BÁO CÁO KHOA HỌC: "Nuôi cấy tế bào Nhân sâm Panax ginseng trong bioreactor: Vai trò của ôxy trong sản xuất sinh khối và sản phẩm ginsenoside" pot

20.8%-50% along with purified air, on high-density cell culture of Panax ginseng were investigated in an balloon type bioreactor 5 l capacity and containing 4 l of MS medium.. A low 20.

Nuôi cấy tế bào Nhân sâm Panax ginseng trong

bioreactor: Vai trò của ôxy trong sản xuất sinh khối và sản phẩm ginsenoside

Ảnh hưởng của nồng độ ôxy (20.8-50%) đến quá trình nuôi

cấy tế bào Panax ginseng đã được nghiên cứu trong

bioreactor hình cầu với dung tích 5l có chứa 4l môi trường

MS Ở nồng độ ôxy 40% đã cho thấy là thích hợp cho việc sản xuất sinh khối và sản phẩm ginsenoside với kết quả thu được là 12.8 g/L sinh khối khô, và 4.5 mg/g SKK

ginsenoside vào ngày thứ 25 Các nồng độ ôxy 20.8, 30 và 50% là không thích hợp cho quá trình nuôi cấy tế bào cũng như sự tích lũy sản phẩm ginsenoside Kết quả trên đây cũng chỉ ra rằng ôxy là nhân tố hữu hiệu trong sản xuất sinh khối và sản phẩm ginsenoside ở quy mô lớn hơn

I ABSTRACT

The effects of oxygen supply within the range of

20.8%-50% (along with purified air), on high-density cell culture

of Panax ginseng were investigated in an balloon type

bioreactor (5 l capacity and containing 4 l of MS medium)

A 40% oxygen supply was found optimal for the

production of cell mass and ginsenoside with

corresponding values of 12.8 g l-1 DW, 4.5-mg/g DW on day 25, respectively A low (20.8%, 30%) and well as high concentration of oxygen (50%) supply was unfavorable to the cell cultures and they affect the cell growth and

ginsenoside accumulation The results indicate that oxygen

is a key factor in scaling up the process of suspension

cultures of Panax ginseng and supplementation of oxygen

is useful for efficient large-scale production of ginsenosides

by the submerged cultures

II INTRDUCTION

Plants are an abundant source of a large number of useful products including pharmaceutical and food additives

Plant cell cultures are an alternative source to whole plant for the production of high-value secondary metabolites During the past decade, a considerable progress has been made to stimulate formation and accumulation of

secondary metabolites using plant cell cultures (Rao and

Ravishankar, 2002) Ginseng (Panax ginseng C A Meyer),

a member of Araliaceae, is traditionally considered one of

the most potent medicinal plants Ginsenosides have been regarded as the most important active components in

ginseng roots and are attributed with cardio-protective,

immunomodulatory, anti-fatigue, and hepato-protective physiological and pharmacological effects (Zhang and

Zhong, 1997)

In recent years, plant cells are cultured in large-scale

bioreactors for production of secondary metabolites

including pharmaceuticals, pigments, and other chemicals (Rao and Ravishankar, 2002) Growth and accumulation of secondary metabolites in large-scale bioreactors is

influenced by various factors such as shear stress, oxygen

supply, and gas composition A conventional stirred-tank bioreactor can produce a high shear region, while in many cases airlift and bubble column reactors are used for

providing shear environment compared to turbine-agitated reactors As reported, oxygen supply is also significant in affecting secondary metabolites formation in cell cultures (Gao and Lee, 1992; Zhong et al., 1993; Han and Zhong, 2003) Gas exchange between the gas and liquid phases is another important factor that may affect the scale-up of plant cell cultures In bioreactors, forced aeration is needed

to supply oxygen and to improve fluid mixing However, it may also lead to the removal of some known (such as CO2 and ethylene) or unknown gaseous compounds Such

gaseous metabolites were proven or suggested to be

important for cell growth and/or synthesis of secondary metabolites in plant cell cultures (Gao and Lee, 1992)

The concentration of dissolved oxygen can be easily

controlled in bubble/airlift bioreactors and interaction

between O2 supply, cell growth and metabolite biosynthesis

can be observed In this study, we have used balloon type bioreactors for cell cultures of ginseng and the interaction between oxygen supply, cell growth, and ginsenoside production was investigated The significance of gas

control during bioreactor culture has been established and this study is considered useful for biotechnological

application ginseng cell cultures to the production of

ginsenosides on a large scale

III MATERIALS AND METHODS

Induction and proliferation of callus

Six-year-old fresh ginseng roots (Panax ginseng C A

Meyer) were sterilized and cultured as described by Yu, (2000)

Bioreactor cultures

A five-liter capacity balloon type bioreactors were used containing 4 l of MS (Murashige, Skoog, 1962) medium working volume with the culture condition as described by Thanh et al., (2004; 2005) to increase the biomass Sixty grams cell fresh weight per liter was added as inoculum In the bubble bioreactor, a sinter glass was used for aeration, and the airflow rate was adjusted during cultivation to

homogenous mixing state To investigate the effects of

different levels of oxygen in the inlet air, air was mixed with different concentrations of oxygen i.e., 20.8%

(control), 30%, 40%, and 50% The schematic diagram of the whole experimental system is shown in Fig 1 The

cultivation temperature was controlled at 25±2oC and

continuous darkness was maintained Three identical

cultivation vessels were operated under each condition, and the cultivation data shown represent average values with standard deviations The bioreactor cultures were

maintained up to 30 days

Sampling and analyses of cell weight, medium sugar,

conductivity

A sample of 30 ml of cell culture was taken once from each bioreactor at an interval of every five days The cell

suspensions were filtered and washed several times with distilled water for the measurement of cell weights (fresh and dry weights) The culture supernatants were used for analysis residual sugar, using HPLC by following

analytical procedures described by Zhang and Zhong,

(1997) and Woragidbum-rang et al., (2001) The electrical conductivity was from the exhausted medium using

conductivity meter Wiss-teelm-werkstalten model LF-54 (WTW GmbH, Wielhalm, Germany)

Fig 1 Schematic diagram of the balloon type bioreactor culture system used in the present study: a body of a

balloon type bioreactor, b air vent, c inoculum port, d sampling port, e medium exchange port, f air flow meter,

g membrane filter, h water column, i air compressor, j air reservoir, k air cooler, l filter system, m air dryer, n

oxygen tank

Determination of ginsenoside content

Ginsenoside (saponin) content was determined by HPLC, and the details have been described elsewhere (Furuya and Yoshikawa, 1987; William and John, 1996)

IV RESULTS AND DISCUSSION

Effect of oxygen concentration on cell growth

Fig 2 shows the growth kinetics of P ginseng cells in 4 l

balloon type bioreactors as influenced by four different levels of oxygen supply The cell growth and biomass

accumulation is gradually increased with lapse of time and optimum biomass accumulation reached after 25 days

Similar growth kinetics pattern was reported in P

notoginseng in shake flask, centrifugal impeller bioreactor

and turbine reactors cultures (Zhong et al., 1999) The

maximum fresh weight with the supply of 20.8% oxygen

(control) was 267 g l-1 and corresponding dry weight was 11.5 g l-1 (Fig 2A-B) It was found that optimum

accumulation of fresh (316 g l-1) and dry biomass (12.8 g l -1

) was with the supply 40% oxygen in the bioreactors The biomass accumulation comparatively declined with the increase in oxygen concentration to 50% (255 g l-1 FW and 9.0 g l-1DW)

Fig 2 Time profiles of fresh cell weight (A), dry cell

weight (B) in high-density culture

of Panax ginseng cells in a 5 l balloon type bioreactor

Electrical conductivity measurements (EC) have been used

as an indirect method of biomass estimation in continuous

on line monitoring of plant cell cultures in bioprocess

engineering studies for its accuracy and efficiency (Ryu et al., 1994) The electrical conductivity of the medium, also which reflects the uptake of medium salts (ions) by the cultured cells and linear decrease, was observed with

increase in cell density during cultivation (Fig 3A) In the cell cultures, which were supplied with 40% oxygen,

showed a decrease in EC values from initial value of 5.6 mS/cm to 1.23 mS/cm (Fig 3A) At the beginning of

cultivation, the cell growth was slow and in a lag phase, and subsequently cells involved in division and

multiplication and hence due to the active metabolic uptake

of the medium ions by the cultured cells Similarly,

observations were recorded with P notoginseng (Zhong et al., 1999) and rice (Wen and Zhong, 1996) suspension

cultures

Time profiles of medium sugar consumption at different levels O2 supply are shown in Fig 3B After inoculation, cells in all cases gradually consumed sugar and residual sugar concentration was almost exhausted when cell

growth reached peak The growth yield (on sucrose) at 50%

O2 supply was lower than that of control (11.5 g l-1 versus 9.5 g l-1 on day 25) and it means that carbon flux was

altered by O2 concentration A similar phenomenon has also been reported during cell culture in Catharathus roseus

(Tate and Payne, 1991) and in P notoginseng (Han and

Zhong, 2003)

Fig 3 Time profiles of medium conductivity (A), residual sugar (B) in high-density cultures

of Panax ginseng cells in a 5 l balloon type bioreactor

Effect of oxygen concentration on metabolite

production

The kinetic profile of total ginsenosides (saponin)

production is shown in Fig 4 Highest saponin

accumulation was on day 20 to 25 and later it declined Saponin content at 50% O2 supply was lower than that of control (20% O2 supply) The maximum total saponin

concentrations were 3.8 mg/g DW, 4.4 mg/g DW, 4.5 mg/g

DW and 2.85 mg/g DW at 20.8%, 30%, 40% and 50% O2 supply, respectively (Fig 4) Highest saponin production was with 40% O2 supply and lowest with 50% O2 supply

Supplementation of oxygen to the high-density suspension cultures significantly affects the accumulation of

ginsenosides and these results are concurrence with earlier published reports (Gao and Lee, 1992; Zhong et al., 1993)

High cell density and fluid viscosity could significantly reduced oxygen transfer efficiencies in bioreactors and conventional way of improving oxygen transfer rate is to

increase agitation speed and/or aeration rate (Huang and Chou, 2000) However, these approaches have several

limitations, such as high power consumption, cell damage due to mechanical shear stress, potential reduction of

productivity because of the stripping of CO2 and other

essential volatiles from the system An alternative approach

is improving the quality of incoming air by with oxygen concentration In the present experiment we have

supplemented the incoming air with different ratios of pure oxygen, which facilitates oxygen transfer rates, improves the accumulation of biomass of cultured cell and in turn accumulation of metabolites

Fig 4 Kinetics of production of ginseng saponin of Panax

ginseng cells in high-density bioreactor cultivations

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