• Microbiology and Biotechnology Letters
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• v.19 no.2
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• pp.109-115
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• 1991

Twenty-five fungal strains producing an extracellular soybean milk clotting enzyme were isolated from 146 soil samples, and identified as 11 species belonging to six genera of Aspergillus oryzae (5 strains), Aspergillus flavus (2 strains), Aspergillus parasiticus (1 strain), Aspergillus tamarii (2 strains), Aspergillus niger (4 strains), Aspergillus fumigatus (2 strains), Mucor hiemalis (2 strains), Wallemia sebi (4 strains), Scopulariopsis condida (1 strain), Fusarium redolens(1 strain) and Verticillum lecanii (1 strain). Among them, Aspergillus oryzae 020 and Aspergillus tamarii 287 showed relatively high soybean milk clotting activity. The coagulabilities of the enzyme from representative strains of those species decreased as the pH of soybean milk increased from 6.0 to 7.0 The optimum temperature for soybean milk clotting enzymes of those strains were 65$^{\circ}C$.

• Journal of the Korean Society of Food Science and Nutrition
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• v.21 no.4
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• pp.418-422
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• 1992

Mucor mucedo C-7 selected as a potent fungus for producing milk-clotting enzyme was cultured on wheat bran solid medium and the optimum culture conditions for the production of milk clotting enzyme were ot tamed as follows. Amount of water added to wheat bran was 100% to the weight of wheat bran and culture temperature and time was 3$0^{\circ}C$ and 72hrs, respectively. The production of milk-clotting enzyme was markedly increased by the addition of Macllvaine buffer solution (pH4.5) instead of water added to wheat bran solid medium and milk-clotting activity was stable for culture period.

• Microbiology and Biotechnology Letters
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• v.17 no.2
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• pp.109-114
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• 1989

Seventeen bacterial strains producing an extracellular soybean milk clotting enzyme were Isolated from 150 soil samples, and identified as Bacillus cereus(8 strains), Bacillus pumilus(8 strains) and Bacillus licheniformis (1 strain). Among them, Bacillus pumilus strain 118 and Bacillus licheniformis strain 192 showed relatively high soybean milk clotting activity. The coagulability of enzymes from these strains decreased as the pH of soybean milk was increased from 6.0 to 7.0. The optimum temperature for soybean milk clotting activity was $65^{\circ}C$.

• Korean Journal of Food Science and Technology
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• v.3 no.3
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• pp.172-177
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• 1971

The amount of the milk clotting enzyme which is produced by Dothiorella ribis in wheat bran was 950 Soxhlet units per gram of wheat bran. The milk clotting activity of this enzyme was increased by the elevation of clotting temperature and by the increase of the addition of $CaCl_2$ to milk. It was also increased when the pH of milk was lower than that of the fresh milk. When milk was diluted by distilled water, the milk clotting activity of the enzyme was decreased. And its milk clotting activity was good when milk was pasteurized at low temperature. The enzyme of Dothiorella ribis has larger proteolytic activity per Soxhlet unit than that of the milk clotting enzyme of Mucor pusillus Lindt. This enzyme was rather stable between pH 6 and pH 8 when it was conditioned for ten minutes. The heat stability of enzyme was tested by treating it under the condition for $10{\sim}60$ minutes. And the enzyme was stable under the temperature of $45^{\circ}C$. Also the recovery of protein as a form of curd was 76.2 percent to the total protein content of milk.

• Microbiology and Biotechnology Letters
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• v.18 no.5
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• pp.449-453
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• 1990

This study was attempted to obtain the efficient milk-clotting enzyme from microorganisms as a rennet subtitute. Fungi which showed the formation ability of the milk-clotting enzyme were selected out from samples of soil hay and wastes etc. Among these isolated fungi, strain no. C-7 which had presented higher value in the ratio of milk-clotting activity to proteolytic activity was selected. The hyphae of this strain was white to gray and no septa. A single sporangiophore which stand erectly above growing hyphae was monomucor type without branching. A globose sporangium was developed at the tip of each sporangiophore. The suitable temperature and pH for the growth of no. C-7 was 20-$30^{\circ}C$ and pH 3.0-8.0 respectively. These morphological and physiological characteristics implied that strain no. C-7 was Mueor mucedo.

• Applied Biological Chemistry
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• v.12
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• pp.7-11
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• 1969

Mucor-rennin, the crystalline milk-clotting enzyme, isolated from Mucor pusillus var. Lindt, has an acid protease activity. The optimum pH for the digestion of k-casein is 4.5, while that for hemoglobin digestion is 4.0. The skim milk solution was easily clotted acidic solution than alkalin solution, and the milk clotting activated by Ca ion. The enzyme was heat stable against heat from pH 4.0 to 6.0 but was more stable at pH 5.0. The activity of the enzyme at pH 5.0 did not decrease at 30 C for 15 days and the activity was not effected by sodium propionate and salicilic acid. Therefore, the enzyme of liguid type could store for a long time and could be transported from Erzyme production Co. to Manufacture of cheese Co. by adding the antiesptic and by adjusting pH to 5.0.

• Korean Journal of Agricultural Science
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• v.14 no.1
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• pp.144-155
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• 1987

A potent fungus producing milk clotting enzyme with fairly weak proteolytic activity was isolated from various soil and sewage, which the selected strain, SA-101, was identified as Mucor sp. with microbiological characteristics. Its milk clotting enzyme production was maximized when grown on 10g of wheat bran media added to 8ml of tap water containing 0.1M HCl for 60hrs at $30^{\circ}C$. This enzyme production was stimulated by addition of 6% lactose, 0.05% NaCl and reached a maximal level of 9810 unit/g wheat bran. The crude enzyme product could be produced effectively by salting out with ammonium sulfate fractionation and lyophilization. The ratio of milk clotting activity to proteolytic activity of crude enzyme product was lower than Hansen rennet, but resembled to Meito rennet. The optimal temperature of milk clotting activity of crude enzyme product was abound $60^{\circ}C$ on a substrate of 10% reconstituted skim milk containing 1/100M $CaCl_2$.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.10
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• pp.1369-1379
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• 2010

This study investigated purification and milk-clotting activity of the enzymes produced by Bacillus subtilis var, natto and Rhizopus oligosporus compared with that of commercial rennet. The clotting time, viscosity, tension and microstructure of the curd and electrophoretic patterns of milk proteins were determined. The milk-clotting activity/proteolytic activity ratios (MCA/PA ratio) of B. subtilis, R. oligosporus and commercial rennet were also compared. The results revealed that the curd formed by the commercial rennet had the highest viscosity and curd tension and the shortest clotting time among the three enzymes. However, curd produced by Rhizopus enzymes was ranked as second. From the MCA/PA ratio and electrophoretogram analyses it could be concluded that the enzyme produced by B. subtilis had the highest proteolytic activity, while the commercial rennet had the highest milk-clotting activity. Observations of microstructures of SEM showed that the three-dimensional network for curd formed by commercial rennet was denser, firmer and more smooth. The milk-clotting activity, specific activity, purification ratio and recovery of the purified enzymes produced by both the tested organisms were also determined with ion exchange chromatography and gel filtration.

• Korean Journal of Food Science and Technology
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• v.3 no.2
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• pp.89-93
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• 1971

Microorganisms producing milk-clotting enzyme were isolated from 1,506 strains which were collected from soil on the various places of Korea, and from strains which were already identified. Dothiorella ribis was taken as a good strain producing milk-clotting enzyme. When it is cultured on wheat bran, the optimum experimental conditions for the production of milk-clotting enzyme were consequently obtained as follows: 1) $30{\sim}35^{\circ}C$ of temperature and 4.0 of pH. 2) $60{\sim]80%$ of cultivating water to the weight of wheat bran. 3) addition of $(NH_4)_2SO_4$ as a nitrogen source, $NaCl\;and\;KH_2PO_4$ as an inorganic salt, and 3% of sucrose as a carbon source. 4) four days for a period of cultivation.

• Journal of Microbiology and Biotechnology
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• v.23 no.8
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• pp.1084-1091
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• 2013

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 $Na_2HPO_4$ 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 $FeSO_4{\cdot}7H_2O$, 0.1 g/l of $MgSO_4{\cdot}7H_2O$, 0.1 g/l of $MnSO_4{\cdot}2H_2O$, 0.1 g/l of $ZnSO_4{\cdot}7H_2O$, 1.52 g/l of $Na_2HPO_4$, 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.