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Microbiological characteristics of ‘‘androlla’’, a Spanish traditional

FOOD

MICROBIOLOGY

Food Microbiology 24(2007)52–58

Microbiological characteristics of ‘‘androlla’’,a Spanish traditional

pork sausage

Mar?a C.Garc?a Fonta

n,Jose M.Lorenzo,Ana Parada,Inmaculada Franco,Javier Carballo ?A

′rea de Tecnolog?′a de los Alimentos,Facultad de Ciencias de Orense,Universidad de Vigo,32004Orense,Spain Received 3December 2005;received in revised form 17March 2006;accepted 17March 2006

Available online 21April 2006

Abstract

Counts of total aerobic mesophilic micro?ora,lactic acid bacteria,salt-tolerant micro?ora,Enterobacteriaceae ,enterococci,moulds and yeasts,and staphylococci,and some physico-chemical parameters (total solids,NaCl and nitrate contents and pH and a w values)were determined in 20units of ‘‘androlla’’,a traditional dry-fermented sausage made in the NW of Spain.

In general,high counts of all the investigated microbial groups were observed,with average values of 8.9970.46log cfu/g for the total aerobic mesophilic micro?ora,9.1170.16log cfu/g for the lactic acid bacteria,6.8770.68log cfu/g for the salt-tolerant micro?ora,2.8071.85log cfu/g for the Enterobacteriaceae ,3.2571.86log cfu/g for the enterococci,4.3071.73log cfu/g for the moulds and yeasts,and 3.6270.60log cfu/g for the staphylococci.

From MRS agar,SPC agar+7.5%NaCl,VRBG agar,and KAA agar,10colonies were randomly taken from each androlla unit and from each culture medium.A total of 200strains per culture medium were then identi?ed using the classical methods.

Among the isolates from MRS agar,Lactobacillus sakei predominated,followed by Lactobacillus curvatus ,Lactobacillus alimentarius and Lactobacillus plantarum .Of the 200isolates obtained from SPC agar+7.5%NaCl,only 56strains belonged to the Staphylococcaceae or Micrococcaceae families.Among the Staphylococcaceae ,Staphylococcus xylosus was the main species,followed by Staph.epidermidis ;Staph.equorum ,Staph.capitis and Staph.saprophyticus were isolated in very low proportions.Among the Micrococcaceae ,Micrococcus luteus predominated,followed by Micrococcus lylae ,Kocuria varians and Kocuria kristinae.Of the 150isolates obtained from VRBG agar,Hafnia alvei was the main species,followed by Serratia liquefaciens and Enterobacter amnigenus ;six isolates were identi?ed as Salmonella .Among the 190isolates obtained from KAA agar,122were considered enterococci;20isolates were identi?ed as Enterococcus faecium ,one as Enterococcus faecalis and 101as Enterococcus inter faecalis-faecium .

Keywords:Androlla;Dry-fermented sausages;Lactic acid bacteria;Salt-tolerant micro?ora;Enterobacteriaceae ;Enterococci

1.Introduction

During the manufacture of dry-fermented sausages,chemical and physico-chemical modi?cations occur,espe-cially dehydration,fermentation of carbohydrates and acidi?cation,development of a typical colour,lipolysis

and oxidation of lipids,and proteolysis (Ordo n ez et al.,

1999).These changes,which are responsible for the arganoleptic characteristics of the ?nal products are,in part,due to the activity of the different microbial groups

(above all lactic acid bacteria,Micrococcaceae and Staphylococcaceae )which develop in sausages during the

fermentation/ripening stage (Lu cke,1984;Ordo

n ez et al.,1999).Also,the lactic acid bacteria guarantee the safety of the product through the production of antimicrobial compounds of different nature as lactic acid,acetic acid,hydrogen peroxide,carbon dioxide and bacteriocins (Aymerich et al.,2000),although they also inhibit the growth of undesirable microorganisms descending the pH and competing for the nutrients (Schillinger and Lu cke,1990).

Androlla is a traditional raw-cured sausage elaborated in the NW of Spain that has a good appreciation among the

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consumers and a great installation in the local markets.At the present,this sausage is elaborated in a handmade or semi-industrial way,following absolutely empiric tradi-tional procedures;due to this,the obtained?nal product possesses a quite heterogeneous quality and in many occasions does not offer a total sanitary guarantee.

The studies carried out on androlla until now only refer to the biochemical characteristics of the?nal product (Lorenzo et al.,2000)and to the biochemical changes during the manufacture(Franco et al.,2002).There is a lack of information about the microbiological character-istics of this sausage.

The aim of this article is to know the counts of the different microbial groups of technological and hygienic interest in the?nal product ready for consumption,and to know the identity of the microorganisms belonging to these microbial groups,as well as the abundance of each species, and constitutes a preliminary study of microbial character-ization of this sausage.

2.Material and methods

2.1.Samples

In order to carry out this study,20androlla samples, made by20different pork meat industries,were taken. Each sample consisted of one entire androlla piece.The average quantities of the ingredients used in the prepara-tion of the mix used in the manufacture of the androlla units used in this study(expressed as kg/100kg of mix) were:ribs with their?eshy parts,60.0;pork jowl,8.0;lean pork,22.0;pork back fat,5.0;salt,2.0;sweet paprika,2.4; spicy paprika,0.4;garlic,0.1;marjoram,0.1.Sugars and LAB starter cultures were not added.Meat was ground, ribs were cut into pieces of approximately3–4cm length, and the ingredients were minced for15min.The resulting mix was left standing for12h at201C and was then stuffed into natural casing of 6.5cm in diameter in units of 20–25cm length.Ripening was carried out in storerooms at 121C and75%of relative humidity.The pieces were smoked during the?rst8days of ripening using oak wood. The androlla units used in this study were collected after 20–30days of ripening.Samples were transported to the laboratory under refrigeration(below41C).

2.2.Microbiological analysis

In each androlla unit,after aseptically removing and discarding the outer casing and the bones,25g of the edible part were aseptically taken and homogenized with100mL of sterile0.1%peptone water also containing0.85%NaCl and1%Tween80as emulsi?er,at40–451C for2min in a Masticator blender(IUL Instruments,Barcelona,Spain), thus making a1/5dilution.Successive decimal dilutions were prepared by mixing10mL of the previous dilution with90mL sterile0.1%peptone water.

Total aerobic mesophilic micro?ora was enumerated in Standard Plate Count Agar(Oxoid,Unipath Ltd., Basingstoke,UK),after incubation at301C for48h;lactic acid bacteria in De Man,Rogosa,Sharpe(MRS)agar (Oxoid)acidi?ed until pH5.6with acetic acid and plus 0.2%of potassium sorbate,after incubation at301C for5 days;salt-tolerant micro?ora in Standard Plate Count agar (Oxoid)+7.5%NaCl,after incubation at301C for48h; Enterobacteriaceae in violet red bile glucose(VRBG)agar (Oxoid)after incubation at371C for24h;enterococci in kanamycin aesculin azide(KAA)agar(Oxoid)incubated for24h at371C;moulds and yeasts in Sabouraud dextrose agar+chloramphenicol(Pronadisa,Madrid,Spain)incu-bated at241C for5days and staphylococci in Baird Parker agar(Oxoid)incubated at371C for24h.From each sample and on each culture medium,1mL of each dilution was inoculated in duplicate on plates and mixed before solidi?cation.Plates of MRS agar and VRBG agar were covered with a layer of the same culture medium before incubation.After incubation,plates with30–300colonies were counted.

2.3.Isolation and identi?cation of strains

From MRS agar,SPC agar+7.5%NaCl,VRBG agar and KAA agar,10colonies were randomly taken from each androlla unit and from each culture medium with the aid of a Harrison disk(Harrigan and McCance,1976).A total of200strains per culture medium(20units of androlla?10colonies per unit)which were then puri?ed. The isolates from MRS agar were puri?ed by four alternate subcultures in MRS agar and MRS broth(Oxoid).Isolates from SPC agar+7.5%NaCl were puri?ed in Brain Heart Infusion(BHI)agar and BHI broth(Oxoid).Isolates from KAA agar and from VRBG agar were puri?ed in Nutrient agar–Nutrient broth(Oxoid).The puri?ed strains were, afterwards,maintained atà801C using a20%of glycerol as cryoprotector agent.

The Gram-positive,catalase-negative rods(lactobacilli) were characterized according to the methods and criteria of Sharpe(1979),and Kandler and Weiss(1986).The following tests were carried out on each isolate:CO2 production from glucose;L-arginine hydrolysis;growth at 15and451C;utilization of L-arabinose,inulin,lactose, melibiose,D-raf?nose,L-rhamnose,D-sorbitol,L-sorbose, sucrose,and D-xylose.

Gram-positive,catalase-positive cocci grouped in pairs, tetrads or irregular clusters,were considered to belong to the Micrococcaceae or Staphylococcaceae families and were identi?ed following the methods and criteria described by Baird-Parker(1979),Devriese et al.(1985),and Schleifer (1986).The following tests were carried out on each isolate: oxidation and fermentation of glucose and mannitol; assimilation of glycerol in aerobic conditions;growth in furazolidone agar;growth in lysostaphin agar;colony pigmentation in BHI agar;growth on7.5%,10%and15% NaCl agar;characteristics of growth in Baird-Parker agar;

M.C.Garc?′a Fonta′n et al./Food Microbiology24(2007)52–5853

esculin hydrolysis;L-arginine hydrolysis;growth at101C; acetoin production;nitrate reduction;presence of oxidase, alkaline phosphatase,urease,arginine dihydrolase,or-nithine decarboxilase,b-galactosidase,arginine arylami-dase,pyrrolydonil arylamidase,b-glucuronidase;novobiocin resistance;acid production from N-acetyl-glucosamine, L-arabinose,D-cellobiose,D-fructose,D-glucose,maltose, D-mannitol,D-mannose,D-raf?nose,D-ribose,sucrose, D-trehalose and D-turanose.

Gram-negative,catalase-positive,oxidase-negative short rods(Enterobacteriaceae)were identi?ed according to the methods and criteria of Lapage et al.(1979)and Brenner (1984).The following tests were carried out in each isolate:motility;growth and characteristics of the growth on MacConkey agar;methyl red and Voges–Proskauer reactions;gas production from D-glucose and lactose;citrate,gluconate and malonate utilization; indole and H2S production;NO3àreduction to NO2à;NO2àreduction to N2gas;gelatin liquefaction;presence of urease,lysine decarboxilase,arginine dihydrolase, ornithine decarboxylase,b-galactosidase,and tryptophane deaminase;fermentation of amygdalin,L-arabinose, D-glucose,inositol,D-mannitol,melibiose,L-rhamnose, D-sorbitol and sucrose.

Gram-positive,catalase-negative cocci,grouped in pairs or short chains,homofermentative,which grew at 10,40and,generally,at451C,generally survived after heating at601C during30min,grew in a6.5%NaCl, at a pH9.6and in40%bile,and formed red or pink colonies generally with a yellowish halo in KF Streptococcus agar(Difco Laboratories,Detroit,MI, USA),were considered as enterococci and were classi?ed according to the methods and criteria described by Sharpe(1979),Collins et al.(1984),Colman and Ball(1984),Mundt(1986),and Devriese et al.(1987). The following tests were carried out on each isolate:growth at501C;hydrolysis of L-arginine;reduction of0.1% methylene blue milk;growth in0.04%potassium tellurite broth;utilization of L-arabinose,arbutin,melezitose, melibiose,sorbitol,L-sorbose,D-raf?nose,L-rhamnose, starch and sucrose.

2.4.Chemical analysis

Moisture,NaCl and nitrate contents,and pH and a w values were determined using the methods cited by Franco et al.(2002).All chemical determinations were made in duplicate in each sample.

2.5.Statistical analysis

Statistical correlations between the physico-chemical parameters determined and log counts of the different microbial groups enumerated were carried out by Pearson’s correlation coef?cient,using the computer programme Statistica5.1for Windows(Statsoft Inc,1996,Tulsa,OK, USA).3.Results and discussion

3.1.Counts of the different microbial groups and its relationship with physico-chemical parameters

Table1shows the counts of the different microbial groups in the units of androlla.The values of the physico-chemical parameters analysed are shown in Table2.In general,the units of analysed androlla showed high counts of all the investigated microbial groups,which indicates a high contamination of the raw materials used in their manufacture.In each microbial group,a great variability of the counts was observed between the different units of androlla;this circumstance re?ects the unequal contamina-tion of the product in the different industries,consequence of a different effectiveness of the hygienic care observed during the production processes.

The total viable mesophilic counts showed an average value of8.99log cfu/g.This value was similar to those observed by Dolazo et al.(1998)in Galician chorizo and by Dom?nguez et al.(1989)at the end of the ripening process of the chorizo of Leo n,around a log unit higher than those observed by Gonza lez and D?ez(2002)in chorizo,by Serrano Moreno(1979)in salchicho n and by Metaxopou-los et al.(2001)and Samelis et al.(1998)in Greek traditional salami,and around two log units higher than those observed by Lizaso et al.(1999)in salchicho n at the end of the ripening process.

Table1

Counts(log cfu/g)of the different microbial groups in androlla(n?20)

Microbial group Average Minimum Maximum SD

Total aerobic mesophilic?ora8.997.819.520.46 Lactic acid bacteria9.118.789.360.16 Salt-tolerant?ora 6.87 5.788.360.68 Enterobacteriaceae 2.80– 4.93 1.85 Enterococci 3.25– 6.03 1.86 Moulds and yeasts 4.30 1.60 6.99 1.73 Staphylococci 3.62 2.36 4.740.60

SD,Standard deviation.

(–)Absence in0.2g.

Table2

Physico-chemical parameters of androlla(n?20)

Parameter Average Minimum Maximum SD

Total solids(TS)(%)62.154.6576.36 6.60 Moisture(%)37.923.6445.35 6.60 pH 5.39 5.08 5.700.19 a w0.9020.8020.9320.041 Nitrate(ppm)35.5815.8868.8215.08 NaCl(%) 1.950.73 3.070.71 NaCl(%T.S.) 3.14 1.24 4.85 1.11 NaCl a 5.43 1.7910.82 2.66 SD,Standard deviation.

a Expressed as g NaCl/100g of moisture.

M.C.Garc?′a Fonta′n et al./Food Microbiology24(2007)52–58 54

In agreement with the results obtained by other authors in previous studies carried out on other raw-cured sausages (Dom?nguez et al.,1989;Dolazo et al.,1998;Samelis et al., 1998;Lizaso et al.,1999;Metaxopoulos et al.,2001; Gonza lez and D?ez,2002),the lactic acid bacteria were the major microbial group.The average counts of lactic acid bacteria observed in our study(9.11log cfu/g)basically coincide with those observed in different varieties of chorizo(Dom?nguez et al.,1989;Dolazo et al.,1998; Gonza lez and D?ez,2002)and they are lightly higher (around one log unit)to those observed in Greek salami (Samelis et al.,1998;Metaxopoulos et al.,2001),Italian salami(Coppola et al.,2000)and salchicho n(Lizaso et al., 1999).Serrano Moreno(1979)observed lower values(on average6.76log cfu/g)at the end of the ripening process of salchicho n.

As expected in products with a moderate salt content, the salt-tolerant micro?ora presented notably lower counts than those of the lactic acid bacteria.The average value of the counts of the salt-tolerant micro?ora in androlla (6.87log cfu/g)is,nevertheless,higher to those reported in other works carried out on raw-cured sausages(Dolazo et al.,1998;Samelis et al.,1998;Lizaso et al.,1999; Metaxopoulos et al.,2001;Gonza lez and D?ez,2002).Our values were quite similar to those observed by Dom?nguez et al.(1989)in chorizo of Leo n and they were fairly lower to those observed by Serrano Moreno(1979)in salchicho n. The Enterobacteriaceae counts in the20units of studied androlla ranged from absence in0.2g of sample up to 4.93log cfu/g.The differences in the Enterobacteriaceae counts among the different units of androlla can be a consequence of the grade of initial contamination of the meat used in their manufacture or of differences in the manufacture process.These differences could also be related to the environmental factors that determine their disappearance along the ripening process.This possibility seems to be supported by the good correlation observed in this work between the log counts of Enterobacteriaceae and the moisture contents(r?0:681,P o0:01),NaCl contents (expressed as g NaCl/100g of moisture)(r?à0:849, P o0:001),and a w values(r?0:774,P o0:001).

The enterococci counts also varied in the units of androlla from absence in0.2g up to6.03log cfu/g.The average value(3.26log cfu/g),and the individual values of each unit of androlla are inside the wide range of enterococci counts reported in the literature for different raw-cured sausages(Kalojanov and Dimitrova,1990; Dolazo et al.,1998;Samelis et al.,1998;Natola et al., 2002).The enterococci counts in the different units of androlla also re?ect different degrees of contamination of the raw materials during the processing.

The counts of moulds and yeasts were also very variable with values between1.60and6.99log cfu/g and an average of4.30log cfu/g.This average value is quite similar to those reported in Italian salami type Naples(Coppola et al., 1995)and in Greek traditional salami(Samelis et al.,1998; Metaxopoulos et al.,2001).Average moulds and yeast counts in androlla were lightly higher than those reported in Galician chorizo(Dolazo et al.,1998)and quite lower than those reported in chorizo of Leo n(Dom?nguez et al., 1989).

Finally,the staphylococci counts ranged from2.36to 4.74log cfu/g,with an average value of 3.62log cfu/g. Samelis et al.(1998)and Metaxopoulos et al.(2001) studied the presence of coagulase-positive staphylococci in Greek salami using the same culture medium(Baird-Parker agar)and they obtained counts noticeably lower than ours (lower than2log cfu/g at the end of the ripening process in most of the studied batches).

3.2.Species isolated and identi?ed from the different culture media

Table3shows the identity of the isolates carried out in the different culture media.

All the identi?ed strains isolated from MRS agar(a total of197)turned out to be lactobacilli.

Lactobacillus sakei and Lactobacillus curvatus,also in the same order of quantitative importance,have been de-scribed as the major lactobacilli species in other raw-cured sausages as chorizo(Santos et al.,1998),salchicho n (Hugas,1994)and Italian sausages(Torriani et al.,1994). The homofermentative lactobacilli also prevailed in Italian salami type Naples(Coppola et al.,2000),being also in this case Lactobacillus sakei the most abundant species,as well as in Greek salami(Samelis et al.,1998).

The predominance of L.sakei over L.curvatus is very frequent in raw-cured sausages subjected to a spontaneous (not controlled)fermentation–maturation(Vogel et al., 1993;Hugas et al.,1995).Possibly,as Samelis et al.(1998) indicated,L.sakei is better adapted to the ecosystem represented by the fermented sausages and to its special environmental conditions.

L.alimentarius and L.plantarum,the species isolated from androlla in smaller proportions,have also been isolated by other authors in different raw-cured sausages (Hugas,1994;Torriani et al.,1994;Samelis et al.,1998). Of the200isolates obtained from SPC agar+7.5% NaCl,only56(28%of the isolates)were considered as belonging to the Staphylococcaceae or Micrococcaceae families.

Staphylococcus xylosus was also described as the major Staphylococcaceae species in other raw-cured sausages as the salami type Naples(Pirone and Manganelli,1990; Coppola et al.,2000),other types of Italian sausages (Torriani et al.,1994)and different varieties of chorizo (Garc?a-Varona et al.,2000).This species is one of the most resistant to unfavorable environmental conditions (McMeekin et al.,1987)and it is very diffused in the animal mammals and in the environment.

Other staphylococci species identi?ed by us in small proportion were Staph.epidermidis,Staph.equorum,Staph. capitis and Staph.saprophyticus.These staphylococci species have been identi?ed,in similar proportions to ours,

M.C.Garc?′a Fonta′n et al./Food Microbiology24(2007)52–5855

in other works carried out on different raw-cured sausage varieties(Torriani et al.,1994;Coppola et al.,2000; Garc?a-Varona et al.,2000).Even,Staph.saprophyticus has been described as the major staphylococci species in Greek traditional salami(Samelis et al.,1998).

It is not frequent to isolate microorganisms of the Micrococcaceae family in high proportions from raw-cured sausages.In fact,in some studies carried out in this type of meat products all the isolates carried out belonged to the family Staphylococcaceae(Hugas and Roca,1997;Garc?a-Varona et al.,2000).Micrococcus luteus,nevertheless,has also been identi?ed,although in small proportions,in chorizo from Cantimpalos and from Guijuelo(Iberian pig) (Daporta Pad?n,1988).

Of the150isolates obtained from VRBGA(because absence of Enterobacteriaceae in0.2g in5units of androlla analysed was observed),124were considered to be Enterobacteriaceae.

Hafnia alvei was the major Enterobacteriaceae species. This species was also the major one in‘‘chorizo de cebolla’’manufactured by industrial procedures(Castan o et al., 2002)and it has also been isolated by Silla Santos (1998)in several types of Spanish sausages.Noveir et al. (2000)isolated H.alvei as the main Enterobacteriaceae species from different foods of animal origin.This species has also been isolated in a majority way from fresh meat vacuum packed that had suffered putrefaction (Bersani et al.,1999).

Serratia liquefaciens was isolated in a very high propor-tion,and in nine of the20androlla units.S.liquefaciens is a psychrotrophic species broadly distributed in the environ-ment(Brenner,1984).This species has also been isolated from other raw fermented sausages(Encinas,1993;Sanz, 2000;Castan o et al.,2002).It can survive under adverse conditions,being isolated from several fermented sausages, even in the last stages of the ripening process(Encinas, 1993).S.liquefaciens has an important proteolytic activity and it has been associated with alterations of meat products such as the blackening of precooked tripes vacuum packed and conserved under refrigeration(Canto-ni et al.,1997).

Enterobacter amnigenus has also been isolated from industrially manufactured‘‘chorizo de cebolla’’(Castan o et al.,2002)and from fresh vegetables(Fujisawa and Mori, 1992).It possesses as peculiarity the capacity to form dextran and heteropolysaccharides from sucrose(Tallgren et al.,1999);this property could generate muddiness in the products with an important presence of this micro-organism.

The species belonging to the genus Serratia and Enterobacter are quite frequent in raw-cured sausages, being majority in some types such as chorizo from Cantimpalos(Sanz,2000).

Six isolates were identi?ed as Salmonella:three as Salmonella choleraesuis,two as S.arizonae and an isolate was not identi?ed reliably at species level.S.choleraesuis has also been isolated from another Spanish traditional sausage(Castan o et al.,2002)in the?rst stages of the ripening process.

Anyway,the presence of Salmonella in androlla should not present health risks,since this product is consumed after cooking.Salmonella,obviously,does not survive after heat treatments so intense.Moreover,some studies (Wei-Li-Ho and Cheng-Chun-Chou,2000)have demon-strated that the citotoxins from S.choleraesuis are totally destroyed after1min at1001C or5min at801C.

Of the190isolates obtained from KAA agar(because absence in0.2g was observed in one unit of androlla analysed),122were considered enterococci.Of these122 strains,20were identi?ed as Enterococcus faecium,one as E.faecalis and101strains showed intermediate character-istics between E.faecalis and E.faecium and they were considered as Enterococcus inter faecalis-faecium.

Table3

Identity of the isolates obtained from the androlla units in the different culture media

Identi?cation No.of

isolates Identi?cation No.of

isolates

Isolates from MRS agar Isolates from VRBG agar

Lactobacillus sakei87Hafnia alvei67 Lactobacillus

curvatus

50Serratia liquefaciens23 Lactobacillus

alimentarius

41Serratia marcescens7

Lactobacillus plantarum 8Enterobacter

amnigenus

Lactobacillus spp.11Enterobacter

cancerogenus

1 Isolates lost3Enterobacter cloacae1

Enterobacter

gergoviae

Isolates from SPA agar+75%NaCl

Salmonella choleraesius

3 Salmonella arizonae2

Staphylococcus

xylosus

25Salmonella spp.1

Staphylococcus epidermidis 3Morganella

wisconsensis

Staphylococcus equorum 2Klebsiella

pneumoniae

Staphylococcus

capitis

1Citrobacter braakii1

Staphylococcus

saprophyticus

1Citrobacter youngae1 Micrococcus luteus19Providencia rettgeri1 Micrococcus lylae2Rahnella aquatilis1 Kocuria varians1

Kocuria kristinae1Isolates from KAA

agar

Isolates lost1

Enterococcus

faecium

Enterococcus

faecalis

Enterococcus inter faecalis-faecium 101

M.C.Garc?′a Fonta′n et al./Food Microbiology24(2007)52–58 56

Although E.faecalis is the enterococci species more broadly diffused(Murray,1990),Devriese et al.(1995) isolated E.faecium as the major Enterococcus species in meat,which coincides with our results. Acknowledgements

Financial support for this work was provided by the Xunta de Galicia(the Regional Government),Grant PGIDIT05PXIB38303PR.

Mar?a C.Garc?a Fonta n was supported by a Pre-doctoral fellowship from the Universidad de Vigo.Jose M. Lorenzo was supported by a Pre-doctoral fellowship from the Xunta de Galicia.

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