Effect of fluidised bed drying on ginsenoside content in hairy root cultures of Panax ginseng C.A. Meyer

Korean Ginseng ( Panax ginseng C.A. Meyer) is a high‐value herb with many pharmacological benefits due to its primary active compound, ginsenosides. The most ginsenosides are known to be thermolabile and susceptible to degradation at high‐temperature processing. Our previous studies revealed that the optimum parameters related to the P. ginseng tissue culture protocol, particularly for hairy root propagation of Cultured Roots of Mountain Ginseng (CRMG)‐88, was using a lab‐scale bioreactor. The next stage involves screening for a suitable post‐harvest treatment, i.e., drying, will be production of the best quality ginsenoside content. This study therefore aimed to examine the ginsenoside content by using a fluidised bed dryer (FBD) on the ginseng roots. Our results showed that FBD produced a significantly higher of total ginsenoside content (5.386 ± 1.167%), compared to control (3.750 ± 0.641%). FBD‐dried CRMG‐88 also appeared lighter in colour and more voluminous with a Loss on Drying (LOD) of 6.448 ± 1.900%. This study concluded that fluidised bed drying is superior in retaining ginsenoside content and has the potential for large‐scale application.


Introduction
Ginseng is widely distributed in dozens of countries and is in great demand on the world market.The trend of in creasing ginseng consumption is due to growing aware ness of healthy food cultures.Several attempt to produce high quality of ginseng are expanding towards conven tional products, such as improvement of root culture us ing bioreactor (Dannis Yuda Kusuma et al. 2023).Pro cessed ginseng products are becoming an alternative of consumers due to their greater practicality and enhanced functional value (Vol et al. 2013; Baeg 2022).
The main active compound of ginseng is ginsenosides, which are known as triterpenoid saponins, has many med ical benefits, including antiinflammatory activity (Kim et al. 2017), mental health promotion (StLaurent and Hammami 2022), anticancer activity (Hu et al. 2019; Zhang et al. 2019a), memory loss improvement (An et al. 2019), neuroprotective ability (Zhang et al. 2016), and antifatigue activity (Shin et al. 2019).Aside from genetic and environmental factors (Chen et al. 2019; Lee et al. 2019; Zhang et al. 2019b), processing can change the gin senoside content in ginseng, which in turn can affect its functional properties (Koh et al. 2015; Jang et al. 2017; Zheng et al. 2017; Jang et al. 2018).
Food drying is essential in safeguarding product safety, as ingredients with a high water content are always susceptible to contamination (Nazghelichi et al. 2010).Nevertheless, a balanced drying technique focuses not only on suppressing spoilage but also on conserving the vital elements in a product.Raw ginseng root has a rich moisture content and often heatdried to be more effi cient.It is indisputable that ginsenosides are thermolabile compounds and susceptible to random conversion to their derivatives at high temperatures (Kim et al. 2020).This creates inconsistency in product composition, leading to a decrease in quality.
Our research group has worked intensely with gin seng to build an optimal in vitro system from cultivation to postharvest.The development of suitable drying methods is imperative to ensure stable product composition.The existing ovendrying process relies on heat convection, which tends to dry slowly and can potentially induce the degradation of heatsusceptible contents.Several research findings have indicated a loss of phytochemical content at tributable to oven treatment.The carotenoid levels of cel ery leaves were reported to be dramatically reduced when drying at 70 °C for 4 h (Kamel et al. 2013).In another Setiabudi et al. Indonesian Journal of Biotechnology 28(3), 2023, 137-142 case, the total phenolic and antioxidant content of Cosmos caudatus showed a remarkable decrease after heating at 44.5 °C for 4 h (Mediani et al. 2014).
A fluidised bed dryer (FBD) offers an alternative dry ing method.The working principle is fluidisation, where solids interact with hot gas at high pressure to become par tially suspended in gas form (Doymaz and Ismail 2010).Several studies have demonstrated the effectiveness of FBD on beetroot (Kumar Y 2015) and mint leaves (Mote vali et al. 2016).However, to our knowledge, the litera ture contains only limited information regarding the effect of FBD on Panax ginseng, especially regarding its ther molabile ginsenoside.
Based on its potential, the current study intends to ex amine the effect of FBD on the ginsenoside composition of ginseng tissue culture.It is expected that a shorter con tact time between the roots and hot gas will minimise the decomposition of ginsenosides.

Materials and Methods
The plant material used was a hairy root culture of Panax ginseng C.A. Meyer, initially provided by Hanbang Bio Laboratory (Kyung Hee University, South Korea).The cell line used was a developmental collection called Cul tured Roots of Mountain Ginseng (CRMG)88.The re search was conducted at Kalbe UBAYAHanbang Bio Laboratory (Surabaya, Indonesia).

Production of CRMG-88
Production was carried out in an 18 L bioreactor sys tem (BRBIO180, Hanbang Bio Inc., South Korea).The growth medium was prepared through modifications from previous studies (Chandra et al. 2021).The previously op timised "B" formula, which comprises a combination of modified Schenk and Hildebrandt basal medium and auxin hormones, was applied to the concentrated media.How ever, the dilution level of the concentrated media was in creased to reach a final volume of 15 L. Furthermore, ster ilization using an autoclave (Hankuk, HKAC200P, Ko rea) was modified with the temperature of 121 °C for 72 min at a pressure of 1.5 atm.Meanwhile, seeding and in cubation proceeded without modification.The 150 g of fresh roots were used as inoculum seeds and the growing conditions were maintained at 21 °C to 25 °C, 500 lux of light intensity with 10/14 h photoperiod (day/night), for 7 weeks at 40 to 50% humidity.

Harvest and drying of CRMG-88
After the incubation period, the roots were harvested and rinsed three times using tap water, and then once with reverse osmosis water.The draining efficiency was el evated using a washing machine (Mito, WM1, Indone sia) for 2 × 5 min.The samples used were taken from three separate month harvest periods.In this study, sep arate fluidised bed and ovendrying treatments were ap plied for each sample.In the FBD, fresh CRMG88 was placed evenly on the porous plate of an FBD (made lo cally, 585 watts, two m•s 1 airflows) and set at 55 °C.The CRMG88 was turned over periodically during the dry ing process to ensure every part of the roots was dry.The FBD is generally able to dry 500 g of fresh roots in 3-4 h in a single run.Drying using a universal oven (Mem mert, UF45, German) was conducted at 60 °C, 60% fan, and 60% flap (Chandra et al. 2021).One tray containing approximately 1.5 kg of fresh roots will dry within 9-10 h of oven drying.Both drying treatments were performed until the CRMG88 reached a Loss on Drying (LOD) value of 5-10%.LOD was measured using a moisture analyser (Sartorius, MA150, German).

Extraction and sample preparation of CRMG-88
The dried roots were extracted gradually based on an ear lier procedure (Chandra et al. 2021).The first step utilised the Soxhlet method to extract 5 g of dried root in 300 mL of 80% methanol for 2 h.The resulting extract was dried with a rotary evaporator (Buchi, R300, German) and re constituted in 20 mL of reverse osmosis water.The second step was liquid-liquid extraction using watersaturated bu tanol at a ratio of 1:1.The organic phase was collected by centrifugation at 8000 rpm (1 rpm = 1/60 Hz) for 15 min.The second step was then repeated with fresh solvent until a clear organic phase was obtained.The resulting extract was evaporated again.

Ginsenosides analysis
Ginsenoside levels were analysed using highperformance liquid chromatography (HPLC) (Agilent, 1260 Infinity II, USA).Samples were dissolved in 50 mL HPLCgrade methanol and filtered with a 0.2 μm PTFE filter.A Sym metry Shield RP18 column 3.5 μm with a size of 4.6 × 150 mm was used, while the eluent was water pH 2.0/acetoni trile (A).The elution gradient was set as follows: 8 min at 80% A (isocratic); 8 min to 40 min from 80% to 60% A; 40 min to 45 min from 60% to 40% A; 45 min to 47 min from 40] to 0% A; 47 min to 52 min at 0% A (isocratic); 52 min to 55 min from 0% to 80% A. The injected sample volume was 20 L, and the absorbance was measured using a Diode Array Detector at a wavelength of 205 nm.Quan titative interpretations were made by comparing the peak areas of 14 types of ginsenosides with their corresponding reference standards, which are listed in Table 2.
The data obtained were analysed by IBM SPSS v. 25 software using an independent ttest for normal distribu tion.The significance of the difference between the data is indicated by a pvalue < 0.05 (Huang et al. 2023).

Results and Discussion
Recent studies have indicated a divergence in the abun dance of ginsenoside types from the two drying treatments.Quantitative variations of ginsenosides are represented by differences in peaks in the ginsenoside profiles of FBD and oven drying, as shown in Figure 1.A significant dif ference (p = 0.033) was observed in the total ginsenoside content among two drying treatments, with FBD yielding 40% higher ginsenoside content compared to oven drying, as shown in Table 1.Fluidised bed drying produced higher   total ginsenoside levels (5.386 ± 1.167%) than oven dry ing (3.750 ± 0.641%).At the individual level, there were a significant decrease in protopanaxatriol (PPT) groups, such as Re, in the ovendrying treatments compared to flu idised bed drying, as shown in Table 2.A similar find ing was reported whereby Re decreased gradually during the first four days of heating with a temperature of 80 °C (Kim et al. 2020).Ginsenoside Re has been studied for its potential healthpromoting properties, including anti inflammatory, antioxidant, and neuroprotective effects.It is also believed to contribute to the adaptogenic properties of ginseng, which may help the body adapt to stress and improve overall wellbeing (Jang et al. 2018).Other re search has reported that in the steaming process at 100 °C, Re undergoes a series of hydrolysis starting from Re → Rg1 or Rg2 → F1 → PPT (Yao et al. 2021).Otherwise, the reaction can run from Re → Rh1 → PPT, as shown in Figure 2. A significant decrease was also observed in the pro topanaxadiol (PPD) group, namely Rb1, F2, and CK, of ovendried CRMG88.This effect could be explained by the conversion mechanism described previously (Yao et al. 2021).In this case, the hydrolysis continuously proceeds from Rb1, Rb2, and Rc → Rd → F2 → CK or Rh2 → PPD, as shown in Figure 3.The number of upstream molecules will eventually decline as molecular breakdown continues toward the pathway.This mechanism is justified by the evidence that the related ginsenosides were also reduced in this current study, except for Rh2.Thus, the detected ginsenoside change phenomenon may refer to the find ing that oven drying at 60 °C sufficiently induces thermal decomposition, which converted the related ginsenosides into their simplest form (PPT/PPD) or possibly other novel ginsenosides that were not included in the measurement standards in the current research.Both possibilities may have contributed significantly to the total ginsenoside per

centage.
FBDdried CRMG88 was found to give a LOD of 6.448 ± 1.900% in drying for 224 ± 26 min and produced a yield of 4.350 ± 0.372%, as shown in Table 3.Our opti misation showed that a fresh weight of 450 g is the maxi mum capacity of the FBD.A higher weight tends to cause uneven heat distribution.However, this smallscale FBD is capable of delivering the desired LOD in a short time.Oven drying presented a LOD and yield of 4.986 ± 1.403% and 5.428 ± 0.393%, respectively, while the drying time was 543 ± 57 min.The oven has a 6 kg fresh weight ca pacity in a single run with four trays.The appearance of the two dryers can be seen in Figure 4.The condition of the roots often determines the degree of dryness attained.
If the roots are dense, they may not dry properly.
In terms of physical appearance, the CRMG88 result ing from FBD was a light golden yellow colour (Figure 5a), while oven drying produced a darker brown colour (Figure 5b).This can be understood based on the fact that heat transfers slowly in an oven and can lead to a burning effect on the drying material to produce a dark colour over time.Meanwhile, in fluidised bed drying, based on the shorter drying contact time (faster heat transfer), the natu ral colour of the material is maintained during the process.In addition, the CRMG88 subjected to fluidised bed dry ing had a voluminous look, while it tended to deflate and harden with oven drying.Fluidised bed drying resulted in a better total ginseno side content and physical appearance compared to oven drying.As such, drying in an FBD would be more suit able for largescale continuous production as it is also su perior in terms of maintenance cost, temperature require ment (lower), and drying rate.

Conclusions
Based on the results of our research, it can be concluded that drying using an FBD is preferable to oven drying based on consideration of the total ginsenoside content and the physical appearance of the drying results.However, efficiency tests and validation of largescale FBD opera tions in the KUH Lab are yet to be conducted.Moreover, comprehensive future studies regarding the heat transfor mation of ginsenosides are required.

FIGURE 1
FIGURE 1 HPLC chromatograms showing the ginsenoside profiles of CRMG-88 prepared under different conditions.a) Fluidised bed drying.b) Oven drying.

TABLE 1
Effect of fluidised bed drying and oven drying on total ginsenoside percentage.

TABLE 2
Content change of ginsenosides in fluidised bed drying and oven drying.