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Effect of ellagitannin acyl hydrolase, xylanase and cellulase on ellagic acid production from cups extract of valonia acorns
Process Biochemistry 42 (2007) 1291–1295 www.elsevier.com/locate/procbio
Effect of ellagitannin acyl hydrolase, xylanase and cellulase on ellagic acid production from cups extract of valonia acorns Wen Huang a, Hai Niu b,*, Zhenshan Li c, Wensheng Lin, Guohua Gong a, Wang Wang a a
Institute for Nanobiomedical Technology and Membrane Biology, State Key Lab of Biotherapy of Human Diseases, Cancer Center, West China Hospital, West China Medical School, Sichuan University, Keyuan 4 lu No.1, Gaopeng Avenue, Gaoxin District, Chengdu 610041, China b Mathematical College, Sichuan University, Chengdu 610064, China c Department of Environmental Engineering, Peking University, Beijing 100871, China Received 27 October 2006; received in revised form 26 May 2007; accepted 11 June 2007
Abstract Ellagic acid production from cups extract of valonia acorns by pure and mixed cultures of Aspergillus oryzae and Trichoderma reesei was investigated. Ellagitannin acyl hydrolase and xylanase as well as cellulase during the pure and mixed cultures were also determined. The results revealed that mixed culture could produce higher ellagic acid yield (23%) than either of pure culture. And it was found that the three enzymes from mixed culture appeared synergistic effect on ellagic acid production. Statistical analysis showed that ellagic acid yield was correlated very well with the three enzymes activities, resulting in the model for ellagic acid production with high R2 value of 0.998 and significant level p < 0.004. # 2007 Elsevier Ltd. All rights reserved. Keywords: Ellagitannin acyl hydrolase; Ellagic acid; Valonia acorn; Aspergillus oryzae; Trichoderma reesei; Synergistic effect
1. Introduction Ellagitannins are a large group of polyphenolic compounds being widely distributed in higher plants (Fig. 1a) [1,2]. The compounds display an enormous structural variability due to the manifold possible sites for the linkage of ellagic acid unit with the glucose moiety [3]. Increasing interest in the biological and pharmacological roles of these polyphenolic compounds in the therapeutic effects has led to a rapid growth of knowledge in this area [4,5]. Ellagic acid, the primary hydrolyzate of ellagitannins, is of particular attention as it has been reported to have many medically important properties as antioxidant, antimicrobial, antiviral and antitumor [6,7]. However, the applications of ellagic acid have not been fully exploited because of its high cost of production. Some attempts have been carried out to obtain ellagic acid by acidic action [8]. Nevertheless, it suffered from the
* Corresponding author. Tel.: +86 28 89859857; fax: +86 28 85405007. E-mail addresses: [email protected] (W. Huang), [email protected] (H. Niu). 1359-5113/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2007.06.013
unspecific reactions yielding numerous structurally closely related reaction products and unspecific byproducts. This has resulted in a focus on biological methods. The hydrolytic efficiency of ellagitannins for ellagic acid production by biomethod requires the synergistic action of an appropriate enzyme system to break down chemical linkages such as ester, C-glucosidic and C–C bonds, which are presenting in ellagitannin monomer and its high polymers [3]. Cleavage of ester bond commonly use tannase acyl hydrolases (EC 3.1.1.20), which are inducible enzymes mainly produced by microorganisms [9–13], and can cleaves ester and depside bonds to free ellagic acid. The decompositions of C–C and Cglycosidic bonds have been involved in capacities of cellulase and xylanase from many microorganisms [14,15]. To convert ellagitannins into ellagic acid, commercial enzymes could be used. The process, however, is not considered economical because the cost of commercial enzymes remains very high. If the enzymes could be produced directly from ellagitannins fermentation and then be applied to produce ellagic acid production, the cost of enzymes could be significantly reduced which, at the same time, enhance potential profitability of ellagic acid production.
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W. Huang et al. / Process Biochemistry 42 (2007) 1291–1295 Fungal cells were sub-cultured in a shaker (at 120 rpm, HZS-H super water bath shaker, China) at 30 8C for subgenerations with the mycelium being used for inoculums.
2.3. Culture conditions Medium composition in each flask was 7 g L1 of ellagitannins of supplemented with 0.5 g L1 of K2HPO4, 0.5 g L1 of KH2PO4, 2 g L1 of NaNO3, 0.5 g L1 of KCl, 0.5 g L1 of MgSO4, 0.01 g L1 of FeSO4, 2 mL L1 of tween-80 and distilled water. The medium was autoclaved at 121 8C for 15 min. For pure culture, either A. oryzae or T. reesei (at 10% inoculums ratio, v/v) was inoculated into each flask, respectively. For mixed culture, A. oryzae and T. reesei inoculums were inoculated into each flask simultaneously. Ellagic acid production and enzymes activities (ellagitannin acyl hydrolase, xylanase and cellulase) in pure and mixed cultures processes were determined.
2.4. Ellagitannins determination
Fig. 1. Typical ellagitannin monomer and ellagic acid.
Based on previous research, Aspergillus oryzae, which could yield higher ellagic acid with high levels of both ellagitannin acyl hydrolase and xylanase activities [16], and Trichoderma reesei, which could effectively degrade ellagitannins with higher level of cellulase activity, were selected for further investigation. The work aims at establishing a cost effective process for ellagic acid production from cups extract of valonia acorns by A. oryzae and T. reesei. 2. Materials and methods 2.1. Ellagitannins extraction Dried cups of valonia acorns of oak from Henan province of Mid-China were ground to dust with a milling cutter. Finely ground dust was extracted with acetone-water solution (7:3, v/v) at room temperature three times (each time for 24 h). After filtration, the acetone was removed by vacuum distillation, and the aqueous residue evaporated to dryness.
2.2. Microorganism A. oryzae, was kindly provided by College of Biological Science, China Agricultural University, Beijing, China. Trichoderma reesei 3.3711, briefly named T. reesei, was bought from the type culture collection center of Chinese Academy of Science in Beijing. They were named A. oryzae YH344 and T. reesei MH-1 3882, respectively, after acclimated in sterilized ground dust of dried cups of valonia acorns. A. oryzae YH344 and T. reesei MH-1 3882 were stored at 4 8C on agar (1.5%) slants of MY medium (2% malt extract, 0.2% yeast extract) and potato dextrose agar slant, respectively [17,18]. To prepare the inoculums, the spores in the slant were suspended in 2 mL medium (106 to 107 spores mL1) and transferred into a 250 mL Erlenmeyer flask containing 50 mL medium. The subculture medium was Mandel salt solution [19] supplemented with 2 mL L1 tween-80, 1 g L1 peptone, and 10 g L1 glucose.
Ellagitannins was determined based on the international standard method [20,21]. The analysis process could be simply described as follows. Firstly, analytical liquid, which contained 0.4% of dried cups extract of valonia acorns, was prepared for total solid, water soluble matter and non-ellagitannin. (1) 50 mL of the analytical liquid was directly evaporated to dryness and weight for total solid. (2) 75 mL of the analytical liquid was filtrated. The filtrate evaporated to dryness and weight for water soluble matter, which could be computed according to equation as: water soluble matter (%) = (dried filtrate weight/50 1000)/cups extract weight 100. (3) 100 mL of the analytical liquid was added into hide powder. After filtration, the filtrate evaporated to dryness and weight for nonellagitannin content, which could be obtained according to following equations: non-ellagitannin (%) = [G1 20 1.2 (100% G2 20 1.2 0.075)]/ W 100, where G1 was the dried filtrate weight (g), G2 was the dried control (distilled water) weight (g), W was the dried cups extract weight (g), 1.2: diluted coefficient of water in the hide powder and 0.075 was the maximum value of water soluble matter in the hide powder (g/1000 mL). Finally, the ellagitannins content can be computed as following equation: ellagitannins content (%) = water soluble matter non-ellagitannin.
2.5. Ellagic acid determination HPLC quantitative determination for ellagic acid was performed in the reverse phase on a HPLC/Diode Array (HP1100, a diode array detector G1306 A, Agilent Technologies, America) with detection at 255 nm and 366 nm (these wavelengths correspond to the optimal absorption of ellagic acid) [22]. Culture broth was centrifuged (F2402H AllegraTM X-22R Cenrifuge) and the residue was washed by water three times. The washed residue was diluted 100–500 times using methanol for the quantitative estimation of ellagic acid. The diluted sample adjusted to pH of 8.5 was filtered (0.2 mm), and then injected (100 mL) onto a SUPELCOSIL LC-18 reverse-phase column (15 cm 4.6 mm, 5 mm). The mobile phase composition was optimized and the best composition obtained was water-methanol (20:80, v/v). The flow-rate was 1.0 mL min1. The regression equation for ellagic acid concentration was y = 0.64x1 + 0.18x2 + 0.13, where y is ellagic acid concentration (mg mL1); x1 and x2 are absorbencies of ellagic acid at 255 nm and 366 nm, respectively. The correlation coefficient for the standard curve was 0.999 in the ellagic acid concentration range 8– 20 mg mL1. The linear relationship between absorbency and concentration of ellagic acid was highly significant ( p < 0.0001). Ellagic acid weight and ellagic acid yield were computed according to following equations: ellagic acid weight (mg) = ellagic acid concentration diluted times of sample culture broth volume; ellagic acid yield (%) = ellagic acid weight after fermentation/initial ellagitannins weight.
2.6. Enzyme activity assay 2.6.1. Ellagitannin acyl hydrolase Two milliliters of culture broth was withdrawn at regular intervals of 12 h and analyzed for ellagitannins acyl hydrolase activity. The broth was filtered and the mycelium washed with citrate buffer (0.05 M, pH 5.5) twice. The
W. Huang et al. / Process Biochemistry 42 (2007) 1291–1295 washed mycelium was ground with quartz sand in a mortar for 10 min. The entire operation was carried out after pre-cooling the suspension to 4 8C, such that the temperature of the mixture does not exceed 10 8C. The suspension was centrifuged at 15,000 rpm (F2402H AllegraTM X-22R Cenrifuge) for 25 min and the supernatant containing ellagitannin acyl hydrolase was recovered. The debris was washed with buffer to recover the enzyme bound to the residue and the washing was mixed with the supernatant. The supernatant—the enzymatic extract was subsequently analyzed for ellagitannin acyl hydrolase activity. The enzyme activity was determined using castalagin as substrate, which can be hydrolyzed by the enzyme to form castalin and ellagic acid. Castalagin (1.8 mM final concentration) was prepared in citrate buffer (pH 5.0) and incubated with an appropriate amount of enzyme at 37 8C for 30 min in a water bath. The reaction was stopped by boiling for 10 min to denaturalize the enzyme. The effect of the boiling treatment for ending the enzymatic reaction on analysis of the enzyme activity was also considered in our experiment. The results showed that 98% ellagic acid was released from castalagin before boiling treatment. Therefore, the effect of the treatment was insignificant on analysis of the enzyme activity. One unit (IU) of activity was defined as the amount of enzyme releasing 1 mmol of ellagic acid per min at pH 5.0 and 37 8C. 2.6.2. Xylanase Two milliliters of culture broth withdrawn at regular intervals of 12 h were filtered. The suspension was then centrifuged at 10,000 rpm for 10 min and the resulting liquid—the enzymatic extract was subsequently analyzed for xylanase activity. Xylanase activity was assayed using xylan as substrate. 2 mL reaction mixture, containing 1 mL of appropriately diluted enzyme solution and 1 mL of a 0.5% (w/v) suspension of xylan in 0.05 M citrate buffer, was made at pH 5.4. The mixture was incubated at 50 8C for 10 min and the reaction stopped by the addition of 2 mL of TCA (0.3 N). One enzyme activity unit was defined as the amount of enzyme that released 1 mmol xylose from xylan per min under the assay conditions. 2.6.3. Cellulase 2 mL of culture broth withdrawn at regular intervals of 12 h were filtered. The suspension was then centrifuged at 10,000 rpm for 10 min and the resulting liquid—the enzymatic extract was subsequently analyzed for cellulase activity. Cellulase solution and carboxymethyl cellulose (CM cellulose) were prepared in citrate phosphate buffer (0.1 M, pH 5.0 assay buffer). Two milliliters of CM cellulose was incubated with 1 mL of enzyme solution at 50 8C for 10 min. The reaction was stopped by the addition of 2 mL of 0.3 M trichloroacetic acid (TCA). The reducing sugar generated as a result of CM-cellulose hydrolysis was estimated by the DNS the method [19]. Results were expressed as glucose concentration using a calibration curve. One enzyme activity unit was defined as the amount of enzyme that released 1 mmol of glucose per min under the assay conditions.
2.7. Model for estimating effects of the enzymes on ellagic acid yield To estimate effects of ellagitannin acyl hydrolase, xylanase and cellulase on bioconversion of ellagitannins to ellagic acid by mixed culture, a polynomial regression model based on experimental results was established as follows: y ¼ b12 x1 x2 þ b13 x1 x3 þ b23 x2 x3 þ b1 x1 þ b2 x2 þ b3 x3 þ b0
2.8. Statistical analysis Data are presented as mean standard deviation. The results were statistically analyzed by analysis of variance (ANOVA) followed by Fisher test. Differences were considered significant for p < 0.05.
3. Results and discussion 3.1. Enzyme activity and ellagic acid yield by pure and mixed cultures During fermentations of cups extract of valonia acorns containing ellagitannins of 62% by pure and mixed culture of A. oryzae and T. reesei, ellagic acid yields, together with ellagitannin acyl hydrolase, xylanase and cellulase activities, were determined. It can be found in Fig. 2a–d that mixed culture could produce more than 22% of ellagic acid with the higher of levels of ellagitannin acyl hydrolase, xylanase and cellulase activities, while pure culture A. oryzae 16% of ellagic acid with higher levels of ellagitannin acyl hydrolase and xylanase activities but low of cellulase activity, and T. reesei 7% of ellagic acid with relatively high cellulase activity but low ellagitannin acyl hydrolase and xylanase activities. The results suggest that the mixed culture could yield more appropriate enzyme system for ellagic acid production than each of pure culture does. Meanwhile, it can be also noted in Fig. 2a and b that although ellagitannin acyl hydrolase activity seems to be more important for ellagic acid production than the other two enzymes activities, xylanase and cellulase activities also appear closely correlated with ellagic acid accumulation. 3.2. Relationship between ellagic acid production and enzymes Ellagic acid yield and enzymes activities were collected during the fermentation process by mixed culture of A. oryzae and T. reesei. These data, as shown in Table 1, were fitted into a polynomial regression model Eq. (3). ANOVA was used to evaluate the adequacy of the fitted model. Ellagitannin acyl hydrolase, xylanase and cellulase activities and ellagic acid yield in Table 1 were the means of three duplicates and their standard deviation were less than 3.65 IU mL1. The results showed a high reproducibility of the three enzymes activities.
(1)
where y is the estimated dependent variable as ellagic acid yield; x1, x2 and x3 are the levels of the independent variables such the factors as ellagitannin acyl hydrolase, cellulase and xylanase; b1, b2 and b3 are the linear terms; b12, b13 and b23 are the interaction terms of the factors; b0 is a constant. If levels values of factors all appear in the range 0–1, it will be favorable the appraisal of the effects of independent variables. For the aim, the levels of the independent variables involved in the research can be transformed by standardization Eq. (2) stated as xmin x x0 ¼ max xmin x
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(2)
where x value is the level of each independent variables (ellagitannin acyl hydrolase, cellulase or xylanase); x0 is the standardized level of each independent variable.
y0 ¼ 48:57x01 48:99x02 þ 8:56x03 45:79x01 x03 þ 42:05x02 x03 þ 14:75
(3)
As evident from Eq. (3), individual effects of the three enzymes activities and interactive effects of cellulase with ellagitannin acyl hydrolase and xylanase appeared to be significant on the ellagic acid accumulation. Statistical analysis proved that the individual and interactive effects as that from Eq. (3) were significant ( p < 0.05). The results imply that ellagitannin acyl hydrolase is important for ellagic acid production, while xylanase and celullase could effectively improve bioconversion of ellagitannins to ellagic acid.
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Fig. 2. Evolutions of ellagic acid yield (a), ellagitannin acyl hydrolase (b), xylanase (c) and cellulase (d) activities on time course of pure and mixed cultures.
The correlation coefficient (R2) of Eq. (3) was 0.998. F tests showed that the equation had a significance of 99.6% ( p < 0.004). All of these indicated that the model was reliable in reflecting the relationship between the three enzymes activities and ellagic acid yield.
The mixed culture of A. oryzae and T. reesei could remarkably enhance ellagic acid accumulation and produce higher levels of ellagitannin acyl hydrolase, xylanase and cellulase activities, compared with either of pure culture. The statistical analysis manifested that the ellagic acid yield were
Table 1 Standardized and experimental values of the three enzymes activities and experimental and theoretical values of ellagic acid yield on time course of mixed culture of A. oryzae and T. reesei Time (h)
Ellagic acid yield (%) c
Experimental value of enzyme activity (IU mL1)b
Standardized value of enzyme activitya x01
x02
x03
x1
x2
x3
y
y0
12 24 36 48 60 72 84 96
0.00 0.92 1.00 0.88 0.81 0.63 0.39 0.13
0.14 1.00 1.00 0.93 0.79 0.46 0.30 0.00
0.00 0.61 0.75 0.88 0.74 0.93 1.00 0.72
9.35 1.35 31.05 2.03 32.91 1.65 30.09 2.08 28.55 1.79 24.22 2.02 18.60 1.40 12.52 1.15
24.09 1.99 55.90 2.29 55.85 3.65 53.17 2.90 48.12 3.22 36.06 2.77 30.22 2.19 18.97 1.99
6.56 0.29 14.95 0.87 16.95 1.10 18.75 1.39 16.79 1.27 19.46 1.69 20.40 1.09 16.53 0.50
7.91 1.14 15.42 0.98 18.09 0.72 18.24 1.06 19.17 1.56 21.75 1.35 22.49 2.20 22.84 1.53
7.89 15.62 17.95 18.42 18.86 21.94 22.31 22.94
Mean S.D.
0.60 0.38
0.58 0.40
0.70 0.31
23.41 1.68 8.94 0.35
40.30 2.62 14.88 0.61
16.30 1.02 4.31 0.46
18.24 1.32 4.89 0.46
18.24 4.88
a b c
x01 ; x02 and x03 are standardized values of ellagitannin acyl hydrolase, xylanase and cellulase activities, respectively. x1, x2 and x3 are experimental values corresponding to x01 ; x02 and x03 , respectively. y and y0 are experimental and theoretical values of ellagic acid yield, respectively.
W. Huang et al. / Process Biochemistry 42 (2007) 1291–1295
significantly correlated with ellagitannin acyl hydrolase, xylanase and cellulase activities. The research indicted that an appropriate enzyme system, resulting in high level of ellagic acid yield, is very important for bioconversion of ellagitannins to ellagic acid production from the forest byproduct.
[9]
[10]
Acknowledgements [11]
Financial support of this research was from the National Key Technologies R&D Program of China with grant no. 2001BA804A21. Sincere thanks are to Dr. Jinshui Yang at China Agricultural University for providing the microbial culture and technical assistance in the experimental studies.
[12]
[13]
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Effect of ellagitannin acyl hydrolase, xylanase and cellulase on ellagic acid production from cups extract of valonia acorns Wen Huang a, Hai Niu b,*, Zhenshan Li c, Wensheng Lin, Guohua Gong a, Wang Wang a a
Institute for Nanobiomedical Technology and Membrane Biology, State Key Lab of Biotherapy of Human Diseases, Cancer Center, West China Hospital, West China Medical School, Sichuan University, Keyuan 4 lu No.1, Gaopeng Avenue, Gaoxin District, Chengdu 610041, China b Mathematical College, Sichuan University, Chengdu 610064, China c Department of Environmental Engineering, Peking University, Beijing 100871, China Received 27 October 2006; received in revised form 26 May 2007; accepted 11 June 2007
Abstract Ellagic acid production from cups extract of valonia acorns by pure and mixed cultures of Aspergillus oryzae and Trichoderma reesei was investigated. Ellagitannin acyl hydrolase and xylanase as well as cellulase during the pure and mixed cultures were also determined. The results revealed that mixed culture could produce higher ellagic acid yield (23%) than either of pure culture. And it was found that the three enzymes from mixed culture appeared synergistic effect on ellagic acid production. Statistical analysis showed that ellagic acid yield was correlated very well with the three enzymes activities, resulting in the model for ellagic acid production with high R2 value of 0.998 and significant level p < 0.004. # 2007 Elsevier Ltd. All rights reserved. Keywords: Ellagitannin acyl hydrolase; Ellagic acid; Valonia acorn; Aspergillus oryzae; Trichoderma reesei; Synergistic effect
1. Introduction Ellagitannins are a large group of polyphenolic compounds being widely distributed in higher plants (Fig. 1a) [1,2]. The compounds display an enormous structural variability due to the manifold possible sites for the linkage of ellagic acid unit with the glucose moiety [3]. Increasing interest in the biological and pharmacological roles of these polyphenolic compounds in the therapeutic effects has led to a rapid growth of knowledge in this area [4,5]. Ellagic acid, the primary hydrolyzate of ellagitannins, is of particular attention as it has been reported to have many medically important properties as antioxidant, antimicrobial, antiviral and antitumor [6,7]. However, the applications of ellagic acid have not been fully exploited because of its high cost of production. Some attempts have been carried out to obtain ellagic acid by acidic action [8]. Nevertheless, it suffered from the
* Corresponding author. Tel.: +86 28 89859857; fax: +86 28 85405007. E-mail addresses: [email protected] (W. Huang), [email protected] (H. Niu). 1359-5113/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2007.06.013
unspecific reactions yielding numerous structurally closely related reaction products and unspecific byproducts. This has resulted in a focus on biological methods. The hydrolytic efficiency of ellagitannins for ellagic acid production by biomethod requires the synergistic action of an appropriate enzyme system to break down chemical linkages such as ester, C-glucosidic and C–C bonds, which are presenting in ellagitannin monomer and its high polymers [3]. Cleavage of ester bond commonly use tannase acyl hydrolases (EC 3.1.1.20), which are inducible enzymes mainly produced by microorganisms [9–13], and can cleaves ester and depside bonds to free ellagic acid. The decompositions of C–C and Cglycosidic bonds have been involved in capacities of cellulase and xylanase from many microorganisms [14,15]. To convert ellagitannins into ellagic acid, commercial enzymes could be used. The process, however, is not considered economical because the cost of commercial enzymes remains very high. If the enzymes could be produced directly from ellagitannins fermentation and then be applied to produce ellagic acid production, the cost of enzymes could be significantly reduced which, at the same time, enhance potential profitability of ellagic acid production.
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W. Huang et al. / Process Biochemistry 42 (2007) 1291–1295 Fungal cells were sub-cultured in a shaker (at 120 rpm, HZS-H super water bath shaker, China) at 30 8C for subgenerations with the mycelium being used for inoculums.
2.3. Culture conditions Medium composition in each flask was 7 g L1 of ellagitannins of supplemented with 0.5 g L1 of K2HPO4, 0.5 g L1 of KH2PO4, 2 g L1 of NaNO3, 0.5 g L1 of KCl, 0.5 g L1 of MgSO4, 0.01 g L1 of FeSO4, 2 mL L1 of tween-80 and distilled water. The medium was autoclaved at 121 8C for 15 min. For pure culture, either A. oryzae or T. reesei (at 10% inoculums ratio, v/v) was inoculated into each flask, respectively. For mixed culture, A. oryzae and T. reesei inoculums were inoculated into each flask simultaneously. Ellagic acid production and enzymes activities (ellagitannin acyl hydrolase, xylanase and cellulase) in pure and mixed cultures processes were determined.
2.4. Ellagitannins determination
Fig. 1. Typical ellagitannin monomer and ellagic acid.
Based on previous research, Aspergillus oryzae, which could yield higher ellagic acid with high levels of both ellagitannin acyl hydrolase and xylanase activities [16], and Trichoderma reesei, which could effectively degrade ellagitannins with higher level of cellulase activity, were selected for further investigation. The work aims at establishing a cost effective process for ellagic acid production from cups extract of valonia acorns by A. oryzae and T. reesei. 2. Materials and methods 2.1. Ellagitannins extraction Dried cups of valonia acorns of oak from Henan province of Mid-China were ground to dust with a milling cutter. Finely ground dust was extracted with acetone-water solution (7:3, v/v) at room temperature three times (each time for 24 h). After filtration, the acetone was removed by vacuum distillation, and the aqueous residue evaporated to dryness.
2.2. Microorganism A. oryzae, was kindly provided by College of Biological Science, China Agricultural University, Beijing, China. Trichoderma reesei 3.3711, briefly named T. reesei, was bought from the type culture collection center of Chinese Academy of Science in Beijing. They were named A. oryzae YH344 and T. reesei MH-1 3882, respectively, after acclimated in sterilized ground dust of dried cups of valonia acorns. A. oryzae YH344 and T. reesei MH-1 3882 were stored at 4 8C on agar (1.5%) slants of MY medium (2% malt extract, 0.2% yeast extract) and potato dextrose agar slant, respectively [17,18]. To prepare the inoculums, the spores in the slant were suspended in 2 mL medium (106 to 107 spores mL1) and transferred into a 250 mL Erlenmeyer flask containing 50 mL medium. The subculture medium was Mandel salt solution [19] supplemented with 2 mL L1 tween-80, 1 g L1 peptone, and 10 g L1 glucose.
Ellagitannins was determined based on the international standard method [20,21]. The analysis process could be simply described as follows. Firstly, analytical liquid, which contained 0.4% of dried cups extract of valonia acorns, was prepared for total solid, water soluble matter and non-ellagitannin. (1) 50 mL of the analytical liquid was directly evaporated to dryness and weight for total solid. (2) 75 mL of the analytical liquid was filtrated. The filtrate evaporated to dryness and weight for water soluble matter, which could be computed according to equation as: water soluble matter (%) = (dried filtrate weight/50 1000)/cups extract weight 100. (3) 100 mL of the analytical liquid was added into hide powder. After filtration, the filtrate evaporated to dryness and weight for nonellagitannin content, which could be obtained according to following equations: non-ellagitannin (%) = [G1 20 1.2 (100% G2 20 1.2 0.075)]/ W 100, where G1 was the dried filtrate weight (g), G2 was the dried control (distilled water) weight (g), W was the dried cups extract weight (g), 1.2: diluted coefficient of water in the hide powder and 0.075 was the maximum value of water soluble matter in the hide powder (g/1000 mL). Finally, the ellagitannins content can be computed as following equation: ellagitannins content (%) = water soluble matter non-ellagitannin.
2.5. Ellagic acid determination HPLC quantitative determination for ellagic acid was performed in the reverse phase on a HPLC/Diode Array (HP1100, a diode array detector G1306 A, Agilent Technologies, America) with detection at 255 nm and 366 nm (these wavelengths correspond to the optimal absorption of ellagic acid) [22]. Culture broth was centrifuged (F2402H AllegraTM X-22R Cenrifuge) and the residue was washed by water three times. The washed residue was diluted 100–500 times using methanol for the quantitative estimation of ellagic acid. The diluted sample adjusted to pH of 8.5 was filtered (0.2 mm), and then injected (100 mL) onto a SUPELCOSIL LC-18 reverse-phase column (15 cm 4.6 mm, 5 mm). The mobile phase composition was optimized and the best composition obtained was water-methanol (20:80, v/v). The flow-rate was 1.0 mL min1. The regression equation for ellagic acid concentration was y = 0.64x1 + 0.18x2 + 0.13, where y is ellagic acid concentration (mg mL1); x1 and x2 are absorbencies of ellagic acid at 255 nm and 366 nm, respectively. The correlation coefficient for the standard curve was 0.999 in the ellagic acid concentration range 8– 20 mg mL1. The linear relationship between absorbency and concentration of ellagic acid was highly significant ( p < 0.0001). Ellagic acid weight and ellagic acid yield were computed according to following equations: ellagic acid weight (mg) = ellagic acid concentration diluted times of sample culture broth volume; ellagic acid yield (%) = ellagic acid weight after fermentation/initial ellagitannins weight.
2.6. Enzyme activity assay 2.6.1. Ellagitannin acyl hydrolase Two milliliters of culture broth was withdrawn at regular intervals of 12 h and analyzed for ellagitannins acyl hydrolase activity. The broth was filtered and the mycelium washed with citrate buffer (0.05 M, pH 5.5) twice. The
W. Huang et al. / Process Biochemistry 42 (2007) 1291–1295 washed mycelium was ground with quartz sand in a mortar for 10 min. The entire operation was carried out after pre-cooling the suspension to 4 8C, such that the temperature of the mixture does not exceed 10 8C. The suspension was centrifuged at 15,000 rpm (F2402H AllegraTM X-22R Cenrifuge) for 25 min and the supernatant containing ellagitannin acyl hydrolase was recovered. The debris was washed with buffer to recover the enzyme bound to the residue and the washing was mixed with the supernatant. The supernatant—the enzymatic extract was subsequently analyzed for ellagitannin acyl hydrolase activity. The enzyme activity was determined using castalagin as substrate, which can be hydrolyzed by the enzyme to form castalin and ellagic acid. Castalagin (1.8 mM final concentration) was prepared in citrate buffer (pH 5.0) and incubated with an appropriate amount of enzyme at 37 8C for 30 min in a water bath. The reaction was stopped by boiling for 10 min to denaturalize the enzyme. The effect of the boiling treatment for ending the enzymatic reaction on analysis of the enzyme activity was also considered in our experiment. The results showed that 98% ellagic acid was released from castalagin before boiling treatment. Therefore, the effect of the treatment was insignificant on analysis of the enzyme activity. One unit (IU) of activity was defined as the amount of enzyme releasing 1 mmol of ellagic acid per min at pH 5.0 and 37 8C. 2.6.2. Xylanase Two milliliters of culture broth withdrawn at regular intervals of 12 h were filtered. The suspension was then centrifuged at 10,000 rpm for 10 min and the resulting liquid—the enzymatic extract was subsequently analyzed for xylanase activity. Xylanase activity was assayed using xylan as substrate. 2 mL reaction mixture, containing 1 mL of appropriately diluted enzyme solution and 1 mL of a 0.5% (w/v) suspension of xylan in 0.05 M citrate buffer, was made at pH 5.4. The mixture was incubated at 50 8C for 10 min and the reaction stopped by the addition of 2 mL of TCA (0.3 N). One enzyme activity unit was defined as the amount of enzyme that released 1 mmol xylose from xylan per min under the assay conditions. 2.6.3. Cellulase 2 mL of culture broth withdrawn at regular intervals of 12 h were filtered. The suspension was then centrifuged at 10,000 rpm for 10 min and the resulting liquid—the enzymatic extract was subsequently analyzed for cellulase activity. Cellulase solution and carboxymethyl cellulose (CM cellulose) were prepared in citrate phosphate buffer (0.1 M, pH 5.0 assay buffer). Two milliliters of CM cellulose was incubated with 1 mL of enzyme solution at 50 8C for 10 min. The reaction was stopped by the addition of 2 mL of 0.3 M trichloroacetic acid (TCA). The reducing sugar generated as a result of CM-cellulose hydrolysis was estimated by the DNS the method [19]. Results were expressed as glucose concentration using a calibration curve. One enzyme activity unit was defined as the amount of enzyme that released 1 mmol of glucose per min under the assay conditions.
2.7. Model for estimating effects of the enzymes on ellagic acid yield To estimate effects of ellagitannin acyl hydrolase, xylanase and cellulase on bioconversion of ellagitannins to ellagic acid by mixed culture, a polynomial regression model based on experimental results was established as follows: y ¼ b12 x1 x2 þ b13 x1 x3 þ b23 x2 x3 þ b1 x1 þ b2 x2 þ b3 x3 þ b0
2.8. Statistical analysis Data are presented as mean standard deviation. The results were statistically analyzed by analysis of variance (ANOVA) followed by Fisher test. Differences were considered significant for p < 0.05.
3. Results and discussion 3.1. Enzyme activity and ellagic acid yield by pure and mixed cultures During fermentations of cups extract of valonia acorns containing ellagitannins of 62% by pure and mixed culture of A. oryzae and T. reesei, ellagic acid yields, together with ellagitannin acyl hydrolase, xylanase and cellulase activities, were determined. It can be found in Fig. 2a–d that mixed culture could produce more than 22% of ellagic acid with the higher of levels of ellagitannin acyl hydrolase, xylanase and cellulase activities, while pure culture A. oryzae 16% of ellagic acid with higher levels of ellagitannin acyl hydrolase and xylanase activities but low of cellulase activity, and T. reesei 7% of ellagic acid with relatively high cellulase activity but low ellagitannin acyl hydrolase and xylanase activities. The results suggest that the mixed culture could yield more appropriate enzyme system for ellagic acid production than each of pure culture does. Meanwhile, it can be also noted in Fig. 2a and b that although ellagitannin acyl hydrolase activity seems to be more important for ellagic acid production than the other two enzymes activities, xylanase and cellulase activities also appear closely correlated with ellagic acid accumulation. 3.2. Relationship between ellagic acid production and enzymes Ellagic acid yield and enzymes activities were collected during the fermentation process by mixed culture of A. oryzae and T. reesei. These data, as shown in Table 1, were fitted into a polynomial regression model Eq. (3). ANOVA was used to evaluate the adequacy of the fitted model. Ellagitannin acyl hydrolase, xylanase and cellulase activities and ellagic acid yield in Table 1 were the means of three duplicates and their standard deviation were less than 3.65 IU mL1. The results showed a high reproducibility of the three enzymes activities.
(1)
where y is the estimated dependent variable as ellagic acid yield; x1, x2 and x3 are the levels of the independent variables such the factors as ellagitannin acyl hydrolase, cellulase and xylanase; b1, b2 and b3 are the linear terms; b12, b13 and b23 are the interaction terms of the factors; b0 is a constant. If levels values of factors all appear in the range 0–1, it will be favorable the appraisal of the effects of independent variables. For the aim, the levels of the independent variables involved in the research can be transformed by standardization Eq. (2) stated as xmin x x0 ¼ max xmin x
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(2)
where x value is the level of each independent variables (ellagitannin acyl hydrolase, cellulase or xylanase); x0 is the standardized level of each independent variable.
y0 ¼ 48:57x01 48:99x02 þ 8:56x03 45:79x01 x03 þ 42:05x02 x03 þ 14:75
(3)
As evident from Eq. (3), individual effects of the three enzymes activities and interactive effects of cellulase with ellagitannin acyl hydrolase and xylanase appeared to be significant on the ellagic acid accumulation. Statistical analysis proved that the individual and interactive effects as that from Eq. (3) were significant ( p < 0.05). The results imply that ellagitannin acyl hydrolase is important for ellagic acid production, while xylanase and celullase could effectively improve bioconversion of ellagitannins to ellagic acid.
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W. Huang et al. / Process Biochemistry 42 (2007) 1291–1295
Fig. 2. Evolutions of ellagic acid yield (a), ellagitannin acyl hydrolase (b), xylanase (c) and cellulase (d) activities on time course of pure and mixed cultures.
The correlation coefficient (R2) of Eq. (3) was 0.998. F tests showed that the equation had a significance of 99.6% ( p < 0.004). All of these indicated that the model was reliable in reflecting the relationship between the three enzymes activities and ellagic acid yield.
The mixed culture of A. oryzae and T. reesei could remarkably enhance ellagic acid accumulation and produce higher levels of ellagitannin acyl hydrolase, xylanase and cellulase activities, compared with either of pure culture. The statistical analysis manifested that the ellagic acid yield were
Table 1 Standardized and experimental values of the three enzymes activities and experimental and theoretical values of ellagic acid yield on time course of mixed culture of A. oryzae and T. reesei Time (h)
Ellagic acid yield (%) c
Experimental value of enzyme activity (IU mL1)b
Standardized value of enzyme activitya x01
x02
x03
x1
x2
x3
y
y0
12 24 36 48 60 72 84 96
0.00 0.92 1.00 0.88 0.81 0.63 0.39 0.13
0.14 1.00 1.00 0.93 0.79 0.46 0.30 0.00
0.00 0.61 0.75 0.88 0.74 0.93 1.00 0.72
9.35 1.35 31.05 2.03 32.91 1.65 30.09 2.08 28.55 1.79 24.22 2.02 18.60 1.40 12.52 1.15
24.09 1.99 55.90 2.29 55.85 3.65 53.17 2.90 48.12 3.22 36.06 2.77 30.22 2.19 18.97 1.99
6.56 0.29 14.95 0.87 16.95 1.10 18.75 1.39 16.79 1.27 19.46 1.69 20.40 1.09 16.53 0.50
7.91 1.14 15.42 0.98 18.09 0.72 18.24 1.06 19.17 1.56 21.75 1.35 22.49 2.20 22.84 1.53
7.89 15.62 17.95 18.42 18.86 21.94 22.31 22.94
Mean S.D.
0.60 0.38
0.58 0.40
0.70 0.31
23.41 1.68 8.94 0.35
40.30 2.62 14.88 0.61
16.30 1.02 4.31 0.46
18.24 1.32 4.89 0.46
18.24 4.88
a b c
x01 ; x02 and x03 are standardized values of ellagitannin acyl hydrolase, xylanase and cellulase activities, respectively. x1, x2 and x3 are experimental values corresponding to x01 ; x02 and x03 , respectively. y and y0 are experimental and theoretical values of ellagic acid yield, respectively.
W. Huang et al. / Process Biochemistry 42 (2007) 1291–1295
significantly correlated with ellagitannin acyl hydrolase, xylanase and cellulase activities. The research indicted that an appropriate enzyme system, resulting in high level of ellagic acid yield, is very important for bioconversion of ellagitannins to ellagic acid production from the forest byproduct.
[9]
[10]
Acknowledgements [11]
Financial support of this research was from the National Key Technologies R&D Program of China with grant no. 2001BA804A21. Sincere thanks are to Dr. Jinshui Yang at China Agricultural University for providing the microbial culture and technical assistance in the experimental studies.
[12]
[13]
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