Bioreactor type affects the accumulation of phenolic acids and flavonoids in microshoot cultures of Schisandra chinensis (Turcz.) Baill.

Microshoots of the East Asian medicinal plant species Schisandra chinensis (Chinese magnolia vine) were grown in bioreactors characterized by different construction and cultivation mode. The tested systems included two continuous immersion systems—a cone-type bioreactor (CNB) and a cylindric tube bioreactor (CTB), a nutrient sprinkle bioreactor (NSB), and two temporary immersion systems (TIS)—RITA® and Plantform. Microshoots were grown for 30 and 60 days in the MS medium enriched with 1 mg l−1 NAA and 3 mg l−1 BA. The accumulation of two groups of phenolic compounds: phenolic acids and flavonoids in the bioreactor-grown S. chinensis biomass, was evaluated for the first time. In the microshoot extracts, seven phenolic acids: chlorogenic, gallic, p–hydroxybenzoic, protocatechuic, syringic, salicylic and vanillic, and three flavonoids: kaempferol, quercitrin and rutoside, were identified. The highest total amount of phenolic acids (46.68 mg 100 g−1 DW) was recorded in the biomass maintained in the CNB for 30 days. The highest total content of flavonoids (29.02 mg 100 g−1 DW) was found in the microshoots maintained in the NSB for 30 days. The predominant metabolites in all the tested systems were: gallic acid (up to 10.01 mg 100 g−1 DW), protocatechuic acid (maximal concentration 16.30 mg 100 g−1 DW), and quercitrin (highest content 21.00 mg 100 g−1 DW). The influence of bioreactor type on the accumulation of phenolic acids and flavonoids in microshoot cultures of Schisandra chinensis was proven and optimized.


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biotechnology. The results of such studies are important from a practical point of view and the prospects of producing valuable, biologically active metabolites in such systems continue to draw scientists' attention (Verpoorte et al. 2002;Karuppusamy 2009). The latest studies have demonstrated the possibility of large-scale production of metabolites like: artemisinin (antimalarial drug) in cultures of Artemisia annua grown in a specially-designed stirred tank bioreactor, plumbagin (anticancer compound) in Plumbago rosea cultures maintained in a customized reaction kettle (Jose et al. 2016), and steviol glycosides (low-calorie glucoside sweeteners) in Stevia rebaudiana cultures grown in a temporary immersion bioreactor (Vives et al. 2017). In all studies involving large-scale in vitro systems, the selection of proper bioreactor type and optimization of process parameters is crucial for maximizing secondary metabolite production.
The role of plant-derived phenolic substances in diverse areas such as medicine, cosmetology and food industry, is invaluable. Secondary metabolites like flavonoids and phenolic acids exhibit many important, scientifically proven, biological activities, e.g. antioxidant (Young and Woodside 2001; Krishnaiah et al. 2011), anti-inflammatory (Zhang and Tsao 2016), or anticancer (Roleira et al. 2015). These compounds are used either in pure form or as constituents of herbs, plant extracts and functional foods.
In the current study, highly differentiated microshoot cultures of rare, East Asian plant species-Schisandra chinensis (Turcz.) Baill. (Chinese magnolia vine) were investigated as a source of flavonoids and phenolic acids. S. chinensis fruits constitute raw pharmacopoeial material and are especially well-known in the traditional medicine of East-Asian counties. However, they have also been included in current phytotherapy all over the world (Barnes and Anderson 2007;World Health Organization 2007; European Directorate for the Quality of Medicines 2017; Szopa et al. 2017a). Their medicinal properties, e.g. hepatoprotective, immunostimulant, adaptogenic and anticancer, are determined mainly by the presence of dibenzocyclooctadiene type lignans (aka "schisandra lignans"), a Schisandra genus-specific group of secondary metabolites (Opletal et al. 2004;Szopa et al. 2017a). However, recent studies have demonstrated that phenolics present in S. chinensis act synergistically with lignans, thus serving as co-adjuvants and increasing biological activities of the plant (Szopa and Ekiert 2012;Cheng et al. 2013;Mocan et al. 2014). The compounds of particular interest are phenolic acids and flavonoids which themselves exhibit beneficial biological effects.
In this study, in vitro microshoot cultures of S. chinensis were evaluated for the production of phenolic compounds in five bioreactors designed for differentiated plant in vitro cultures (systems nomenclature after Kim et al. 2004;Steingroewer et al. 2013). Of these, three were custom-made systems: a cone-type balloon bioreactor (CNB) a cylindrical tube bioreactor (CTB) with internal rack for biomass immobilization and a nutrient sprinkle bioreactor (NSB), and two were commercially available, temporary immersion bioreactors-RITA ® (Vitropic, France) and Plantform (PlantForm, Sweden) (Fig. 1). The construction details and setup of the bioreactors had been described by us previously (Szopa et al. 2017c). To our knowledge, this is the first work aimed at optimizing conditions for favourable biomass growth and phenolic acids and flavonoids production in bioreactorgrown S. chinensis microshoots.
As described previously, the microshoots were cultivated in the Murashige & Skoog (MS) medium (Murashige and Skoog 1962) supplemented with 1.0 mg l −1 1-naphthalene acetic acid (NAA) and 3.0 mg l −1 6-benzyladenine (BA), which had previously been demonstrated to provide the best biomass growth and secondary metabolites production in S. chinensis in vitro cultures Ekiert 2013, 2015;. All bioreactor systems were inoculated at 3/100 w/v ratio which corresponds to 15 g of microshoots per 500 ml of medium in CNB, CTB, NSB and Plantform bioreactors, and 6 g of microshoots per 200 ml of medium for RITA ® container. The cultures were grown in constant artificial light (Philips fluorescent white lamps, 90 ± 2 μmol m −2 s −1 ), at 24 ± 2 °C. The bioreactor cultures were maintained for 30 and 60 days and the collected biomasses were evaluated for growth parameters including fresh (FW) and dry (DW) weight, growth index (Gi), accumulated dry biomass (ADB) and specific growth rate (µ). The recorder values and formulae used are presented in Table 1. Afterwards, the microshoots and corresponding media samples (40 ml) were freeze dried (Lyovac GT2, Finland) and subjected to phytochemical analysis. The bioreactor experiments were done in four repetitions.
The lyophilized and pulverized biomass samples (0.5 g from each bioreactor, four samples for each bioreactor type) were extracted with methanol (50 ml) for 2 h under reflux condenser (Harborne 1984). The extracts were transferred to crystallizers through a filter paper (Whatman paper) and left at room temperature to evaporate the solvent. The dry residue was dissolved in 2 ml of methanol (HPLC grade purity, Merck) and subjected to HPLC analysis.

Bioreactor type
Growth period (days) In all the types of bioreactors employed, S. chinensis microshoots showed vigorous growth, and no necrosis or medium browning was observed (Fig. 1). After the biomasses were collected, their growth parameters were determined (Table 1). The Gi parameter ranged from 2.53 to 3.49 and from 6.73 to 10.78 for microshoots cultivated for 30 and 60 days, respectively. Of the custom made systems, the NSB yielded the highest ADB after 60 days of cultivation. In terms of Gi, ADB and biomass productivity, the TIS bioreactors performed substantially better than other systems employed. The highest biomass increments were obtained with RITA ® bioreactors which was reflected by high values of all growth parameters, most notably ADB and biomass productivity (Table 1).
Based on HPLC-DAD quantitative analyses, the total contents of phenolic acids varied from 19.16 mg 100 g −1 DW (CTB, 60-day growth period) to 46.68 mg 100 g −1 DW (CNB, 30-day growth period). Comparing the amounts of phenolic acids, we found that the highest total quantities were obtained in biomass of the cultures grown over 30-day growth periods in all the tested types of bioreactor. The maximal total amount of phenolic acids achieved in biomass extracts from the CNB bioreactor was, respectively, 1.50, 1.18, 1.43 and 1.35 times higher than in the CTB, NSB, RITA ® and Plantform bioreactors ( Table 2).
The total amounts of estimated flavonoids ranged from 12.90 mg 100 g −1 DW (CTB, 60-day growth period) to 29.02 mg 100 g −1 DW (NSB, 30-day growth period). Comparing the amounts of flavonoids in the respective systems, we found that the highest total contents were obtained in microshoots grown over 30-day growth periods in all the tested types of bioreactor. The maximal total amount of flavonoids achieved in biomass extracts from the NSB bioreactor was, respectively, 1.40, 1.32, 1.36 and 1.42 times higher than in the CNB, CTB, RITA ® and Plantform bioreactors (Table 3).
In all experiments, none of the estimated phenols were found in the media samples, indicating that the investigated compounds were stored solely intracellularly.
Our study proved that biomass growth and secondary metabolite contents in S. chinensis microshoots depended on the type of bioreactor used and its mode of operation, including medium circulation and aeration, which is in agreement with previous reports (Liu et al. 2003;Zobayed et al. 2004;Paek et al. 2005), including our work on the production of dibenzocyclooctadiene type lignans in bioreactor-grown S. chinensis microshoots (Szopa et al. 2017c). In that study, the best conditions were provided by the Plantform bioreactor, in which, during a 30-day growth period, microshoots had accumulated the highest amounts of lignans (546.98 mg 100 g −1 DW), with gomisin A, deoxyschisandrin and schisandrin as the major constituents (67.86, 77.66 and 118.59 mg 100 g −1 DW, respectively) (Szopa et al. 2017c). As mentioned before, the pharmacological action of S. chinensis is the result of synergistic action of all the plant's components; in this case, phenolic compounds seem to play a major role besides lignans (Mocan et al. 2014;Szopa et al. 2017b). We had proved before that different types of in vitro cultures of S. chinensis were able to produce phenolic acids and flavonoids, and that their yield depended on the in vitro conditions used, such as the basal media formulation, concentration of phytohormones, duration of the growth period, lighting conditions, as well as the mode of cultivation (agar, agitated, stationary liquid) Ekiert 2012, 2016;Szopa et al. 2017b).
Under the current study, we made the first step towards scaling-up the production of phenolic compounds using S. chinensis microshoot cultures. Of the tested systems, the RITA ® TIS bioreactor provided the highest biomass yields (Table 1). However, the total phenolic acids content was considerably greater in the CNB (Table 2), while that of flavonoids in the NSB (Table 3). In the CNB system, aeration-induced mechanical stress caused by tissue-medium contact resulted in lower biomass growth (Gi equal to 3.01 and 7.90, after 30-and 60-day growth cycles, respectively) ( Table 1), but also in higher production of phenolic acids (Table 2). Phenolic compounds are the plant's "stress metabolites", hence the high production (Halliwell 2003(Halliwell , 2007Akula and Ravishankar 2011). Moreover, it is worth noting that the 'bubble (air-lift) bioreactors' had been successfully applied before for the production of caffeic acid derivatives in Echinacea purpurea adventitious root cultures (Jeong et al. 2009), ginsenosides in adventitious root cultures of Panax ginseng (Kim et al. 2004) as well as phenolic acids and flavonoids in Eleutherococcus senticosus somatic embryos (Shohael et al. 2006).
In this study, the gas-phase nutrient sprinkle bioreactor (NSB) proved to be the best for flavonoid production ( Table 3). The characteristic feature of this bioreactor design is the lack of mechanical stress exerted on growing biomass, as well as good gas circulation and nutrient accessibility (Steingroewer et al. 2013;Georgiev et al. 2014). Similar to our study, the correlative experiment on Artemisia annua in vitro shoots had showed an improvement in artemisinin output in a gas-phase bioreactor (Liu et al. 2006). Moreover, shoots grown in a nutrient sprinkle bioreactor have been employed for effective biomass growth and production of phenolic compounds, expressed as total phenolic acids and flavonoids, in medicinal plants such as Rehmannia glutinosa (Piątczak et al. 2014) and Scutellaria alpina (Grzegorczyk-Karolak et al. 2017).
To conclude, our work showed that S. chinensis in vitro microshoots grown in different types of bioreactors (Fig. 1,  Table 1), could provide biologically active phenolic compounds. Moreover, we proved, for the first time, the influence of bioreactor type on the phenolic acid and flavonoid production in S. chinensis biomass (Tables 2 and 3). The obtained results are thus a good starting point for further studies, involving scale-up experiments.