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(India), and was routinely grown on Emerson's YpSs ... ª 2008 The Society for Applied Microbiology, Journal of Applied Microbiology 105 (2008) 1858–1865.
Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Phytase production by Sporotrichum thermophile in a cost-effective cane molasses medium in submerged fermentation and its application in bread B. Singh and T. Satyanarayana Department of Microbiology, University of Delhi South Campus, New Delhi, India

Abstract

Keywords bread, Ca-alginate, cane molasses, immobilization, phytase, Sporotrichum thermophile, surfactants, water activity. Correspondence Tulasi Satyanarayana, Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi – 110 021, India. E-mail: [email protected]

2008 ⁄ 0316: received 23 February 2008, revised 15 April 2008 and accepted 7 May 2008 doi:10.1111/j.1365-2672.2008.03929.x

Aims: Phytase production by Sporotrichum thermophile in a cost-effective cane molasses medium in submerged fermentation and its application in bread. Methods and Results: The production of phytase by a thermophilic mould S. thermophile was investigated using free and immobilized conidiospores in cane molasses medium in shake flasks, and stirred tank and air-lift fermenters. Among surfactants tested, Tweens (Tween-20, 40 and 80) and sodium oleate increased phytase accumulation, whereas SDS and Triton X-100 inhibited the enzyme production. The mould produced phytase optimally at aw 0Æ95, and it declined sharply below this aw value. The enzyme production was comparable in air-lift and stirred tank reactors with a marked reduction in fermentation time. Among the matrices tried, Ca-alginate was the best for conidiospore immobilization, and fungus secreted sustained levels of enzyme titres over five cycles. The phytic acid in the dough was efficiently hydrolysed by the enzyme accompanied by the liberation of soluble phosphate in the bread. Conclusions: The phytase production by S. thermophile was enhanced in the presence of Tween-80 in cane molasses medium. A peak in enzyme production was attained in 48 h in the fermenter when compared with that of 96 h in shake flasks. Ca-alginate immobilized conidiospores germinated to produce fungal growth that secreted sustained levels of phytase over five cycles. The bread made with phytase contained reduced level of phytic acid and a high-soluble phosphate. Significance and Impact of the Study: The phytase accumulation by S. thermophile was increased by the surfactants. The sustainability of enzyme production in stirred tank and air-lift fermenters suggested the possibility for scaling up of phytase. The bread made with phytase contained low level of antinutrient, i.e. phytic acid.

Introduction Phytase (myo-inositol hexakisphosphate 3-phosphorylase, EC 3Æ1Æ3Æ8.) catalyses the sequential release of phosphate from phytic acid (myo-inositol hexakisphosphate), the organic stored form of phosphorus present in various seeds and grains commonly used as raw materials in foods and feeds. Phytase reduces the antinutritional properties of phytic acid and eutrophication, caused by the excretion of undigested phytic acid by monogastrics 1858

because of the lack of adequate levels of phytase in their digestive tracts (Vohra and Satyanarayana 2003; Greiner and Konietzny 2006). Membrane proteins are tightly bound to the lipids by a combination of hydrophobic and ionic bonds, and therefore, more drastic procedures have to be applied for breaking these bonds, such as the use of detergents and aqueous organic solvents to enhance the secretion of microbial enzymes (Rega et al. 1967; Shibuya et al. 1968; Uma Maheswar Rao and Satyanarayana 2003). The permeabilization

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B. Singh and T. Satyanarayana

of membrane facilitates the secretion of proteins. Modification of the environment of the microbes by adding salts, polyalcohols, surfactants, sugars and polymers is an approach used for enhancing the production, stability and activity of enzymes (Uma Maheswar Rao and Satyanarayana 2003; Sharma et al. 2005). Fungi have been reported to possess complex nutrient utilization schemes, which ensure expression of required catabolic pathways under changing environmental conditions (Sharma et al. 2005). The microbial cells are commonly entrapped in a gel matrix through which substrates and products diffuse easily. Agar, agarose, j-carrageenan, collagen, alginate, chitosan or cellulose have been used for immobilizing microbes by entrapment. Some of these are expensive and also have weak mechanical strength. Alginate has been used for the entrapment of animal cells, mitochondria, chloroplasts, protoplasts and red blood cells (Park and Chang 2000). The mild conditions for immobilization and its simplicity are some of the reasons for choosing calcium alginate as the immobilization matrix. In the present investigation, an attempt has been made to select a suitable surfactant and immobilization matrix, which could support fungal growth as well as a high phytase production by Sporotrichum thermophile. Materials and methods Source of strain and culture conditions The thermophilic mould, S. thermophile BJTLR50 was isolated from a soil sample collected from Rohtak, Haryana (India), and was routinely grown on Emerson’s YpSs (Emerson 1941) agar medium. Phytase production The phytase was produced in 250-ml Erlenmeyer flasks containing 50 ml of the cane molasses medium (cane molasses 7%, MgSO4Æ7H2O 0Æ1% (NH4)2SO4 0Æ4%) with different surfactants (0Æ2%, v ⁄ v) or different concentrations of Tween-20 and Tween-80 and inoculated with 1 ml of conidiospore suspension (1 · 107 CFU ml)1) prepared from 6-day-old culture of S. thermophile on Emerson’s YpSs agar. The flasks were incubated at 45C in an incubator shaker and agitated at 250 rev min)1 for 4 days. The cultures were harvested and cell-free culture filtrates were used in phytase assays. Effect of Tween-80 and water activity on fungal growth and phytase production The mould was cultivated in optimized medium with or without Tween-80 up to 8 days. A set of flasks was har-

Phytase production by S. thermophile in cane molasses medium

vested at regular intervals and assayed for phytase, and determined fungal biomass and total sugar consumption. The mould was also cultivated in 50, 100, 200 and 400 ml of the production medium with Tween-80 (0Æ4% v ⁄ v) in 250-ml, 500-ml, 1-l and 2-l Erlenmeyer flasks, respectively. The effect of water activity (aw) on phytase production was studied by adjusting aw values (0Æ95–0Æ70) of the medium according to Grajek and Gervais (1987) using glycerol as a water activity depressant. Immobilization of fungal spores The conidiospore suspension of a 6-day-old culture was prepared in normal saline containing 0Æ1% (v ⁄ v) Tween80, washed and mixed with sterile Na-alginate (4%) solution. The mixture was extruded through a sterile syringe needle into a chilled sterile solution of 0Æ2 mol l)1 CaCl2 aseptically with constant stirring (Quan et al. 2003). The spherical beads of an average diameter of 3 mm were formed. The beads were cured in the same solution for 1 h, washed with sterile double distilled water and stored at 4C until use. In preparing other matrices, 4% of j-carrageenan, agar and 10% of polyacrylamide (non-denaturing) were used. The gels were mixed well with spore suspension and poured into sterile petriplates, allowed to harden and cut into blocks (5 · 5 · 3 mm3). Fifty Ca-alginate beads ⁄ 40 blocks of other matrices (1 · 107 CFU) were used for inoculating 50 ml medium in 250-ml Erlenmeyer flasks. All experiments were carried out in triplicate and their mean values are presented. Phytase assay Phytase was assayed by determining the amount of inorganic phosphate liberated according to Fiske and Subbarow (1925) using sodium phytate as the substrate at 60C and pH 5Æ0 (Singh and Satyanarayana 2006, 2008a,b). One unit of phytase is defined as the amount of enzyme required to liberate 1 nmol of inorganic phosphate per second per millilitre under the assay conditions. Biomass, total sugar, inorganic phosphate, soluble protein and phytic acid estimation Fungal biomass was estimated gravimetrically by filtering the culture broth through a pre-weighed dry Whatman No. 1 filter paper circles. The mycelium was thoroughly washed with double distilled water and dried at 80C to constant weight. Freshly prepared anthrone reagent was used to estimate total sugars in the culture filtrate according to Brink et al. (1960). Phytic acid content of the bread was determined by extracting with 0Æ2 N HCl with

ª 2008 The Authors Journal compilation ª 2008 The Society for Applied Microbiology, Journal of Applied Microbiology 105 (2008) 1858–1865

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Phytase production by S. thermophile in cane molasses medium

constant shaking at 200 rev min)1 for 1 h. The suspension was centrifuged at 8000 g for 15 min and the supernatant was used for estimating phytic acid according to Haug and Lantzsch (1983). Reducing sugars and soluble protein were determined according to Miller (1959) and Lowry et al. (1951), respectively. The inorganic phosphate was estimated according to Fiske and Subbarow (1925).

B. Singh and T. Satyanarayana

cutting. This process was performed with phytase of S. thermophile alone and together with a-amylase of Geobacillus thermoleovorans. The bread prepared under normal conditions without these enzymes (but with commercial enzymes, xylanase and amylase 500 U each) was treated as the control. The breads were assessed for phytate content, soluble inorganic phosphate, reducing sugars and soluble protein.

Scanning electron microscopy of alginate beads

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Wheat flour (500 g) was mixed with dry yeast (4Æ5 g), 1Æ5% NaCl, 2Æ0% sucrose, 10 ml sunflower oil and phytase 500 U ⁄ 250 U of phytase + 65 U of a-amylase, blended with 60% water, and mixed mechanically for 30 min to produce dough (Uma Maheswar Rao and Satyanarayana 2007). This was allowed to undergo proofing by fermentation for an extended period of up to 40 min, followed by baking at 250C for 30–40 min, shaping and

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The role of Sporotrichum thermophile phytase in bread-making

Sporotrichum thermophile secreted phytase optimally at pH 5Æ0 and 45C after 4 days in cane molasses medium. Among different surfactants tested, a high phytase accumulation was recorded in a medium containing non-ionic detergents (Tweens) when compared with that in control (Fig. 1). The enzyme titre was higher in Tween-80 than that in Tween-20 and Tween-40. While Triton X-100, a non-ionic detergent, and SDS, an anionic detergent inhibited enzyme production. The phytase titres were high in the presence of 0Æ4% Tween-80 (10 177 U l)1) and 0Æ5% Tween-20 (8487 U l)1). Tween-80 enhanced phytase production by the mould with out affecting the growth of the mould (Fig. 2a,b). The enzyme production in the medium containing Tween-80 was sustainable in shake flasks of varied volumes (Table 1). A high phytase production and fungal growth was achieved at aw of 0Æ95 and it declined drastically below this value (Table 2). There were no growth and phytase production at or below 0Æ85 aw.

C

The phytase production was studied in a 22-l Biostat C air-lift fermenter (B. Braun, Melsungen, Germany). The fermenter containing 10-l medium (g l)1: cane molasses 70; MgSO4Æ7H2O 1Æ0; (NH4)2SO4 4Æ0 and Tween-80 4Æ0) was operated at 45C, 2 vvm of aeration and the pH was maintained at 5Æ0 ± 0Æ05 with sterile 2 N HCl ⁄ NaOH. The samples were withdrawn at the desired intervals aseptically and the culture filtrates were used for the determining phytase titre, biomass, total sugars and inorganic phosphate. In the second fermentation experiment, the air was supplemented with pure oxygen in order to maintain the pO2 level of the medium at 100%. The samples were withdrawn at regular intervals for analysis. Under similar conditions, fermentation was also conducted in the stirred tank bioreactor with aeration rate of 1 vvm and 250 rev min)1 agitation, without maintaining pH of medium.

Effect of surfactants and water activity on phytase production

Tr it

Fermentation in air-lift and stirred tank bioreactors

Results

Phytase production (U l–1)

Fungal growth in and on the alginate beads was visualized by scanning electron microscopy. The beads were washed with phosphate buffer (0Æ1 mol l)1, pH 7Æ2), fixed for 4 h in glutaraldehyde (2Æ5%) and then washed again with phosphate buffer. This was followed by dehydration in ascending grades of alcohol (30%, 50%, 70%, 80%, 90%, 100%) each for 30 min. The samples were dried in 1,1,1,3,3,3,-hexamethyldisilazane (HMDS) and mounted on stubs with silver glue for conduction, and examined under the scanning electron microscope (Philips SEM-50B).

Surfactants (0·2%) Figure 1 Effect of surfactants on phytase production by Sporotrichum thermophile.

ª 2008 The Authors Journal compilation ª 2008 The Society for Applied Microbiology, Journal of Applied Microbiology 105 (2008) 1858–1865

B. Singh and T. Satyanarayana

Phytase production by S. thermophile in cane molasses medium

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Figure 3 Phytase production by Sporotrichum thermophile agitated fermenter in optimized cane molasses medium. Biomass (g l–1)

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Figure 2 (a) Growth profile and phytase production by Sporotrichum thermophile with out Tween-80. (b) Effect of Tween-80 on growth profile and phytase production.

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Phytase production (U l)1) ± SD

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Medium (ml)

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10 Biomass (g l–1)

Table 1 Effect of Tween-80 (0Æ4%) on phytase production in shake flasks of varied volumes and in a 22l fermenter

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Figure 4 (a) Phytase production by Sporotrichum thermophile in airlift fermenter in cane molasses medium. (b) Phytase production by Sporotrichum thermophile in air-lift fermenter in cane molasses medium supplied with pure oxygen.

*Fermenter.

Table 2 Effect of water activity on growth and phytase production by Sporotrichum thermophile Water activity (aw)

Phytase production (U l)1) ± SD

Biomass (g l)1)

0Æ95 0Æ92 0Æ87 0Æ85 0Æ80

10 120 8247 5520 82 0

7Æ54 4Æ80 3Æ43 0Æ46 0

± ± ± ±

482Æ6 230Æ3 286Æ9 4Æ1

± ± ± ±

0Æ14 0Æ21 0Æ09 0Æ02

Phytase production in stirred tank and air-lift fermenters The phytase production by S. thermophile was comparable in 22-l stirred tank (Fig. 3) and air-lift (Fig. 4) fermenters. A peak in phytase production was attained in 48 h in the fermenter when compared with that of 96 h in shake flasks (Fig. 4a). Sparging of the medium with air supplemented with oxygen did not exert any observable effect

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Phytase production by S. thermophile in cane molasses medium

on phytase production. There was, however, a slight improvement in the fungal biomass (Fig. 4b). Immobilization of conidiospores for phytase production Phytase production by immobilized fungus within different gels was studied in batch fermentation. Ca-alginateentrapped conidiospores germinated to produce fungal growth that secreted higher enzyme titres than those entrapped in other matrices (Table 3) and comparable with free cells. Phytase production was slightly higher with alginate beads than the free spores. Therefore, Ca-alginate was selected for further experiments. Among different concentrations of sodium alginate, 4% supported high phytase production when compared with lower and higher concentrations (Table 4). Increasing the sodium alginate concentration above 4Æ0% decreased the phytase production. A peak in the phytase production was achieved after 96 h by immobilized spores as with free spores (Table 3). The sustained levels of phytase titres were achieved up to five repeated batch fermentations, which declined thereafter. Mycelia grew densely around and within the alginate matrix. The diameter of the beads increased gradually with every fermentation cycle and the mycelial growth outside the bead was noticeable with the naked eye. The scanning electron micrographs further confirmed luxuriant mycelial growth inside and outside the alginate matrix (Fig. 5), without any trace of conidiospores. Table 3 Phytase production by Sporotrichum thermophile immobilized in different matrices up to three repeated batch fermentations Phytase production (U l)1 ± SD)* Matrix

1st Cycle

2nd Cycle

Free cells Agar Ca-alginate j-Carrageenan Polyacrylamide

9896 8187 9968 9067 7470

9467 8924 10 170 9645 7200

± ± ± ± ±

424Æ6 220Æ3 422Æ8 392Æ0 198Æ5

± ± ± ± ±

3rd Cycle 250Æ2 301Æ2 488Æ3 276Æ8 180Æ1

8260 8246 10 056 8877 7290

± ± ± ± ±

150Æ2 262Æ8 450Æ3 269Æ0 176Æ2

*Mean of three values. Each cycle = 96 h.

Table 4 Effect of sodium alginate production up to three repeated cycles Sodium alginate (%) 2 4 6

on

phytase

Phytase production (U l)1 ± SD)* 1st Cycle

2nd Cycle

3rd Cycle

8570 ± 320Æ8 10 089 ± 402Æ7 8875 ± 275Æ0

8879 ± 189Æ5 11 024 ± 352Æ2 8578 ± 200Æ8

8235 ± 300Æ0 10 206 ± 522Æ8 8144 ± 188Æ0

*Mean of three values.

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The role of Sporotrichum thermophile phytase in bread-making The supplementation of dough with phytase from S. thermophile resulted in the liberation of higher inorganic phosphate (3Æ2 mg g)1 bread), higher reducing sugars (42Æ4 mg g)1 bread) and higher soluble protein (7Æ86 mg g)1 bread) than the control bread made with commercial enzymes (1Æ02, 28Æ4 and 5Æ3 mg g)1 bread, respectively). The addition of a-amylase and phytase to the dough further improved the quality and properties of the bread when compared with the control bread prepared using commercial enzymes (Table 5). Discussion Surfactants are known to affect the growth and enzyme production in fungi (Nampoothiri et al. 2004), and therefore, have been used in biotechnology for improving the yield of a number of enzymes produced by fermentation. Among surfactants, a high phytase titre was attained in the medium containing sodium oleate and non-ionic detergents (Tween-20, 40 and 80). However, Triton X-100 (a non-ionic detergent) and SDS (an anionic detergent) inhibited enzyme production in S. thermophile. This could be due to increased viscosity of the medium by high concentration of the surfactants that results in decreased oxygen transfer rate or due to the toxic effect of surfactants (Uma Maheswar Rao and Satyanarayana 2003). Detergents are known to solubilize membrane proteins that lead to an increase in cell membrane permeability with concomitant enhancement in the secretion of biomolecules (Ne’eman et al. 1971; Uma Maheswar Rao and Satyanarayana 2003). Similarly, Tweens as well as oleic acid increased phytase production by Aspergillus niger (El-Batal and Abdel Karem 2001) and A. carbonarius (Ebune et al. 1995), while Triton X-100 exerted a negative effect. Tween-20 and Tween-80 have also been reported to enhance phytase accumulation in a thermophilic mould Thermoascus aurantiacus (Nampoothiri et al. 2004). The production of laccase by a Ganoderma sp. kk-02 (Sharma et al. 2005) and a-amylase by G. thermoleovorans (Uma Maheswar Rao and Satyanarayana 2003) was also improved by Tween-40. The fungal growth in S. thermophile was not so much affected by the surfactant, suggesting the possible role of cell permeabilization in the enhanced enzyme secretion. The production of phytase in the medium containing Tween-80 (0Æ4%, v ⁄ v) was not affected when the mould was grown in Erlenmeyer flasks up to 1 l capacity, but declined at higher volume. This could be due to improper mixing of nutrients and inadequate aeration on increasing the volume of the medium (Uma

ª 2008 The Authors Journal compilation ª 2008 The Society for Applied Microbiology, Journal of Applied Microbiology 105 (2008) 1858–1865

B. Singh and T. Satyanarayana

Phytase production by S. thermophile in cane molasses medium

(b)

(a)

(c)

10 µm

200 µm

10 µm

Figure 5 (a) Scanning electron micrograph showing the single calcium alginate bead (34·) (b) luxuriant mycelial growth around the bead (500 · ) and (c) inside the alginate matrix (500 · ). Table 5 Comparison of properties of breads prepared by the action of Sporotrichum thermophile phytase together with a-amylase of Geobacillus thermoleovorans (test bread) and bread prepared by commercial enzymes (control bread)

Property of bread Weight of dough (g) Dough rise (cm) Weight of bread (g) Bread moisture (%) Height of bread (cm) Soluble protein content (mg g)1 bread) Reducing sugars (mg g)1 bread) Inorganic phosphate (mg g)1 bread) Phytic acid reduction (%)

Test bread

Control bread

472 5Æ0 437 40 8Æ95 8Æ8

474 5Æ13 437 35 8Æ9 6Æ24

51Æ68

42Æ81

1Æ56

0Æ540

39Æ2

12Æ0

Maheswar Rao and Satyanarayana 2003; Singh and Satyanarayana 2008a). The addition of glycerol as a depressant of water activity decreased phytase production drastically. A high phytase production was attained at aw 0Æ95. Fungal growth as well as phytase production declined at lower aw values. A similar observation was made in mycelial growth and exopectinase production by A. niger in SSF as well as Smf (Godinez et al. 2001). Sporotrichum thermophile also secreted a high titre of phytase in SSF at aw 0Æ95 (Singh and Satyanarayana 2006), thus confirming that the mould is desiccation sensitive. A comparable enzyme production was attained in 0Æ25– 2 l flasks, and there was a slight enhancement in the 22-l fermenter with concomitant reduction in the fermentation time as reported earlier (Kumar et al. 2007; Singh and Satyanarayana 2008a). The enzyme titres were almost similar in air-lift and stirred tank bioreactors. Similarly, glucoamylase production by a thermophilic mould

Thermomucor indicae-seudaticae was sustainable in stirred tank and air-lift fermenters with concomitant reduction in fermentation time (Kumar et al. 2007). A resurgence in the use of immobilized whole cells and biocatalysts has been witnessed in the past few years, as it is a very useful technique that permits efficient reuse of cells and improves cost-effectiveness of the process (Hemachander et al. 2001). Immobilization involving entrapment of cells in different polysaccharides has been the most successful approach, which allows the retention of cell viability and activity by making the required nutrients available for growth leading to high cell densities (Bucke 1987; D’Souza 1989). Ca-alginate was found to be the most suitable matrix for entrapment of S. thermophile conidiospores for phytase production. Ca-alginate is probably the best polysaccharide for cell immobilization because of its simplicity and being food grade polysaccharide, which could find potential applications in food biotechnological processes (D’Souza 1989). Alginate has been successfully utilized for immobilization of Candida krusei cells for phytase production (Quan et al. 2003). Lower enzyme yields in S. thermophile spores entrapped in other gel matrices could be attributed to mechanical and diffusion limitations in these matrices (D’Souza 1989). The concentration of alginate is known to affect the growth and enzyme production by the immobilized cells (Blandino et al. 2000). In this investigation, 4% alginate was found to be the most suitable for phytase production as reported by Quan et al. (2003) for phytase production by C. krusei cells. Similarly, Bacillus licheniformis 44MB82 immobilized in 4% secreted high titre of a-amylase (Dobreva et al. 1996). This is explained by the fact that with the increase in sodium alginate beyond 4%, the thickness of the bead increased (Blandino et al. 2000), and this is presumably as a result of increase in the number of biopolymer molecules per unit solution and the binding sites for Ca2+, leading to densely cross-linked gel structure that causes diffusional resistance. This results in

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lower-product formation as nutrients and substrates are restricted to diffuse into the cells. When only 2% sodium alginate concentration was used, the beads produced were too soft, and thus were easily disrupted because of their low mechanical strength causing spore leakage. The enzyme production by immobilized spores was sustainable over five successive batches and declined thereafter, as reported in glucoamylase production by A. phoenicus (Kuek 1991) and over eight batches by T. indicae-seudaticae (Kumar and Satyanarayana 2007). The heavy mycelial growth around the bead might cause resistance to nutrients and oxygen transfer, and therefore, lower enzyme titres after five batches. Scanning electron microscopy revealed extensive mycelial growth inside and around the beads, thereby confirming sufficient availability of oxygen and nutrients, which might have become limiting after five batches of fermentation leading to lower phytase titres. There was no sporulation throughout the repeated batch fermentations suggesting that stress conditions did not set in beads because of the supply of fresh nutrients and removal of any toxic metabolites produced during fermentation. The stress conditions are known to trigger sporulation in microbes. The supplementation of phytase of S. thermophile to the dough increased the inorganic phosphate, reducing sugar and soluble protein in the bread when compared with bread prepared with commercial enzymes. The addition of a-amylase of G. thermoleovorans with phytase to dough resulted in improved qualities of bread with concomitant reduction in phytic acid content (39Æ2%). The shelf life of bread was improved by the supplementation of dough with a-amylase of G. thermoleovorans and the enzyme was found to be useful in preventing the staling of the bread (Uma Maheswar Rao and Satyanarayana 2007). Phytase promoted the fermentation process, improvement in the shape, a slight increase in specific volume, and also better crumb structures (Haros et al. 2001a,b). Additionally, the phytate content in the bread was reduced by the addition of phytase. A process for the preparation of chapathi dough with reduced phytic acid levels was developed using a mutated strain of the yeast Candida versatilis, which brought down 10–45% reduction in phytate levels (Bindu and Varadaraj 2005). Sesame oil cake (Singh and Satyanarayana 2006) and soymilk (Singh and Satyanarayana 2008b) were also dephytinized by the phytase of S. thermophile. This clearly suggested that S. thermophile phytase could be useful in ameliorating the nutritional value of food and feed ingredients. The inclusion of different surfactants such as Tween-80 in the medium enhanced phytase accumulation by S. thermophile. The reduction in water activity of the medium drastically affected the enzyme yield as well as fungal growth. A comparable phytase production was 1864

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attained in air-lift and stirred tank bioreactors. The fungus was immobilized by entrapping conidiospores in the Ca-alginate gel that secreted sustained phytase over five repeated cycles. The enzyme degraded phytic acid efficiently in the dough during bread-making with concomitant liberation of inorganic phosphate. The enzyme, being thermostable, acid stable and protease insensitive, can find applications in food and feed industries. Acknowledgements The authors wish to thank Mr Pathania and Mr Girish of All India Institute of Medical Sciences, New Delhi for carrying out scanning electron microscopy and Mr Vijay Kumar Gupta (Tushar Nutritive Food Industry, New Delhi) for extending help in assessing the applicability of phytase in bread-making. BS gratefully acknowledges the financial assistance from the Council of Scientific and Industrial Research, New Delhi, India during the course of this investigation. References Bindu, S. and Varadaraj, M.C. (2005) Process for the preparation of Chapathi dough with reduced phytic acid level. United States Patent Application #20050048165. Blandino, A., Macıas, M. and Cantero, D. (2000) Glucose oxidase release from calcium alginate gel capsules. Enzyme Microb Technol 27, 319–324. Brink, R.H., Dubach, P. and Lynch, D.L. (1960) Measurement of carbohydrate in soil hydrolysates with anthrone. Soil Sci 89, 157–166. Bucke, C. (1987) Cell immobilization in calcium alginate. In Methods in Ezymology, Vol 135B. ed. Mosbach, K. pp. 175–189. New York: Academic press. D’Souza, S.F. (1989) Immobilised cells: techniques and applications. Indian J Microbiol 29, 83–117. Dobreva, E., Ivanova, V., Tonkova, A. and Radulova, E. (1996) Influence of the immobilization conditions on the efficiency of a-amylase production by Bacillus licheniformis. Process Biochem 313, 229–234. Ebune, A., Al-Asheh, S. and Duvnjak, Z. (1995) Effects of phosphate, surfactants and glucose on phytase production and hydrolysis of phytic acid in canola meal by Aspergillus ficuum during solid-state fermentation. Bioresour Technol 54, 241–247. El-Batal, A.I. and Abdel Karem, H. (2001) Phytase production and phytic acid reduction in rapeseed meal by Aspergillus niger during solid state fermentation. Food Res Intern 34, 715–720. Emerson, R. (1941) An experimental study of the life cycles and taxonomy of Allomyces. Lloydia 4, 77–144. Fiske, C.H. and Subbarow, Y. (1925) The colorimetric determination of phosphorous. J Biol Chem 65, 375–380.

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and enzymic activities of Acholeplasma laidlawii membrane proteins. Biochim Biophys Acta 249, 169–179. Park, J.K. and Chang, H.N. (2000) Microencapsulation of microbial cells. Biotechnol Adv 18, 303–319. Quan, C.S., Fan, S.D. and Ohta, Y. (2003) Immobilization of Candida krusei cells producing phytase in alginate gel beads: an application of the preparation of myo-inositol phosphates. Appl Microbiol Biotechnol 62, 41–47. Rega, A.F., Weed, R.I., Reed, C.F., Berg, E.G. and Rothsteen, H. (1967) Changes in the properties of human erythrocyte membrane protein after solubilization by butanol extraction. Biochim Biophys Acta 147, 297–312. Sharma, K.K., Kapoor, M. and Kuhad, R.C. (2005) In vivo enzymatic digestion, in vitro xylanase digestion, metabolic analogues, surfactants and polyethylene glycol ameliorate laccase production from Ganoderma sp. kk-02. Lett Appl Microbiol 41, 24–31. Shibuya, I., Honda, H. and Maruo, B. (1968) Stepwise solubilization of chloroplast lamellae by a non-ionic detergent P-40. J Biochem 64, 571–576. Singh, B. and Satyanarayana, T. (2006) Phytase production by thermophilic mould Sporotrichum thermophile in solid state fermentation and its application in dephytinization of sesame oil cake. Appl Biochem Biotechnol 133, 239–250. Singh, B. and Satyanarayana, T. (2008a) Phytase production by Sporotrichum thermophile in solid state fermentation and its applications. Bioresour Technol 99, 2824–2830. Singh, B. and Satyanarayana, T. (2008b) Improved phytase production by a thermophilic mould Sporotrichum thermophile in submerged fermentation due to statistical optimization. Bioresour Technol 99, 824–830. Uma Maheswar Rao, J.L. and Satyanarayana, T. (2003) Enhanced secretion and low temperature stabilization of a hyperthermostable and Ca2+-independent a-amylase of Geobacillus thermoleovorans by surfactants. Lett Appl Microbiol 36, 191–196. Uma Maheswar Rao, J.L. and Satyanarayana, T. (2007) Improving production of hyperthermostable and high maltose-forming a-amylase by an extreme thermophile Geobacillus thermoleovorans using response surface methodology and its applications. Bioresour Technol 98, 345–352. Vohra, A. and Satyanarayana, T. (2003) Phytases: microbial sources, production, purification and potential biotechnological applications. Crit Rev Biotechnol 23, 29–60.

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