Development of improved process with treatment of cellulase for isolation of ampelopsin from dried fruits of Ampelopsis grossedentata

Abstract

The commercial method for isolation of ampelopsin, one of the most common flavonoids isolated from the plant species Ampelopsis grossedentata, is a simple hydrothermal extraction at high temperature. To develop an improved process to isolate ampelopsin, the effects of treatment of cellulase on hydrolysis of the dried fruit of A. grossedentata were investigated. The treatment of cellulase was found to decrease the temperature and time for hydrolysis of the dried fruit of A. grossedentata. The conditions of the filter press and continuous flow centrifuge for removal of insoluble materials from the hydrolysate of the dried fruit of A. grossedentata were optimized. The recovery yield of ampelopsin from the dried fruits of A. grossedentata was 39.4%, as determined by HPLC chromatographic analysis. A safe and economical process at low temperature with treatment of cellulase for the isolation of ampelopsin was developed in this study.

Full Article

Development of Improved Process with Treatment of Cellulase for Isolation of Ampelopsin from Dried Fruits of Ampelopsis grossedentata

Wa Gao,^a Sang-Un Lee,^a Jianghong Li,^b and Jin-Woo Lee ^a,*

The commercial method for isolation of ampelopsin, one of the most common flavonoids isolated from the plant species Ampelopsis grossedentata, is a simple hydrothermal extraction at high temperature. To develop an improved process to isolate ampelopsin, the effects of treatment of cellulase on hydrolysis of the dried fruit of A. grossedentata were investigated. The treatment of cellulase was found to decrease the temperature and time for hydrolysis of the dried fruit of A. grossedentata. The conditions of the filter press and continuous flow centrifuge for removal of insoluble materials from the hydrolysate of the dried fruit of A. grossedentata were optimized. The recovery yield of ampelopsin from the dried fruits of A. grossedentata was 39.4%, as determined by HPLC chromatographic analysis. A safe and economical process at low temperature with treatment of cellulase for the isolation of ampelopsin was developed in this study.

Keywords: Ampelopsin; Isolation; Cellulase; Ampelopsis grossedentata

Contact information: a: Department of Biotechnology, College of Natural Resources and Life Science, Dong-A University, Busan 604-714, Korea; b: College of Plant Science & Technology, Huazhong Agricultural University, Wuhan, Hebei 430070, China; *Corresponding author: jwlee@dau.ac.kr

INTRODUCTION

Ampelopsin (3,5,7,3’,4’,5’-hexahydroxyl 2,3 dihydrogen flavanonol) is one of the most common flavonoids isolated from the plant species Ampelopsis grossedentata, known as rattan tea in China (Woo et al. 2012). Ampelopsin is known to have a broad range of functional compounds that have antioxidant, antitumor, and hepatoprotective effects (Kou and Chen 2012; Ye et al. 2015). The commercial method for the isolation of ampelopsin from A. grossedentata is simple hydrothermal extraction at temperature of approximately 100 °C for 24 h (Zhong et al. 2014). Because of this high temperature, the development of an improved process is needed for safety and economical aspects (Yoo et al. 2006).

Enzymatic saccharification of lignocellulosic materials can be accomplished through a complex reaction that involves three different types of cellulases: endoglucanase (carboxymethylcellulase), exocellobiohydrolase (avicelase), and β-glucosidase (Jo et al. 2008; Lee et al. 2008). Rice hulls, as lignocellulosic materials, have been hydrolyzed for production of fermentable sugars by commercial cellulases, in which the major cellulase was carboxymethylcellulase (CMCase) (Wei et al. 2009, 2010). In this study, cellulases were used for hydrolysis of the dried fruits of A. grossedentata to develop a safe and economical process for the enhanced recovery yield of ampelopsin (Lee et al.2010).

Simple procedures involving liquid-liquid extraction, synthetic adsorbent treatment, and low-pressure chromatography have been investigated for recovery of ampelopsin (Yoo et al. 2006). In recent years, membrane technology has been widely used for isolation and/or purification of functional materials (Yun et al. 2015). However, membranes made of polymeric structures have some disadvantages for stable operation. Polymeric membranes cannot be utilized at elevated temperatures or drastic chemical conditions (Kujawski et al. 2016). To overcome their disadvantages, numerous studies and development of inorganic membranes such as ceramic membranes have been performed recently (Yun et al. 2015). Due to their excellent chemical resistance to inorganic acids and oxidants, the tolerance to high temperatures and pressures, and longer life span, application of ceramic membranes has increased over the last decades (Loganathan et al. 2015; Ramakrishnan et al. 2015). Ceramic ultrafiltration for the enhanced recovery of ampelopsin was applied in this study (Gringer et al. 2015).

EXPERIMENTAL

Materials

The dried fruits of A. grossedentata were purchased from a domestic company (Korea Biosolution Ltd. Co., Busan, Korea). They were air-dried in an oven at 70 °C for 12 h before being milled in a hammer mill; particles smaller than 40 mesh were collected for further use in experiments (Wei et al. 2009).

Thermal Treatment for Hydrolysis of Dried Fruits of A. grossedentata

The dried fruits of A. grossedentata were hydrolyzed by thermal treatment with or without cellulase (Wei et al. 2010). The temperatures for hydrolytic reaction were 40, 50, 60, 70, and 80 °C. The cellulases used in this study were purified from Bacillus amyloliquefaciens DL-3 (Jo et al. 2008; Lee et al. 2008), Bacillus velezensis A-68 (Kim et al. 2013; Gao et al. 2014), and Cellulophaga lytica LBH-14 (Gao et al. 2012, 2013). The activities of cellulases as carboxymethylcellulases were measured and adjusted to 75 U/mL before the experiment (Lee et al. 2010).

HPLC Analysis of Ampelopsin

The isolated ampelopsin was analyzed by an AUURA Bio-LC system (SYSBIOLC002A, Berlin Germany) equipped with an AS autosampler, pulsed amperometry gold electrode, and Rheodyne injector. The HPLC system was equipped with a C₁₈ reversed phase column (250 mm x 4.6 mm, 5 μm) in conjunction with a precolumn and a UV-visible detector. The mobile phase was composed of CH₃OH and H₂O (60:40), and pH was adjusted to 3 with phosphoric acid (Zga et al. 2009). The size of the injection loop was 20 μL, and the temperature of the heated column was 25 °C (Wei et al. 2010).

Filter Press and Continuous Flow Centrifugation

A filter press (Plate & Frame Type Filter Press, Korea Filter Co., Ltd. Seoul Korea) was used for removal of insoluble materials after hydrolysis of the dried fruit A. grossedentata. It had two chambers; the filter area and volume of each chamber were 0.11 m² and 1.7 L (Kim et al. 2011). A continuous flow centrifuge (Tubular Type Centrifuge, Kokusan Chemical Co. Ltd., Tokyo, Japan) was used for removal of insoluble materials after hydrolysis. It had a bowl with a capacity of 9.0 L and a diameter of 12.2 cm. Its rotational speed and relative centrifugal force were fixed at 12,000 rpm and 11,500 x g, respectively.

Ultrafiltration with Ceramic Membrane

An ultrafiltration (UF) system with tubular ceramic membranes made of titanium dioxide (TiO₂) was supplied by TAMI Industries (Nyons, France). This system consisted of a collecting tank (100 L), a recirculating pump, and the UF membrane inside stainless steel housing. The feed solution flowed along the length of the membrane and was divided into permeate or retentate, which returned to the collecting tank (Gringer et al. 2015). The filtration area and maximal pressure of ceramic membrane were 0.35 m² and 10 bars. Two tubular ceramic membranes, with molecular weight cut offs (MWCOs) of 300 and 15 kDa, respectively, were used for recovery of reducing sugars.

Analytical Methods

The chemical compositions of the dried fruit of A. grossedentata were determined by the Feed & Food Nutrition Research Center at Pukyong National University in Korea, using the National Renewable Energy Laboratory, Laboratory Analytical Procedure, Technical Reports (NREL/TP) (Dagnino et al. 2013). Reducing sugars were determined by the DNS method (Miller 1959). Ampelopsin (dihydromyricetin as commercial name) was purchased from Sigma-Aldrich (USA).

RESULTS AND DISCUSSION

Effect of Temperature on Hydrolysis of Dried Fruits of A. grossedentata

The effect of temperature on the hydrolysis of the dried fruit of A. grossedentata was investigated. As shown in Table 1, the dried fruit of A. grossedentata consisted of crude fiber, crude lipid, crude protein, ash, and carbohydrate. Its carbohydrate composition was 57.6%. The concentration of the dried fruit of A. grossedentata used for the experiment was 40 g/L. The temperature for thermal hydrolysis ranged from 40 to 80 °C. As shown in Fig. 1, an increase in temperature for thermal hydrolysis resulted in higher conversion rate of reducing sugars. The highest conversion rates of reducing sugars from the dried fruit of A. grossedentata and its carbohydrates were 34.2% and 59.4%, respectively.

Table 1. Chemical Compositions of Dried Fruits of A. grossedentata

Effect of Cellulase on Hydrolysis of Dried Fruits of A. grossedentata

The effect of cellulase on the hydrolysis of the dried fruit of A. grossedentata was investigated. The cellulase produced by B. amyloliquefaciens DL-3 was in this experiment, and its CMCase activity in the reaction mixture was adjusted to be 75 U/mL. As shown in Fig. 2, an increase in temperature with treatment of cellulase for thermal hydrolysis also resulted in higher conversion rate of reducing sugars.

The highest conversion rates of reducing sugars from the dried fruit of A. grossedentata and its carbohydrates were 33.1% and 57.5%, respectively.

Fig. 1. Effect of temperature on hydrolysis of dried fruits of A. grossedentata without treatment of cellulase (●; 40 °C, ■; 50 °C, ▲; 60 °C, ▼; 70 °C, and ◆; 80 °C)

Fig. 2. Effect of temperature on hydrolysis of dried fruits of A. grossedentata with treatment of cellulase (●;40 °C, ■; 50 °C, ▲; 60 °C, ▼; 70 °C, and ◆; 80 °C)

The preferred substrate for production of cellulases by B. amyloliquefaciens DL-3 and B. velezensis A-68 was rice hulls (Kim et al. 2013; Gao et al. 2014), whereas that by C. lytica LBH-14 was rice bran (Gao et al. 2012, 2013). The conversion rates of reducing sugars without treatment of cellulase were compared with those with treatment of cellulase. As shown in Fig. 3, the final conversion rates of reducing sugars without treatment of cellulase after 24 h were similar to those with treatment of cellulase. However, the time and temperature to reach certain conversion rates of reducing sugars with treatment of cellulase were shorter and lower than those without treatment.

Comparison of Various Cellulases for Hydrolysis

The hydrolytic abilities of cellulases produced by B. amyloliquefaciens DL-3, B. velezensis A-68, and C. lytica LBH-14 were compared. The cellulases produced by Bacillus amyloliquefaciens DL-3, Bacillus velezensis A-68, and Cellulophaga lytica LBH-14 were used in this experiment, and their activities of CMCase in the reaction mixture were adjusted to be 75 U/mL. The optimal temperature for the enzymatic reaction of CMCase produced by B. amyloliquefaciens DL-3 was 50 °C (Lee et al.2008).

Fig. 3. Comparison in hydrolysis of dried fruits of A. grossedentata without (●) and with treatment of cellulase (○) at various temperatures; reaction temperature at (A) 40 °C, (B) 50 °C, (C) 60 °C, (D) 70 °C, and (E) 80 °C

The concentrations of the dried fruits of A. grossedentata ranged from 40 to 120 g/L. The reaction temperature and time were 50 °C and 12 h. As shown in Fig. 4, the conversion rate of reducing sugars with treatment of cellulase produced by B. amyloliquefaciens DL-3 was higher than those by B. velezensis A-68 and C. lytica LBH-14. As shown in Fig. 5, the maximal amount of reducing sugars was obtained when the concentration of dried fruits was 120 g/L. However, the highest conversion rate of reducing sugars was obtained when it was 40 g/L. Based on the conversion rate and production of reducing sugars, the concentration of the dried fruits of A. grossedentata and reaction time were set at 60 g/L and 6 h, respectively, for the following experiments.

Fig. 4. Comparison of cellulases produced by B. amyloliquefaciens DL-3 (■), B. velezensis A-68 (□), and C. lytica LBH-14 (▨) in hydrolysis of dried fruits of A. grossedentata (Different letters above bars mean that each value with a different letter is significantly different at p < 0.05)

Fig. 5. Effect of amount of dried fruit of A. grossedentata on its hydrolysis with treatment of cellulase produced by B. amyloliquefaciens DL-3 (●; 40 g/L, ■; 60 g/L, ▲; 80 g/L, ◆; 100 g/L, and ○; 120 g/L); (A) reducing sugars and (B) conversion rate

Effect of Diatomite on Removal of Insoluble Materials after Hydrolysis

Filtration for isolation is widely used in many areas of industries (Lihong et al. 2011). Diatomite as a filter aid is needed to improve the efficiency of filtration operation (Du et al. 2011). The effect of diatomite concentration in the filter press on removal of insoluble materials after thermal hydrolysis with treatment of cellulase was investigated. The concentration of the dried fruits of A. grossedentatafor thermal hydrolysis was 60 g/L. The reaction temperature and time were 50 °C and 6 h. The final concentration of diatomite mixed with the hydrolyzed fruits of A. grossedentata ranged from 0.0 to 10%. The volume of reaction mixture for thermal hydrolysis was 60 L. As shown in Fig. 6, higher concentration of diatomite resulted in more removal of insoluble materials. However, recovery yield of reducing sugars in the filtrate decreased with increasing concentration of diatomite. The optimal concentration of diatomite was set at 3.0% based on removal of insoluble materials and recovery yield of reducing sugars. Statistical analyses of Duncan’s multiple range test were conducted using the Statistical Program for Social Science (SPSS version 12, SPSS Co., Chicago, USA).

Effect of Flow Rate on Removal of Insoluble Materials after Hydrolysis

Continuous flow centrifugation (CFC) offers recoveries equivalent to filtration with improved economy and ease of use (Higgins et al. 2003). The effects of flow rate of CFC on removal of insoluble materials after thermal hydrolysis with treatment of cellulase were investigated. The flow rate into the continuous flow centrifuge ranged from 0.0 to 10.0 L/min. As shown in Fig. 7, a lower flow rate of the continuous flow centrifuge resulted in more removal of insoluble materials. The highest removal rate was 82.6% when the flow rate was 2 L/min, at which the removal of insoluble materials and the recovery rate of reducing sugars were 86.8% and 99.8%, respectively.

Fig. 6. Effect of diatomite on removal of insoluble materials after hydrolysis of dried fruits of A. grossedentata (■; removal of insoluble materials and □; recovery yield)

Fig. 7. Effect of flow rate on removal of insoluble materials after hydrolysis of dried fruits of A. grossedentata (■; removal of insoluble materials and □; recovery yield)

Recovery of Ampelopsin using Ultrafiltration System

The filtrate after removal of insoluble materials using the filter press and continuous flow centrifuge was applied in an ultrafiltration system to isolate ampelopsin. The filtrate was first divided by the membrane with the molecular weight cut off (MWCO) of 300 kDa. The permeate with molecular weight less than 300 kDa was divided by the membrane with the 15-kDa MWCO. As shown in Table 2, the concentrations of reducing sugars and ampelopsin in the permeate after filtration with the membrane with MWCO of 15 kDa were 13.3 and 0.23 g/L, respectively. The recovery yields of reducing sugars and ampelopsin from the dried fruits of A. grossedentata were 33.9% and 39.5%, respectively. The recovery yield of ampelopsin throughout the ceramic filtration was relative to that of reducing sugars. The molecular weights of major reducing sugars seemed to be similar to that of ampelopsin.

Table 2. Recovery Yields of Reducing Sugars and Ampelopsin using Ceramic Ultrafiltration (UF) System

The HPLC chromatograms of the hydrolysate of dried fruits of A. grossedentata with cellulase treatment and the ampelopsin isolated from the hydrolysate using an ultrafiltration system after the filter press and continuous flow centrifugation are shown in Fig. 8. The relatively large peak between 3.0 and 3.4 min can be attributed to the absorption peak of ampelopsin. The peak of ampelopsin was identified by comparison of chromatographic retention time with that of the ampelopsin standard (Zhong et al. 2014).

Fig. 8. HPLC analysis of (A) ampelopsin with concentrations ranging from 0.05 to 0.40 mg/L, (B) hydrolysate of dried fruits of A. grossedentata, and (C) ampelopsin isolated from hydrolysate

CONCLUSIONS

1. The process developed in this study with treatment of cellulases decreased in temperature and time for hydrolysis of the fruits of A. grossedentata. The time and temperature of this process were 6 h and 50 °C whereas those of the conventional method without treatment of cellulases were 24 h and 100 °C.
2. The safe and economic process at lower temperature and shorter time for extraction with treatment of cellulase for isolation of ampelopsin can be directly applied on the industrial scale.
3. The maximal amount of reducing sugars was obtained when the concentration of dried fruits was 120 g/L, whereas the highest conversion rate of reducing sugars was obtained when it was 40 g/L.
4. Higher concentration of diatomite in the filter press resulted in more removal of insoluble materials from the hydrolysate of the fruits of A. grossedentata. Lower flow rate of the continuous flow centrifuge also resulted in more removal of insoluble materials. The optimal concentration of diatomite mixed with hydrolysate and flow rate of continuous flow centrifuge were 3.0% and 2 L/min, respectively, based on removal of insoluble materials and recovery yield of reducing sugars.
5. The recovery yields of reducing sugars and ampelopsin from the dried fruits of A. grossedentatausing the ceramic ultrafiltration system were 33.9% and 39.5%, respectively. The ampelopsin isolated from the dried fruits of A. grossedentata was identified with the absorption peak in the HPLC chromatogram.

ACKNOWLEDGMENTS

This paper was financially supported by Dong-A University Research Fund.

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Article submitted: September 15, 2015; Peer review completed: January 9, 2016; Revised version received and accepted: January 19, 2016; Published: February 1, 2016.

DOI: 10.15376/biores.11.1.2712-2722