Production of Lipase from Geotrichum candidum Using Corn Steep Liquor in Different Bioreactors

The production of an extracellular lipase using corn steep liquor (CSL) as the nitrogen source in the cultivation of Geotrichum candidum NRRLY‐552 was evaluated. The optimized conditions in shake flasks were CSL, 8.0 % w/v, soybean oil, 0.6 % w/v, pH 7.0, 30 °C, 250 rpm, and 48 h, resulting in a maximum lipase productivity of 0.438 U mL^?1 h^?1(U = the amount of enzyme required to liberate 1 μmol of fatty acid per minute). Scale‐up was evaluated with airlift and stirred tank reactors; the best conditions, respectively, were 1 vvm(volume of gas per volume of medium per minute) of aeration which resulted in 0.535 U mL^?1 h^?1 (32 h) and 1 vvm and 300 rpm resulting in 0.563 U mL^?1 h^?1 (16 h). To facilitate downstream processes, lipase production was also evaluated using CSL previously clarified with activated charcoal resulting in 0.275 U mL^?1 h^?1 (24 h) using 12 % (w/v) of clarified CSL in shake flasks. The obtained results showed that CSL leads to similar productivity compared to peptone using the same microorganism under similar conditions. In addition the cost of fermentation medium using CSL is much lower because it is a very inexpensive by‐product from corn processing.     

Optimization of pullulan production from synthetic medium by Aureobasidium pullulans in a stirred tank reactor by response surface methodology

The production of pullulan from synthetic medium by Aureobasidium pullulans P56 in a stirred tank fermenter was investigated. The kinetics of polysaccharide, pullulan and biomass production was determined. Response surface methodology was used to investigate the effects of three factors (initial sugar concentration, aeration rate and agitation speed) on the concentration of pullulan in batch cultures of A pullulans. In the experiments, the range of values used for the three variables described were; 30–70 g dm^?3 initial sugar concentration, 200–600 rpm agitation speed and 1.0–3.0 vvm aeration rate. No previous work has used statistical analysis in determining the interactions among these variables in pullulan production. Results of the statistical analysis showed that the fit of the model was good in all cases. Aeration rate, agitation speed and sugar concentration had a strong linear effect on pullulan concentration. Moreover, pullulan concentration was significantly influenced by the negative quadratic effects of the given variables and by their positive or negative interactions with the exception that the interaction between agitation speed and aeration rate was insignificant (P > 0.05). Maximum pullulan concentration of 17.2 g dm^?3 was obtained at the optimum levels of process variables (initial sugar concentration 51.4 g dm^?3, aeration rate 2.36 vvm, agitation speed 345.3 rpm). These values were obtained by fitting of the experimental data to the model equation. Scanning electron microscope (SEM) photographs of polysaccharide particles containing different concentrations of pullulan were also taken to observe the morphological differences of the samples. Copyright © 2005 Society of Chemical Industry     

Determination of agitation and aeration conditions for scale-up of cellulolytic enzymes production by <Emphasis Type="Italic">Trichoderma inhamatum</Emphasis> KSJ1

10 to 35 L jar fermentation scale-up cultures were performed to determine the optimum agitation and aeration rates in the cellulolytic enzymes production culture by Trichoderma inhamatum KSJ1. The optimum agitation rate in the 35 L jar fermenter was provisionally determined to be 150 rpm by using a geometrically resembled scale up method from the 10 L jar fermenter. The optimum aeration rate was determined to be 0.5 vvm by applying the mean values of superficial velocity and vvm. The DO (Dissolved Oxygen) concentration of the culture liquid was maintained below the critical DO concentration (2.336 mg/L) at 150 rpm in the 35 L jar fermenter. To increase the DO above the critical DO concentration, the agitation rate was increased from 150 to 200 rpm, with the aeration rate maintained at 0.5 vvm. As a result, the DO was maintained above critical DO concentration. The OUR (Oxygen Uptake Rate) and k _L a values were 0.91 mg-DO/L·min and 11.1 hr⁻¹, respectively. The amylase and FPase (filter paper activity) activities were 4.48 and 0.74 U/mL, respectively, in the 35 L jar fermenter, which was comparable to that in the 10 L fermenter (4.2 and 0.5 U/mL, respectively). Therefore, the scale-up conditions, 0.5 vvm and 200 rpm, were concluded to be the optimum aeration and agitation rates in the 35 L jar fermenter.     

Optimization of the production of chitosan from beet molasses by response surface methodology

Chitosan was produced by Rhizopus oryzae 00.4367 in shake flask culture and a stirred tank fermenter. Synthetic medium, treated and untreated beet molasses were used as cultivation media in shake flask cultures. In the stirred tank fermenter, the cultivation media were synthetic medium and untreated beet molasses. Shake flask culture containing untreated molasses with a sugar concentration of 40 g dm^?3 produced the maximum chitosan yield (961 mg dm^?3). Chitosan concentration reached its maximum value at the late exponential growth phase of R oryzae. In all experiments almost 8–10% of biomass and 32–38% of alkali‐insoluble material was extracted as chitosan. A central composite design was employed to determine the optimum values of process variables (aeration rate, agitation speed and initial sugar concentration) leading to maximum chitosan concentration in the stirred tank fermenter. In all cases, the fit of the model was found to be good. Aeration rate, agitation speed and initial sugar concentration had a strong linear effect on chitosan concentration. Moreover, the concentration of chitosan was significantly influenced by the negative quadratic effects of the given variables and by their positive or negative interactions. A maximum chitosan concentration of 1109.32 mg dm^?3 was obtained in untreated molasses medium containing an initial sugar concentration of 45.37 g dm^?3 with an aeration rate and agitation speed of 2.10 vvm and 338.93 rpm, respectively. Copyright © 2004 Society of Chemical Industry     

Scaling up the Bioconversion of Cheese Whey Permeate into Fungal Oil by Mucor circinelloides

The bioconversion of hydrolyzed whey permeate into an oil-rich biomass by Mucor circinelloides was scaled up from 250 mL to 4 L with the use of an aerated stirred tank bioreactor. Biomass production and oil accumulation were strongly influenced by agitation speed (99–451 rpm) and aeration rate (0.29–1.70 vvm). Higher agitation and aeration rates (e.g., >400 rpm, >1 vvm), resulted in significantly higher biomass yield due to increased oxygen transfer capabilities and better mixing. Additionally, oil accumulation in the fungal biomass was related to high agitation (>400 rpm), while aeration (0.5–1.5 vvm) had no significant effect within the range evaluated. The predictive model was validated at the optimal conditions of 450 rpm and 1 vvm. Maximum biomass yield of 10.7 g L⁻¹ and lipid content of 32% dry biomass were achieved during 120 hours of fermentation. Simultaneous optimization of agitation and aeration in a bioreactor was found to not only improve fungal growth but also lipid content (24% vs. 32%), lipid yield (2.2 vs. 3.1 g L⁻¹) and γ-linoleic production (73–464 mg L⁻¹) compared to that of shake-flask. This study resulted in a scaled-up and optimized fermentation process that increased production of M. circinelloides biomass for subsequent use as raw material for food, feed, and fuel applications. This signifies a starting point for further studies aimed at assessing the development of a fully functioning fungi-to-food/fuel system on an industrial scale for several agricultural streams.     

Combined Effect of Agitation/Aeration and Fed‐Batch Strategy on Ubiquin‐ one‐10 Production by Pseudomonas diminuta

The effects of aeration rate and agitation speed on ubiquinone‐10 (CoQ10) submerged fermentation in a stirred‐tank reactor using Pseudomonas diminuta NCIM 2865 were investigated. CoQ10 production, biomass formation, glycerol utilization, and volumetric mass transfer coefficient (k_La) were affected by both aeration and agitation. An agitation speed of 400 rpm and aeration rate of 0.5 vvm supported the maximum production (38.56 mg L^–1) of CoQ10 during batch fermentation. The fermentation run supporting maximum production had an k_La of 27.07 h^–1 with the highest specific productivity and CoQ10 yield of 0.064 mg g^–1h^–1 and 0.96 mg g^–1 glycerol, respectively. Fermentation kinetics performed under optimum aeration and agitation showed the growth‐associated constant (a = 5.067 mg g^–1) to be higher than the nongrowth‐associated constant (β = 0.0242 mg g^–1h^–1). These results were successfully utilized for the development of fed‐batch fermentation, which increased the CoQ10 production from 38.56 mg L^–1 to 42.85 mg L^–1.     

Effect of the aeration rate on pullulan production and fermentation broth rheological properties in an airlift reactor

The effect of aeration rate on pullulan production and the rheological properties of the fermentation broth in an airlift reactor was investigated. An airlift fermenter was shown to be an appropriate fermentation system for the production of pullulan. A maximum pullulan concentration (30 kg m^?3), biomass concentration (6.0 kg m^?3), pullulan yield (60%, w/w) and sugar utilization (100%, w/w) was obtained at an aeration rate of 2 vvm. The mycelium and the yeast‐like cells were the morphological forms responsible for pullulan production. The highest polysaccharide concentration was obtained when the mycelial forms and the yeast‐like cells were 60% and 40% (w/w) of the total biomass, respectively. The apparent viscosity of the broth was increased with the increase of the aeration rate from 1 to 2 vvm and then decreased at higher vvms. On the other hand, the dissolved oxygen concentration and the volumetric mass transfer coefficient continually increased with the increase of the aeration rate. The mycelial forms and the production of extracellular polysaccharide were responsible for the non‐Newtonian flow behaviour of the fermentation broth. The rheological behaviour can be characterized by a power law type of equation. The relationship between shear rate/shear stress and shear rate/apparent viscosity showed a non‐Newtonian behaviour of the fermentation broth. © 2001 Society of Chemical Industry     

A comprehensive study of lipase production by Yarrowia lipolytica CECT 1240 (ATCC 18942): from shake flask to continuous bioreactor

BACKGROUND: Lipases are commercially important enzymes, and the development and optimization of their production processes are of great interest. The diversity of behaviours between strains stresses the need for research on this topic, especially when bioreactor culture is considered. The study of a continuous operating mode is especially attractive, since very scarce information is available on its application to microbial lipases production. RESULTS: Lipase production in submerged cultures of Yarrowia lipolytica CECT 1240 (ATCC 18 942) has been investigated. Significant lipolytic activity (over 700 U dm^?3), mostly extracellular and membrane‐bound, was obtained in shake flasks using medium supplemented with olive oil. The culture was carried out in air‐lift and stirred tank bench‐scale bioreactors and the latter was selected. The influence of aeration and agitation rates was assessed in batch cultures, and agitation from 400–700 rpm and low aeration rates (i.e. 0.2 vvm) are recommended. Batch, fed‐batch and continuous operation were investigated, and regular enzyme production (up to 600 U dm^?3) was achieved with the latter. CONCLUSION: Lipase production by the selected strain was successfully carried out in shake flasks and bench‐scale bioreactors. After studying batch, fed‐batch and continuous processes, continuous culture in a stirred tank bioreactor was found best in terms of regular enzyme production, exceptionally good operational stability and good fitting of the results to mathematical models. Copyright © 2009 Society of Chemical Industry     

Production of antifungal lipopeptide from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> in submerged fermentation using shake flask and fermentor

Optimization of the production of antifungal lipopeptide, iturin A, by B. subtilis was carried out in submerged cultural fermentation. In a shake-flask experiment, response surface methodology (RSM) was employed to optimize the cultivation conditions of Bacillus subtilis S3 for the enhancement of iturin A production. The optimal conditions for iturin A production obtained from RSM were pH 6.0, 0.93% maltodextrin, 1.11% glucose, 0.72% corn steep powder (C.S.P), 1.5 mM MgSO₄, 0.75 mM KH₂PO₄, rotation speed 180 rpm and area of aeration 4.35 cm². 57% increase of iturin A productivity (from 47.19 mg/L to 74.22 mg/L) was observed using the one-factor-at -a-time technique; however, 180% increase of iturin A productivity (from 47.19 mg/L to 132.23 mg/L) was observed with RSM. The iturin A production was further studied in a 5-L fermentor with a variation of agitation speed, aeration and baffles. In the 5-L fermentor, it was found that increased agitation speed improved the growth of B. subtilis and the production of iturin A. Aeration at 2 vvm gave excellent production of iturin A (175.52 mg/L) at 6 d of fermentation. The addition of baffles in the fermentor has significantly influenced the oxygen mass transfer coefficient (KLa) and iturin A production.     

Production and quality analysis of Pr55gag particles produced in baculovirus-infected insect cells

In this work Sf-9 cells previously adapted to SF900II and EXCELL 401 serum-free media (SFM) were grown and infected at different agitation rates in order to study the effects of power input upon cell growth, infection and production of Pr55gag particles in both SFM. Maximum cell concentration increased from 3×10⁶ to 6×10⁶ cells cm⁻³ and 6·5×10⁶ cells cm⁻³ when the agitation increased from 80 to 250 rpm or sparged aeration (0·01 vvm at 170 rpm) is used, clearly indicating that cell growth is limited by gas transfer. The specific productivity increased 3·5-fold when the agitation rate was increased from 80 to 170 rpm, indicating that in SFM cell infection is also limited by gas transfer. The highest product concentration was obtained in SF900II at 120 h post-infection (hpi). The product quality analysis showed that SF900II is the best medium for production of Pr55gag particles and that a careful optimisation of the harvest time is required. The maximum product titre was obtained at 120 hpi, 48 h after the achievement of the highest quality. © 1998 SCI     

Aeration and mixing in vortex fermenters

The overall apparent volumetric gas—liquid mass transfer coefficient (k₉₅a) and the mixing time (t₉₅) were determined in a 240 dm³ vortex aerated fermenter over stirrer speed and air flow ranges of 300–800 rpm and 10–45 normal dm³ min^?1, respectively. The mass transfer data obtained in an aqueous salt solution (2.5 kg m^?3 NaCl in water) compared well with the measurements in a fermentation medium used in culture of certain microaerophilic bacteria. Over the ranges examined, the gas-liquid mass transfer coefficient depended only on air flow rate; the dependence was linear with flow. Mixing time declined with increasing agitation according to a power-law relationship. The mixing and mass transfer characteristics of the vortex aerated system were compared with that of a ‘standard’ stirred tank fermenter (27 dm³). The mixing time variations with respect to agitation rate were remarkably similar for the two types of fermenters examined.     

Xylitol production by Candida tropicalis from corn cob hemicellulose hydrolysate in a two‐stage fed‐batch fermentation process

BACKGROUND: Xylitol, a sugar alcohol widely used in food and pharmaceutical industries, can be produced through biological reduction of xylose present in hemicellulose hydrolysates by Candida tropicalis. However, the aeration rate and by‐products originating from hemicellulose hydrolysis strongly inhibit the production of xylitol in a fermentation process. A two‐stage fed‐batch fermentation system was developed to reduce these inhibitory effects and to improve xylitol production from corn cob hemicellulose hydrolysates by C. tropicalis. RESULTS: Results of batch fermentations indicated that high xylitol production could be obtained from C. tropicalis at an initial xylose concentration of 80 g L^?1 in corn cob hydrolysate medium at an aeration rate of 0.4 vvm at the micro‐aeration stage. In the two‐stage fed‐batch fermentation process, 96.5 g L^?1 xylitol was obtained after 120 h, giving a yield of 0.83 g g^?1 and a productivity of 1.01 g L^?1 h^?1, which were 12.16% and 65.57% higher than those in a batch fermentation. CONCLUSION: High xylitol production can be achieved in a two‐stage fed‐batch fermentation process, in which the negative effects of aeration rate and inhibitory compounds on xylitol formation can be considerably reduced. Copyright © 2011 Society of Chemical Industry     

Enhancement of Candida parapsilosis catalyzing deracemization of (R,S)‐1‐phenyl‐1, 2‐ethanediol: agitation speed control during cell cultivation

BACKGROUND: Microbial cells have been used widely in biosynthesis of chiral alcohols. However, research concerning the effect of oxygen supply in cultivation on biocatalytic activity of whole cells in organic synthesis is limited. This study improved the reaction efficiency of Candida parapsilosis catalyzing (R,S)‐1‐phenyl‐1,2‐ethanediol (PED) deracemization by controlling agitation during cell cultivation. RESULTS: The increase of dissolved oxygen concentration by adjusting agitation speed from 200 to 300 rpm at aeration rate 1.5 vvm significantly improved the cell growth of C. parapsilosis and the activities of two key enzymes involved in deracemization. (S)‐PED with higher optical purity of 98.23%e.e. and yield of 82.94% was formed. Compared with the initial fermentation conditions at aeration rate 0.75 vvm and agitation speed 200 rpm, enhanced oxygen supply conditions afforded better cells for highly efficient conversion under higher substrate concentration. CONCLUSION: Oxygen supply had a significant effect on cell growth and catalytic activity of C. parapsilosis catalyzing asymmetric oxidoreduction. By conveniently controlling agitation in cultivation, cell activity and key enzymes production for a complex reaction of concurrent tandem oxidation and reduction processes can easily be conducted, which could help to cultivate cells catalyzing synthesis of interested chiral compounds. Copyright © 2008 Society of Chemical Industry     

In‐line mixing for production of citric acid by Candida lipolytica grown on n‐paraffins

This study reports on the effects of internal fermenter and external in‐line agitation and fed‐batch mode of operation on citric acid production from Candida lipolytica using n‐paraffin as the carbon source. An optimum range of fermenter agitation speeds in the range 800–1000 rpm corresponding to Reynolds numbers of 50433–62947 (based on initial batch conditions) seemed to give the best balance between substrate utilization for biomass growth and citric acid production. Proof of concept evidence is presented that indicates that an external in‐line agitator could be used in place of high speed internal agitation to increase citric acid production. However, more work is required to optimize the external agitator concept. Application of multiple fed‐batch feedings can be used to extend the batch fermentation and increase final citric acid concentrations and product yield. Experiments were conducted implementing a three‐cycle fed‐batch process which increased overall citric acid yields to 0.8–1.0 g citric acid g^?1 n‐paraffin, approximately 200% improvement from those found in the normal batch process. The three‐cycle fed‐batch mode of operation also increased the final citric acid concentration to 42 g dm^?3 from about 6 g dm^?3 for normal batch operation. Increased citric acid concentrations in three‐cycle fed‐batch mode was achieved at longer fermentation times. Copyright © 2004 Society of Chemical Industry     

Modulated gluconic acid production from immobilized cells of Aspergillus niger ORS‐4.410 utilizing grape must

BACKGROUND: Gluconic acid (GA) production by immobilized cells of mutant Aspergillus niger ORS‐4.410 on polyurethane sponge (PUS) and calcium‐alginate (Ca‐alginate) was evaluated in repeated batches of solid state surface fermentation (SSF) and submerged fermentation (SmF) conditions, respectively, utilizing rectified grape must as carbon source. RESULTS: The passive immobilization of cells in fermentation medium solid support of having 0.4 cm³ cube size, 4% spore suspension, 0.6 g inoculum of PUS immobilized cells at 32 °C and 2.0 L min^?1 resulted in the maximum GA production (88.16 g L^?1) with a 92.8% yield, while the Ca‐alginate matrix with a 0.5 cm diameter bead size, 2–3% spore suspension, 15 g inoculum at 34 °C and 150 rpm agitation speed revealed 67.19 g L^?1 GA with a 85.2% yield. Repeated use of PUS showed higher levels of GA (110.94 g L^?1) in the third–fourth fermentation cycles with 95–98% yield and 22.50 g L^?1 d^?1 productivity under SSF that was 2.5‐fold higher than the productivity obtained from a typical fermentation cycle, and 54% greater than the productivity obtained with repetitive use of Ca‐alginate immobilized cells of A. niger under SmF. CONCLUSION: Using immobilized cells of A. niger in PUS, the rectified form of grape must can be utilized for GA production as an alternative source of carbohydrate by replacing the conventional fermentation conditions. Copyright © 2008 Society of Chemical Industry     

长川霉素发酵条件的优化

考察了发酵过程中接种、培养基、搅拌速度、通气量对长川霉素(Changchuanmycin)发酵的影响，通过实验确定了250L发酵罐的批式发酵条件为，以5％～10％的接种量在36～40h时接种，搅拌速度发酵前期为200rpm，然后逐步提高至400rpm；通气量发酵前期为0．6vvm，中期为1．0vvm，后期为0．6vvm；发酵过程中溶氧控制在30％以上，可以使发酵单位达到380μg/ml。     

Effect of oxygen transfer rates on alcohols production by Candida guilliermondii cultivated on soybean hull hydrolysate

BACKGROUND: In this research the use of soybean hull hydrolysate (SHH) as substrate for xylitol and ethanol production using an osmotolerant strain of Candida guilliermondii was studied. The production of alcohols was investigated in batch cultivations in which the variable parameter was the volumetric oxygen mass transfer coefficient (k_La) obtained from three different conditions of air supply: anaerobic (150 rpm, no aeration); microaerobic (300 rpm, 1 vvm), and aerobic (600 rpm, 2 vvm), corresponding to k_La values of 0; 8; and 46 h^?1, respectively. RESULTS: SHH, although presenting a very high osmotic pressure (1413 mOsm kg^?1), was completely metabolized under aerobic conditions with high biomass productivities of 0.49 g cells (L h)^?1, with little formation of ethanol. Xylitol was produced under microaeration, with product yield of 0.22 g g^?1 xylose, with the formation of glycerol as a by‐product. No xylose was metabolized under anaerobic conditions, but ethanol was produced from hexoses with high product yields of 0.5 g g^?1. CONCLUSION: These results suggest that the hydrolysis of soybean hull and its conversion to ethanol and other alcohols could be an important use of this agro‐industrial waste, which could be used for biofuel, xylitol or biomass production, depending on the aeration conditions of the cultures. Copyright © 2008 Society of Chemical Industry     

Inoculum strategies for Penicillium simplicissimum lipase production by solid‐state fermentation using a residue from the babassu oil industry

Two alternative inoculation strategies for lipase production by the fungus Penicillium simplicissimum were tested in solid‐state fermentation using a residue from the babassu oil industry (babassu cake). Conventional spore inoculation was compared with fungal pellets grown in liquid medium and with inocula consisting of fermented cake. Fungal pellets delayed lipase production whereas fermented cake accelerated enzyme synthesis, yielding a productivity of 0.45 U g^?1 h^?1, which is equivalent to the highest values obtained with conventional inocula. Therefore, a 2² factorial design was used to determine the best conditions for lipase production with fermented cake as inoculum strategy, varying the inoculum propagation time and inoculum concentration. Lipase activity and productivity reached 30 U g^?1 and 0.63 U g^?1 h^?1, respectively, with 10% inoculum and 36 h. Thus, fermented cake inocula increased production 1.5‐fold with 10 times fewer spores than in conventional inoculation, indicating that fermented solids are an interesting alternative for inoculum development in solid‐state fermentation, mainly for large‐scale processes. Copyright © 2007 Society of Chemical Industry     

Assessment of Relevant Factors Influencing Lipolytic Enzyme Production by Thermus thermophilus HB27 in Laboratory‐Scale Bioreactors

Nowadays, there is not much information on the large‐scale production of thermostable lipolytic enzymes by thermophilic organisms. Therefore, in this study the lipolytic enzyme production by Thermus thermophilus HB27 in laboratory‐scale bioreactors was undertaken. In order to determine the most suitable bioreactor, two configurations were investigated: stirred‐tank and airlift bioreactor. It was demonstrated that the stirred‐tank configuration led to the highest lipolytic extracellular activities, about 2.3‐fold higher than those found in the corresponding cultivation in the airlift bioreactor. On the other hand, the influence of several factors such as culture nutrients concentration, aeration, and agitation rate on the production of thermophilic lipolytic enzymes in a 5‐L stirred‐tank bioreactor was assayed. At reduced nutrients concentration (50 % with respect to the basal medium), a higher product/biomass yield was attained, without any operational problems. From the relationship between mass transfer coefficient (K_La), aeration, and agitation rates it was concluded that there is a lesser dependence on the aeration than the agitation rate. In addition, the mechanical stirring of the bioreactor could tear the membranes that contain the rotund bodies often found in cultures of thermophilic microorganisms, thus increasing the extracellular enzyme production.     

The effect of D‐glucose on milk clotting activity of Mucor miehei in a chemostat with biomass retention

In this study microbial production of rennin from a commercial strain of Mucor miehei (NRRL 3420) has been accomplished in a continuously fed fermenter. The effects of feed D ‐glucose concentration (2.5–30 g dm⁻³) on milk clotting activity and on other system variables were investigated at optimum mixing and dilution rates of 400 rpm and 0.0052 h⁻¹, respectively, without pH control. Maximum milk clotting activity (1.24 IU cm⁻³) occurred when the feed D ‐glucose concentration was 7.5 g dm⁻³. Enzyme production continued for 500 h, producing a total milk clotting activity of 10 230 IU. At the maximum milk clotting activity point, total protein concentration, milk clotting and proteolytic activities were analysed and compared with those of a commercial rennet. The ratio of milk clotting activity to proteolytic activity, and specific milk clotting activity were determined as 1.55 × 10⁻³ IU PU⁻¹ and 5.28 IU mg⁻¹ medium protein, respectively, denoting similar characteristics to a commercial rennet after concentration of the fermentation medium. © 1999 Society of Chemical Industry