Phytase production by a thermophilic mould Sporotrichum thermophile in solid state fermentation and its potential applications

Phytase production by a thermophilic mould Sporotrichum thermophile Apinis was investigated in solid state fermentation (SSF) using sesame oil cake as the substrate. Scanning electron microscopy of the fermented sesame oil cake revealed a dense growth of the mould with abundant conidia. Glucose, ammonium sulphate and incubation period were identified as the most significant factors by Plackett-Burman design. The optimum values of the critical components determined by central composite design of response surface methodology for the maximum phytase production were glucose 3%, ammonium sulphate 0.5% and incubation period 120 h. An overall 2.6-fold improvement in phytase production was achieved due to optimization. Highest enzyme production (348.76 U/g DMR) was attained at a substrate bed depth of 1.5 cm in enamel coated metallic trays. The enzyme liberated inorganic phosphate from wheat flour and soymilk with concomitant dephytinization and liberation of soluble inorganic phosphate.     

High level phytase production by <Emphasis Type="Italic">Aspergillus niger</Emphasis> NCIM 563 in solid state culture: response surface optimization,up-scaling,and its partial characterization

Phytase production by Aspergillus niger NCIM 563 was optimized by using wheat bran in solid state fermentation (SSF). An integrated statistical optimization approach involving the combination of Placket–Burman design (PBD) and Box–Behnken design (BBD) was employed. PBD was used to evaluate the effect of 11 variables related to phytase production, and five statistically significant variables, namely, glucose, dextrin, NaNO₃, distilled water, and MgSO₄·7H₂O, were selected for further optimization studies. The levels of five variables for maximum phytase production were determined by a BBD. Phytase production improved from 50 IU/g dry moldy bran (DMB) to 154 IU/g DMB indicating 3.08-fold increase after optimization. A simultaneous reduction in fermentation time from 7 to 4 days shows a high productivity of 38,500 IU/kg/day. Scaling up the process in trays gave reproducible phytase production overcoming industrial constraints of practicability and economics. The culture extract also had 133.2, 41.58, and 310.34 IU/g DMB of xylanase, cellulase, and amylase activities, respectively. The partially purified phytase was optimally active at 55°C and pH 6.0. The enzyme retained ca. 75% activity over a wide pH range 2.0–9.5. It also released more inorganic phosphorus from soybean meal in a broad pH range from 2.5 to 6.5 under emulated gastric conditions. Molecular weight of phytase on Sephacryl S-200 was approximately 87 kDa. The K _m and V _max observed were 0.156 mM and 220 μm/min/mg. The SSF phytase from A. niger NCIM 563 offers an economical production capability and its wide pH stability shows its suitability for use in poultry feed.     

Statistical optimization of a thermostable and neutral glucoamylase production by a thermophilic mold <Emphasis Type="Italic">Thermomucor indicae-seudaticae</Emphasis> in solid-state fermentation

Glucoamylase production by a thermophilic mold Thermomucor indicae-seudaticae was optimized in solid-state fermentation (SSF) by conventional one variable at a time approach and further statistically using response surface methodology (RSM). Glucoamylase secretion was strongly affected by three variables (moisture ratio, inoculum level and incubation time), and therefore, these three factors were further optimized using response surface methodology. The glucoamylase production in flasks containing wheat bran, under the conditions optimized by RSM, was 455 ± 23 U/g of dry moldy bran (DMB), while the predicted value by a polynomial model was 433.30 U/g DMB. The enzyme titre (455 ± 23 U/g DMB) attained in the validation experiment of this investigation is higher than those reported in the literature. When the large-scale production was attempted in enamel trays, a marginally lower enzyme titres were attained. An overall 1.8-fold increase in glucoamylase production was achieved in SSF due to statistical optimization in comparison with that of one variable at a time approach (250 ± 13 U/g DMB). A 10-fold enhancement in glucoamylase production was recorded in SSF as compared to that in submerged fermentation.     

Response surface methodology for the optimization of <Emphasis Type="Italic">α</Emphasis>-amylase production by <Emphasis Type="Italic">Streptomyces</Emphasis> sp. ML12 using agricultural byproducts

Amylases constitute one of the most important groups of enzymes for commercial use. In the present study, production of α-amylase was optimized using a newly isolated actinobacterial strain from the coral reef environment of the Gulf of Mannar Biosphere Reserve, India. It was identified as Streptomyces sp. ML12 based on chemotaxonomy, cultural and morphological characteristics, carbon source utilization and 16S rRNA gene sequencing. Fermentation variables were selected in accordance with the Plackett-Burman design and were optimized by response surface methodology. Five significant variables (rice bran and wheat bran — both agricultural byproducts, sodium chloride, magnesium sulphate and incubation period) were selected for the optimization via central composite design. The optimal features were rice bran (5.5 g/100 mL), wheat bran (5.3 g/100 mL), sodium chloride (2.8 g/100 mL), magnesium sulphate (1.4 g/100 mL) and 8 days of incubation period. Optimization of the medium with the above tested features increased the amylase yield by 4.4-fold.     

Optimization of medium composition for alkali-stable xylanase production by Aspergillus fischeri Fxn 1 in solid-state fermentation using central composite rotary design

Response surface methodology and central composite rotary design (CCRD) was employed to optimize a fermentation medium for the production of alkali-stable cellulase-free xylanase by Aspergillus fischeri in solid-state fermentation at pH 9.0 with wheat bran as substrate. The four variables involved in this study were sodium nitrite, potassium dihydrogen phosphate, magnesium sulphate and yeast extract. The statistical analysis of the results showed that, in the range studied, only sodium nitrite had a significant effect on xylanase production. The optimized medium containing (in g/l) NaNO(2)-7.0, K2HPO(4)-1.0, MgSO(4)-0.5 and yeast extract-5.0 resulted in 1.9-fold increased level of alkali-stable xylanase (1024 U/g wheat bran) production compared to initial level (540 U/g) after 72 h of fermentation, whereas its value predicted by the quadratic model was 931 U/g. The level of protease activity was considerably decreased in optimized medium, thus helping to preserve the xylanase activity and demonstrating another advantage of applying statistical experimental design.     

Production of high activity thermostable phytase from thermotolerant Aspergillus niger in solid state fermentation

The thermotolerant fungus, Aspergillus niger NCIM 563, was used for production of extracellular phytase on agricultural residues: wheat bran, mustard cake, cowpea meal, groundnut cake, coconut cake, cotton cake and black bean flour in solid state fermentation (SSF). Maximum enzyme activity (108 U g⁻¹ dry mouldy bran, DMB) was obtained with cowpea meal. During the fermentation phytic acid was hydrolysed completely with a corresponding increase in biomass and phytase activity within 7 days. Phosphate in the form of KH₂PO₄ (10 mg per 100 g of agriculture residue) increased phytase activity. Among various surfactants added to SSF, Trition X-100 (0.5%) exhibited a 30% increase in phytase activity. The optimum pH and temperature of the crude enzyme were 5.0 and 50°C respectively. Phytase activity (86%) was retained in buffer of pH 3.5 for 24 h. The enzyme retained 75% of its activity on incubation at 55°C for 1 h. In the presence of 1 mM K⁺ and Zn²⁺, 95% and 55% of the activity were retained. Scanning electron microscopy showed a high density growth of fungal mycelia on wheat bran particles during SSF. Journal of Industrial Microbiology & Biotechnology (2000) 24, 237–243. Received 07 June 1999/ Accepted in revised form 18 December 1999     

Comparison of phytase production on wheat bran and oilcakes in solid-state fermentation by Mucor racemosus

Comparisons were made for phytase production using wheat bran (WB) and oilcakes as substrates in solid-state fermentation (SSF) by Mucor racemosus NRRL 1994. WB was also used as mixed substrate with oil cakes. Sesame oil cake (SOC) served as the best carbon source for phytase synthesis by the fungal strain as it gave the highest enzyme titres (30.6 U/gds). Groundnut oil cake (GOC) also produced a reasonably good quantity of enzyme (24.3 U/gds). Enzyme production on WB was surprisingly much less (almost 3.5 times less in comparison to SOC). Mixing WB with SOC (1:1 ratio) resulted in better phytase activity (32.2 U/gds). Optimization of various process parameters such as incubation time, initial moisture content and inoculum concentration was carried out using the single variable mode optimization technique. Under optimized conditions, the production of phytase reached 44.5 U/gds, which was almost 1.5-fold higher than the highest yield obtained with any individual substrate used in this study and was more than 4-fold higher than that obtained from WB.     

Optimizing the fermentation conditions and enhanced production of keratinase from Bacillus cereus isolated from halophilic environment

Keratinase degrading Bacillus cereus was isolated from the halophilic environment in Tamilnadu, India and keratinase production was optimized using wheat bran substrate. Of the screened bacterial isolates, four were found to have the ability to produce keratinolytic enzyme. The process parameters were optimized using one-variable-at-a-time approach and response surface methodology. Supplementation of 1% lactose supported more keratinase production (120?U/g). Among the selected nitrogen sources, addition of casein significantly enhanced maximum keratinase production (132.5?U/g). Among the ions, manganese chloride significantly enhanced keratinsase production (102.6?U/g), however addition of zinc sulphate and copper sulphate decreased keratinase production. The maximum keratinase production was obtained in the wheat bran medium containing 1% lactose, 0.5% manganese with 80% moisture (292?U/g). Statistics based contour plots were generated to explore the variations in the response surface and to find the relationship between the keratinase yield and the bioprocess conditions.     

A marked enhancement in phytase production by a thermophilic mould Sporotrichum thermophile using statistical designs in a cost-effective cane molasses medium

AIMS: Statistical optimization of phytase production by a thermophilic mould Sporotrichum thermophile in a cost-effective cane molasses medium. METHODS AND RESULTS: Sporotrichum thermophile secreted phytase in cane molasses medium at 45 degrees C and 250 rev min(-1) after 5 days. The important factors identified by Plackett-Burman design (magnesium sulfate, Tween 80, ammonium sulfate and incubation period) were further optimized by response surface methodology (RSM). An overall 107% improvement in phytase production was achieved due to optimization. Supplementation of the medium with inorganic phosphate repressed the enzyme synthesis. When inorganic phosphate was reduced from the cane molasses medium by treatment with calcium chloride, the enzyme production increased. The phytase activity was not affected by the enzyme treatment with trypsin and pepsin. CONCLUSIONS: A twofold increase in phytase production was achieved due to optimization using statistical designs in a cost-effective cane molasses medium. SIGNIFICANCE AND IMPACT OF THE STUDY: Phytase production was doubled due to optimization. The enzyme, being resistant to trypsin and pepsin, thermostable and acid stable, can find application in animal feed industry for improving nutritional status of the feed and combating environmental phosphorus pollution.     

Phytase from Citrobacter koseri PM-7: Enhanced production using statistical method and application in ameliorating mineral bioaccessibility and protein digestibility of high-phytate food

The present study was aimed at enhancing phytase (Phy-Ck) production from Citrobacter koseri PM-7 using response surface methodology (RSM) and improving the bioaccessibility of minerals (Fe and Zn) and protein digestibility in high-phytate food using Phy-Ck. A five-variable and three-level central composite design of RSM using wheat bran (6.681%, w/v), inoculum level (2.5%, v/v), and triton X-100 (0.2%, v/v) resulted in up to 5.57-fold (1.047 U/ml) improvement in Phy-Ck yield from C. koseri PM-7 when compared with fermentation media I and II. The model was successfully validated in the design space by taking a random set of variable combinations. Treatment of high-phytate food with partially purified Phy-Ck showed improvement in mineral bioaccessibility maximally for defatted sesame flour (DSF) (Fe 45.5%; Zn 50.7%) followed by wheat flour (WF) (Fe 13.5%; Zn 14.4%), green gram flour (GGF) (Fe 0.7%; Zn 3.8%) and defatted groundnut flour (DGF) (Zn 5.6%). The in vitro protein digestibility (IVPD) of WF increased from 48.83 to 65.04%, GGF from 45.04 to 57.12%, and DSF from 47.34 to 55.7% after Phy-Ck treatment.     

Improved phytase production by a thermophilic mould Sporotrichum thermophile in submerged fermentation due to statistical optimization

Culture variables affecting phytase production by a thermophilic mould Sporotrichum thermophile in submerged fermentation were optimized. Soluble starch, peptone, Tween-80 and sodium phytate were identified by Plackett-Burman design as the most significant factors to affect phytase production. The 2(4) full factorial central composite design of response surface methodology was applied for optimizing the concentrations of the significant variables and to delineate their interactions. Starch, Tween-80, peptone and sodium phytate at 0.4%, 1.0%, 0.3% and 0.3% supported maximum enzyme titres, respectively. An overall 3.73-fold improvement in phytase production was achieved due to optimization. When sodium phytate was substituted with wheat bran (3%), the phytase titre in the former was comparable with that in the latter.     

A bioprocess for the production of phytase from Schizophyllum commune: studies of its optimization, profile of fermentation parameters, characterization and stability

Schizophyllum commune produces phytase through solid-state fermentation using different agroindustrial residues. After optimization of phytase production, a maximal level of phytase (113.7 Units/gram of dry substrate) was obtained in wheat bran based medium containing 5% sucrose, 50% humidity, 7.5% of biomass at 33 °C pH 7.0 during 72 h and a 285% improvement in enzyme titre was achieved. Analysis of fermentation parameters profile for phytase production showed the highest productivity (1.466 Units/gram of dry substrate/hour) in 66 h of fermentation. Phytase has an optimal pH of 5.0, an optimal temperature of 50 °C and K (m) and V (max) values of 0.16 mM and 1.85 μmol mL(-1) min(-1), respectively. Phytase activity was stimulated essentially in the presence of K(+), Ca(2+), Mg(2+), Mn(2+), Zn(2+), Cu(2+), Fe(2+), Fe(3+), Co(2+), Ni(2+), acetate and citrate at concentrations of 1 mM. Phytase had the best shelf life when stored at a cooling temperature, maintaining 38% of its initial activity after 112 days of storage, and still presenting enzymatic activity after 125 days of storage. Stability studies of phytase performed in aqueous enzyme extracts showed satisfactory results using polyethyleneglycol 3350, carboxymethylcellulose, methylparaben, mannitol and benzoic acid in concentrations of 0.25, 0.025, 0.025, 0.25, and 0.0025%, respectively. PEG 3350 was shown to be the best stabilizing agent, resulting in 109% of phytase activity from the initial crude extract remaining activity in after 90 days.     

Enhanced endoxylanase production by Myceliophthora thermophila using rice straw and its synergism with phytase in improving nutrition

The goal of the present investigation was to attain the enhanced production of endoxylanase in submerged fermentation using different approaches followed by its utility in improving nutrition of wheat and rice flours along with phytase. Myceliophthora thermophila BJTLRMDU3 produced 51.70 U/mL of xylanase using rice straw as a substrate after optimization with ‘one variable at a time’ approach. After Plackett-Burman design study, sodium nitrate, K₂HPO₄ and Tween 20 were selected as critical factors and further optimized by response surface methodology. Increased xylanase production (80.15 U/mL) was attained with 2.5 % (w/v) sodium nitrate, 1.25 % (w/v) K₂HPO₄, and 2 % (v/v) Tween 20 at 40 °C. An overall 1.5-fold increase in xylanase production was achieved after statistical optimization. Applicability of M. thermophila xylanase (200 U/g flour) alone and in combination with phytase (15 U/g flour) from Aspergillus oryzae SBS50 in wheat and rice flours showed enhancement in nutritional qualities of both flours. About 45.67 %, 29.73 %, and 107.91 % increase in reducing sugars, soluble proteins and inorganic phosphate, respectively in wheat flour, while 94.16 %, 134.52 %, and 473.33 % increase in reducing sugars, soluble proteins and inorganic phosphate, respectively in rice flour was achieved at 60 °C and pH 5.0 by synergistic action of xylanase and phytase as compared to control having only xylanase.     

A cost-effective cane molasses medium for enhanced cell-bound phytase production by Pichia anomala

AIM: Formulation of an inexpensive cane molasses medium for improved cell-bound phytase production by Pichia anomala. METHODS AND RESULTS: Cell-bound phytase production by Pichia anomala was compared in synthetic glucose-beef extract and cane molasses media. The yeast was cultivated in 250 ml flasks containing 50 ml of the medium, inoculated with a 12 h-old inoculum (3 x 10(6) CFU ml(-1)) and incubated at 25 degrees C for 24 h at 250 rev min(-1). Different cultural parameters were optimized in cane molasses medium in batch fermentation. The cell-bound phytase content increased significantly in cane molasses medium (176 U g(-1) dry biomass) when compared with the synthetic medium (100 U g(-1) dry biomass). In fed-batch fermentation, a marked increase in biomass (20 g l(-1)) and the phytase yield (3000 U l(-1)) were recorded in cane molasses medium. The cost of production in cane molasses medium was pound 0.006 per 1000 U, which is much lower when compared with that in synthetic medium (pound 0.25 per 1000 U). CONCLUSIONS: An overall 86.6% enhancement in phytase yield was attained in optimized cane molasses medium using fed-batch fermentation when compared with that in synthetic medium. Furthermore, the production in cane molasses medium is cost-effective. SIGNIFICANCE AND IMPACT OF THE STUDY: Phytase yield was improved in cane molasses when compared with the synthetic medium, and the cost of production was also significantly reduced. This enzyme can find application in the animal feed industry for improving the nutritional status of feed and combating environmental pollution.     

OPTIMIZATION OF SOLID-STATE FERMENTATION FOR PHYTASE PRODUCTION BY Thermomyces lanuginosus USING RESPONSE SURFACE METHODOLOGY

A strain of Thermomyces lanuginosus, isolated from hot spring water in Turkey, was studied for optimization of phytase production using solid-state fermentation. Effects on fermentation of different production parameters such as substrate type, moisture, culture time, and inoculum size were investigated using a one-factor-at-a-time approach. Central composite design (CCD) of response surface methodology was applied for the optimization of four factors (culture temperature, initial pH, aeration area, age of seeding culture) that were affecting phytase production by Thermomyces lanuginosus in rice bran. Maximum phytase activity was achieved by using rice bran. The optimum levels of variables that supported maximum enzyme activity were moisture 70%, culture time 7 days, inoculum size 40%, culture temperature 55°C, initial pH 7.5, aeration area 30%, age of seeding culture 5 days, sucrose 1%, and ZnSO₄ 2.5 mM. An overall 10.83-fold enhancement in phytase activity (0.30 to 3.248 U) was attained due to the optimization.     

In vivo enzymatic digestion, in vitro xylanase digestion, metabolic analogues, surfactants and polyethylene glycol ameliorate laccase production from Ganoderma sp. kk-02

AIMS: The effect of in vivo enzymatic digestion (IVED), in vitro xylanase digestion (IVXD), metabolic analogues, surfactants and polyethylene glycol (PEG) on laccase production from Ganoderma sp. kk-02 was studied. METHODS AND RESULTS: An acidic laccase producing Ganoderma sp. kk-02 produced 16.0 U ml(-1) and 365.0 U g(-1) of laccase, when grown under submerged (SmF) and solid state (SSF) fermentation conditions, respectively. Modification of the substrate (wheat bran) molecular architecture by IVED and IVXD increased subsequent laccase production from Ganoderma sp. kk-02 by 1.31-fold (21.0 U ml(-1)) (SmF); 2.21-fold (810.0 U g(-1)) (SSF) and 1.10-fold (18.0 U ml(-1)) (SmF); 1.78-fold (650.0 U g(-1)) (SSF) when compared with untreated wheat bran. Further enhancement in laccase yield under SmF and SSF was obtained when IVED treated wheat bran was used in conjunction with amino acids [DL-tryptophan, 2.66-fold (56.0 U ml(-1)) SmF; 2.86-fold (2324.0 U g(-1)) SSF], vitamins [biotin, 1.71-fold (36.0 U ml(-1)) SmF; 3.06-fold (2483.0 U g(-1)) SSF], surfactants [Tween-40, 1.85-fold (39.0 U ml(-1)) SmF; 2.25-fold (1828.0 U g(-1)) SSF], and PEG [PEG 6000, 1.93-fold (40.0 U ml(-1)) SmF; 1.58-fold (1284.0 U g(-1)) SSF]. CONCLUSIONS: The IVED of substrate (wheat bran) facilitated hyper laccase production in presence of additives from Ganoderma sp. kk-02. SIGNIFICANCE AND IMPACT OF THE STUDY: The study highlights a new methodology viz. IVED for concomitant and economic production of diverse enzymes using the same substrate. The hyper laccase levels obtained could improve the economic competitiveness of environmentally benign processes applied in varied industries. The work also provides an insight into the regulation of complex metabolic pathways governing the expression of extra cellular proteins from white-rot fungi.     

Optimization of phytase production by Penicillium purpurogenum GE1 under solid state fermentation by using Box–Behnken design

Phytase production by Penicillium purpurogenum GE1 isolated from soil around bean root nodules was investigated by solid state fermentation (SSF) using mixed substrates consisted of corn cob and corn bran. The SSF conditions were optimized by using one-variable–at-a-time strategy. The optimum conditions for phytase production were at 27 °C, pH 8 and 66% moisture content. The study of different carbon and nitrogen sources revealed that glucose and peptone registered the highest enzyme productivity (92 ± 5.6 U/g ds, 125 ± 4.9 U/g ds). Among different surfactants, maximum phytase productivity was observed with Tween 80 at 0.001 concentrations (170 ± 4.2 U/g ds). A Box–Behnken design was employed to investigate the optimization of the most significant variables affecting the enzyme production. Maximal phytase production was detected after the addition of (g/5 g ds): 0.75 glucose, 0.375 peptone and 0, 01 tween 80. This result represented an improvement in phytase production of 2.6 folds when compared to that previously obtained using the basal medium under the same cultivation conditions. The generated model was found to be very adequate for phytase production (90% accuracy) as the experimental value was 444 ± 3.5 U/g ds compared to 401 U/g ds for the predicted value. In brief, the production of phytase using corn cob and corn bran is a novel and cheap way for the production of this important enzyme and opens a new way for researchers to discover and explore this arena.     

Potential of Aspergillus oryzae CFTRI 1480 for producing proteinase in high titres by solid state fermentation

Aspergillus oryzae CFTRI 1480, an isolate from a spoiled moist sample of casein, produced 59,105 units of an extracellular proteinase/g dry mouldy bran (DMB) at 72 h in an arbitrarily formulated wheat bran medium in a solid state fermentation system. The enzyme production was significantly affected by mineral salt content and pH of the liquid used for moistening the wheat bran. Enzyme titres were enhanced 1.34-fold with the addition of 0.4% corn starch. Optimization of key parameters, i.e., initial moisture content, age and size of inoculum, increased the enzyme production to 191,869 units/g DMB and reduced the fermentation time to 48 h. Such high titres in a simple medium, surpassing most of the literature reports, indicate the industrial importance of the culture. The properties of acetone-precipitated enzyme, viz, the optimum pH of 10.0, more than 95% activity between pH 7.0 and 10.0, temperature optimum at 55° C and more than 90% activity between 10 and 27°C, are similar to those of commercially available fungal proteinases employed in animal feed, leather and other industries. Correspondence to: B. K. Lonsane     

绿色木霉ZY-1固态发酵产纤维素酶

利用筛选的绿色木霉ZY-1（Trichoderma viride ZY-1）固态发酵产纤维素酶,采用稻草和麸皮为底物,考察稻草与麸皮比例随发酵时间对产酶的影响。结果表明：底物中,在m（稻草）：m（麸皮）为0：5和1：4时,发酵48h,pH保持4．5左右,还原糖量急剧上升,胞外蛋白产量最低;仅以稻草作底物时,整个发酵过程中pH约为7,还原糖量最低,胞外蛋白产量较高而滤纸酶活、羧甲基纤维素酶（CMCase）和β-葡萄糖苷酶（β-Gase）酶活均较低;在m（稻草）：m（麸皮）为3：2时,发酵96h,滤纸酶活达最大值5．01U／g干曲;m（稻草）：m（麸皮）为1：4时,发酵96h,β-Gase酶活达最大值4．6U／g干曲;m（稻草）：m（麸皮）为4：1时,发酵72h,CMCase酶活达最大值6．01U／g干曲。因此,底物中存在适量的稻草和麸皮有利于Trichoderma viride ZY—1产纤维素酶。     

Production of phytase by Mucor racemosus in solid-state fermentation

Phytase production was studied by three Mucor and eight Rhizopus strains by solid-state fermentation (SSF) on three commonly used natural feed ingredients (canola meal, coconut oil cake, wheat bran). Mucor racemosus NRRL 1994 (ATCC 46129) gave the highest yield (14.5 IU/g dry matter phytase activity) on coconut oil cake. Optimizing the supplementation of coconut oil cake with glucose, casein and (NH(4))(2)SO(4), phytase production in solid-state fermentation was increased to 26 IU/g dry matter (DM). Optimization was carried out by Plackett-Burman and central composite experimental designs. Using the optimized medium phytase, alpha-amylase and lipase production of Mucor racemosus NRRL 1994 was compared in solid-state fermentation and in shake flask (SF) fermentation. SSF yielded higher phytase activity than did SF based on mass of initial substrate. Because this particular isolate is a food-grade fungus that has been used for sufu fermentation in China, the whole SSF material (crude enzyme, in situ enzyme) may be used directly in animal feed rations with enhanced cost efficiency.