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food and bioproducts processing x x x ( 2 0 1 3 ) xxx–xxx

Contents lists available at ScienceDirect

Food and Bioproducts Processing

j ourna l ho me page: www.elsev ier .com/ locate / fbp

otential use of different agroindustrial by-products asupports for fungal ellagitannase production underolid-state fermentation

uan Buenrostro-Figueroaa, Alberto Ascacio-Valdésa, Leonardo Sepúlvedaa,eynaldo De la Cruza, Arely Prado-Barragánb, Miguel A. Aguilar-Gonzálezc,aúl Rodrígueza, Cristóbal N. Aguilara,∗

Food Research Department, School of Chemistry, Universidad Autónoma de Coahuila, Saltillo, 25280, Coahuila, MexicoDepartment of Biotechnology, Universidad Autónoma Metropolitana, Iztapalapa, 09340, MexicoCINVESTAV-IPN, Ramos Arizpe, 25900, Coahuila, Mexico

a b s t r a c t

Ellagitannase is a novel enzyme responsible for biodegradation of ellagitannins and ellagic acid production. Ellagic

acid is a bioactive compound with great potential in food, pharmaceutical and cosmetic industries. This work

describes the ellagitannase enzyme production from partial purified ellagitannins as inducers by Aspergillus niger

GH1 grown on solid-state fermentation. Solid-state fermentation was carried out on four different lignocellulosic

materials (sugarcane bagasse, corn cobs, coconut husks and candelilla stalks) as matrix support and production of

ellagitannase enzyme was evaluated. All lignocellulosic materials were characterized in terms of water absorption

index and critical humidity point. The best lignocellulosic materials for ellagitannase production were sugarcane

bagasse and corn cobs (1400 U L−1 and 1200 U L−1, respectively). The lowest values were obtained with candelilla

stalks (500 UL-1). The highest specific productivity was obtained with corn cobs (2.5 U mg−1 h−1) which enable increase

ellagitannase productivity up to 140 times. Corn cobs have great potential as support matrix for production of fungal

ellagitannase in SSF.

© 2013 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

Keywords: Ellagitannins; Ellagic acid; Biodegradation; Agro industrial by-products; Aspergillus niger GH1; Corn cobs

(Ascacio-Valdés et al., 2010; Robledo et al., 2008; Seeram et al.,

. Introduction

lagitannins are an important group of phytochemicalompounds with great value in food, pharmaceutical andosmetic industries. The ellagitannins (ETs) are water-oluble hydrolysable polyphenolic compounds (Wilson andagerman, 1990). These are considered secondary plantetabolites found in cytoplasm and cell vacuoles (Khadem

nd Marles, 2010). ETs presence has been principally describedn leaves, stalks, husks of some fruits, flowers, etc. (Ascacio-aldés et al., 2011). When ET’s are exposed to acidic or basictrong conditions, the ester bounds are hydrolyzed and theexahydroxydyphenic acid group (HHDP) is released, which

Please cite this article in press as: Buenrostro-Figueroa, J., et al., Potentiaellagitannase production under solid-state fermentation. Food Bioprod Pr

pontaneously rearranged to form a stable and insoluble

∗ Corresponding author. Tel.: +52 844 416 1238; fax: +53 844 415 9534.E-mail address: cristobal.aguilar@uadec.edu.mx (C.N. Aguilar).Received 3 January 2013; Received in revised form 8 May 2013; Accept

960-3085/$ – see front matter © 2013 The Institution of Chemical Engittp://dx.doi.org/10.1016/j.fbp.2013.08.010

dilactone, commonly named ellagic acid (Aguilera-Carbóet al., 2008b; Gross, 2009)

Recent studies on sources and biological properties ofellagic acid showed its relevant bioactivity, such as antiox-idant, anti-inflammatory, antiviral, antimicrobial, antimu-tagenic, antitumoral and anticarcinogenic, among others(Ascacio-Valdés et al., 2011). Ellagic acid is present in con-siderable amounts in cranberry (Vattem and Shetty, 2003),raspberry (Koponen et al., 2007) and pomegranate fruits(Aguilera-Carbó et al., 2008a; Robledo et al., 2008; Seeram et al.,2005). Several authors have reported the recovery of high lev-els of ETs with a high purity degree from pomegranate husks

l use of different agroindustrial by-products as supports for fungalocess (2013), http://dx.doi.org/10.1016/j.fbp.2013.08.010

ed 18 August 2013

2005).

neers. Published by Elsevier B.V. All rights reserved.

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The studies on ETs hydrolysis have permitted establishedsome control parameters of ellagic acid production usingstrong acids or bases, however this method generate highcosts and high volumes of chemical wastes. Information aboutETs biodegradation is scarce and confuse. However, in recentyears some enzymatic studies have offered some clues toelucidate the degradation pathway of ETs. Action of someenzymes, such as �-glucosidase (Vattem and Shetty, 2002,2003), tannase, polyphenol oxidase (Shi et al., 2005), cellulase(Huang et al., 2007), valonea tannin hydrolase (Huang et al.,2005) and ellagitannase (Aguilera-Carbó et al., 2009) has beenreported.

Microbial hydrolysis of ETs using enzymes has been poorlyevaluated in SSF, and the first reports hypothesized a syn-ergistic action of different enzymes (Aguilera-Carbó et al.,2008b). Recently, Ascacio-Valdés et al. (2013) reported thatAspergillus niger GH1 was able to grown in solid state fermen-tation and produce the ellagitannase enzyme. The authorspropose that the ester bonds among HHDP group and glyco-sides are degraded by ellagitannase which has high specificity,and this enzymatic activity allows the EA accumulation.

Solid-state fermentation (SSF) consists of microbial growthand product formation on surface and inside of a porous solidmatrix, in absence or near absence of free water (Barrios-González, 2012). Substrate must contain enough moisture toallow microbial growth and metabolism, simulating naturalgrowth conditions (Orzúa et al., 2009). Water availability inSSF is a critic limiting point which has an important influ-ence on microbial growth and metabolism. This availability ofwater corresponds to water activity (aW), a physico-chemicalparameter defined as the relative humidity of the gaseousatmosphere in equilibrium with the substrate. Chemical com-position and particle structure of the supports used in SSFhave a determinant influence in the value of aW whichcan range from 0.80 to 0.99 to permit an efficient fungalmetabolism (Martins et al., 2011). A great variety of ligno-cellulosic materials (LM) have been tested as solid supportsfor SSF, including coffee by-products (Machado et al., 2012),rice bran and wheat bran (Khandeparkar and Bhosle, 2006);sugarcane bagasse and agave (Hernández-Salas et al., 2009;Pandey et al., 2000); mango peels (Buenrostro-Figueroa et al.,2010), grape skins (Botella et al., 2007; Rodríguez et al., 2010),cranberry pomace (Vattem and Shetty, 2003), pomegranatepeels (Robledo et al., 2008); corn cobs (Mussatto et al., 2009b)and coconut husks (Orzúa et al., 2009) among others. Sev-eral of these by-products have been used as supports and/orsubstrates for production of metabolites of industrial impor-tance, such as organic acids, antibiotics, pigments, flavor andaroma compounds, bioactive molecules and a great variety ofenzymes (Martins et al., 2011). The aim of this study was pro-duce ellagitannase enzyme by growing Aspergillus niger GH1 onSSF using different agroindustrial by products as supports andpartially purified ellagitannins extracted from pomegranatepeels as carbon source.

2. Materials and methods

2.1. Materials preparation and physico-chemicalcharacterization

The agro industrial by-products used in this study werecollected from different Mexican agricultural regions and

Please cite this article in press as: Buenrostro-Figueroa, J., et al., Potentiaellagitannase production under solid-state fermentation. Food Bioprod Pr

included: sugarcane (Saccharis officinalis) bagasse (SB), corn(Zea mays) cobs (CC), coconut (Cocos nucifera) husk (CH) and

candelilla (Euphorbia antisyphilitica) stalks (CS). All of themwere grinded up to particle size of 0.85 mm of diameter. Beforeuse, the matrix supports were pre-treated by boiling during10 min and washed three times with distilled water. Supportwas dried at 60 ◦C until constant weight is reached (Mussattoet al., 2009b). Physical and chemical tests consisted of waterabsorption index (WAI) (Orzúa et al., 2009), critical humiditypoint (CHP) (Mussatto et al., 2009a) and packing density (PD)determination (Santomaso et al., 2003). For WAI determina-tion, the sample (1.5 g) was placed in 50 mL centrifuge tubeand 15 mL of distilled water was added. The sample wasstirred for 1 min at room temperature (25 ◦C) and centrifugedat 3000 g for 10 min. The supernatant was discarded, and theWAI was calculated from the weight of the remaining gel andexpressed as g gel/g dry weight. The CHP was estimated byadding 1 g of sample in a thermo-balance at 120 ◦C for 60 min.PD was calculated by placing 10 g of sample in standard gradu-ated cylinders and clamped to a shaker and vertically agitateduntil no change in volume during 5 min was observed.

To be used as matrix support, the materials were pre-treated by boiling during 10 min, washed three times withdistilled water, and subsequently dried at 60 ◦C for 24–48 h(Mussatto et al., 2009a). Prior to use, all LM were autoclavedat 121 ◦C for 15 min.

2.2. Fungal strain and cell culture

Aspergillus niger GH1 strain (Food Research Department Collec-tion, Universidad Autonoma de Coahuila, Mexico) was used.The strain has been previously isolated, characterized andidentified (Cruz-Hernández et al., 2005), highlighting their abil-ity to degrade ellagitannins (Robledo et al., 2008; Sepúlvedaet al., 2012). The strain was maintained at −40 ◦C in glycerol-skimmed milk. Spores of A. niger GH1 were activated in potatodextrose agar (PDA-Bioxon) medium at 30 ◦C for five days. Theculture spores were harvested with sterile solution of 0.01%Tween-80 and counted in a Neubauer® chamber.

2.3. SSF conditions

Ellagitannase production experiments were performed in60 mL sterile columns (100% polypropylene) considered asbioreactor, which were aseptically packed an homogeneousmixture containing the following fermentable mass: 3 g ofeach support (SB, CC, CH and CS) was mixed with 7 mLof Pontecorvo culture medium (Aguilera-Carbó et al., 2009)with the following composition (g L−1): NaNO3 (6.0), KH2PO4

(1.52), KCl (0.52), MgSO4·7H2O (0.52), ZnSO4 (0.001), FeCl3(0.85) and trace metals (1 mL L−1). The trace metals solutioncontained (mg L−1) Na2B4O7·10H2O (10.0), MnCl2·4H2O (50.0),Na2MoO4·2H2O (50.0) and CuSO4·5H2O (250.0). Pomegranatehusk ellagitannins (PHE) supplied by the Bioprocess Labora-tory of the Food Research Department (School of Chemistry,Universidad Autonoma de Coahuila, Mexico) were used ascarbon source and ellagitannase inducer. The medium pHwas adjusted to 9 and then autoclaved (1.1 kg/m3, 121 ◦C)for 15 min. PHE (30 g L−1) were added to the culture brothwhen the temperature was between 35–40 ◦C. Final pH was6.5. The fermentable mass was aseptically inoculated with2 × 107 spores/g of support. The SSF was carried out at 30 ◦Cfor 32 h (Ascacio-Valdés et al., 2013). Forced air was not supple-mented for aeration of column bioreactor. Enzymatic extract

l use of different agroindustrial by-products as supports for fungalocess (2013), http://dx.doi.org/10.1016/j.fbp.2013.08.010

(EE) was obtained by adding 7 mL of 50 mM citrates buffer pH5 to each reactor. Fermented material was compressed and

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Table 1 – Water absorption index (WAI), critical humiditypoint (CHP) and packing density (PD) of differentagro-industrial wastes*.

Support WAI (g/g dw) CHP (%) PD (g/cm3)

Coconut husk (CH) 12.09a 16b 0.82b

Corn cobs (CC) 2.97cd 27c 0.74a

Sugarcane bagasse (SB) 9.46ab 12a 0.83b

Candelilla stalks (CS) 3.14c 29.5cd 0.86c

*There are no significant differences among the same letters.

ltered through 0.45 filter (MilliporeTM). Ellagitannase activ-ty, soluble protein and biomass were determined on the EE.olumetric enzyme productivity (g.L−1 h−1) was calculated ashe ratio among the higher ellagitannase activity value andermentation time (EAHP = EAH/t).

.4. Analytical procedures

rotein content was analyzed using bovine serum albuminolution at 100 ppm (10 mg in 100 mL of 50 mM citrate buffer pH). For assay 100 �L of sample was added with 1000 �L of Brad-ord reagent. The samples were shaken and allowed to rest five

inutes. Absorbance was recorded at 595 nm (Bradford, 1976).Fungal biomass was assayed following glucosamine

ethod reported by Blix (1948). Samples were hydrolyzed inrder to release the glucosamine from cell wall, the pyrroleompound formed when combined with acetylacetone reactsith p-dimethylaminobenzaldehyde forming a red compoundetected at 530 nm. A calibration curve (0–200 mg.mL−1 oflucosamine) was carried out at the same experimental con-itions than the samples. Glucosamine associated with fungalrowth was determined to obtain the biomass content (mg g−1

f sample).Ellagitannase activity was assayed according to (Ascacio-

aldés et al., 2013). Pomegranate husk ellagitannins [PEH1 mg.mL−1) in 50 mM citrate buffer pH 5] were used as enzymeubstrate. PEH contain punicalagin and punicalin, both ofhem are ellagitannins molecules that releases ellagic acidhen are subjected to hydrolysis. An enzyme treatment

ontrol (1000 �L ECG + 50 �L 50 mM citrate buffer pH 5), anxtract treatment control (1000 �L of 50 mM citrate bufferH 5 + 50 �L of enzymatic extract) and the reaction mixture

1000 �L PEH + 50 �L to enzymatic extract) were prepared. Allnzymatic preparations were allowed to react for 10 min at 60◦

in a water bath (Sheldon manufacturing m. 1225). The reac-ion was terminated by adding 1050 �L of absolute ethanol.hen samples were sonicated for 25 min, filtered through.45 �m membrane units (Millex®) and collected in vials.llagic acid quantification was carried out by HPLC (High Per-ormance Liquid Chromatography) equipment (Varian ProStarystem) with a Diode Array Detector (PDA ProStar) to 254 nm,ccording to Ascacio-Valdés et al. (2010), under the followingperation conditions: 5 �m Optisil ODS column (250 × 4.6 mm),ow rate of 1 mL min−1, sample volume of 10 �L, 30 ◦C in col-mn for 40 min, with acetonitrile and 3% acetic acid as mobilehase. Ellagic acid (0–500 ppm) stock solution was prepared foralibration curve.

One ellagitannase enzymatic unit was defined as thenzyme amount needed to release 1 �mol of ellagic acid perin under the above conditions.

.5. Scanning electron microscopy

isualization of spore and fungal growth was done using ahillips XL30-ESEM (Environmental Scanning Electron Micro-cope). Samples dehydrated were coated with electrolytic gold99.99% purity) in a vacuum evaporator Jeol Model JEE-400. Allamples were analyzed under vacuum with a GSE (Gaseousecondary Electron) detector.

.6. Experimental design and data analysis

Please cite this article in press as: Buenrostro-Figueroa, J., et al., Potentiaellagitannase production under solid-state fermentation. Food Bioprod Pr

he effect of support on ellagitannase production was evalu-ted under a completely randomized design. The treatments

were sugarcane bagasse (SB), coconut husk (CH), corn cobs (CC)and candelilla stalks (CS). Biomass production, protein solu-ble, ellagitannase, volumetric and specific productivities weredeterminated. All treatments were realized by three replica-tions. Data were analyzed by ANOVA using SAS 9.0, whenneeded mean treatments were compared using Tukey’s multi-ple range procedure. A p-value of less than 0.05 was regardedas significantly different.

3. Results and discussion

3.1. Physico-chemical characterization

The SSF process requires the use of low water content mate-rials to facilitate fungal growth and development, due to thetype of support plays an important role in yielding the highergrowth rates of microorganisms (Manpreet et al., 2005). WAIand CHP are parameters highly relevant when evaluatingthe potential of different materials used as support in SSF(Mussatto et al., 2009b; Orzúa et al., 2009). WAI indicates sam-ple capacity to absorb water, depending on the availability ofhydrophilic groups which bind water molecules, and on the gelforming capacity of macromolecules (Mussatto et al., 2009a).The highest WAI value was found in CH, which was four timeshigher than those obtained for CC and CS. SB was three timeshigher than CC and CS. No significant differences (p ≤ 0.05)were observed between CH and SB values (Table 1). Accordingto (Robledo et al., 2008), materials with high WAI are preferredsince facilitate microorganism growth and development. ForCC, WAI are similar to those reported by Orzúa et al. (2009).For CH, WAI are similar to those reported by Mussatto et al.(2009b).

CHP represents the amount of water linked to the sup-port, which cannot be used by the microorganism for theirmetabolic functions. The materials must have low CHP to facil-itate microorganism culture. High values of CHP can affectmicroorganism growth because a high proportion of wateris bonded to the support material, and consequently, micro-bial species development will be affected, due to free wateris not much (Martins et al., 2011). Moo-Young et al. (1983)recommended a maximum limit of CHP at 40% for A. nigerstrains in SSF, due to the need for modification of the mois-ture content in relation to the absorbed media. Table 1 showsthe CHP values obtained in the present work for each agro-industrial by-product assay. All the supports tested have CHPvalues under 40%, limit recognized for the growth of A. nigerin SSF (Moo-Young et al., 1983), however those obtained in CHand SB are up two times lower than those obtained in CC andCS. CHP are lower than those previouslly reported for CC andCH (Mussatto et al., 2009b; Orzúa et al., 2009). The high WAI

l use of different agroindustrial by-products as supports for fungalocess (2013), http://dx.doi.org/10.1016/j.fbp.2013.08.010

and low CHP values found in CH and SB make it an excellent

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Fig. 1 – Kinetics of: (a) biomass production, (b) ellagitannase activity, (c) volumetric productivity and (d) specific productivityobtained by SSF with A. niger GH1 using as supports coconut husk (CH), candelilla stalks (CS), sugarcane bagasse (SB) andcorn cobs (CC).

materials to be used as a support on SSF, compared with CCand CS.

Packing density (PD) is another important parameter toevaluate for the SSF processes, due to provides the materialcompaction degree, therefore, the available space for mass andenergy transfer (Chávez-González et al., 2010). The lowest PDvalue (Table 1) was obtained with CC (0.74 g cm−3). There areno difference in PD values among CH and SB. A high value ofPD was found in CS, which might to produce problems in massand energy transference.

3.2. Ellagitannase production in SSF

Supports can also provide carbon and nutriment during thefermentation process, or as bonding surface for fungus inva-sion in the impregnated support with culture medium. Fungiare microorganisms easily adaptable to SSF since their hyphaecan grow over the particles surface and even penetrate theintra-individual spaces and colonize the solid support sub-strate (Graminha et al., 2008). A. niger GH1 was able togrow invading and penetrating the different supports, asso-ciated with ellagitannins biodegradation by the ellagitannaseenzyme.

In this work, A. niger GH1 presents a fast biomass produc-tion reaching the maximum value (400 mg g−1) in SB, this valueis two-fold better than those obtained with CH, CS and CC(Fig. 1(a)). This difference may be attributed to presence ofother compounds in the SB after the wash process, such aspolysaccharides and monomeric sugars, which are prefer tothe fungus growth.

Please cite this article in press as: Buenrostro-Figueroa, J., et al., Potentiaellagitannase production under solid-state fermentation. Food Bioprod Pr

The support-substrate invasion was associated to ellagi-tannase activity (Fig. 1(b)). It was observed that ellagitannase

activity appeared after 8 h of culture, reached the maximumenzyme activity among 16 and 24 h for all tested supports,after this the enzymatic activity decreases, maybe attributedto production of other enzymes, such as proteases, whichmay generate enzyme degradation during the ellagitanninsdegradation process. Maximum ellagitannase activities werefound in SB and CC (1404.01 U L−1 and 1179.98 U L−1 respec-tively), higher than CH (925 U L−1) and 2.5 times more thanthat obtained using CS (650 U L−1). No significant differencesbetween CC and SB at 24 h (p ≤ 0.05) were observed.

The lowest value of ellagitannase activity was found in CS,may be due to the high CHP value which limits availability offree water for fungus growth and metabolism. On the otherhand, PD value found in CS was higher than those observedfor all others supports (Table 1). An increase in packing densitymay cause a reduction in the void space between particles anda concomitant reduction in the area of exchanged with thesurrounding atmosphere (Barrios-González et al., 1993). WithCH was observed the highest enzyme volumetric productivity(115.62 U L h−1), two more times than CC and SB, and up to fourtimes higher than CS (Fig. 1(c)).

The use of purified pomegranate ellagitannins as ellagi-tannase inducers in SSF with A. niger GH1 fungus allowed toobtain high enzymatic titers and good specific productivity.The best specific productivity was obtained using CC, beingtwo more times than SB and CH at the same fermentationtime (Fig. 1(d)).

Environmental scanning electron microscopy demon-strated that A. niger GH1 grew invading and penetrating thecorn cobs support (Fig. 2). Pinto et al. (2012) reported that

l use of different agroindustrial by-products as supports for fungalocess (2013), http://dx.doi.org/10.1016/j.fbp.2013.08.010

corn cob is composed by three different layers, clearly differ-ent among them. Corn cobs present a porous particle layer

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Fig. 2 – Scanning electronic microscope images of the fungus Aspergillus niger GH1 growth on corn cobs particles. (A)Without fungus; (B), (C) and (D): with fungus.

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processes using impregnated supports with culture mediumis important to take into account aspects such as cost and

Fig. 3 – Selection index (-) for use of sugarcane bagasse

Fig. 2(A)). After fermentation, mycelial growth was observedn and inside corn cob cavities (Fig. 2(B)). Furthermore, it wasbserved other microstructure with regular geometric shapend regular alveolar forms (Fig. 2(C)). In this figure, it wasbserved spores of A. niger GH1, which possess a rough struc-ure surface (Fig. 2(D)).

This support has been reported to promote high enzy-atic production values. Evaluating fructooligosaccharides

nd ˇ-fructofuranosidase production by A. japonicus on differ-nt lignocellulosic materials as support, Mussatto et al. (2009b)chieved the highest productivity values using CC under sub-erged fermentation (SmF). There are a few reports about

iodegradation of pomegranate ellagitannins by microorgan-sms, however in recent times, important works have beeneveloped. Ellagitannase activity and productivities valuesbtained in this study are 31 and up to 140 more times thanhose reported by Aguilera-Carbó (2009) whom used SSF ofomegranate husks and the same A. niger GH1 strain. Thisould be explained by the fact that they used a complexubstrate which consists in compounds such as polysaccha-ides, proteins, monomeric sugars and of course, polyphenols,here the fungus metabolized other molecules such as sugars

eaving for later time polyphenols and therefore the enzymesesponsible for the degradation of the latter will be expressedn different time of culture.

Previously, ellagic acid production has been reported byspergillus oryzae and Trichoderma reesei in SmF, using as sub-trate and carbon source valonea extracts (Huang et al., 2007),hom obtained 300 U L−1 ellagitannin acyl hydrolase activity,

his value was lower than that obtained with candelilla stalks

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n this study and up to 4.7 times lower than that achieved withC and SB. Due to the obtained results, a selection criterion

(S) was established to choose the best support. The responsevariables [ellagitannase activity (ea), specific activity (sa), vol-umetric productivity (vp) and specific productivity (sp)] wereweighted according to their importance in the process (S = [1.5(ea) + 2.0 (sa) + 2.5 (vp) + 4.0 (sp)]). The highest value was givento the specific productivity (U mg protein h−1) as an indicatorof process efficiency and purity of the enzyme obtained. Allsupports were suitable for use in ellagitannase production bySSF with A. niger GH1 (Fig. 3), excepts candelilla stalks. No sig-nificant differences between SB, CH and CC were found. In SSF

l use of different agroindustrial by-products as supports for fungalocess (2013), http://dx.doi.org/10.1016/j.fbp.2013.08.010

(SB), coconut husk (CH), corn cobs (CC) and candelilla stalks(CS) as supports in ellagitannase production by SSF.

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availability of support, environmental impact of the solids pro-duced, as wells as process cost and product.

The use of SB, CH and CC as a matrix for the culture mediaabsorption and attachment matrix for the fungal growth pro-vides important advantages, such as higher production titersand productivity. However, the use of CC may significantlydecrease costs production, due to high specific productivityat short times compared with the other materials evaluated,facilitates the recovery of interest compounds, and can bereused, eliminating the needed time for fungus growth. Thenext step will be evaluated these fermented materials in theellagitannins hydrolysis for continuous ellagic acid produc-tion.

4. Conclusions

According to the obtained results from the physico-chemicalanalysis (WAI, CHP and PD), all tested agro-industrial by-products (sugarcane bagasse, coconut husk, corn cobs andcandelilla stalks) have great potential to be used as supportin SSF process.

A. niger GH1 secreted in higher amounts the ellagitannaseenzyme, in order to break the ester bonds among HHDP groupand glycosides to release ellagic acid. This suggests the induc-tion of ellagitannase by partially purified ellagitannins. Corncobs, sugarcane bagasse and coconut husk can be used asexcellent support in SSF for the ellagitannase production withhigh titers. However the use of corn cobs provides the highestspecific productivity at shorter times, which may decreasesthe production costs.

The development of a bioprocess for ellagitannase produc-tion would offer economic and environmental advantages inellagic acid production compared with chemical methods.

Acknowledgement

Juan Buenrostro wants to thanks the Mexican Council of Sci-ence and Technology (CONACyT) for the scholarship assignedfor their postgraduate studies in the program of Food Scienceand Technology, UAdeC.

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