Letters in Applied Microbiology 2005, 41, 6976

doi:10.1111/j.1472-765X.2005.01725.x

Characterization of a xylanase from the newly isolated thermophilic Thermomyces lanuginosus CAU44 and its application in bread making
Z.Q. Jiang, S.Q. Yang, S.S. Tan, L.T. Li and X.T. Li
Department of Biotechnology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China
2004/0864: received 25 July 2004, revised 5 November 2004 and accepted 14 December 2004

ABSTRACT
Z . Q . J I A N G , S . Q . Y A N G , S . S . T A N , L . T . L I A N D X . T . L I . 2005.

Aims: A xylanase from the newly isolated thermophilic fungus, Thermomyces lanuginosus CAU44, was characterized and evaluated for its suitability in bread making. Methods and Results: Xylanase was puried 35-fold to homogeneity with a recovery yield of 328%. It appeared as a single protein band on SDS-PAGE gel with a molecular mass of c. 256 kDa. The puried xylanase had an optimum pH of 62, and it was stable over pH 56103. The optimal temperature of xylanase was 75C and it was stable up to 65C at pH 62. Study was further carried out to investigate the effect of the puried xylanase on the properties of wheat bread and its staling during storage. Conclusions: The puried xylanase from T. lanuginosus CAU44 was stable up to 65C and had a broad pH range. The presence of thermostable xylanase during bread making led to an improvement of the specic bread volume and better crumb texture. Besides, addition of xylanase provided an anti-staling effect. Signicance and Impact of the Study: The xylanase from the newly isolated Thermomyces lanuginosus CAU44 shows great promise as a processing aid in the bread-making industry. Keywords: anti-staling, bread making quality, specic volume, Thermomyces lanuginosus, thermostable, xylanase.
nez-Anaya and Jime nez 1997; Hilhorst in the bakeries (Mart et al. 2002). Endo-1,4-b-xylanase (EC 32.18) may be used to improve our bread making characteristics and bread quality parameters by helping to break down polysaccharides in the dough (Courtin and Delcour 2002). The efciency of xylanases of Aspergillus niger var. awamori in improving the quality of bread was seen with the increase in the specic bread volume. This was further enhanced when amylase was used in combination with the xylanase (Maat et al. 1992). Addition of the puried 22-kDa xylanase from Aspergillus sp. caused a 30% increase in bread volume (Norma and Guillermo 2003). The most important change associated with bread staling is the gradual increase in the rmness of crumb (Scanlon and Zghal 2001). It is also known that xylanases have an anti-staling action during bread storage but their action is not clear (Haros et al. 2002). The effect of xylanase action on doughs and bread is different, probably because AXs in our vary with wheat varieties (Saulnier

INTRODUCTION The main nonstarch polysaccharides in wheat our are arabinoxylans (AXs), which occur as minor components of wheat our (23%, dry basis). However, they play an important role in dough rheology and bread quality (Courtin and Delcour 2002). Numerous studies on the functional role of AXs in dough development and their effect on bread properties have been performed in the past decades (Biliaderis et al. 1995; Denli and Ercan 2001; Courtin and Delcour 2002). Starch and nonstarch carbohydrates hydrolysing enzymes are commonly used in the bread making industry as bread improvers (McCleary 1986; Si 1997). There is currently much interest in using nonstarch polysaccharide hydrolysing enzymes, especially xylanases,
Correspondence to: Prof. Dr Lite Li, PO Box 294, China Agricultural University, No.17 Qinghua Donglu, Haidian District, Beijing 100083, China (e-mail: llt@cau.edu.cn or zhqjiang@cau.edu.cn).

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et al. 1995), as well as the fact that the specicity of xylanases towards different substrates is also variable. Also other factors, such as wheat xylanase inhibitors, gluten proteins, fat content and the type of bread-making process, etc. may have some impact on the functionality of xylanases (Courtin and Delcour 2002; Frederix et al. 2004). Many researchers have investigated the effects of the mesophilic xylanases on bread making, but comparatively little has been done about the thermostable xylanases (Jorgensen et al. 1997; Laurikainen et al. 1998; Norma and Guillermo 2003). Thermomyces lanuginosus strains are particularly interesting producers of thermostable xylanases from the industrial point of view, because of the fact they excrete high level of xylanases into the medium (Singh et al. 2003). Efcient production of xylanolytic enzymes from T. lanuginosus has been described in a number of studies (Alam et al. 1994; Puchart et al. 1999; Singh et al. 2000). Until the present study, the properties and application of xylanases from T. lanuginosus strains have not been investigated in detail a 1996; Bennett et al. (Anand et al. 1990; Cesar and Mrs 1998; Lin et al. 1999; Singh et al. 2003; Xiong et al. 2004). Hence, the main purpose of the present study was to investigate the properties of a puried xylanase from the newly isolated T. lanuginosus CAU44. The effect of xylanase on bread quality and bread staling, namely crumb rmness during storage, was also studied. The results here suggest that this thermostable xylanase from T. lanuginosus CAU44 could be of commercial interest in bread-making industry.

Bacterial strains and growth conditions Thermomyces lanuginosus CAU44 was isolated from the soil samples of Sinkiang Province (China). The strain was identied by The Institute of Microbiology of Chinese Academy of Sciences (IMCAS, Beijing). Stock cultures were maintained on potato dextrose agar (PDA) at 4C and were transferred every 67 weeks. PDA plates were incubated at 40C for 45 d and stored at 4C until use. For xylanase production, the basal medium of ask culture contained (g l)1): corncob xylan, 20; yeast extract, 10; tryptone, 10; (NH4)2SO4, 2; MgSO47H2O, 03; FeSO4, 03; and CaCl2, 03. The initial pH of the medium was adjusted to 70 and not further controlled. An agar block (1 cm2) of an actively growing 5-d-old culture of the strain was used to inoculate the growth medium (100 ml) in 300 ml Erlenmeyer asks. Triplicate cultures were shaken at 200 rev min)1 for 5 d at 50C. Enzyme assay and protein determination Xylanase activity was assayed according to the method of Bailey et al. (1992). The reaction mixture containing 09 ml of 10% (w/v) xylan and 01 ml of a suitably diluted enzyme solution was incubated in 005 mol l)1, pH 60 citrate-phosphate buffer at 50C for 10 min. The reaction was stopped by adding 1 ml of 10% (w/v) dinitrosalicylic acid (DNS). The amount of reducing sugar liberated was determined by DNS method using xylose (Sigma) as the standard. One unit of xylanase activity was dened as the amount of enzyme that produced 1 lmol of xylose equivalent per minute. Protein concentrations were measured by the Lowry method (Lowry et al. 1951) with bovine serum albumin (BSA) as the standard. Specic activities are expressed as units per mg of protein. Purication of xylanase All purication steps were performed at 4C unless stated otherwise. The crude extracellular xylanase was obtained by centrifuging the culture broth at 10 000 g for 10 min at 4C. The crude supernatant was subjected to 3060% ammonium sulphate saturation. The precipitated protein collected by centrifugation (10 000 g) was dissolved in 002 mol l)1 sodium acetate buffer. Further purication was performed by gel ltration followed by ion exchange chromatography. A 05-ml sample from the 30 to 60% ammonium sulphate precipitation fraction was loaded on a Sephacryl S-100 column (40 cm 10 cm) and equilibrated with 002 mol l)1 sodium acetate buffer, pH 65, and the proteins were eluted at a ow rate of 030 ml min)1. All active fractions were combined. A 80-ml solution was applied to a Q-Sepharose column (20 cm 10 cm) equilibrated with 0025 mol l)1 potassium phosphate buffer, pH 65. The bound xylanase was eluted with

M A T E R I A LS A N D M E T H O D S Materials The Sephacryl S-100 HR and Q-Sepharose Fast Flow were from Pharmacia (Pharmacia, Uppsala, Sweden). Birchwood xylan, beechwood xylan, oat-spelt xylan, laminarin, lichenan, locust bean gum and carboxymethylcellulose (low viscosity) were purchased from Sigma (St Louis, MO, USA). Articial substrates (p-nitrophenyl derivatives) such as pNP-b-D-xylopyranoside, pNP-b-D-glucopyranoside, pNPb-D-cellobioside, pNP-a-L-arabinofuranoside and pNP-b-Dmannopyranoside were also from Sigma. Corncob xylan was made by the method of Pellerin et al. (1991). Waterextractable arabinoxylans (WE-AX) was isolated from wheat our as described by Izydorczyk et al. (1990). Waterunextractable arabinoxylans (WU-AX) was prepared from wheat our by the method of Gruppen et al. (1990). Commercial bread our with 126% moisture, 065% ash and 142% protein was obtained from Beijing Heng Tong Foods Co., LTD (Beijing, China). Vegetable oil, dehydrated yeast (Swallow, France), milk powder, commercial sugar and salt were obtained from local market. All other chemicals used were analytical grade reagents unless otherwise stated.

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005 mol l)1 NaCl gradient at a ow rate of 05 ml min)1. The active fractions were combined and dialysed against the same buffer and the dialysate was used as the nal preparation for further experiments. This purication step yielded one xylanase active fraction, of which the homogeneity was checked by SDS-polyacrylamide gel electrophoresis (PAGE). SDS-PAGE SDS-PAGE was performed using 125% (w/v) acrylamide gels as described by Laemmli (1970). Protein bands were visualized by Coomassie brilliant blue R-250 staining. The molecular weight standard used was the low molecular weight calibration kit (Amersham Biosciences, Buckinghamshire, UK): phophorylase b (970 kDa), albumin (660 kDa), ovalbumin (450 kDa), carbonic anhydrase (300 kDa), trypsin inhibitor (201 kDa), and a-lactalbumin (144 kDa). Biochemical characterization of puried xylanase The optimum pH for xylanase activity was studied in several different buffers (005 mol l)1) for pH 25115: citratephosphate buffer for pH 2561; MES [2-(N-morpholino)ethane sulfonic acid] buffer for pH 5171; MOPS [3-(N-morpholino)-propane sulfonic acid] buffer for pH 6282; Tris-HCl buffer for pH 7090; CAPS [(cyclohexylamino)-1-propanesulphonic acid] buffer for pH 93115). To determine the pH stability, the puried xylanase was incubated in different buffers as mentioned above (nal concentration 005 mol l)1) at 50C for 30 min, and then the remaining activities of these treated enzyme were measured by the standard assay procedure. The optimum temperature for xylanase activity was determined by incubation of the enzyme in 005 mol l)1 MOPS (pH 62) at different temperatures (30100C). For thermal stability determination, the puried xylanase in 005 mol l)1 MOPS (pH 62) was incubated at different temperatures for 30 min. After cooling the treated enzymes on ice for 30 min, the residual xylanase activities were measured according to the standard assay method. Substrate specicity and hydrolysis property of puried xylanase To determine the substrate specicity of the enzyme, the puried xylanase was incubated with 10 mg ml)1 of each substrate in 005 mol l)1 MOPS buffer (pH 62) at 50C for 10 min. Amount of reducing sugars produced was estimated using the DNS method as described above. Activities towards p-nitrophenyl derivatives were measured by the rate of p-nitrophenol formed during hydrolysis of 0004 mol l)1 of the substrates in 005 mol l)1 MOPS buffer (pH 62) at 50C for 60 min, and detected by spectrophotometry at

410 nm. One unit (U) of activity was dened as the amount of enzyme releasing 1 lmol of p-nitrophenol per min under the above conditions (Lachke 1988). For enzymatic hydrolysis of AX, the reaction mixture consisted of 40 mg of wheat WE-AX or WU-AX solubilized in 20 ml of 005 mol l)1 MOPS buffer (pH 62) and 10 U enzyme was incubated at 50C. Aliquots were removed after 15 min, 30 min, 1 h, 2 h, 4 h and 8 h of hydrolysis. Products of xylan hydrolysis were analysed by thin-layer chromatography (TLC) on a silica gel plate (Merck Silica Gel 60F 254; E. Merk, Darmstadt, Germany). Puried b-1,4-xylooligosaccharides were kindly supplied by Prof. Kusakabe of Tsukuba University (Japan). Bread-making procedure The bread dough formula consisted of wheat our (100 g), dehydrated yeast (20 g), milk powder (50 g), salt (15 g), sugar (20 g), vegetable oil (30 g) and water (60 ml). The dough was mixed for 10 min, rested for 5 min, divided (50 g), kneaded and then rested (15 min). Then the divided dough were sheeted and rolled, proofed (up to three times the initial dough volume, at 38C, 80% relative humidity). Baking was performed at 200C for 15 min. The puried xylanase was incorporated at levels of 25100 ppm (mg kg)1 our basis) to the our before the mixing. The baking test was repeated two times. After baking, loaves were cooled for 2 h at room temperature (22C). Bread volume and crumb texture assessment Bread quality was evaluated by determining the weight, volume, specic volume and crumb rmness of the bread. Loaf volume measurements were taken 2 h after baking by rapeseed displacement method. Bread-crumb texture was measured as the maximum deformation of a 20-mm-thick slice on an Instron Tensile/Compression Testing machine (Model 4411; Instron Corp., Canton, MA, USA) equipped with a cylindrical stainless steel probe having a diameter of 13 mm. Bread samples were packaged in polypropylene bags and stored at room temperature for staling study. Bread was sliced, and deformation strength values were registered only from the three central slices by measuring the force required to compress each slice to three-quarters of its original thickness at a crosshead speed of 1 mm s)1.

RESULTS Purication of xylanase from T. lanuginosus CAU44 A xylanase from the newly isolated strain CAU44 was puried using a procedure which included ammonium

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Purication step Crude supernatant 3060% Ammonium sulfate precipitation Sephacryl S-100 Q-Sepharose fast ow

Total activity (U)* 29733 24489 22166 10652

Protein (mg) 3937 1574 1439 404

Specic activity (U mg)1) 7552 15558 1851 26366

Purication factor (-fold) 1 206 255 350

Recovery (%) 100 824 6145 328

Table 1 Summary of xylanase purication from Thermomyces lanuginosus CAU44

*Activity was measured in 005 mol l)1 citrate-phosphate buffer (pH 60) at 50C using 10% (w/v) birchwood xylan as substrate by the DNS method. The protein was measured by the Lowry method with BSA as the standard.

sulfate precipitation, gel ltration and ion exchange chromatographies. The summary of the xylanase purication is presented in Table 1. The xylanase was puried to 35-fold apparent homogeneity with a recovery yield of 328% (Fig. 1). The specic activity increased during the purication steps from 7552 to 26366 U mg)1. Effect of pH and temperature on the activity and stability of xylanase The puried xylanase was most active at pH 62 (Fig. 2a). It retained more than 80% of its activity at 50C for 30 min when tested in the pH range of 56 to 103 (Fig. 2b). The xylanase exhibited its optimal activity at 75C (Fig. 3a). It was stable up to 65C for 30 min (Fig. 3b).

Substrate specicity and hydrolysis property of puried xylanase The puried xylanase was assayed for hydrolytic activity against a variety of natural and synthetic substrates. The enzyme showed a high specicity towards different xylans

(a) 100 90 80 70 60 50 40 30 20 10 0

Relative activity (%)

970 kDa 660 kDa 450 kDa (b) 100 90 80 70 60 50 40 30 20 10 0 Relative activity (%) M 1 2 3 4 M

7 8 pH

9 10 11 12

300 kDa

201 kDa

144 kDa

7 8 pH

9 10 11 12

Fig. 1 Purication of xylanase from Thermomyces lanuginosus CAU44. Lanes M, low molecular weight calibration kit; lane 1, crude extract; lane 2, fraction of 3060% ammonium sulphate precipitation; lane 3, fraction of Sephacryl S-100 column; lane 4, fraction of Q-Sepharose Fast Flow column

Fig. 2 Optimal pH (a) and pH stability (b) of the puried xylanase from Thermomyces lanuginosus CAU44. The inuence of pH on xylanase activity was determined using 005 mol l)1 different buffers. The remaining activity was measured after incubation for 30 min at 50C over various pH ranges. Buffer used: (r) citrate, ( ) MES, ( ) MOPS, (() Tris-HCl, and ()) CAPS

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(a) 100 90 80 70 60 50 40 30 20 10 0 30

Table 2 Substrate specicity of the puried xylanase Relative Specic activity (U mg)1) activity (%) 26366 17823 36517 14043 20776 11127 100 676 1385 533 788 422

Substrate* Birchwood xylan Beechwood xylan Oat-spelt xylan Corncob xylan Water-extractable arabinoxylans Water-unextractable arabinoxylans

Relative activity (%)

40

50 60 70 80 Temperature (C)

90

100

(b) 100 90 Relative activity (%) 80 70 60 50 40 30 20 10 0 30 40 50 60 70 80 Temperature (C) 90 100

*No activity was observed for Avicel, carboxymethylcellulose, lter paper, starch, laminarin, and locust bean gum. No activity was observed for pNP-b-D-xylopyranoside, pNP-b-D-glucopyranoside, pNP-b-D-cellobioside, pNP-a-L-arabinofuranoside and pNP-b-Dmanopyranoside at 50C for 60 min. The protein was measured by the method of Lowry et al. (1951), using BSA (bovine serum albumin) as the standard.

Effect of xylanase on bread quality Effect of the puried xylanase (at 26366 U mg)1) at various levels on the specic volume of 50-g rolls made from wheat our is shown in Fig. 4a. Addition of the xylanase improved the specic volume with increasing enzyme concentration up to 40 ppm. The highest specic volume rose to 414% when 40 ppm of the xylanase was present. The presence of the xylanase also modied the texture of the fresh bread crumb (Fig. 4b). The crumb rmness of the fresh breads decreased from 786 to 929% of the bread without the enzyme. The bread-crumb rmness decreased by up to 214% with 40 ppm of the xylanase addition. Addition of the xylanase also improved cell structure of the breads. The coarse and nonhomogeneous crumb cell structure of the control sample become progressively ner and more homogeneous at the 5100 ppm xylanase addition levels (Fig. 5). Effect of xylanase on bread shelf life The staling process of the control and xylanase-added crumbs was monitored by measuring crumb rmness. The added xylanase decreased the bread rming of all samples during storage (Fig. 6). Although rmness increased (almost linearly) over the 7-day storage for both control and xylanase-added crumbs, the latter were found to be less rm in all cases. The rming rate was reduced from 107 N d)1 in the control loaf to 08043 N d)1 in xylanaseadded loaves. Thus, addition of xylanase reduced the rming rate and provided an anti-staling effect. DISCUSSION Xylanase of T. lanuginosus CAU44 was puried to homogeneity with the 328% yield in the present investigation.

Fig. 3 Optimal temperature (a) and thermal stability (b) of the puried xylanse from Thermomyces lanuginosus CAU44. The optimal temperature was measured at different temperatures. For determination of thermal stability, the residual activity of the treated xylanase was measured after 30 min preincubation at different temperatures at pH 62

tested (Table 2). The highest activity (36517 U mg)1 and 1385%) was observed with oat-spelt xylan. Xylanase did not show any activity towards Avicel, carboxymethylcellulose, lter paper, starch and locust bean gum. No detectable activity towards pNP-b-D-xylopyranoside, pNP-b-D-glucopyranoside, pNP-b-D-cellobioside or pNP-b-D-mannopyranoside and pNP-a-L-arabinofuranoside was observed. Like xylanases from other T. lanuginosus strains, the puried xylanase only hydrolysed xylan and was free from all other enzyme activities examined including those of carboxymethylcellulase, b-xylosidase, b-D-glucosidase, b-cellobiosidase, b-D-mannosidase and a-arabinofuranosidase (Singh et al. 2003). The hydrolysis properties of the isolated wheat WE-AX and WU-AX were further studied using the xylanase. Similar hydrolysis pattern was observed in the hydrolysis of wheat WE-AX and WU-AX (data not shown). While increase of xylobiose was observed at the initial stage of hydrolysis, the xylose appeared to increase after 1 h of hydrolysis, suggesting the xylanase is an endoxylanase.

2005 The Society for Applied Microbiology, Letters in Applied Microbiology, 41, 6976, doi:10.1111/j.1472-765X.2005.01725.x

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(a) 150 Relative specific volume (%) 125 100 75 50 25 0

25

5 10 20 40 60 Enzyme dosage (ppm)

100

(b) 100 Relative crumb firmness (%)

75

50

25

0 0 25 5 10 20 40 60 100 Enzyme dosage (ppm)

Fig. 5 Effect of the xylanase on bread volume. (a) Photograph of bread loaves. (b) Photograph of cut loaves from the same breads. From left to right: control loaf, loaf with the puried xylanase 5, 10, 20, 40, 60 and 100 ppm (mg kg)1)

Crumb firmness (N)

Fig. 4 Effect of xylanase dosages on the specic volume (a) and crumb rmness (b) of fresh bread. The puried xylanase was added at 25 100 ppm (mg kg)1). At least three measurements were carried out for each sample

110 100 90 80 70 60 50 40 30 20 0 1 2 3 4 Time (d) 5 6 7

The molecular mass of xylanase from T. lanuginosus was found to be in the range of 225290 kDa (Singh et al. 2003). The apparent molecular mass of xylanase found in the strain (256 kDa) (Fig. 1) is similar to the value of 255 kDa for the strains DSM 5826 and DSM 10635 (Cesar a 1996; Xiong et al. 2004) and 257 kDa reported and Mrs for the strain ATCC 46882 (Bennett et al. 1998). The molecular mass is higher than the value of 225 kDa xylanase from the strain (Griffon and Maublanc) Bunce (Anand et al. 1990) and 236 kDa xylanase from the strain SSBP (Lin et al. 1999). The optimal pH for puried xylanase was pH 62 compared with pH 6070 from other strains of T. lanuginosus (Singh et al. 2003). Like xylanases characterized from different strains of T. lanuginosus, the xylanase was also stable over a wide pH range. However, the pH stability (pH 56103) of the puried xylanase was higher than pH 5090 of T. lanuginosus strains DSM 5826 and ATCC a 1996; Bennett et al. 1998) and 4682 (Cesar and Mrs pH 6090 of the strain (Griffon and Maublanc) Bunce

Fig. 6 Antistaling effect of xylanase-added bread crumbs stored at room temperature over a period of 7 d. At least three measurements were carried out for each sample. Enzyme dosage (ppm): (() control, ( ) 25, (r) 5, ( ) 10, (+) 20, ( ) 40, () 60 and ( ) 100

(Anand et al. 1990). The xylanase was optimally active at 75C compared with the 6070C from most strains of T. lanuginosus. The optimal temperature of xylanase in this study was similar to those of xylanases from the strains ATCC 46882 (Bennett et al. 1998) and SSBP (Lin et al.

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1999). Xylanases produced by T. lanuginosus strains have good thermostability so far (Singh et al. 2003). The thermal stability of xylanase of present investigation was similar to another xylanase from the strain ATCC 46882, which was almost completely stable up to 60C for 5 h (Bennett et al. 1998). This xylanase was stable up to 65C and retained 865% of its activity at 70C after 05 h of incubation. Thus, its thermal stability is an attractive feature for industrial applications. The important nding in this study is that the increase in the specic volume of bread by the xylanase was c. 30%, which was c. 10% higher than that obtained with the other xylanases reported (McCleary 1986; Maat et al. 1992; nez-Anaya and Jime nez 1997; Norma and Guillermo Mart 2003). The temperature prole at the center of the crumb was monitored in bread baking, and a typical baking process (i.e. three stages) was observed (data not shown). A possible explanation is that the reported xylanases act mainly on the dough at the rst stage (c. 60C) and it maintained its activity even at early second stage because this enzyme retained 865% of its activity at 70C after 05 h of incubation. Thus, the xylanase can enhance the breakdown of AXs efciently at the increased temperature, resulting in more oven spring during baking. The optimum enzyme level was 40 ppm for the best loaf volume and rmness values. The crumb rmness showed a 214% decrease, indicating that the improvement in the specic volume lowered the crumb rmness. Many results indicated that the use of xylanases could reduce initial crumb rmness nez-Anaya and Jime nez 1997; Laurikainen et al. (Mart 1998). At the same time, the deleterious effects of over dosage of some xylanases on loaf crumb structure are observed (McCleary 1986; Maat et al. 1992; Si 1997). However, this trend was not obvious in this study. Firmness of crumb is one of the most evident changes observed during bread storage. The inuence of xylanases on the process of bread staling is still being debated. Information on this aspect is confusing possibly because of the variety of xylanases exist. Some results indicated that added xylanases (or hemicellulases or pentosanases) do not modify the crumb-rming rate, but decrease the initial crumb rmness, possibly by increasing the loaf volume (Rouau et al. 1994). However, the results in other studies show that the addition of xylanases can decrease the staling nez-Anaya and Jime nez 1997; Laurikarate of bread (Mart inen et al. 1998). It has also been reported that xylanases are among different carbohydrases that exerted the greatest effect on the anti-staling during bread storage (Haros et al. 2002). All added xylanase retarded the staling rate of wheat breads by 1940% in this study. Besides, addition of the xylanase also increased the moisture content of the bread (data not shown). The moisture content of the fresh breads increased from 303 to 345% compared with 294%

moisture content of the bread without the enzyme. The staling rate was retarded possibly because of the breakdown of the polysaccharide network and the presence of more hygroscopic oligosaccharides. Hence, xylanase possibly induced retardation of the bread staling by reducing the initial crumb rmness and the rming process during storage. ACKNOWLEDGEMENTS This work was nancially supported by The National High Technology Research and Development Program of China (863 program, no. 2003AA214020). REFERENCES
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