Production Of Milk-clotting Enzyme Research Articles

Isolation and production optimization of a novel milk-clotting enzyme Bacillus velezensis DB219

The milk-clotting enzyme (MCE) is a crucial ingredient in cheese manufacture. Due to the limits of traditional MCE, finding viable substitute is a pressing issue. This study aims to isolate and identify a wild strain with high milk-clotting activity (MCA) and low proteolytic activity (PA) and optimize the fermentation conditions for MCE production. A strain of Bacillus velezensis DB219 with high MCA/PA value (9.2) was isolated from dairy soil (Wuchang, Heilongjiang, China) and identified through 16S rRNA from 40 strains. The optimal wheat bran, carbon, nitrogen, inoculum size, volume and initial pH were 60 g/L, soluble starch 12.5 g/L, corn steep liquor 3 g/L, 5%, 40 mL and 6.15, respectively for improving DB219 MCE production through single factor experiment. The wheat bran concentration, corn steep liquor concentration and volume were the most critical factor and their changed range was determined through Plackett–Burman design and the steepest ascent/descent experiments. The response surface analysis experiment of three factors and three levels was conducted by Box–Behnken design. The theoretical optimal fermentation conditions for DB219 MCE were as follows: wheat bran concentration 60.14 g/L, soluble starch 12.5 g/L, corn steep liquor 3 g/L, inoculum size 5%, volume 40.08 mL and initial pH 6.15. DB219 MCE achieved the maximal MCA (3164.84 SU/mL) that was 101.9% of the predicted value (3104.49 SU/mL) and 4.3-fold higher than the control.

Statistical optimization of B. subtilis MK775302 milk clotting enzyme production using agro-industrial residues, enzyme characterization and application in cheese manufacture

Abstract The agricultural and industrial wastes are environmental pollutants. So, there is an increasing demand for more efficient exploitation of these wastes and their conversion into useful products. Recently, the limited availability of calf rennet led to a search for rennet substitutes for cheese manufacture. In this study, Bacillus subtilis subsp. subtilis strain 168 (B. subtilis MK775302) was isolated from the marine sponge Pseudoceratina Arabica and it was used for milk clotting enzyme (MCE) production using some agro-industrial wastes. The production medium containing agro-industrial residues was optimized by using Plackett-Burman (PB) and central composite designs (CCD). After optimization, the MCE productivity was increased by 2.3-fold compared to the non-optimized medium. The enzyme showed maximum activity at 70 °C, pH 5.0. Ultrafilterated (UF) white soft cheese production was studied by using the crude extract of B. subtilis MK775302 in a comparison with commercial rennet. The cheese produced by the crude extract of B. subtilis MK775302 had a higher ripening index, higher acidity, higher flavor intensity, and acceptable organoleptic score. Therefore, this study offered the production of a promising rennet substitute that is eco-friendly with low production costs and competes with the commercial coagulants for the production of acceptable UF white soft cheese.

Fermentation conditions of serine/alkaline milk-clotting enzyme production by newly isolated Bacillus licheniformis BL312

This study was conducted to find a microbial milk-clotting enzyme (MCE) with a high and stable milk-clotting activity (MCA) to proteolytic activity (PA) ratio suitable for the cheese industry. Microbial strains were isolated from soil suspensions cultured in solid casein medium. 16S rDNA of representative isolates were sequenced to identify the microbial species. Nutrition and fermentation conditions were systematically examined to optimize MCA of the selected MCE. Protease inhibitors were used to identify the type of MCE. The casein hydrolysis was analyzed through reversed-phase HPLC (RP-HPLC) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The Bacillus licheniformis BL312 was identified from 50 bacterial strains. BL312 MCE achieved a maximal MCA (460 ± 15 SU/mL) at 48 h that was 2.7-fold higher than the control, and the MCA/PA ratio (9.0) and pH (6.6) remained stable throughout the fermentation process. Medium containing 30 g/L wheat bran shorts, 5 g/L glucose, and 3 g/L corn steep liquor was sufficient for optimal BL312 MCE production. Fermentation conditions of an inoculum size of 7.0% (v/v), fermentation temperature of 37 °C, agitation speed of 210 rpm, and initial pH 6.6 were required to achieve maximal MCA. BL312 MCE was inhibited by phenylmethanesulfonyl fluoride (PMSF) and high concentrations of ethylenediaminetetraacetic acid (EDTA) (5–25 mM). The αs-casein (αs-CN) and β-casein (β-CN) hydrolysates generated by BL312 MCE and calf rennet were different. BL312 MCE is a serine/alkaline protease that exhibits high MCA and various hydrolysis for caseins in comparison with calf rennet.

Detection of Milk Clotting Enzyme Produced by Lactic Acid Bacteria Isolated from Fermented Food

Lactic acid bacteria (LAB) are known to produce extracellular enzymes that could be exploited for use in the dairy industry. Two LABs isolated from fermented foods (paste shrimp) were evaluated for extracellular proteinase production, milk clotting activity (MCA) and proteolytic activity (PA). The two isolates were identified by (API 50CHL) as Pediococcus acidilactici SH, and Lactobacillus paracasei CF1. These LABs showed proteolytic activity using skim milk agar as indicated by the visible clear zones developed in skim milk agar. P. acidilactici SH showed high MCA 50 SU/ml with PA 1.9 U/ml while L. paracasei CF1 showed MCA 44 SU/ml) with PA 3.5 U/ml using trypticase peptone as a nitrogen source in enzyme production media. MCA was not observed when the LABs were grown in MRS broth. This study indicates the possibility of exploiting LAB from food sources for the production of milk-clotting enzymes for dairy production.

Fungal strain selection for protease production by solid-state fermentation using agro-industrial waste as substrates

Screening of the fungal strains, Aspergillus niger, Trichoderma harzianum, and Rhizopus microsporus var. chinensis for protease production was realised by monitoring the presence of clear zones on skimmed milk agar plates. Additionally, screening of the radial growth on vanilla waste, cane bagasse, and pineapple crown plates was performed. Radial growth data of the fungal strains on skimmed milk agar and agro-industrial waste plates were adjusted to the Gompertz model. The results indicate that R. microsporus var. chinensis and pineapple crown were adequate for the production of milk-clotting enzymes by solid-state fermentation. Crude enzyme extract was produced via solid-state fermentation using two particle sizes [2–2.6 mm (SSF1) and a mixture of less than 1 mm with 2–2.6 mm, at a ratio of 1:1(SSF2)]. The highest milk-clotting and proteolytic activities were shown within 12 h and 48 h of fermentation for SSF1 and SSF2, respectively. The optimum milk-clotting activity presented by the crude enzyme extract was at pH 6.5, 40 °C, and 0.04 M CaCl2. The ratio of milk-clotting activity to proteolytic activity (9.7) presented by the crude enzyme extract indicates potential for use as a calf rennet substitute.

APPLICABILITY OF WOOL COVALENT BONDED Bacillus circulans 25 CELLS FOR MILK-CLOTTING ENZYME PRODUCTION BY BATCH, REPEATED BATCH AND CONTINUOUS PROCESS

Abstract The production of milk-clotting enzyme (MCE) by free and immobilized Bacillus circulans 25 cells was investigated. The investigation evaluates cell immobilization through entrapment and covalent binding to different carriers. B. circulans 25 cells immobilized covalently via glutaraldehyde (cross-linker) to natural wool exhibited higher effectiveness factor (0.76) compared to other carriers. Immobilized cells produce maximum level of MCE and highest ratio of milk-clotting activity/proteolytic activity (MCA/PA) after 24 h using 0.4g wet weight cells/g wool. In batch operation, decreasing biomass loading to 0.2 g enhanced the MCA/PA ratio and specific productivity by 5.94 and 59.88%, respectively. Immobilized cells on natural wool as an effective and suitable carrier were able to produce the same level of MCA, productivity and MCA/PA ratio for 9 repeated cycles (216 h). Under continuous operation immobilized cells in a packed-bed bioreactor were able to keep producing MCA with MCA/PA ratio for about 7 days at the same level. Moreover, continuous operation demonstrates a very good productivity of 0.385 KU/L/h, which is higher than the other production systems by 7.0-11.7 fold. Immobilized B. circulans 25 cells proved to be fully capable of continuous MCE production in a packed-bed bioreactor.

Optimization of milk-clotting enzyme production by Bacillus amyloliquefaciens SP1 isolated from apple rhizosphere

Present study aims to isolate and optimize fermentation conditions of milk-clotting enzyme producing rhizobacteria Bacillus amyloliquefaciens SP1. A bacterium producing an extracellular milk-clotting enzyme (MCE) was isolated from the rhizosphere of the planted population of apple trees growing at Distt. Chamba of Himachal Pradesh, India. According to morphological, physiological, biochemical, and molecular characterization, isolate was identified as B. amyloliquefaciens. Single-factor testing was used to study the optimum physical conditions and nutritional parameters for production of the MCE per proteolysis activity which insures its usefulness as new source of milk coagulant for cheese making. The optimum conditions for production of the milk-clotting enzyme were: temperature, 30 °C; inoculum size, 1 %; and initial pH of the medium, 6.0. The maximum milk-clotting activity and milk-clotting activity per proteolysis activity were found using soyabean as nitrogen source and sucrose as carbon source. These optimized conditions resulted in a 1.9-fold increase in production of the milk-clotting enzyme. This study reported a plant growth promoting rhizobacteria, i.e., B. amyloliquefaciens as source of milk-clotting enzyme which has potential as a calf rennet substitute.

Optimization of Upstream Process Parameters for Enhanced Production of Thermostable Milk Clotting Enzyme from Bacillus Subtilis MTCC 10422

AbstractThe process protocol of high titer milk clotting enzyme (MCE) production from Bacillus subtilis was delineated through optimizing different energy sources and growth conditions like incubation period, temperature and pH. The culture broth showed maximum production of MCE at pH 6.0 and temperature 40C after 48 h of incubation with the yield of 191.55 U/mL. Quantitative comparison of MCE activity before and after optimization revealed 7.46‐fold increase in MCE production after the process conditions were standardized. The characteristics of MCE viz. broad pH and temperature stability could make it useful as a new source of milk coagulant in dairy processing.Practical ApplicationsEach microbial strain is unique in its molecular, biochemical and metabolic enzyme production properties. These production characteristics would have to offer competitive advantages over the existing products of un‐optimized medium. This warrants thorough study of isolated microbial species under variable growth conditions. Fungal MCEs have been widely reported. The Milk Clotting Activity (MCA) and MCA per Proteolytic Activity (PA) index of the bacterium under study are comparable to that of many fungal reports and better than quite a few bacterial MCEs. Bacillus subtilis can be a promising microorganism for MCE production and has a good probability of successful use in cheese production.

Optimization of the production and characterization of milk-clotting enzyme from Bacillus subtilis isolated from marine sponge

Background and objectives Milk-clotting enzyme (MCE) has important applications in the dairy industry and in the cheese-manufacturing process. Because of the increase in cheese consumption and the low supply of calf rennets, there is a need for a suitable rennet substitute from an appropriate source. The present investigation aims at microbial production of MCE and medium optimization for maximal enzyme production by the most potent strain. Partial purification and the properties of the partially purified enzyme are also studied. Materials and methods In the present study several microorganisms were tested for production of the MCE. MCE/caseinase ratio was investigated and was used as the key parameter for selection of the most potent strain and for medium optimization. Medium optimization experiments were carried out in an attempt to increase the enzyme productivity by the most potent strain. The produced enzyme was partially purified using ammonium sulfate at 50% concentration and the properties of the partially purified enzyme were investigated. Results and conclusion Among all the tested bacteria the marine sponge Pseudoceratina arabica isolated bacterium (isolate I) showed the highest milk-clotting activity. This isolate was used for MCE production throughout this study and it was identified as Bacillus subtilis KU710517. Maximum enzyme productivity (581.8 U/ml) and maximum MCE/caseinase ratio were obtained using a medium containing 50 g/l wheat bran with xylose and yeast extract as carbon and nitrogen sources. For the partially purified enzyme, the optimal temperature and pH were 85°C and 5.0, respectively. The enzyme was thermally stable at 45°C and retained 100% activity after 90 min. At 50°C for the same period of time it retained about 82% activity. However, at 60°C, the enzyme lost about 70% of its original activity after 30 min. The activation energy (Ea) of the enzyme was calculated as 6.95 kcal/mol. Km and Vmax values were 4.6 mg/ml and 2933 U/mg protein, respectively.

Production of a Milk-Clotting Enzyme by Glutinous Rice Fermentation and Partial Characterization of the Enzyme

Fermentation parameters affecting a milk-clotting enzyme (MCE) production by glutinous rice fermentation were evaluated using selected rice starters called Jiuqu. The MCE was produced under the following conditions: rice inoculation starter at 3%, fermentation time of 5 days, temperature at 30C, initial rice medium pH of 5.0, rice concentration of 70% and rotation speed of 120 rpm. The enzyme preparation obtained by 70% ethanol precipitation of the culture supernatant had milk-clotting activity (MCA) of 6,160 Su/g and a C/P ratio (the ratio between MCA and proteolytic activity) of 10.81 at a recovery rate of 44.82%. The molecular mass of the MCE was 39.7 kDa. MCE production was optimal at 35C and a pH of 5.4. The presence of Na+, K+, Mg2+, Ca2+ and Al3+ stimulated enzyme activity. The Km and Vmax values were 22.90 g/L and 21.11 mL/s, respectively, with skim milk as the substrate. Practical Applications In the last two decades, different sources of milk-clotting enzymes (MCEs) have been explored to meet the increasing demand for cheese production worldwide. This study describes a MCE isolated by ethanol fractionation from glutinous rice wine fermented by Jiuqu. The MCE, with a relatively high C/P ratio, is potentially applicable as a coagulant in cheese making. The property of thermal instability of the enzyme is advantageous as it would be inactivated during curd scalding (at about 50C), thus formation of bitter-taste peptides caused by residual proteolytic activity of the enzyme during cheese ripening could be prevented. In addition, the optimal temperature (35C) of the MCE is within the range of temperature (30–35C) generally used during milk coagulation processes. Therefore, the results suggest that a MCE that could have potential application in cheese production may be obtained by glutinous rice fermentation under the conditions of this study.

English

Some microorganisms have the ability to produce enzymes that could clot milk and used as a substitute for calf rennet. Strains of lactic acid bacteria (LAB) could produce proteolytic enzymes that may have the potential to be used as a source of milk clotting enzyme (MCE). In the present study, LAB isolated from shrimp paste identified as Pediococcus acidilactici SH, was evaluated for milk clotting activity (MCA), proteolytic activity (PA) and enzyme production using different sources of nitrogen. Casein, tryptophan, trypticase peptone and tryptone soya were added to the enzyme production media, and casein was found to increase the MCA to 75.0 SU ml-1 at pH 6.0 at 50°C. SDS-PAGE of the partially purified enzyme solution produced a band with a molecular weight of approximately 29 kDa. This study indicates the possibility of exploiting LAB from food sources for the production of MCEs for dairy production.   Keywords: Lactic acid bacteria (LAB), milk clotting activity, nitrogen source, Pediococcus acidilactici.

Statistical Optimization of Medium Components for Milk-Clotting Enzyme Production by Bacillus amyloliquefaciens D4 Using Wheat Bran-an Agro-Industry Waste

In this paper, two statistical methods were applied to optimize medium components to improve the production of the milk-clotting enzyme by Bacillus amyloliquefaciens D4. First, wheat bran juice, skim milk powder, and Na2HPO4 were shown to have significant effects on D4 enzyme production using the Plackett-Burman experimental design. Subsequently, an optimal medium was obtained using the Box-Behnken method, which consisted of 3.31 g/l of skim milk powder, 5.0 g/l of sucrose, 0.1 g/l of FeSO4·7H2O, 0.1 g/l of MgSO4·7H2O, 0.1 g/l of MnSO4·2H2O, 0.1 g/l of ZnSO4·7H2O, 1.52 g/l of Na2HPO4, and 172.45 g/l of wheat bran juice. With this optimal medium, the milk-clotting enzyme production was remarkably enhanced. The milk-clotting enzyme activity reached 3,326.7 SU/ml after incubation of 48 h, which was 1.76-fold higher than that of the basic medium, showing that the Plackett-Burman design and Box-Behnken response surface method are effective to optimize medium components, and B. amyloliquefaciens D4 possessed a high rennet-producing capacity in the optimal medium.

Isolation of clt Genes Encoding milk clotting enzyme and characterization of milk clotting enzyme from new isolate Streptomyces pseudogriseolusNRC-15

Research focused on isolation and characterization on new strain of Streptomyces pseudogrisiolus from Egyptian soil have proteinases which possesses activity (MCE) identified by 16S rRNA with universal primer and submit in genbank with accession number AB739622. In this work, study optimizes the extraction of milk-clotting enzyme (MCE) according to its adequacy to be employed as substitute of rennin. The organism grew well in medium 3 after 4days of incubation at temperatures of 35° ±2, pH 6.0. Cultural and environmental conditions for milk clotting enzyme production by Streptomyces were studied. Glucose (3%, w/v) and ammonium sulphate (0.2%w/v), were found to be the best C and N sources for milk clotting enzyme production respectively. Partially purified and characterized. MCE was obtained by fractional precipitation with ammonium sulphate, followed by the chromatography of the most active fraction on Sephadex G-100 with 20.39 purification fold and recovery about 80%. The maximum enzyme activity was at pH (6.5) and 45oC. The clotting activity of the purified enzyme was stimulated with increasing CaCl2 concentration up to 10mM. However, a gradual reduction of the activity was observed by increasing NaCl concentration between 4-8%. Whereas, Cu +2 and Zn +2 significant inhibition it. Interestingly MnCl2 and MgSO4 were found to have stimulatory effect, which has been rarely reported. Also study includes some kinetics calculation about MCE. Gene control MCE expression was detected by specific primer design using DNA star program, sequence illustrate gene found clt a and clt b, and submitted in genbank as new sequence with accession number AB739621. The milk clotting enzyme (MCE) obtained from S. pseudogrisiolus has potential as calf rennet substitutes in dairy products and it can be used in lypholyzed form as tablets in pharmaceutical products for patients with digestive system.

Statistical Optimization of Culture Conditions for Milk-Clotting Enzyme Production by Bacillus Amyloliquefaciens Using Wheat Bran-An Agro-Industry Waste

In order to improve the production of the milk-clotting enzyme under submerged fermentation, two statistical methods were applied to optimize the culture conditions of Bacillus amyloliquefaciens D4 using wheat bran as nutrient source. First, initial pH, agitation speed, and fermentation time were shown to have significant effects on D4 enzyme production using the Plackett-Burman experimental design. Subsequently, optimal conditions were obtained using the Box-Behnken method, which were as follows: initial pH 7.57, agitation speed 241rpm, fermentation time 53.3h. Under these conditions, the milk-clotting enzyme production was remarkably enhanced. The milk-clotting enzyme activity reached 1996.9SU/mL, which was 2.92-fold higher than that of the initial culture conditions, showing that the Plackett-Burman design and Box-Behnken response surface method are effective to optimize culture conditions. The research can provide a reference for full utilization of wheat bran and the production of milk-clotting enzyme by B. amyloliquefaciens D4 under submerged fermentation.

Optimization of the production and characterization of milk clotting enzymes by Bacillus subtilis natto

Suitable medium for production of milk clotting enzyme (MCE) by Bacillus subtilis (natto) Takahashi in submerged liquid-state fermentation was screened, the nutrient factors affecting MCE production was optimized by response surface methodology. The MCE production by B. subtilis (natto) Takahashi was increased significantly by 428% in the optimal medium developed. The MCE was filtered and concentrated by ultrafiltration. The retentate after tandem filtration carried out with the combined membranes of MWCO 50kDa and 5 kDa showed two major bands between 25kDa and 30kDa on SDS-PAGE, and the MCA and MCA/PA improved significantly in comparison with those in the initial broth. The crude enzyme thus obtained showed MCA and MCA/PA ratio of 48,000 SU/g and 6,400, which are commensurate with those (MCA 26,667 SU/g and MCA/PA 6,667) of the commercial rennet. It had optimal pH and temperature at pH 6 and 60°C, and showed excellent pH and thermal stability.

A two-stage oxygen supply control strategy for enhancing milk-clotting enzyme production by Bacillus amyloliquefaciens

To enhance the yield and productivity of milk-clotting enzyme (MCE) by Bacillus amyloliquefaciens, a two-stage oxygen supply control strategy was proposed and successfully applied in the MCE fermentation. During the first 16 h, KLa was controlled at 72.2 h−1 to obtain high cell growth rate (v) and MCE activity (MCA) productivity (r MCA). Subsequently, KLa was controlled at 33.9 h−1 to maintain high specific MCA productivity (q MCA). Using this strategy, MCA peaked at 36 h with the MCA of 6,590.41 SU ml−1, which was 18 h earlier than other investigated processes. The concept and results described represent the basis of an industrial scale-up process to achieve high MCE yield, MCA productivity and MCA/proteolytic activity.

Comparative evaluation of Bacillus licheniformis 5A5 and Aloe variegata milk-clotting enzymes

The properties of a milk clotting enzyme (MCE) produced by bacteria (Bacillus licheniformis 5A5) were investigated and compared to those of rennet extracted from a plant (Aloe variegata). Production of MCE by B. licheniformis 5A5 was better in static than in shaken cultures. Maximum activity (98.3 and 160.3 U/ml) of clotting was obtained at 75oC and 80oC with bacterial and plant rennet, respectively. In the absence of substrate, the clotting activity of Aloe MCE was found to be less sensitive to heat inactivation up to 80oC for 75 min, retaining 63.8% of its activity, while bacterial MCE was completely inhibited. CaCl2 stimulated milk clotting activity (MCA) up to 2% and 1.5% for bacterial and plant enzymes. NaCl inhibited MCA for both enzymes, even at low concentration (1%). Plant MCE was more sensitive to NaCl at 3% concentration it retained 30.2% of its activity, whereas bacterial MCE retained 64.1%. Increasing skim milk concentration caused a significant increase in MCA up to 6% for both enzymes. Mn2+ stimulated the activity of bacterial and plant enzymes to 158.6 and 177.9%, respectively. EDTA and PMSF increased the activity of plant MCE by 34.4 and 41.1%, respectively, which is higher than those for the bacterial MCE (19.1 and 20.9%). Some natural materials activated MCE, the highest activation of bacterial MCE (128.1%) was obtained in the presence of Fenugreek (with acid extraction). However Lupine Giza 1 (with neutral extraction) gave the highest activation of plant MCE (137.9%). All extracts from Neem plant increased MCA at range from 105.6% to 136.4%. Plant MCE exhibited much better stability when stored at room temperature (25-30oC) for 30 days, retaining 51.2% of its activity. Bacterial MCE was highly stabile when stored under freezing (-18oC), retaining 100% of its activity after 30 days. Moreover, bacterial MCE was highly tolerant to repeated freezing and thawing without loss of activity for 8 months.

English

Three strains, Bacillus subtilis B1, B. subtilis B18 and Bacillus thuringiensis B12,were screened from wheat bran to produce milk-clotting enzyme. Among them, B. subtilis B1 exhibited considerable milk-clotting activity with low proteolytic activity. After response surface methodology optimization, milk-clotting activity wasimproved from 782 SU ml-1 to 1129.05 ± 74.55 SU mL-1 with a small inoculum size (0.130%, v/v). The optimized medium contained glucose (16.2 gL-1), wheat bran (30 gL-1), NaCl (5 gL-1), MgSO4·7H2O (5 gL-1), KH2PO4 (2 gL-1) and CaCO3 (3 gL-1). The milk-clotting enzyme from B. subtilis B1 has the optimum pH 5.5 and CaCl2concentration 50 mmol L-1. It is completely inactivated after 5 min at 70°C. The result showed that B. subtilis B1 was a promising strain for industrial milk-clotting enzyme production.   Key words: Milk-clotting enzyme, Bacillus subtilis, submerged fermentation, wheat bran.

Fermentation parameter and partial biochemical characterisation of milk clotting enzyme from Chinese distiller’s yeast

A strain F34 producing milk clotting enzyme with high yield was obtained from Chinese distiller’s yeast, using casein plate prescreening and fermentation reselection. The optimum fermentation medium for milk clotting enzyme production was as follows: 5.0% rice flour and bran digest, 5.0% bean flour digest, 0.5% CaCl2, 0.5% KCl, initial pH 4.5. The optimal fermentation parameters was at 34°C for 60h. The enzyme action was optimal at 50°C and at about pH 5.5. In the absence of substrate the enzyme showed good stability at 40°C and pH 3.5. Ca2+, Zn2+, Fe3+ and Fe2+ showed stimulation on the milk clotting activity; K+ and Mg2+ showed weak stimulation; Na+, Cu2+, Co2+ and Li2+ showed inhibition. The results indicate that this milk clotting enzyme may partially substitute calf rennet in the cheese manufacture.

Milk-clotting enzymes produced by culture of Bacillus subtilis natto

Abstract Factors affecting the production of milk-clotting enzyme (MCE) by Bacillus subtilis (natto) Takahashi, a ready available commercial natto starter, were studied. Remarkable milk-clotting activity (MCA), 685.7 SU/ml or 12,000 SU/g, was obtained when the bacteria were cultivated in the medium containing sucrose (50 g/L) and basal salts at pH 6, 37 °C with shaking at 175 rpm for 1 day. The MCA and MCA/PA ratio of the crude enzyme obtained are comparable with those of Pfizer microbial rennin and Mucor rennin. The crude enzyme showed excellent pH and thermal stability; it retained 96% of MCA after incubation for 40 min at 40 °C and retained more than 80% of its activity between pH 4 and pH 7 for more than 30 min at 30 °C. The MCE of B. subtilis (natto) Takahashi has potential as calf rennet substitutes.