Microbial production of tannase: an enzyme with potential use in food industry

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Microbial production of tannase: an enzyme with potential use in food industry
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  Lebensm.-Wiss. u.-Technol. 37 (2004) 857–864 Microbial production of tannase: an enzyme with potential use infood industry Ruth Belmares a , Juan Carlos Contreras-Esquivel a , Ra ! ul Rodr ! ıguez-Herrera a ,Ascensi ! on Ram ! ırez Coronel b , Crist ! obal Noe Aguilar a, * a Food Research Department, School of Chemistry, Universidad Autonoma de Coahuila, Unidad Saltillo, Blvd. Venustiano Carranza, P.O. BOX 252,ZIP 2500, Coahuila, Mexico b Institut de Recherche pour le De´ veloppement, Laboratorio de Microbiologie, Universite´  de Provence, ESIL, Case 925, Avenue de Luminy, 132888,Marseille, Cedex 9, France Received 18 March 2004; received in revised form 9 April 2004; accepted 13 April 2004 Abstract Tannase catalyses the hydrolysis of gallic acid esters and hydrolysable tannins. This enzyme is produced by plants andmicroorganisms and it is industrially used as catalysts in the manufacture of gallic acid. Also, it is potentially used in beverage andfood processing. Two critical factors, production costs and insufficient knowledge of the basic characteristics, physicochemicalproperties, catalytic characteristics, regulation mechanisms and potential uses, limit the use of tannase at the industrial level. Thiswork reviews the state of critical aspects related to the tannase, emphasizing aspects such as sources, substrates, metabolic regulationmechanisms, physicochemical properties, inhibitors, production, applications and potential uses. r 2004 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. Keywords:  Tannase; Tannins; Regulation mechanism; Properties; Production; Applications 1. Introduction Tannase or tannin acyl hydrolase (EC, 3.1.1.20)catalyses the hydrolysis reaction of the ester bondspresent in the hydrolysable tannins and gallic acid esters.Its production at industrial level is in a microbial wayusing submerged culture (SmC), where the activity isexpressed mainly of intracellular form, implying addi-tional costs in its production (Lekha & Lonsane, 1994). Tannase is recently commercialized by Biocon (India),Kikkoman (Japan) ASA Specilaeznyme GmbH (Ger-many) and JFC GmbH (Germany) with differentcatalytic units depending of the product presentation.However, several studies have reported interestingadvantages between the tannase produced by solid stateculture (SSC) in relation with that produced by SmC.On this topic there are few reports, but they are clearlyinteresting (Barthomeuf, Regerat, & Pourrat (1994); Garc ! ıa-Pen ˜a (1996); Chaterjee, Dutta, Banerjee, & Bhattacharyya, 1996; Lekha & Lonsane, 1997; Garc ! ıa-Pen ˜a et al., 1999; Ram ! ırez-Coronel, Viniegra-Gonz ! alez,& Augur, 1999; Aguilar, Augur, Viniegra-Gonz ! alez, &Favela, 1999; Aguilar, Augur, Favela, & Viniegra- Gonz ! alez, 2001a,b; Aguilar, Favela-Torres, Viniegra- Gonz ! alez, & Augur, 2002; Van de Lagemaat & Pyle, 2001). In these, attractive advantages indicated, are thehigh-production titles (up to 5.5 times more than inSmC), the extracellular nature of the enzymes and thestability to wide pH and temperature ranges (Lekha &Lonsane, 1994). Aguilar et al. (1999) reported produc- tivities of 6.667 UE/Lh and 1.275 UE/Lh for SSC andSmC, respectively, the tannase activity maximumexpressed intracellularly is also 18 times more in SSCthan in SmC, while the extracellular activity is 2.5 timeshigher in SSC that in SmC.At the moment, the biggest commercial applicationsof the tannase are given in the elaboration of instantaneous tea or of acorn liquor and in the ARTICLE IN PRESS *Corresponding author. Tel.: +52-844-416-9213; fax: +52-844-439-0511. E-mail address:  cag13761@mail.uadec.mx (C.N. Aguilar).0023-6438/$30.00 r 2004 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.doi:10.1016/j.lwt.2004.04.002  production of the gallic acid (Coggon, Graham, &Sanderson, 1975; Chae & Yu, 1983; Pourrat, Regerat, Pourrat, & Jean, 1985; Garc ! ıa-N ! a jera, 2002), which is animportant intermediary compound in the synthesis of the antibacterial drug, trimetroprim, used in thepharmaceutical industry (Sittig, 1988) and also in thefood industry; gallic acid is a substrate for the chemicalor enzymatic synthesis of the propylgallate, a potentantioxidant. Also, the tannase is used as clarifying agentin some wines, juices of fruits and in refreshing drinkswith coffee flavour (Lekha, Ramakrishna, & Lonsane, 1993).In the case of the wines, it is important to state thatthe main tannins present are catechins and epi-catechins,which can get a complex with galacto-catechins andothers galloyl-derivated. The amount of catechin inwhite wines is around of 10 to 50mg/l, while in otherwines it can reach 800mg/l (Ribereau-Gayon, 1973).Fifty percent of the colour of the wines is due to thepresence of the tannins, however, if these compoundsare oxidized to quinones by contact with the air it couldbe formed an undesirable turbidity, presenting severequality problems. The use of the tannase can be asolution to these problems.In the manufacture of beer, the tannase could be usedsince the tannins are present in low quantities. When theproteins of the beer are in considerably high quantitiesan undesirable turbidity is presented by the accomplish-ing with these tannins. This problem could be resolvedwith the employment of the tannase. The tanneryeffluents contains high amounts of tannins, mainlypolyphenols, which are dangerous pollutants, for thisreason the use of the tannase represents a cheaptreatment and cash for the removal of these compounds. 2. Tannase substrates Tannins are widely distributed in different parts(bark, needles, heartwood, grasses, seeds and flowers)of vascular plants. They are a group of complexoligomeric chains substances characterized by thepresence of polyphenolic compounds. They have mole-cular weight higher than 500, reaching values above20000kDa. One of the major characteristic of tannins isits ability to form strong complexes with protein andother macromolecules such as starch, cellulose andminerals. Tannins are classified in two major groups:hydrolysable and condensed tannins (Lekha & Lonsane, 1997; Aguilar & Gutie  ´rrez-S ! anchez, 2001).The hydrolysable tannins are constituted by severalmolecules of organic acids, such as gallic, ellagic, digallicand chebulic acids, esterified to a molecule of glucose.Molecules with a core of quinic acid instead of glucosehave been also considered as hydrolysable tannins.Fig. 1 presents some examples of hydrolysable tannins(Mueller-Harvey, 2001).In order to maintain its binding capacity, gallotanninsmust have more than two gallic acid constituentsesterified to the glucose core. Hydrolysable tannins canbe easily hydrolysed under mild acid or alkalineconditions; with hot water or enzymatically (L ! opez-R ! ıos, 1984).Condensed tannins or Proanthocyanidins (Fig. 2) arecomplex compounds constituted by flavonoid groups(from 2 to 50) which are considered not to behydrolysable. Their major constituents are cyanidinand delphinidin which are responsible for the astringenttaste of fruit and wines (Sanchez, 2001).The negative effect of tannins in animal nutrition isdue to their capacity to bind macromolecules renderingthem undigestible (Mendez, 1984). Tannins form stablecomplex with enzymes and minerals required for theruminal microorganisms. They are also responsible of abitter taste, which considerably reduces the feed intake.However, low tannin concentrations in feed havedemonstrated to increase nitrogen assimilation inruminants, rendering higher growth rates and milkproduction (Nip & Burns, 1969). 3. Tannase Tannase catalyses the breakdown of hydrolysabletannins such as tannic acid, methygallate, ethyl gallate, n -propylgallate and isoamylgallate. A typical reaction of tannase is the hydrolytic cleavage of (-)epigallocatechin-3-ol gallate (Fig. 3) (Bajpai & Patil, 1997; Lekha & Lonsane, 1997).Although tannase is present in plants, animals andmicroorganisms, it is mainly produced by microorgan-isms (Ayed & Hamdi, 2002; Nishitani & Osawa, 2003). Table 1 presents some of the microorganisms studies fortannase production.Tannase production and application have beenextensively studied, studies related to strain isolationand improvement, process development and applicationof tannases have conducted to a great number of scientific publications and patents. Table 2 presentssome of the published patents regarding tannaseproduction and application. 4. Production of tannase Filamentous fungi of the  Aspergillus  genus have beenwidely used for tannase production. Although tannaseproduction by  Aspergillus  can occur in the absence of tannic acid, this fungi tolerates tannic acid concentra-tions as high as 20% without having a deleterious effecton both growth and enzyme production. Studies on ARTICLE IN PRESS R. Belmares et al. / Lebensm.-Wiss. u.-Technol. 37 (2004) 857–864 858  ARTICLE IN PRESS OOCOHOHOHOOHOHOHOOCOOOCOHOHOHO CO OHOHOHCO OHOOHOHOHOHCOOHOOHHOHOOHHOHOC=OOOCOOCOCOC=OOOH OHHOOH OHOHOHOOHOO   OHOHOCOOHOHOHCOOHHOHOHOHOHOCOOHHOHO (A)(B)(C)(D)(E) Fig. 1. Hydrolysable tannins and some of their constituents. A. Gallotannin, B. Ellagitannin, C. Ellagic acid, D. Hexahydroxyphenic acid and E.Gallic acid. HOOCOH O H OO H O H O H O H O H O H O H O H O H O H O H O H O H O H O HOCOO Fig. 3. Typical reaction of tannase. OOHOHOHHOHOHOHO OHOHOHOHOHOOHOHOO Fig. 2. Condensed tannins or Proanthocyanidins. R. Belmares et al. / Lebensm.-Wiss. u.-Technol. 37 (2004) 857–864  859  tannase production by  Aspergillus  have been carried outon submerged and solid state cultures. Depending on thestrain and the culture conditions, the enzyme can beconstitutive or inducible, showing different productionpatterns. Phenolic compounds such as gallic acid,pyrogallol, methyl gallate and tannic acid inducestannase synthesis (Bajpai & Patil, 1997). However, the induction mechanism has not been demonstrated andthere is some controversy about the role of some of thehydrolysable tannins constituents on the synthesis of tannase (Deschamps, Otuk, & Lebeault, 1983). For instance, gallic acid, one of the structural constituents of some hydrolysable tannins, such as tannic acid, has beenreported as an inducer of tannase synthesis undersubmerged fermentation, whilst it represses tannasesynthesis under solid state fermentation. Nevertheless,independently of the involved mechanism, it has beenwell accepted that due to the complex composition of the hydrolysable tannins, some of their hydrolysisproducts induces tannase synthesis (Aguilar et al., 2002).Addition of carbon sources such as glucose, fructose,sucrose, maltose, arabinose to the culture medium atinitial concentrations from 10 to 30g/l improves tannaseproduction by  Aspergillus niger . Nitrogen requirementscan be supplied by different organic and inorganicsources. Inorganic nitrogen can be supplemented asammonium salts (sulphate, carbonate, chloride, nitrate,monohydrated phosphate) or nitrate salts (sodium,potassium or ammonium). Other nutritional require-ments such as potassium, magnesium, zinc, phosphateand sulphur are supplied as salts. Although  A. niger does not require cofactor supplementation, folic andpantothenic acid are eventually added to the culturemedium. A typical medium for tannase production by Aspergillus niger  is presented in Table 3.Tannase production in submerged culture by  Asper-gillus  sp. is improved at high aeration rates. It isfavoured at 30–33  C and initial pH values from 3.5 to ARTICLE IN PRESS Table 1Microorganisms used for tannase productionBacteria  Bacillus pumilusBacillus polymyxaCorynebacterium  sp Klebsiella pneumoniaeStreptococcus bovisSelenomonas ruminantium Yeast  Candida  sp. Saccharomyces cerevisiaeMycotorula japonica Fungi  Aspergillus nigerAspergillus oryzaeAspergillus japonicusAspergillus gallonycesAspergillus awamori Penicillium chrysogenumRhizopus oryzaeTrichoderma virideFusarium solani Mucor  sp.Table 2Published patents related to tannase production or applicationYear Title Patent No.1974 Conversion of green tea and natural tea leaves using tannase USP38122661975 Production of tannase by  Aspergillus  JP72257861975 Tea soluble in cold water UKP12801351976 Extraction of tea in cold water GP26105331976 Enzymatic solubilization of tea cream USP39594971985 Gallic acid ester(s) preparation EP-1376011985 Preparation of gallic acid esters e.g. propylgallate EP-1376011985 Enzymatic treatment of black tea leak EP1352221987 Preparation of tannase JP622729731987 Manufacturing of tannase with  Aspergillus  JP622729731988 Production of tannase by  Aspergillus oryzae  JP633049811988 Elaboration of tannase by fermentation JP633049811989 Preparation of spray-concrete coating in mining shaft SUP15149471989 Antioxidant catechin and gallic acid preparation JP012686831989 Tannase production by culture of   Aspergillus tamarii   EP-3390111989 New  Aspergillus niger  B1 strain EP3070711989 Tannase production process by  Aspergillus  and its application to obtain gallic acid EP3390111992 Tannase preparation method JP4360684Table 3Typical culture medium for tannase production by  Aspergillus niger Constituent Initial concentration (g/l)Tannins 50Glucose 10(NH 4 ) 2 HPO 4  5K 2 HPO 4  1MgSO 4  7H 2 O 1ZnSO 4  7H 2 O 0.1NaCl 0.005 R. Belmares et al. / Lebensm.-Wiss. u.-Technol. 37 (2004) 857–864 860  6.5. The maximal enzymatic activity is attained after 1 to3 days of cultivation.Tannase production has been mostly studied insubmerged fermentation; however, few studies havebeen also carried out under solid state fermentationconditions. Wheat bran has been used as support andsole source of nutrients for tannase production by Aspergillus niger  in SSC. Innert supports, such as sugarcanne pith or polyurethane foam, impregnated with adefined culture medium have been also used (Aguilaret al., 2001a,b). Two major differences are found whensubmerged and solid state conditions are compared: (i)tannase yield production and productivity are higher inSSC than in SmC; (ii) tannase location under SSCconditions is mostly extracellular, whilst it is bounded tothe mycelium under SmC conditions.It is important to note that such higher tannaseactivity levels in SSC than SmC have been clearlyassociated with the concomitant production of proteo-lytic activities in the last culture system (Aguilar et al.,2002; Viniegra-Gonz ! alez et al., 2003). Also, in SSC thetannase produced exhibits a higher tolerance to widerange of pH and temperature (Lekha & Lonsane, 1994). For optimization of tannase production, Pinto,Bruno, Hamacher, Terzi, and Couri (2003) evaluatedthe tannic acid/wheat bran ratio, different moisturelevels, addition of supplementary nitrogen sources,addition of supplementary phosphate and the concen-tration of supplementary nitrogen and phosphate addedto the medium. Their results showed that best mediumwas with 15% of tannic acid, 37.5% of initial moisture,1.7 ammonium sulphate and 2.0% of sodium phos-phate. The presence of phosphate showed a greatimportance for optimization, because promoted increasein the synthesis level and a very expressive decrease inthe maximum production time, from 72 to 24h of fermentation. The optimized process promoted aincrease 861% in yield and 2783% in productivity. 5. Tannase extraction Tannase extraction strongly depends on the fermenta-tion system used. Since tannase is mostly extracellularwhen produced by SSC, it can be easily extracted withwater or a buffer. Two to three volumes of the agentextraction is well mixed with the fermented mass andpressed to obtain the enzymatic extract. Tannaselocation during its production by SmC depends on thecultivation time (Rajakumar & Nandy, 1983). It is mainly intracellular at the beginning of the culture and itis further secreted to the culture medium. However, upto 80% of tannase remained bounded to the myceliumwhen the maximum overall tannase titter is attained.Bounded tannase can be extracted after cell wallhydrolysis with digestive enzymes such as chitinase.The cells can be also mechanically disrupted to recoverthe bounded tannase. Recently, Ramirez-Coronel,Viniegra-Gonzalez, Darvill, and Augur (2003) producedan extracellular tannase by solid-state cultures of  Aspergillus niger . The enzyme was purified to homo-geneity from the cell-free culture broth by preparativeisoelectric focusing and by FPLC using anion-exchangeand gel-filtration chromatography. SDS-PAGE analysisas well as gel localization studies of purified tannaseindicated the presence of two enzyme forms. 6. Tannase immobilization Since a fraction of the produced tannase remainsbounded to the cell under submerged fermentationconditions, the produced biomass may be recycled as abiocatalyst. Several strategies can be used for tannaseconcentration or purification and immobilization afterextraction from the biomass (submerged fermentation)or from the culture medium (submerged an solid statefermentation). In order to increase the specific activity of the enzymatic preparation, tannase should be concen-trated. For that, classical methods such as salt or solventprecipitation, ultrafiltration followed by ion exchange orsize exclusion chromatography, as well as solventextraction can be used (Lekha & Lonsane, 1994). Once the tannase activity has been concentrated and even-tually purified, it can be immobilized to reuse theenzymatic preparation. Agarose, chitosan, alginate anddifferent derivatized silicious materials can be used fortannase immobilization (Sharma, Bhat, & Gupta, 2002). Abdel-Naby, Sherif, El-Tanash, and Mankarios (1999)immobilized tannase from  Aspergillus oryzae  on variouscarriers, however, that enzyme immobilized on chitosan-glutaraldeheyde showed the highest activity. The boundenzyme retained 20.3% of srcinal specific activity. Onthe other hand, Sharma et al. (2002) immobilizedtannase from  A. niger  on concavalin A-Sepharose viabioaffinity interaction. The immobilized preparationwas quite stable to reuse, there was no loss of enzymeactivity after three cycles and it retained 81% activityeven after the sixth cycle. Ester hydrolysis using theimmobilized enzyme led to a 40% conversion into gallicacid as compared with 30% obtained with the freeenzyme. 7. Properties of tannase The tannase of some  Aspergillus  strains has amolecular weight around 150–350kDa. Their activityand stability pH are 5–6.0 and 3.5–8.0, respectively,whilst optima temperatures from 35  C to 40  C havebeen reported. Tannase is stable for several months at30  C. Tannase produced by  Penicillium  strains present ARTICLE IN PRESS R. Belmares et al. / Lebensm.-Wiss. u.-Technol. 37 (2004) 857–864  861
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