income of farmers is greatly enhanced due to its high economic value compared to other fruit trees and industrial plants. Macadamia nut is a rich source of vitamins (especially tocotrienols and squalene), essential minerals, dietary fibres, proteins, phenolics with antioxidant capacities (Maguire et al., 2004;Wall, 2010;Minh et al., 2018). Macadamia nut is highly valued by its oil content with the high percentage of monounsaturated fatty acids, especially omega-7 palmitoleic acid (Akhtar et al., 2006;Silva et al., 2008;Saez et al., 2014). The oleic acid content is an important indicator reflecting the attributes of macadamia nut (Suporntip et al., 2012). Consumption of macadamia nuts is beneficial for those with cardiovascular ailments (Garg et al., 2003;Amy et al., 2008). Fingerroot (Boesenbergia sp.) belongs to the ginger (Zingiberaceae) family widely cultivated in Vietnam and other Asian countries ( Figure 2). Its rhizome had several slender and long tubers about 1.0-1.5 cm thick in diameter and 5-10 cm long (Tan et al., 2012). Fingerroot rhizome varied pale yellow colour depending on the soil in cultivation, cultivation technique and maturity at harvesting (Iijima and Joh, 2014). Its rhizome is used as folk medicine, culinary spice and seasoning vegetable  (Agus et al., 2014). Boesenbergia rhizome extract is an important seasoning vegetable in preparation of "Nuoc leo" rice noodle soup, a speciality of Khmer people in the Mekong Delta. Boesenbergia rhizome extract contained a high content of essential oils, flavonoids and polyphenols contributing to multiple pharmacological activities such as anti-fungal, anti-bacterial, antiparasitic, anti-ulceration, hepatoprotective, anti-cancer, antioxidant, anti-anthelmintic, anti-inflammatory (Ling et al., 2010;Abdelwahab et al., 2011;Isa et al., 2012;Salama et al., 2013;Chahyadi et al., 2014;Chiang et al., 2017;Yonna et al., 2018;Rosdianto et al., 2020). It is utilised to cure stomach discomfort, aphthous ulcers, dysentery, dry mouth, leucorrhoea (Fahmi et al., 2020). Fingerroot is highly perishable with a short shelf-life after harvesting. Dehydration into powder is the most common practice to stabilise its quality for long-term usage. Fingerroot powder could be utilised for different purposes like soup, spice, food sanitiser, condiment, confectionery, due to its aromatic flavour, which promotes appetite (Fahmi et al., 2020). Isopanduratin A originated from fingerroot is an ideal anti-bacterial agent against cariogenic mutans (Tan et al., 2012), while panduratin A effectively inhibits Escherichia coli and Staphylococcus aureus (Rukayadi et al., 2010).
In order to improve the quality as well as the economic value of the dry-roasted macadamia nut, the purpose of this study was to verify the influence of supplementation of fingerroot rhizome extract powder in different proportions into the dry-roasted macadamia nut to improve its chemical, microbial and sensory properties during storage. Phytochemical constituents in fingerroot rhizome extract would be effective to retard coliform growth and proliferation, inhibit lipid oxidation, enhance overall acceptance of the dry-roasted macadamia nut.

Materials
Raw Boesenbergia sp. rhizomes were purchased from Thu Duc market, Ho Chi Minh City, Vietnam. Raw macadamia nuts were supplied from Dong Nai province, Vietnam. They were washed under clean water to remove dirt and foreign matter. Chemical reagents such as phosphate buffer, acetic acid, chloroform, KI, Na 2 S 2 O 3 , thiobarbituric acid, trichloroacetic acid, and 4-Butoxybenzyl alcohol, petroleum ether, ethanol, phenolphthalein, NaOH were all analytical grade supplied from Fluka (Switzerland), Sigma Aldrich (USA) and Merck (Germany). 3M-Petrifilm coliform count plates were purchased from Van Dai Phat Co. Ltd., Ho Chi Minh City, Vietnam.

The preparation of roasted Macadamia nuts incorporated with Boesenbergia powder
Raw Boesenbergia sp. rhizomes were finely ground by a grinder. The fine powders (100 g) were soaked in ethanol 90% (900 mL) for 20 mins at room temperature at agitation speed 600 rpm. The filtrate was filtered using Whatman No. 2 filter paper with a vacuum pump. The filtrate was concentrated by a vacuum rotary evaporator (RV 3V, IKA, Germany) at 45 o C to obtain the Boesenbergia crude extract. Maltodextrin 12% was mixed with Boesenbergia crude extract. The freeze dehydration was conducted by freeze dryer (model: Coolsafe Superior XS/XL, Labogene, Denmark) with a condenser temperature of -65°C, the pressure of 250 μmHg for 28 hrs. The Boesenbergia powder (BP) was kept in a laminated bag ready for experiments.
Raw macadamia nuts were convective-dried (model: OF-01E/11E/21E, Lab Companion, Korea) at temperature 45 o C for 8 hrs, and then roasted at 145 o C for 15 mins. The roasted macadamia nuts were cooled to room temperature. The roasted macadamia nuts were mixed with Boesenbergia powder in different proportions (0, 1.5, 3.0, 4.5, and 6.0%). These roasted macadamia nut samples previously incorporated by Boesenbergia powder were stored in laminated PET/AL/ PE (Polyethylene Terephthalate/Aluminium/ Polyethylene, supplied from Binh Minh Packaging Joint Stock Company) bag for 12 months. Periodically (3 months), the treated samples were taken to determine coliform, peroxide value, thiobarbituric acid reactive substances content, free fatty acid content, and overall acceptance.

Chemical, microbial and sensory evaluation
Peroxide value or PV (meq/kg) was estimated by weighing 5 g of sample, adding 15 mL acetic acid and 10 mL chloroform, supplemented with 1 mL KI solution. This mixture was kept in a dark place for 15 mins, and a starch indicator is added. The final titration was defined by adding 0.01 N Na 2 S 2 O 3 until colourless.
Thiobarbituric acid reactive substances or TBARS content (mg malonaldehyde/kg) was determined following the 2-thiobarbituric acid spectrophotometric method (Anna et al., 2017). 5 g of sample was mixed with thiobarbituric acid, trichloroacetic acid, and 4-Butoxybenzyl alcohol. This mixture was conditioned in a water bath at 45 o C for 30 mins and then centrifuged (Eppendorf Centrifuge 5920R, Sigma Aldrich, USA) at 4,000 rpm for 5 mins. The absorbance was measured by UV-VIS spectrophotometer (model UV-1800, Shimadzu, Japan) at wavelength 532 nm.
Free fatty acid (FFA) content (% oleic acid) was eISSN: 2550-2166 © 2022 The Authors. Published by Rynnye Lyan Resources FULL PAPER determined following the titration method described by Calvo et al. (2017). Approximately 10 g sample was homogenised with 100 mL of petroleum ether. The mixture was filtered by Whatman paper No. 2 (Sigma Aldrich, USA) to obtain the filtrate. A 10 mL filtrate was taken to determine the residual fat. Another set of 10 mL filtrate was mixed with 5 mL of ethanol. It was titrated with NaOH 0.1 N in the presence of phenolphthalein (1%) until a light pink appearance was achieved. The FFA content was calculated from the following equation: Where v was the volume of the titrant (mL), b was the blank volume (mL), N was the normality of NaOH and w was the weight of the sample (g) Coliform (log CFU/g) was enumerated by 3M Petrifilm coliform count plates. Approximately 5 g of the sample was homogenised with 45 mL of phosphate buffer dilution. Lifting the top film, 1 mL of sample suspension was dispensed onto the centre of the bottom film, leaving the top film down. The counting plates were incubated at 37±1 °C for 24±2 hrs in a horizontal position in an incubator (model IF450, Memmert, Germany). Coliform was identified by red or blue colonies with associated gas bubbles. Coliform was wellenumerated by using a standard colony counter (model SC6Plus, Stuart, UK).
Overall acceptance (score) was determined by a group of specialists using a 9-point Hedonic scale. Panellists of 9 assessors (age 30-40 years old) were previously trained (90 hours) to utilize a 9-point strength ratio to evaluate sweetness, taste, flavour, and colour in dry-roasted nuts. During the training, panellists were individually evaluated to determine the overall panel mean and to ensure that all panellists were able to scale the properties of interest. Following training, panellists discussed together an agreeing mark for each attribute of the dry-roasted nuts, which were prepared directly from dry-roasted nuts and were provided as a reference sample. Panellists were provided samples in an ordinary order to prevent prediction based on the order of sample display. Three to five pickle pieces were set in each sample cup. Panellists examined a set of 5 to 7 samples per session. They were also offered two 2 oz. sample cups specified as the neutral sample to calibrate scoring of taste and texture characteristics on the 9-point scale. Each panellist was guided to first taste the neutral sample, neutralise the panellist's palate, and bite an unidentified roasted sample. Panellists were required to relax in two-minute intervals between two samples to minimize tasting tiredness. They could either swallow or expectorate their samples. Three sensory replications were executed on each sample during the research.
Sensory evaluation was performed on dry-roasted samples, which were preserved at room temperature (Peryam and Girardot, 1952).

Statistical analysis
The experiments were run in triplicate with different groups of samples. The data were presented as mean ± standard deviation. Statistical analysis is performed by Statgraphics Centurion version XVI. The mean value (x̄) and standard deviation (2s) of a set of data were obtained by analysis of random samples estimating the population statistics. Around 95% of results would be expected to lie within the range (x̄±2s) we described the lower and upper bounds of this range as the 95% confidence limits of the results. The differences between the pickling samples were analysed using a one-way analysis of variance (ANOVA). A significant value was set at a 95% confidence interval (p<0.05). If significant differences were found, then post hoc analysis was performed using Duncan's multiple range tests.

Coliform
The effectiveness of Boesenbergia powder in different incorporation ratios (0, 1.5, 3.0, 4.5, 6.0%) on the coliform load of dry-roasted macadamia nut during 12 months of storage is presented in Table 1. There is an increasing trend of coliform load by the time of storage, with the highest accumulation on the control and the lowest on the BP 6%. There is no significant difference in coliform load between the BP 4.5% and BP 6.0%. At the end of 12 months of storage, the coliform load in the dry-roasted macadamia nut treated by BP 4.5% is quite low, with 1.28±0.03 (log CFU/g). According to Southern African Macadamia Growers' Association (SAMAC), coliform should be less than 300 CFU/g in macadamia nuts. Meanwhile, the Brazilian Macadamia Association (ABM) set the maximum limit of coliform at 350 CFU/g in macadamia nuts.
Boesenbergia extract could retard spoilage fungi like Aspergillus flavus, Aspergillus niger, Aspergillus parasiticus, and Fusarium oxysporum with MICs of >10% (v/v), 8% (v/v), 10% (v/v), and <8% (v/v), respectively (Pattaratanawadee et al., 2006). Boesenbergia rhizome extract showed high inhibition of HIV-1 protease with IC 50 values of 18.7 μM (Cheenpracha et al., 2006). Essential oil of fingerroot rhizome is effective against dermatophytes, filamentous fungi and yeast (Jantan et al., 2001). Fingerroot rhizome extract showed the capacity to modify the morphology of the hyphae by disrupting the hard profile (Rasooli et al., 2006). 0.5% of Boesenbergia rhizome extract revealed anti-fungal properties against filamentous fungi of Penicillium sp., Aspergillus sp., Geotrichum sp., Fusarium sp., Aureobasidium sp. after 10 min treatment (Zakuan et al., 2018). Anti-fungal activity of fingerroot rhizome extract is due to 4-hydroxypanduratin, pinostrobin and pinocembrin (Seniya et al., 2013). These substances invade the cell membranes to the core of the cell and modify the critical intercellular attributes (Cristani et al., 2007). Cell disruption due to the structural vulnerability of cell membranes is induced by these substances; moreover, these substances interacted with the hyphal cell wall of fungi and led to the demise of the fungal mycelium (Gill and Holley, 2006).

Peroxide value
The efficacy of Boesenbergia powder in different incorporation ratios (0, 1.5, 3.0, 4.5, and 6.0%) on the peroxide value of the dry-roasted macadamia nut during 12 months of storage is presented in Table 2. There is an increasing trend of peroxide value by the time of storage, with the highest accumulation on the control and the lowest on the BP 6%. There is no significant difference in peroxide value between the BP 4.5% and BP 6.0%. At the end of 12 months of storage, the peroxide value in the dry-roasted macadamia nut treated with BP 4.5% is quite low at 0.78±0.02 (meq/kg). According to the Australian Macadamia Society (AMS), Southern African Macadamia Growers' Association (SAMAC) and Brazilian Macadamia Association (ABM), the peroxide value in macadamia nut should be ≤ 2 meq/kg, ≤ 3 meq/ kg and ≤ 5 meq/kg, respectively. Primary lipid oxidation is followed by the peroxide value (Servet and Hudayi, 2011). The effectiveness of rancidity retardation by Nepenthes extract is similar to other plant extracts. Green tea extract strongly retarded the oxidative rancidity in eel oil (Song and Kim, 2018). The peroxide value of anchovy oil is kept at low content by green tea extract (Kang et al., 2007). Basil leaf essential oil is effective in limiting peroxide value in Sea bass slices (Arfat et al., 2015). Pomegranate peel extract is also appropriated for Nile tilapia fillets to slow down peroxide value (Alsaggaf et al., 2017). Eryngium caucasicum extract is a beneficial substance to decrease peroxide value in Silver carp fillets (Raeisi et al., 2017) (Shindo et al., 2006).

Thiobarbituric acid reactive substances
Different supplementation ratios of Boesenbergia powder (0, 1.5, 3.0, 4.5, and 6.0%) on TBARS of the dry -roasted macadamia nut during 12 months of storage were shown in Table 3. There is an increasing trend of TBARS by the time of storage, with the highest accumulation on the control and the lowest on the BP 6%. There is no significant difference in TBARS between the BP 4.5% and BP 6.0%. At the end of 12 months of storage, the TBARS in the dry-roasted macadamia nut treated with BP 4.5% is quite low, with 0.65±0.03 (mg malonaldehyde/kg). This TBARS value is in the range of the acceptable limit (2 mg malonaldehyde/kg). Thiobarbituric acid reactive substances (TBARS) are also one of the most important indicators to determine the oxidative rancidity of polyunsaturated fatty acids via the formation of malonaldehyde facilitating to release of ketones and aldehydes by peroxidase reaction (Bremner, 2002;Feliciano et al., 2010). TBARS should be below 2 mg malonaldehyde/kg sample to avoid bad smell and poor taste accumulation (Connell, 1990). Panduratin A in Boesenbergia rhizome extract showed a protective effect against oxidative vulnerability by tert-Butylhydroperoxide. Tert-Butylhydroperoxide is an organic hydroperoxidant that triggers fat rancidity through its metabolism to free-radical intermediates, leading to oxidative vulnerability to tissues. Panduratin A in Boesenbergia rhizome extract could minimise malondialdehyde accumulation and glutathione depletion. Intracellular reactive oxygen species accumulation is also decreased by panduratin A treatment (Sohn et al., 2005).

Free fatty acids
Various proportions of Boesenbergia powder (0, 1.5, 3.0, 4.5, and 6.0%) incorporated into the dry-roasted macadamia nut during 12 months of storage on free fatty acid content were noticed in Table 4. There is an increasing trend of free fatty acid value by the time of storage, with the highest accumulation on the control and the lowest on the BP 6%. There is no significant difference in free fatty acid value between the BP 4.5% and BP 6.0%. At the end of 12 months of storage, the free fatty acid value in the dry-roasted macadamia nut treated with BP 4.5% is quite low, with 0.76±0.02 (% oleic acid). Panduratin A in Boesenbergia rhizome extract is a natural AMP-activated protein kinase (AMPK) activator. The trigger of AMPK would accelerate fatty acid oxidation by triggering fatty acid oxidation-related genes. This behaviour would retard fat synthesis by elimination of sterol regulatory elementbinding protein-1c (SREBP-1c) and PPARγ phosphorylation. Panduratin A in Boesenbergia rhizome extract would facilitate AMPK signalling to induce nuclear translocation of AMPKα2, following the activation of PPARα/δ, with LKB1 being the vital mediator of these effects. Trigger of PPARα/δ enhanced fatty acid oxidation (Kim et al., 2011).

Overall acceptance (score)
Overall acceptance of the dry-roasted macadamia nut incorporated by Boesenbergia powder (0, 1.5, 3.0, 4.5, FULL PAPER and 6.0%) during 12 months of storage was reported in Table 5. There is a decreasing trend of the sensory score by the time of storage, with the lowest score on the control and the highest on the BP 6%. There is no significant difference in sensory score between the BP 4.5% and BP 6.0%. At the end of 12 months of storage, the sensory score in the dry-roasted macadamia nut treated with BP 4.5% is very high, with 8.24±0.17 (score). Fingerroot extract showed a higher polyphenol content, ascorbic acid content, DPPH radical scavenging activity, and ABTS radical scavenging activity than those in ginger extract (Lee et al., 2020).

Conclusion
Fingerroot rhizome is normally utilised as food seasoning and ethnomedicine. Boesenbergia sp. revealed a wide range of biological properties. This research has successfully investigated the influence of different ratios of fingerroot rhizome extract powder incorporated into the dry-roasted macadamia nut on its chemical, microbial and sensory attributes during preservation. Results revealed that 4.5% of fingerroot rhizome extract powder is sufficient to retard coliform proliferation, limit the formation of the peroxide value, thiobarbituric acid reactive substances, free fatty acid and maintain the overall acceptance of the dry-roasted macadamia nut for 12 months of storage.

Conflict of interest
The author strongly confirms that this research is conducted with no conflict of interest.