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Utilization of acorn fringe for ellagic acid production by Aspergillus oryzae and Endomyces fibuliger
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Bioresource Technology 99 (2008) 3552–3558
Utilization of acorn fringe for ellagic acid production by Aspergillus oryzae and Endomyces fibuliger Wen Huang a
a,*
, Zhenshan Li b, Hai Niu c, Lulu Li a, Wensheng Lin a, Jinshui Yang
d
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, Chengdu 610041, China b Department of Environmental Engineering, Peking University, Beijing 100871, China c Mathematical College, Sichuan University, Chendu 610064, China d China Agricultural University, Beijing 100083, China Received 2 May 2007; received in revised form 26 July 2007; accepted 27 July 2007 Available online 10 September 2007
Abstract Conversion of acorn fringe extract into ellagic acid production by Aspergillus oryzae and Endomyces fibuliger were investigated. The results showed that ellagic acid production was maximized when co-fermentation of the two fungi was performed at 30 C and pH 5.0 with 5.7 g/l of initial substrate concentration, which were close to the optimal values for both fungi to yield an appropriate consortium of hydrolytic enzymes. Meanwhile, it was found that the co-fermentation could compensate the deficiencies in the level of polyphenol oxidase activity from pure A. oryzae and the levels of ellagitannin acyl hydrolase and b-glucosidase activities from pure E. fibuliger, resulting in. 0.91 g/l of biomass concentration containing 1.84 g/l of ellagic acid. The research not only demonstrates that the co-fermentation is an effective approach to utilize forest byproduct for ellagic acid production, but also provides more evidences for understanding evolution of ellagic acid production with enzymes actions, which is important for process control of ellagic acid production in industrial application. 2007 Elsevier Ltd. All rights reserved. Keywords: Ellagitannin acyl hydrolyase; Ellagic acid; Aspergillus oryzae; Endomyces fibuliger
1. Introduction Acorn fringe of valonia oak is a forestry byproduct containing up to at least 10% by weight of C-glucosidic ellagitannins (Zhentian et al., 1999). The predominant ellagitannins found in acorn fringe are casuarictin, pedunculagin, castalagin and vescalagin, which are polymers of ellagic acid or compounds related to it esterified to a core glucose (Grundhofera et al., 2001). Utilization of acorn fringe is commercially confined almost exclusively to extract ellagitannins as tanning agent, a low valued product about 500$ per ton, which was used for converting hides to leather. (Britta et al., 2002). In *
Corresponding author. Tel.: +86 028 85164074; fax: +86 028 85164073. E-mail address: [email protected] (W. Huang). 0960-8524/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2007.07.047
recent years, the use of the tanning agent in the leather industry is greatly limited due to its stronger astringency and darker color, and is also considered to produce higher COD in the wastewaters of leather industries. High content of ellagitannins, however, enhances the suitability for yielding ellagic acid production with many medically important bioactivities as antioxidant, antimicrobial, antiviral and antitumor properties (Makris et al., 2007; Maksimovic et al., 2005; Masamune et al., 2005), suggesting some alternative utilizations. So far, preparation of ellagic acid mainly uses acidic hydrolysis (Mullen et al., 2002b; Yean-Yean and Philip, 2006). Nevertheless, it suffered from the unspecific reactions yielding numerous structurally closely related products and byproducts. It is well known that biological methods have many advantages. It is technical (high specificity, mild reaction conditions) as well as economic (costs
W. Huang et al. / Bioresource Technology 99 (2008) 3552–3558
of industrial processes). However, very few information on ellgic acid production by biological method using ellagitannins of acorn fringe as substrate, which are wider molecular weights range and more complicated chemical structures than that from fruits and medicinal plant (Nicolas et al., 2004), is available. Based on previous research, Aspergillus oryzae, which could produce higher level of ellagic acid production with higher activities of ellagitannin acyl hydrolase and b-glucosidase (Wen et al., 2002a, 2007), and Endomyces fibulige, which could effectively degrade ellagitannins with a significant level of polyphenol oxidase (Wen et al., 2002b, 2005), were selected for further investigation. The objective of the work was to develop an effective approach to utilize acorn fringe of oak for ellagic acid production by fungi considering operational parameters, enzyme and degrading property of fungi. 2. Methods 2.1. Ellagitannins extraction Dried acorn fringe of valonia oak collected 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 and E. fibuliger were kindly provided by College of Biological Science, China Agricultural University, Beijing, China. They were named A. oryzae YH344 and E. fibuliger HY7688, respectively, after acclimation in sterilized ground dust of dried acorn fringe of oak. They were stored at 4 C on agar (1.5%) slants of MY medium (2% malt extract, 0.2% yeast extract), respectively (Cunrou and Yixiu, 1999). The subculture medium was Mandel salt solution supplemented with tween-80 of 2 ml, peptone of 1 g, and 10 g/l glucose (Mandel and Weber, 1969). Fungal cells were sub-cultured in a shaker (at 150 rpm, HZS-H super water bath shaker, China) at 30 C for subgenerations used for inoculums.
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acid yield, ellagitannins degradation rate and enzyme activity in hydrolysate were determined to evaluate the hydrolysis efficiency. The initial pH was controlled with 5 N aqueous NH4, and 5 N HCI. The medium was autoclaved at 121 C for 15 min. 2.4. Enzyme assay 2.4.1. Ellagitannin acyl hydrolase The enzyme activity was presented as total activities of extracellular and intracellular ellagitannin acyl hydrolases pooled. Two milliliters of culture broth was withdrawn at regular intervals of 12 h and analyzed for ellagitannins acyl hydrolase activity. The broth was ground with quartz sand in a mortar for 10 min. The entire operation was carried out after pre-cooling the suspension to 4 C, such that the temperature of the mixture does not exceed 10 C. The suspension was centrifuged at 15,000 rpm (F1010, AllegraTM X-22R Cenrifuge with max. rcf of 19,926g) for 25 min and the supernatant containing ellagitannin acyl hydrolase was recovered. The supernatant-the enzymatic extract was subsequently determined using castalagin as substrate, which can be hydrolyzed by the enzyme to form castalin and ellagic acid. Castalagin was prepared in citrate buffer (pH 5.0) and incubated with an appropriate amount of enzyme at 37 C for 30 min in a water bath. The reaction was stopped by boiling for 10 min to denaturalize the enzyme. All enzyme assays were performed in triplicate and with appropriate blanks to allow for correction for any background reactions. One unit (IU) of activity was defined as the amount of enzyme releasing 1 lmol of ellagic acid per min at pH 5.0 and 37 C.
2.3. Culture condition
2.4.2. b-glucosidase Two milliliters of the culture broth collected at regular interval of 12 h was filtered through cheesecloth and followed by centrifugation at 15,000 rpm for 10 min to remove debris. The suspension containing b-glucosidase was assayed colorimetrically by measuring the amount of p-nitrophenol released from p-nitrophenyl-b-D-glucoside (Sigma Chemical Co.) used as the substrate (Wood and Bhat, 1988). The assay was carried out in 3 ml of 0.1 M acetate buffer pH 4.8 at 50 C for 30 min. The reaction was stopped with 4 ml of 0.4 M glycine buffer pH 10.8. One unit of activity was the amount of enzyme that released l lmol of p-nitrophenol per min under the assay condition.
Medium composition in each flask was ellagitannins supplemented with K2HPO4 of 0.5 g, KH2PO4 of 0.5 g, MgSO4 Æ 7H2O of 0.2 g, tween-80 of 2 ml, glucose of 2 g and soytone of 2 g/l and distilled water. For pure culture, either A. oryzae or E. fibuliger of 2 ml of a spore suspension (2 · 106 spore/ml) was inoculated into each flask, respectively. For mixed culture, A. oryzae and E. fibuliger inoculums were inoculated into each flask simultaneously. The flasks were incubated in a shaker (at 120 rpm), and ellagic
2.4.3. Polyphenol oxidase Two milliliters of the culture broth collected at regular interval of 12 h was filtered through cheesecloth and followed by centrifugation at 15,000 rpm for 10 min to remove debris. The suspension containing polyphenol oxidase was subsequently analyzed by measuring the increase in absorbancy at 430 nm caused by the oxidation of 1 mM (+)-catechin (Sigma) at 45 C and pH 5.0. One unit of polyphenol oxidase activity was defined as the amount of
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enzyme that caused an increase of 0.1 in the absorbance per min under the assay condition. 2.5. Ellagitannins determination After incubation, the culture filtrates were analyzed for ellagitannins content. The analysis was based on the international standard method described in leather analysis and detection and the method of detection (Leather chemistry department of northwest institute of light industry, 1996; Wen et al, 2005). The degradation rate of total ellagitannins was computed as follows, Degradation rate of ellagitannins (%) = (initial ellagitannins weight – ellagitannins weight after fermentation)/initial ellagitannins weight. 2.6. Ellagic acid determination The HPLC quantitative determination for ellagic acid was analyzed 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. Firstly, sample adjusted to pH of 8.5 was filtered (0.2 lm), and injected then (100 ll) onto a SUPELCOSIL LC-18 reverse-phase column (15 cm · 4.6 mm, 5 lm). 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/min. Ellagic acid was quantified by reference to an ellagic acid calibration curve (these wavelengths correspond to the optimal absorption of ellagic acid (Ho et al., 1999). The yield of ellagic acid is computed as: ellagic acid yield (%) = ellagic acid weight after fermentation/initial ellagitannins weight. 2.7. Experimental design and data analysis A central composite design (CC0318) (Haaland, 1989) was used to obtain the optimal conditions (temperature, pH, and ellagitannins concentration) for ellagic acid production by the pure culture of A. oryzae using acorn fringe extract as substrate. The design matrix was a 23 factorial design combined with four central points and six axial points where one variable was set at an extreme level (±1.68) while the other variable was set at its central point (Table 1). The design involved 15 experimental data points. The central point (run 15) was repeated four times to obtain the standard deviation using S-Plus. Although the standard deviation of the central point could be used to
evaluate the experimental error of the design, consideration that the deviation may vary under different conditions required us to perform runs 1–14 in duplicates and the deviation of each individual run was estimated. The coding unit of variable i was done as follows: xi xcp ; i ¼ 1; 2; 3 x0 ¼ Dxi where xi is the coded level, xi the true value, xcp the true value at central point, and Dxi is the step change of variable i, respectively. The true values of the variables are also given in Table 1. Ellagic acid yield can be written as the function of the independent variables by second-polynomial, i.e., X X X y ¼ a0 þ aii xi þ aii x2i þ aij xi xj ð1Þ where y is the predicted response (ellagic acid yield), the coefficients of the equation, and xi and xj are the coded levels of variables i and j, respectively. After ellagic acid yield from each run was obtained, the response and variables (in coded unit) were correlated by the ‘‘Response Surface Analysis’’ function of the S-Plus software (Insightful Corp, 2006) to obtain the coefficients of Eq. (1). Only the estimates of coefficients with significant levels higher than 99% (i.e., p < 0.01) were included in the final model. The correlation coefficient (R2) and significance of the model were also tested by the S-Plus software. The significance of the model was evaluated by F-test. 3. Results and discussion 3.1. Ellagic acid yield, ellagitannins degradation rate and enzyme activity by A. oryzae and optimization of operation parameters for ellagic acid production As evident from Fig. 1, the maximum ellagic acid yield with 20% of ellagitannins degradation rate by A. oryzae
Table 1 Coded and real levels of the independent variables used in the design to estimate the ellagic acid yield by the pure culture A. oryzae Independent variables
Temperture (C) pH Ellagitannins concentration (g/l)
Factor level –1
0
+1
22 3.8 2
27 4.8 4
32 5.8 6
Fig. 1. Time course of ellagic acid yield, ellagitannins degradation rate and enzyme activity by A. oryzae. Symbols: (m) ellagic acid yield, (s) ellagitannins degradation rate, (j) ellagitannin acyl hydrolase activity and (h) b-glucosidase activity. Data are means of three replicates and error bars show standard deviation.
W. Huang et al. / Bioresource Technology 99 (2008) 3552–3558
were 14%. It may be noted that ellagic acid accumulation in the experiment appear to be closely related to activity of ellagitannin acyl hydrolase, which can cleave ester and depside bonds to free ellagic acid (Purohit et al., 2006; Battestin and Macedo, 2007; Murugan et al., 2007). Meanwhile, it could also be found that ellagic acid yield was varying in parallel with b-glucosidase activity. The result implies that b-glucosidase is also important for ellagic acid production. Optimal experiment for ellagic acid production was performed by central composite design. Ellagic acid yield collected from each of runs 1–14 in Table 2 was the means of two duplicates, while the central point was run in four duplicates (run 15) and its standard deviation was 1.82%. The results showed a high reproducibility of ellagic acid yield as the deviation of each run was less than 3%. The response and variable (in coded unit) in Table 2 were correlated as a second-order polynomial model (Eq. (1)). Table 3 listed the estimates of coefficients and the associated t-values and significant levels. In this work, the estimates with significant level higher than 99% (p < 0.01) were included in the final model (Eq. (1). Thus, the reduced model describing ellagic acid yield as a function of the significant variables (T and pH) was obtained as follows: Yield of ellagic acidð%Þ ¼ 49:55 þ 1:79½T þ 14:53½ pH þ 0:46½EC 2
2
0:03½T 1:41½pH 0:04½EC
2
ð2Þ
Table 2 Central composite design of temperature (T), medium pH, and ellagitannins concentration with ellagic acid yield as responsea Run
Variables
Response
Coded unit
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Mean Std a
T
pH
EC
True value T (C)
pH
ECc (g/l)
Ellagic acid yield (%)
Deviation (%)
1 1 1 1 +1 +1 +1 +1 1.68 +1.68 0 0 0 0 0
1 1 +1 +1 1 1 +1 +1 0 0 1.68 +1.68 0 0 0
1 +1 1 +1 1 +1 1 +1 0 0 0 0 1.68 +1.68 0
22 22 22 22 32 32 32 32 18.6 35.4 27 27 27 27 27
3.8 3.8 5.8 5.8 3.8 3.8 5.8 5.8 4.8 4.8 3.1 6.5 4.8 4.8 4.8
2 6 2 6 2 6 2 6 4 4 4 4 0.6 7.4 4
10.96 11.14 13.35 13.72 12.24 12.23 14.87 14.96 12.11 14.02 8.95 13.44 14.63 14.90 15.30
0.88 0.60 1.30 1.06 0.59 1.45 2.70 1.14 1.82 1.85 0.72 1.23 1.45 2.23 1.45
27 5
5 1
4 2
13.12 1.82
1.36 0.60
Run 1–14 were performed in two duplicate, while run 15 (central point) was performed in four duplicate. b T was incubation temperature c EC was ellagitannins concentration.
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Table 3 Estimates of coefficients of the variables in second-order polynomials and the associated statistical tests Coefficient
Variable
Estimate
t-Value
p-Level
b0 b1 b11 b2 b22 b3 b33 b12 b13 b23
Constant [T] [T]2 [pH] [pH]2 [EC] [EC]2 [T] · [pH] [T] · [EC] [pH] · [EC]
49.549 1.793 0.031 14.526 1.412 0.462 0.044 0.010 0.006 0.018
32.880 27.407 28.084 48.852 51.663 4.475 6.427 2.004 2.415 1.495
0.0000005 0.0000012 0.0000011 0.0000001 0.0000001 0.0065500 0.0013542 0.1013718 0.0604888 0.1951210
The predicted maximum ellagic acid yield from Eq. (2) was 15.91%, which was less than 2% in deviation from the experimental data (17.12%). The correlation coefficient (R2) of Eq. (2) was 0.99. F-test gave the equation a significance of 99% (p < 0.01). All of these indicated that the equation was reliable in reflecting effects of T, pH and ellagitannins concentration on ellagic acid production. Eq. (2) was then used to derive the optimal values of T, pH and ellagitannin concentration. Using S-Plus software, the exact optimal T, pH and ellagitannin concentration values for ellagic acid production were obtained as 29.83 C, 5.75 and 5.7 g/l, respectively. 3.2. Ellagic acid yield, ellagitannins degradation rate and enzyme activity by E. fibuliger Fig. 2 showed that under the optimal temperature and pH being held at 28 C and 5, the maximum ellagic acid yield with 41% ellagitannins degradation rate by E. fibuliger were 7.54% (Wen et al., 2002c). It may be noted in Fig. 2 that ellagitannins degradation rate increased in parallel with activity of polyphenol oxidase, whilst evolution of
Fig. 2. Time course of ellagic acid yield, ellagitannins degradation rate and enzyme activity by E. fibuliger. Symbols: (m) ellagic acid yield, (s) ellagitannins degradation rate, (j) ellagitannin acyl hydrolase activity, (h) b-glucosidase activity and (–) polyphenol oxidase activity. Data are means of three replicates and error bars show standard deviation.
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ellagic acid yield was following the fluctuation of ellagitannin acyl hydrolase activity. The higher degradation rate of ellagitannins by E. fibuliger than that by A. oryzae might be ascribed to the result from action of polyphenol oxidase (lack in pure culture of A. oryzae), which are generally described as importance for degradation of polyphenolic compound (Carrie et al., 2006; Okafor, 2005; Dura´n et al., 2002). The lower ellagic acid yield by E. fibuliger than that by A. oryzae suggests that ellagitannins acyl hydrolase was insufficient in the case of pure culture of E. fibuliger. The results indicate that although E. fibuliger appear a low ability on conversion of ellagitannins to ellagic acid production, however, it could effectively degrade ellagitannins, which is favorable for decreasing steric hindrance of action of ellagitannin acyl hydrolase to release ellagic acid unit. Ellagitannins degradation rate instead of ellagic acid yield, therefore, was selected as a function of culture condition of E. fibuliger in the latter research.
3.3. Ellagic acid production, ellagitannins degradation rate and enzyme activity by co-fermentation of E. fibuliger and A. oryzae The optimal culture conditions for ellagitannins degradation rate by E. fibuliger and for ellagic acid yield by A. oryzae are summarized in Table 4. For the two pure cultures, temperature and pH were very similar, except ellagitannins concentration, which was optimal at 5.7 g/l for ellagic acid production in the case of pure culture A. oryzae and can vary from 4 to 7 g/l without influencing ellagitannins degradation rate in the case of pure culture E. fibuliger. It means that 5.7 g/l concentration could be suitable for both ellagitannins degradation (by E. fibuliger) and ellagic acid accumulation (by A. oryzae). The response surfaces of ellagic acid yield (by A. oryzae) and ellagitannins degradation rate (by E. fibuliger) as functions of T and pH were given in Fig. 3a and b. The plots were hump shaped with a clear peak within the experimental range investigated. More importantly, it was found that both ellagitannins degradation rate and ellagic acid accumulation did not fall steeply when the valTable 4 Summary of optimal conditions for pure cultures of E. fibuliger and A. oryzae Parameters
Fungi culture A. oryzae
E. fibuliger
Objective
Ellagic acid yield (%) 15.91 29.83 5.15 5.75
Ellagitannins degradation rate (%) 48.79 29.92 5.32 2–7
This work
Wood and Bhat (1988)
Optimal value Temperature (C) Initial pH Ellagitannin concentration (g/l) Reference
Fig. 3. Three-dimension surface plot of ellagitannins degradation rate vs. temperature and medium pH of E. fibuliger (a); ellagic acid yield vs. temperature and medium pH of A. oryzae (b). ellagitannins concentration was 5.7 g/l for both of the two pure cultures.
ues of T and pH changed slightly from their best values (Fig. 3a and b). These are desired properties because it means that the ellagitannins degradation rate by E. fibuliger and ellagic acid yield by A. oryzae will remain robust even with slight fluctuations in T and pH. Such a property provides a possibility that when T and pH are set at suboptimal values, which are very close to the optimal values, the resulting ellagitannins degradation rate and ellagic acid yield will insignificantly decrease from their maximum level. In other words, ellagitannins degradation rate by E. fibuliger and ellagic acid production by A. oryzae could be simultaneously maintained at high level by appropriately controlling pH and T.
W. Huang et al. / Bioresource Technology 99 (2008) 3552–3558
Based on the above analysis, 30 C of incubation temperature and 5 of pH and 5.7 g/l of ellagitannins concentration were selected as operation parameters for ellagic acid production in co-fermentation. Fig. 4 showed that the co-fermentation of A. oryzae and E. fibuliger could result in 0.91 g/l of biomass concentration containing 1.83 g/l of ellagic acid. It may be noted in Fig. 5 that the maximum b-glucosidas activity by the cofermentation was slightly lower than that by pure culture A. oryzae (Fig. 3a), and the maximum polyphenol oxidase activity with 38% ellagitannins degradation by the co-fermentation was lower than that by the pure culture E. fibuliger (Fig. 3b). However, ellagic acid yield and ellagitannin acyl hydrolase activity, compared with that by each of pure culture, were significantly high. The increased ellagic acid yield by co-fermentation might be attributed to higher level of ellagitannin acyl hydrolase and appropriate levels of b-glucosidase and pol-
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yphenol oxidase. The results indicate that the hydrolysis efficiency (in term of ellagic acid production) could significantly increased by co-fermentation of A. oryzae and E. fibuliger. 4. Conclusion The present work showed that co-fermentation of E. fibuliger and A. oryzae could effectively increase the utilization of ellagitannins for ellagic acid production, compared with each of pure culture. At the same time, it was found that the deficiencies in levels of ellagitannin acyl hydrolase and b-glucosidase activities by pure culture E. fibuliger and polyphenol oxidase activity by pure culture A. oryzae can be compensated using the co-fermentation by controlling operational condition at 30 C, pH 5.5 with 5.7 g/l of initial ellagitannins concentration, resulting in 32% of ellagic acid yield. The research demonstrates a possible way to develop an efficient hydrolytic system for recovery of higher value product-ellagic acid- from forestry byproduct. Acknowledgements Financial support of this research was from the National Natural Science Foundation of China with Grant No. 40371011. Sincere thanks are to Dr. Jinshui Yang at China Agricultural University for providing the microbial culture and technical assistance in the experimental studies. References
Fig. 4. Fermentation profile of co-fermentation for ellagic acid production. Symbols: (m) ellagic acid, (s) ellagitannins, (h) cell dry wt. Data are means of three replicates and error bars show standard deviation.
Fig. 5. Time course of ellagic acid yield, ellagitannins degradation rate and enzyme activity by co-fermentation. Symbols: (m) ellagic acid yield, (s) ellagitannins degradation rate, (j) ellagitannin acyl hydrolase activity, (h) b-glucosidase activity and (–) polyphenol oxidase activity. Data are means of three replicates and error bars show standard deviation.
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Bioresource Technology 99 (2008) 3552–3558
Utilization of acorn fringe for ellagic acid production by Aspergillus oryzae and Endomyces fibuliger Wen Huang a
a,*
, Zhenshan Li b, Hai Niu c, Lulu Li a, Wensheng Lin a, Jinshui Yang
d
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, Chengdu 610041, China b Department of Environmental Engineering, Peking University, Beijing 100871, China c Mathematical College, Sichuan University, Chendu 610064, China d China Agricultural University, Beijing 100083, China Received 2 May 2007; received in revised form 26 July 2007; accepted 27 July 2007 Available online 10 September 2007
Abstract Conversion of acorn fringe extract into ellagic acid production by Aspergillus oryzae and Endomyces fibuliger were investigated. The results showed that ellagic acid production was maximized when co-fermentation of the two fungi was performed at 30 C and pH 5.0 with 5.7 g/l of initial substrate concentration, which were close to the optimal values for both fungi to yield an appropriate consortium of hydrolytic enzymes. Meanwhile, it was found that the co-fermentation could compensate the deficiencies in the level of polyphenol oxidase activity from pure A. oryzae and the levels of ellagitannin acyl hydrolase and b-glucosidase activities from pure E. fibuliger, resulting in. 0.91 g/l of biomass concentration containing 1.84 g/l of ellagic acid. The research not only demonstrates that the co-fermentation is an effective approach to utilize forest byproduct for ellagic acid production, but also provides more evidences for understanding evolution of ellagic acid production with enzymes actions, which is important for process control of ellagic acid production in industrial application. 2007 Elsevier Ltd. All rights reserved. Keywords: Ellagitannin acyl hydrolyase; Ellagic acid; Aspergillus oryzae; Endomyces fibuliger
1. Introduction Acorn fringe of valonia oak is a forestry byproduct containing up to at least 10% by weight of C-glucosidic ellagitannins (Zhentian et al., 1999). The predominant ellagitannins found in acorn fringe are casuarictin, pedunculagin, castalagin and vescalagin, which are polymers of ellagic acid or compounds related to it esterified to a core glucose (Grundhofera et al., 2001). Utilization of acorn fringe is commercially confined almost exclusively to extract ellagitannins as tanning agent, a low valued product about 500$ per ton, which was used for converting hides to leather. (Britta et al., 2002). In *
Corresponding author. Tel.: +86 028 85164074; fax: +86 028 85164073. E-mail address: [email protected] (W. Huang). 0960-8524/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2007.07.047
recent years, the use of the tanning agent in the leather industry is greatly limited due to its stronger astringency and darker color, and is also considered to produce higher COD in the wastewaters of leather industries. High content of ellagitannins, however, enhances the suitability for yielding ellagic acid production with many medically important bioactivities as antioxidant, antimicrobial, antiviral and antitumor properties (Makris et al., 2007; Maksimovic et al., 2005; Masamune et al., 2005), suggesting some alternative utilizations. So far, preparation of ellagic acid mainly uses acidic hydrolysis (Mullen et al., 2002b; Yean-Yean and Philip, 2006). Nevertheless, it suffered from the unspecific reactions yielding numerous structurally closely related products and byproducts. It is well known that biological methods have many advantages. It is technical (high specificity, mild reaction conditions) as well as economic (costs
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of industrial processes). However, very few information on ellgic acid production by biological method using ellagitannins of acorn fringe as substrate, which are wider molecular weights range and more complicated chemical structures than that from fruits and medicinal plant (Nicolas et al., 2004), is available. Based on previous research, Aspergillus oryzae, which could produce higher level of ellagic acid production with higher activities of ellagitannin acyl hydrolase and b-glucosidase (Wen et al., 2002a, 2007), and Endomyces fibulige, which could effectively degrade ellagitannins with a significant level of polyphenol oxidase (Wen et al., 2002b, 2005), were selected for further investigation. The objective of the work was to develop an effective approach to utilize acorn fringe of oak for ellagic acid production by fungi considering operational parameters, enzyme and degrading property of fungi. 2. Methods 2.1. Ellagitannins extraction Dried acorn fringe of valonia oak collected 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 and E. fibuliger were kindly provided by College of Biological Science, China Agricultural University, Beijing, China. They were named A. oryzae YH344 and E. fibuliger HY7688, respectively, after acclimation in sterilized ground dust of dried acorn fringe of oak. They were stored at 4 C on agar (1.5%) slants of MY medium (2% malt extract, 0.2% yeast extract), respectively (Cunrou and Yixiu, 1999). The subculture medium was Mandel salt solution supplemented with tween-80 of 2 ml, peptone of 1 g, and 10 g/l glucose (Mandel and Weber, 1969). Fungal cells were sub-cultured in a shaker (at 150 rpm, HZS-H super water bath shaker, China) at 30 C for subgenerations used for inoculums.
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acid yield, ellagitannins degradation rate and enzyme activity in hydrolysate were determined to evaluate the hydrolysis efficiency. The initial pH was controlled with 5 N aqueous NH4, and 5 N HCI. The medium was autoclaved at 121 C for 15 min. 2.4. Enzyme assay 2.4.1. Ellagitannin acyl hydrolase The enzyme activity was presented as total activities of extracellular and intracellular ellagitannin acyl hydrolases pooled. Two milliliters of culture broth was withdrawn at regular intervals of 12 h and analyzed for ellagitannins acyl hydrolase activity. The broth was ground with quartz sand in a mortar for 10 min. The entire operation was carried out after pre-cooling the suspension to 4 C, such that the temperature of the mixture does not exceed 10 C. The suspension was centrifuged at 15,000 rpm (F1010, AllegraTM X-22R Cenrifuge with max. rcf of 19,926g) for 25 min and the supernatant containing ellagitannin acyl hydrolase was recovered. The supernatant-the enzymatic extract was subsequently determined using castalagin as substrate, which can be hydrolyzed by the enzyme to form castalin and ellagic acid. Castalagin was prepared in citrate buffer (pH 5.0) and incubated with an appropriate amount of enzyme at 37 C for 30 min in a water bath. The reaction was stopped by boiling for 10 min to denaturalize the enzyme. All enzyme assays were performed in triplicate and with appropriate blanks to allow for correction for any background reactions. One unit (IU) of activity was defined as the amount of enzyme releasing 1 lmol of ellagic acid per min at pH 5.0 and 37 C.
2.3. Culture condition
2.4.2. b-glucosidase Two milliliters of the culture broth collected at regular interval of 12 h was filtered through cheesecloth and followed by centrifugation at 15,000 rpm for 10 min to remove debris. The suspension containing b-glucosidase was assayed colorimetrically by measuring the amount of p-nitrophenol released from p-nitrophenyl-b-D-glucoside (Sigma Chemical Co.) used as the substrate (Wood and Bhat, 1988). The assay was carried out in 3 ml of 0.1 M acetate buffer pH 4.8 at 50 C for 30 min. The reaction was stopped with 4 ml of 0.4 M glycine buffer pH 10.8. One unit of activity was the amount of enzyme that released l lmol of p-nitrophenol per min under the assay condition.
Medium composition in each flask was ellagitannins supplemented with K2HPO4 of 0.5 g, KH2PO4 of 0.5 g, MgSO4 Æ 7H2O of 0.2 g, tween-80 of 2 ml, glucose of 2 g and soytone of 2 g/l and distilled water. For pure culture, either A. oryzae or E. fibuliger of 2 ml of a spore suspension (2 · 106 spore/ml) was inoculated into each flask, respectively. For mixed culture, A. oryzae and E. fibuliger inoculums were inoculated into each flask simultaneously. The flasks were incubated in a shaker (at 120 rpm), and ellagic
2.4.3. Polyphenol oxidase Two milliliters of the culture broth collected at regular interval of 12 h was filtered through cheesecloth and followed by centrifugation at 15,000 rpm for 10 min to remove debris. The suspension containing polyphenol oxidase was subsequently analyzed by measuring the increase in absorbancy at 430 nm caused by the oxidation of 1 mM (+)-catechin (Sigma) at 45 C and pH 5.0. One unit of polyphenol oxidase activity was defined as the amount of
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enzyme that caused an increase of 0.1 in the absorbance per min under the assay condition. 2.5. Ellagitannins determination After incubation, the culture filtrates were analyzed for ellagitannins content. The analysis was based on the international standard method described in leather analysis and detection and the method of detection (Leather chemistry department of northwest institute of light industry, 1996; Wen et al, 2005). The degradation rate of total ellagitannins was computed as follows, Degradation rate of ellagitannins (%) = (initial ellagitannins weight – ellagitannins weight after fermentation)/initial ellagitannins weight. 2.6. Ellagic acid determination The HPLC quantitative determination for ellagic acid was analyzed 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. Firstly, sample adjusted to pH of 8.5 was filtered (0.2 lm), and injected then (100 ll) onto a SUPELCOSIL LC-18 reverse-phase column (15 cm · 4.6 mm, 5 lm). 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/min. Ellagic acid was quantified by reference to an ellagic acid calibration curve (these wavelengths correspond to the optimal absorption of ellagic acid (Ho et al., 1999). The yield of ellagic acid is computed as: ellagic acid yield (%) = ellagic acid weight after fermentation/initial ellagitannins weight. 2.7. Experimental design and data analysis A central composite design (CC0318) (Haaland, 1989) was used to obtain the optimal conditions (temperature, pH, and ellagitannins concentration) for ellagic acid production by the pure culture of A. oryzae using acorn fringe extract as substrate. The design matrix was a 23 factorial design combined with four central points and six axial points where one variable was set at an extreme level (±1.68) while the other variable was set at its central point (Table 1). The design involved 15 experimental data points. The central point (run 15) was repeated four times to obtain the standard deviation using S-Plus. Although the standard deviation of the central point could be used to
evaluate the experimental error of the design, consideration that the deviation may vary under different conditions required us to perform runs 1–14 in duplicates and the deviation of each individual run was estimated. The coding unit of variable i was done as follows: xi xcp ; i ¼ 1; 2; 3 x0 ¼ Dxi where xi is the coded level, xi the true value, xcp the true value at central point, and Dxi is the step change of variable i, respectively. The true values of the variables are also given in Table 1. Ellagic acid yield can be written as the function of the independent variables by second-polynomial, i.e., X X X y ¼ a0 þ aii xi þ aii x2i þ aij xi xj ð1Þ where y is the predicted response (ellagic acid yield), the coefficients of the equation, and xi and xj are the coded levels of variables i and j, respectively. After ellagic acid yield from each run was obtained, the response and variables (in coded unit) were correlated by the ‘‘Response Surface Analysis’’ function of the S-Plus software (Insightful Corp, 2006) to obtain the coefficients of Eq. (1). Only the estimates of coefficients with significant levels higher than 99% (i.e., p < 0.01) were included in the final model. The correlation coefficient (R2) and significance of the model were also tested by the S-Plus software. The significance of the model was evaluated by F-test. 3. Results and discussion 3.1. Ellagic acid yield, ellagitannins degradation rate and enzyme activity by A. oryzae and optimization of operation parameters for ellagic acid production As evident from Fig. 1, the maximum ellagic acid yield with 20% of ellagitannins degradation rate by A. oryzae
Table 1 Coded and real levels of the independent variables used in the design to estimate the ellagic acid yield by the pure culture A. oryzae Independent variables
Temperture (C) pH Ellagitannins concentration (g/l)
Factor level –1
0
+1
22 3.8 2
27 4.8 4
32 5.8 6
Fig. 1. Time course of ellagic acid yield, ellagitannins degradation rate and enzyme activity by A. oryzae. Symbols: (m) ellagic acid yield, (s) ellagitannins degradation rate, (j) ellagitannin acyl hydrolase activity and (h) b-glucosidase activity. Data are means of three replicates and error bars show standard deviation.
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were 14%. It may be noted that ellagic acid accumulation in the experiment appear to be closely related to activity of ellagitannin acyl hydrolase, which can cleave ester and depside bonds to free ellagic acid (Purohit et al., 2006; Battestin and Macedo, 2007; Murugan et al., 2007). Meanwhile, it could also be found that ellagic acid yield was varying in parallel with b-glucosidase activity. The result implies that b-glucosidase is also important for ellagic acid production. Optimal experiment for ellagic acid production was performed by central composite design. Ellagic acid yield collected from each of runs 1–14 in Table 2 was the means of two duplicates, while the central point was run in four duplicates (run 15) and its standard deviation was 1.82%. The results showed a high reproducibility of ellagic acid yield as the deviation of each run was less than 3%. The response and variable (in coded unit) in Table 2 were correlated as a second-order polynomial model (Eq. (1)). Table 3 listed the estimates of coefficients and the associated t-values and significant levels. In this work, the estimates with significant level higher than 99% (p < 0.01) were included in the final model (Eq. (1). Thus, the reduced model describing ellagic acid yield as a function of the significant variables (T and pH) was obtained as follows: Yield of ellagic acidð%Þ ¼ 49:55 þ 1:79½T þ 14:53½ pH þ 0:46½EC 2
2
0:03½T 1:41½pH 0:04½EC
2
ð2Þ
Table 2 Central composite design of temperature (T), medium pH, and ellagitannins concentration with ellagic acid yield as responsea Run
Variables
Response
Coded unit
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Mean Std a
T
pH
EC
True value T (C)
pH
ECc (g/l)
Ellagic acid yield (%)
Deviation (%)
1 1 1 1 +1 +1 +1 +1 1.68 +1.68 0 0 0 0 0
1 1 +1 +1 1 1 +1 +1 0 0 1.68 +1.68 0 0 0
1 +1 1 +1 1 +1 1 +1 0 0 0 0 1.68 +1.68 0
22 22 22 22 32 32 32 32 18.6 35.4 27 27 27 27 27
3.8 3.8 5.8 5.8 3.8 3.8 5.8 5.8 4.8 4.8 3.1 6.5 4.8 4.8 4.8
2 6 2 6 2 6 2 6 4 4 4 4 0.6 7.4 4
10.96 11.14 13.35 13.72 12.24 12.23 14.87 14.96 12.11 14.02 8.95 13.44 14.63 14.90 15.30
0.88 0.60 1.30 1.06 0.59 1.45 2.70 1.14 1.82 1.85 0.72 1.23 1.45 2.23 1.45
27 5
5 1
4 2
13.12 1.82
1.36 0.60
Run 1–14 were performed in two duplicate, while run 15 (central point) was performed in four duplicate. b T was incubation temperature c EC was ellagitannins concentration.
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Table 3 Estimates of coefficients of the variables in second-order polynomials and the associated statistical tests Coefficient
Variable
Estimate
t-Value
p-Level
b0 b1 b11 b2 b22 b3 b33 b12 b13 b23
Constant [T] [T]2 [pH] [pH]2 [EC] [EC]2 [T] · [pH] [T] · [EC] [pH] · [EC]
49.549 1.793 0.031 14.526 1.412 0.462 0.044 0.010 0.006 0.018
32.880 27.407 28.084 48.852 51.663 4.475 6.427 2.004 2.415 1.495
0.0000005 0.0000012 0.0000011 0.0000001 0.0000001 0.0065500 0.0013542 0.1013718 0.0604888 0.1951210
The predicted maximum ellagic acid yield from Eq. (2) was 15.91%, which was less than 2% in deviation from the experimental data (17.12%). The correlation coefficient (R2) of Eq. (2) was 0.99. F-test gave the equation a significance of 99% (p < 0.01). All of these indicated that the equation was reliable in reflecting effects of T, pH and ellagitannins concentration on ellagic acid production. Eq. (2) was then used to derive the optimal values of T, pH and ellagitannin concentration. Using S-Plus software, the exact optimal T, pH and ellagitannin concentration values for ellagic acid production were obtained as 29.83 C, 5.75 and 5.7 g/l, respectively. 3.2. Ellagic acid yield, ellagitannins degradation rate and enzyme activity by E. fibuliger Fig. 2 showed that under the optimal temperature and pH being held at 28 C and 5, the maximum ellagic acid yield with 41% ellagitannins degradation rate by E. fibuliger were 7.54% (Wen et al., 2002c). It may be noted in Fig. 2 that ellagitannins degradation rate increased in parallel with activity of polyphenol oxidase, whilst evolution of
Fig. 2. Time course of ellagic acid yield, ellagitannins degradation rate and enzyme activity by E. fibuliger. Symbols: (m) ellagic acid yield, (s) ellagitannins degradation rate, (j) ellagitannin acyl hydrolase activity, (h) b-glucosidase activity and (–) polyphenol oxidase activity. Data are means of three replicates and error bars show standard deviation.
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ellagic acid yield was following the fluctuation of ellagitannin acyl hydrolase activity. The higher degradation rate of ellagitannins by E. fibuliger than that by A. oryzae might be ascribed to the result from action of polyphenol oxidase (lack in pure culture of A. oryzae), which are generally described as importance for degradation of polyphenolic compound (Carrie et al., 2006; Okafor, 2005; Dura´n et al., 2002). The lower ellagic acid yield by E. fibuliger than that by A. oryzae suggests that ellagitannins acyl hydrolase was insufficient in the case of pure culture of E. fibuliger. The results indicate that although E. fibuliger appear a low ability on conversion of ellagitannins to ellagic acid production, however, it could effectively degrade ellagitannins, which is favorable for decreasing steric hindrance of action of ellagitannin acyl hydrolase to release ellagic acid unit. Ellagitannins degradation rate instead of ellagic acid yield, therefore, was selected as a function of culture condition of E. fibuliger in the latter research.
3.3. Ellagic acid production, ellagitannins degradation rate and enzyme activity by co-fermentation of E. fibuliger and A. oryzae The optimal culture conditions for ellagitannins degradation rate by E. fibuliger and for ellagic acid yield by A. oryzae are summarized in Table 4. For the two pure cultures, temperature and pH were very similar, except ellagitannins concentration, which was optimal at 5.7 g/l for ellagic acid production in the case of pure culture A. oryzae and can vary from 4 to 7 g/l without influencing ellagitannins degradation rate in the case of pure culture E. fibuliger. It means that 5.7 g/l concentration could be suitable for both ellagitannins degradation (by E. fibuliger) and ellagic acid accumulation (by A. oryzae). The response surfaces of ellagic acid yield (by A. oryzae) and ellagitannins degradation rate (by E. fibuliger) as functions of T and pH were given in Fig. 3a and b. The plots were hump shaped with a clear peak within the experimental range investigated. More importantly, it was found that both ellagitannins degradation rate and ellagic acid accumulation did not fall steeply when the valTable 4 Summary of optimal conditions for pure cultures of E. fibuliger and A. oryzae Parameters
Fungi culture A. oryzae
E. fibuliger
Objective
Ellagic acid yield (%) 15.91 29.83 5.15 5.75
Ellagitannins degradation rate (%) 48.79 29.92 5.32 2–7
This work
Wood and Bhat (1988)
Optimal value Temperature (C) Initial pH Ellagitannin concentration (g/l) Reference
Fig. 3. Three-dimension surface plot of ellagitannins degradation rate vs. temperature and medium pH of E. fibuliger (a); ellagic acid yield vs. temperature and medium pH of A. oryzae (b). ellagitannins concentration was 5.7 g/l for both of the two pure cultures.
ues of T and pH changed slightly from their best values (Fig. 3a and b). These are desired properties because it means that the ellagitannins degradation rate by E. fibuliger and ellagic acid yield by A. oryzae will remain robust even with slight fluctuations in T and pH. Such a property provides a possibility that when T and pH are set at suboptimal values, which are very close to the optimal values, the resulting ellagitannins degradation rate and ellagic acid yield will insignificantly decrease from their maximum level. In other words, ellagitannins degradation rate by E. fibuliger and ellagic acid production by A. oryzae could be simultaneously maintained at high level by appropriately controlling pH and T.
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Based on the above analysis, 30 C of incubation temperature and 5 of pH and 5.7 g/l of ellagitannins concentration were selected as operation parameters for ellagic acid production in co-fermentation. Fig. 4 showed that the co-fermentation of A. oryzae and E. fibuliger could result in 0.91 g/l of biomass concentration containing 1.83 g/l of ellagic acid. It may be noted in Fig. 5 that the maximum b-glucosidas activity by the cofermentation was slightly lower than that by pure culture A. oryzae (Fig. 3a), and the maximum polyphenol oxidase activity with 38% ellagitannins degradation by the co-fermentation was lower than that by the pure culture E. fibuliger (Fig. 3b). However, ellagic acid yield and ellagitannin acyl hydrolase activity, compared with that by each of pure culture, were significantly high. The increased ellagic acid yield by co-fermentation might be attributed to higher level of ellagitannin acyl hydrolase and appropriate levels of b-glucosidase and pol-
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yphenol oxidase. The results indicate that the hydrolysis efficiency (in term of ellagic acid production) could significantly increased by co-fermentation of A. oryzae and E. fibuliger. 4. Conclusion The present work showed that co-fermentation of E. fibuliger and A. oryzae could effectively increase the utilization of ellagitannins for ellagic acid production, compared with each of pure culture. At the same time, it was found that the deficiencies in levels of ellagitannin acyl hydrolase and b-glucosidase activities by pure culture E. fibuliger and polyphenol oxidase activity by pure culture A. oryzae can be compensated using the co-fermentation by controlling operational condition at 30 C, pH 5.5 with 5.7 g/l of initial ellagitannins concentration, resulting in 32% of ellagic acid yield. The research demonstrates a possible way to develop an efficient hydrolytic system for recovery of higher value product-ellagic acid- from forestry byproduct. Acknowledgements Financial support of this research was from the National Natural Science Foundation of China with Grant No. 40371011. Sincere thanks are to Dr. Jinshui Yang at China Agricultural University for providing the microbial culture and technical assistance in the experimental studies. References
Fig. 4. Fermentation profile of co-fermentation for ellagic acid production. Symbols: (m) ellagic acid, (s) ellagitannins, (h) cell dry wt. Data are means of three replicates and error bars show standard deviation.
Fig. 5. Time course of ellagic acid yield, ellagitannins degradation rate and enzyme activity by co-fermentation. Symbols: (m) ellagic acid yield, (s) ellagitannins degradation rate, (j) ellagitannin acyl hydrolase activity, (h) b-glucosidase activity and (–) polyphenol oxidase activity. Data are means of three replicates and error bars show standard deviation.
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