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Isolation and Fermentation Condition of Milk-clotting Enzyme Producing Strain from Glutinous Rice Wine
CHINESE JOURNAL OF BIOTECHNOLOGY Volume 24, Issue 6, June 2008 Online English edition of the Chinese language journal RESEARCH PAPER
Cite this article as: Chin J Biotech, 2008, 24(6), 999í1003.
Isolation and Fermentation Condition of Milk-clotting Enzyme Producing Strain from Glutinous Rice Wine Qiaoling Cheng, Xiaojia Bai, and Yanping Wang Tianjin Key Laboratory of Food Nutrition & Safety, Department of Food Engineering & Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China
Abstract: Glutinous rice wine is a traditional food in south of China and it can coagulate milk. It has been proved that its function of coagulating milk is because of the presence of milk-clotting enzyme produced by microorganisms in glutinous rice wine. The aim of this work is to isolate milk-clotting enzyme producing strain from glutinous rice wine and study the fermentation condition. We screened out four bacteria and fungus by gradient dilution. It was proved that mold played the most important role in the production of milk-clotting enzyme. This is further confirmed by casein plate method. The optimization of fermentation conditions revealed that two times concentrated potato medium supplemented with 5% glucose without additional nitrogen was better for production of the enzyme. The enzyme activity was increased 144% under the conditions established. Keywords: glutinous rice wine; milk-clotting enzyme producing strain; isolation; fermentation condition
Introduction Milk-clotting enzyme, the key enzyme in cheese production, is a very important enzyme in the food industry[1]. Chymosin (EC 3.4.23.4) is the traditional milk-clotting enzyme (MCE), obtained from the fourth stomach of the unweaned calf[2]. Problems associated with animal slaughtering have made it necessary to find other alternatives to calf chymosin[3]. It is the most promising direction to use microbial enzymes as substitutes of chymosin[4], because microorganisms grow rapidly, and their growth substrates are relatively inexpensive. However, some amounts of non-enzyme metabolites are formed by the enzymeproducing organisms, residues from the fermentation medium, and the recovery processes. For this reason, several tests, such as oral toxicity, carcinogenicity, and cutaneous toxicity (skin and eye irritation), have to be carried out before a new enzyme product for the food industry can be marketed. Thus, the strains that can be used
are limited to some extent. Glutinous rice wine, a traditional food in south China, can coagulate milk. The milk-clotting enzyme in glutinous rice wine is a kind of metabolite of the microorganisms in fermenting starter[5]. Thus, we do not need to worry about safety problems using strain, which is isolated from glutinous rice wine to produce the milk-clotting enzyme. In the present study, the microorganisms of glutinous rice wine were isolated and purified, and milk-clotting enzyme producing strain was confirmed and the fermentation conditions were investigated. This study lays the foundation for finding a new milk-clotting enzyme producing strain.
1
Materials and methods
1.1 Materials Glutinous rice powder: the fresh glutinous rice was triturated, which was bought from located market; Starters: bought from 4 different factories in Jingzhou,
Received: January 15, 2008; Accepted: March 12, 2008 Supported by: the grant from Science and Technology Supporting Project of National Eleventh Five-Year-Plan (No. 2006BAD04A06). Corresponding author: Yanping Wang. Tel: + 86-22-60601400; E-mail: [email protected] Copyright © 2008, Institute of Microbiology, Chinese Academy of Sciences and Chinese Society for Microbiology. Published by Elsevier BV. All rights reserved.
Qiaoling Cheng et al. / Chinese Journal of Biotechnology, 2008, 24(6): 999–1003
Hubei province; Glutinous rice wine: 4 different starters were cultured to glutinous rice powder, fermented for 2 days, and the glutinous rice wine that possessed the highest milk-clotting activity was selected to study. The other chemicals used were of analytical grade. 1.2 Medium PDA: 200 g of sliced (washed and unpeeled) potatoes were boiled in water for 1 h and then decanting or straining off the broth was done. Dextrose was then added (20 g/L) and the medium was sterilized under 115oC for 20 min. Glutinous rice powder culture: 50 mL of 10 g/100 mL glutinous rice powder solution was added into 250 mL flask, shaken, and then sterilized under 115oC for 20 min. Other cultures were made as described by Du LX[6]. 1.3 Methods 1.3.1 Enzyme assay: The milk-clotting activity (MCA) was determined as described by Arima and Iwasake[7]. 5 mL of 100 g/L skimmed milk was taken in a test tube and the contents were kept under the temperature of 37oC for 5 min. 0.5 mL of diluted glutinous rice wine or fermentation fluid was then added and mixed immediately. The curd formation was observed and when discrete particles were discernible, the end time point was recorded. One milk clotting unit (Soxhelt unit, SU) is defined as the amount of enzyme required to clot 1 mL of 10g/L skimmed milk in 40 min. SU=2400/T×5/0.5×D where, T is the clotting time(s) and D is the dilution factor. Relative unit (RU) indicates the effect of different factors. RU(%)=SU under experiment condition/SU under control condition×100% 1.3.2 Glutinous rice wine preparation: The starter (0.32 g/L) was transferred to the glutinous rice power medium and the flasks were incubated with orbital shaker (140 rpm) for 52 h at 30oC. 1.3.3 Isolation and purification of microorganisms from glutinous rice wine: Glutinous rice wine was diluted by gradient dilution[8]; 100ǂL of 10í4, 10í5, 10í6, 10í7, 10í8 dilutions were separately spread onto minimal medium, complete medium, and PDA medium, and then cultured under 37°C, 30°C, or 28°C, respectively. Different colonies were chosen for further purification after 2 d, 3 d, and 5 d. 1.3.4 Confirmation of milk-clotting enzyme producing strain: (1) Microorganisms counting and MCA assay during fermentation: Diluted glutinous rice wine was spread onto bacterial basal medium, potato medium, and YEPD medium, respectively. The bacteria, mold, and yeast were numbered by colony counting method. MCA assay was carried out following 1.3.1. (2) Confirmation of milk-clotting enzyme producing strain by the casein plate method Inoculated the bacteria, mold, and yeast strains which has been isolated and purified to casein plate by the three points method.
The plates were cultured at 37oC, 28oC, and 30oC, respectively, for 2 d, and the size of the deposit circle was observed. 1.3.5 Optimization of fermentation conditions for milk-clotting enzyme production: (1) Effect of different carbon source on the activity of milk-clotting enzyme produced by mold: 3% (W/V) of glutinous rice powder, soluble starch, and glucose, were respectively added into PDA medium, and sterilized under 115oC for 20min. After cooling, mold spores were inoculated (0.1% V/V) and cultured with orbital shaker (140 rpm) at 30°C; samples were taken at 48h and MCA assay was carried out. (2) Effect of different concentration of glucose in PDA on the activity of milk-clotting enzyme: glucose was added to a final concentration of 2%, 3%, 5%, 7%, 10% (W/V) in PDA medium. Fermentation and MCA assay were carried out following 1.3.5(1). (3) Effect of different nitrogen source on the activity of milk-clotting enzyme produced by mold: 0.5% (W/V) of tryptone, animal protein hydrolysate, soybean protein, (NH4)2SO4, (NH4)3C6H5O7, and NH4NO3 were added into PDA medium, respectively. Fermentation and MCA assay were carried out following 1.3.5(1). (4) Effect of concentration of liquid PDA on the activity of milk-clotting enzyme: PDA medium was concentrated to 2 times, 3 times, and 4 times by Rotary Evaporator; Fermentation and MCA assay were carried out following 1.3.5(1).
2 2.1
Results and discussion
Isolation and purification of microorganisms in glutinous rice wine Four bacteria and fungi were screened out from glutinous rice wine (Fig.1). It can be judged from the colony and morphology of microorganisms that the four strains included one mold, one yeast, and two bacteria. In this study, the microorganisms were isolated from glutinous rice wine, which was fermented, and thus fewer microorganisms were obtained compared with that isolated from the starter. Since in the starter, the microorganisms balanced themselves by rivalizing the environment and space for living, and since the starter was produced in open system, some adventitious microorganism were easy to intrude. During fermenting, the growth of adventitious microorganism can be inhibited by preponderant microorganism; therefore, isolating microorganism from glutinous rice wine can minish the work and find the milk-clotting enzyme producing strain quickly. 2.2 Confirmation of milk-clotting enzyme producing strain 2.2.1 Microorganisms counting and MCA assay during fermentation Microorganisms counting and MCA assay are carried out following 1.3.4(1), and the results are shown in Fig.2.
Qiaoling Cheng et al. / Chinese Journal of Biotechnology, 2008, 24(6): 999–1003
Fig. 1 Morphology of microorganisms isolated from glutinous rice wine
Fig. 2
Relation between the change of bacteria, yeast, mold
counts and milk-clotting activity during fermentation of glutinous rice wine
As seen in Fig.2, there were more bacteria in the initial fermentation period than in the later period. It was possibly because along with the fermentation time increasing, the system acidity increased gradually, which was not suitable for the bacteria to grow; also, with the increase of yeast and mold quantity, the habitat of bacteria was destroyed unceasingly. The mold quantity achieved a peak after 48 h and started to drop, and the yeast quantity also increased until fermentation for 60 h, and then maintained stably. This was mainly because the mold can hydrolyze starch into monosaccharide, which can accelerate yeast to propagate. However, alcohol, one of the yeast metabolites, restrained the propagating of mold, and therefore, the mold quantity started to drop when the yeast increased to a certain level. It is noteworthy that the change tendency of the mold quantity and MCA of glutinous rice wine maintained consistently. After fermenting for 48 h, the mold quantity and MCA of glutinous rice wine achieved their peaks
synchronously. From this result, mold was thought to be the most probable milk-clotting enzyme producing strain by and large. 2.2.2 Confirmation of milk-clotting enzyme producing strain by casein plate method According to 1.3.4(2), four strains were inoculated to the casein plate; two bacteria and the yeast could grow on the plates, but no precipitation circle appeared. Around the mold, a white precipitation circle with diameter of about 1 cm was observed. This result further confirmed that mold was the milk-clotting enzyme producing strain in glutinous rice wine. 2.3 Optimization of fermentation conditions for milkclotting enzyme production The MCA of enzyme produced by microorganism is related with the way and the conditions of fermentation besides being related with the microorganism itself. Generally speaking, the liquid in-depth fermentation is simple to operate, and the automation and control sterile process is easy to realize. This study optimized the fermentation conditions for milk-clotting enzyme production, and enhanced the MCA to a certain extent. 2.3.1 Effect of different carbon source on the activity of milk-clotting enzyme produced by mold: Experiments were carried out according to 1.3.5(1), and results are shown in Fig. 3. As seen in Fig.3, the results indicate that adding glucose into PDA fluid had a very clear function to increase MCA whereas adding glutinous rice powder and soluble starch into PDA fluid resulted in the decrease in MCA slightly. Supplemented with 3% glucose, the enzyme activity was 137% that of the control. This indicated that adding glucose as carbon source could promote mold propagating, and was advantageous for increasing MCA. Therefore, glucose was selected as the additional carbon source. 2.3.2 Effect of different concentration of glucose in PDA on the activity of milk-clotting enzyme: According to 1.3.5(2), the effect of different concentrations of glucose in PDA on the activity of milk-clotting enzyme was investigated, and the results are shown in Fig. 4.
Fig. 3
Effect of different carbon source on activity of milk-clotting enzyme produced by mold
1: blank; 2: glucose; 3: glutinous rice power; 4: soluble starch
Qiaoling Cheng et al. / Chinese Journal of Biotechnology, 2008, 24(6): 999–1003
From Fig.4 it suggested that when 5% glucose was added in PDA, the MCA was maximal. As the glucose concentration continued to increase, the mold was eugonic, but the MCA dropped gradually. This was possibly because the nutrition needed for mold propagating was different from that of enzyme production. The suitable nutritive proportion was the essential condition for mold to produce enzyme. 5% of glucose was chosen for the best concentration. 2.3.3 Effect of different nitrogen source on the activity of milk-clotting enzyme produced by mold: Different nitrogen sources were tested according to 1.3.5(3), and the results obtained are shown in Fig.5. The result shown in Fig.5 indicated that the nutrition of PDA fluid was rich and its nitrogen source could meet the needs of mold. Additional nitrogen source had a certain function to decrease MCA, and inorganic nitrogen source showed stronger inhibitory action than organic nitrogen source. This was mainly related to the nutrient needs, which were provided for enzyme production. Our results show that there was no need to add additional nitrogen source. 2.3.4 Effect of different concentration of liquid PDA on the activity of milk-clotting enzyme: The result shown in Fig.4 indicated that the concentration of glucose in PDA had a very clear influence on MCA. Thus, we tested the effect of other nutrition ingredient in PDA fluid on MCA produced by mold. Experiments were carried out according to 1.3.5(4) and the results are shown in Fig.6.
Fig. 4 Effect of different concentration of glucose in PDA on activity of milk-clotting enzyme
Fig. 6 Effect of different concentration of liquid PDA on activity of milk-clotting enzyme
As seen in Fig. 6, two times of PDA fluid was more advantageous for mold to produce milk-clotting enzyme compared with original liquid PDA. With the increase of PDA concentration, although the mold grew well, the MCA dropped. This was possibly because the nutrition that was provided for mold propagating was different from that for enzyme production. Two times of PDA fluid was the most suitable PDA concentration.
3
Conclusions
This study has isolated milk-clotting enzyme producing strain from glutinous rice wine and has studied the fermentation condition. Four bacteria and fungus were screened out by gradient dilution. The relations between the change of bacteria, yeast, mold counts, and milk-clotting activity during the fermentation of glutinous rice wine were investigated It was proved that mold played the most important role in the production of milk-clotting enzyme. This was further confirmed by the casein plate method. The optimization of the fermentation conditions revealed that two times concentrated potato medium supplemented with 5% glucose without additional nitrogen was better for production of the enzyme. The relative enzyme activity was increased to 144% under the conditions established. Further identification of the mold and optimization of its fermentation process conditions were carried out by our team.
REFERENCES [1] Dao RN. New dairy product of home market-cheese. China Dairy Cattle, 2000, 2: 4748. [2] Zhang FX. Studies on extraction technology of calf rennet. Journal of Northwest Sci-Tech University of Agriculture and Forestry, 2002, 30(1): 2426. Fig. 5
Effect of different nitrogen source on activity of milk-clotting enzyme produced by mold
1: blank; 2: tryptone; 3: animal protein hydrolysate; 4: soybean protein; 5: (NH4)2SO4; 6: (NH4)3C6H5O7; 7: NH4NO3
[3] Meng GZ. Progress in research of milk clotting enzymes. Micorbiology, 1987, 14(2): 3537. [4] Zhou JQ, Lin QL, Zhao MM. Studies on the production of chymosin by microorganisms. China Food Addtives, 2004,
Qiaoling Cheng et al. / Chinese Journal of Biotechnology, 2008, 24(6): 999–1003
2: 69. [5] Liu ZM, Luo CY. Studies on milk-clotting mechanism of
[7] Arimk K, Iwasake K. Milk-clotting enzyme from microorganisms part I screening test and the identification of the
glutinous rice wine. Food Science, 2000, 1(21): 1316.
potent fungus. Agric Biol Chem, 1967, 31(5): 540545.
[6] Du LX. Protocols for Industrial Microbiology. Tianjin:
[8] Zhang JZ. Microbiology Chemotaxonomy. Beijing: China
Tianjin Science and Technology Press, 1992, 8788.
Light Industry Press, 1996, 154156.
Cite this article as: Chin J Biotech, 2008, 24(6), 999í1003.
Isolation and Fermentation Condition of Milk-clotting Enzyme Producing Strain from Glutinous Rice Wine Qiaoling Cheng, Xiaojia Bai, and Yanping Wang Tianjin Key Laboratory of Food Nutrition & Safety, Department of Food Engineering & Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China
Abstract: Glutinous rice wine is a traditional food in south of China and it can coagulate milk. It has been proved that its function of coagulating milk is because of the presence of milk-clotting enzyme produced by microorganisms in glutinous rice wine. The aim of this work is to isolate milk-clotting enzyme producing strain from glutinous rice wine and study the fermentation condition. We screened out four bacteria and fungus by gradient dilution. It was proved that mold played the most important role in the production of milk-clotting enzyme. This is further confirmed by casein plate method. The optimization of fermentation conditions revealed that two times concentrated potato medium supplemented with 5% glucose without additional nitrogen was better for production of the enzyme. The enzyme activity was increased 144% under the conditions established. Keywords: glutinous rice wine; milk-clotting enzyme producing strain; isolation; fermentation condition
Introduction Milk-clotting enzyme, the key enzyme in cheese production, is a very important enzyme in the food industry[1]. Chymosin (EC 3.4.23.4) is the traditional milk-clotting enzyme (MCE), obtained from the fourth stomach of the unweaned calf[2]. Problems associated with animal slaughtering have made it necessary to find other alternatives to calf chymosin[3]. It is the most promising direction to use microbial enzymes as substitutes of chymosin[4], because microorganisms grow rapidly, and their growth substrates are relatively inexpensive. However, some amounts of non-enzyme metabolites are formed by the enzymeproducing organisms, residues from the fermentation medium, and the recovery processes. For this reason, several tests, such as oral toxicity, carcinogenicity, and cutaneous toxicity (skin and eye irritation), have to be carried out before a new enzyme product for the food industry can be marketed. Thus, the strains that can be used
are limited to some extent. Glutinous rice wine, a traditional food in south China, can coagulate milk. The milk-clotting enzyme in glutinous rice wine is a kind of metabolite of the microorganisms in fermenting starter[5]. Thus, we do not need to worry about safety problems using strain, which is isolated from glutinous rice wine to produce the milk-clotting enzyme. In the present study, the microorganisms of glutinous rice wine were isolated and purified, and milk-clotting enzyme producing strain was confirmed and the fermentation conditions were investigated. This study lays the foundation for finding a new milk-clotting enzyme producing strain.
1
Materials and methods
1.1 Materials Glutinous rice powder: the fresh glutinous rice was triturated, which was bought from located market; Starters: bought from 4 different factories in Jingzhou,
Received: January 15, 2008; Accepted: March 12, 2008 Supported by: the grant from Science and Technology Supporting Project of National Eleventh Five-Year-Plan (No. 2006BAD04A06). Corresponding author: Yanping Wang. Tel: + 86-22-60601400; E-mail: [email protected] Copyright © 2008, Institute of Microbiology, Chinese Academy of Sciences and Chinese Society for Microbiology. Published by Elsevier BV. All rights reserved.
Qiaoling Cheng et al. / Chinese Journal of Biotechnology, 2008, 24(6): 999–1003
Hubei province; Glutinous rice wine: 4 different starters were cultured to glutinous rice powder, fermented for 2 days, and the glutinous rice wine that possessed the highest milk-clotting activity was selected to study. The other chemicals used were of analytical grade. 1.2 Medium PDA: 200 g of sliced (washed and unpeeled) potatoes were boiled in water for 1 h and then decanting or straining off the broth was done. Dextrose was then added (20 g/L) and the medium was sterilized under 115oC for 20 min. Glutinous rice powder culture: 50 mL of 10 g/100 mL glutinous rice powder solution was added into 250 mL flask, shaken, and then sterilized under 115oC for 20 min. Other cultures were made as described by Du LX[6]. 1.3 Methods 1.3.1 Enzyme assay: The milk-clotting activity (MCA) was determined as described by Arima and Iwasake[7]. 5 mL of 100 g/L skimmed milk was taken in a test tube and the contents were kept under the temperature of 37oC for 5 min. 0.5 mL of diluted glutinous rice wine or fermentation fluid was then added and mixed immediately. The curd formation was observed and when discrete particles were discernible, the end time point was recorded. One milk clotting unit (Soxhelt unit, SU) is defined as the amount of enzyme required to clot 1 mL of 10g/L skimmed milk in 40 min. SU=2400/T×5/0.5×D where, T is the clotting time(s) and D is the dilution factor. Relative unit (RU) indicates the effect of different factors. RU(%)=SU under experiment condition/SU under control condition×100% 1.3.2 Glutinous rice wine preparation: The starter (0.32 g/L) was transferred to the glutinous rice power medium and the flasks were incubated with orbital shaker (140 rpm) for 52 h at 30oC. 1.3.3 Isolation and purification of microorganisms from glutinous rice wine: Glutinous rice wine was diluted by gradient dilution[8]; 100ǂL of 10í4, 10í5, 10í6, 10í7, 10í8 dilutions were separately spread onto minimal medium, complete medium, and PDA medium, and then cultured under 37°C, 30°C, or 28°C, respectively. Different colonies were chosen for further purification after 2 d, 3 d, and 5 d. 1.3.4 Confirmation of milk-clotting enzyme producing strain: (1) Microorganisms counting and MCA assay during fermentation: Diluted glutinous rice wine was spread onto bacterial basal medium, potato medium, and YEPD medium, respectively. The bacteria, mold, and yeast were numbered by colony counting method. MCA assay was carried out following 1.3.1. (2) Confirmation of milk-clotting enzyme producing strain by the casein plate method Inoculated the bacteria, mold, and yeast strains which has been isolated and purified to casein plate by the three points method.
The plates were cultured at 37oC, 28oC, and 30oC, respectively, for 2 d, and the size of the deposit circle was observed. 1.3.5 Optimization of fermentation conditions for milk-clotting enzyme production: (1) Effect of different carbon source on the activity of milk-clotting enzyme produced by mold: 3% (W/V) of glutinous rice powder, soluble starch, and glucose, were respectively added into PDA medium, and sterilized under 115oC for 20min. After cooling, mold spores were inoculated (0.1% V/V) and cultured with orbital shaker (140 rpm) at 30°C; samples were taken at 48h and MCA assay was carried out. (2) Effect of different concentration of glucose in PDA on the activity of milk-clotting enzyme: glucose was added to a final concentration of 2%, 3%, 5%, 7%, 10% (W/V) in PDA medium. Fermentation and MCA assay were carried out following 1.3.5(1). (3) Effect of different nitrogen source on the activity of milk-clotting enzyme produced by mold: 0.5% (W/V) of tryptone, animal protein hydrolysate, soybean protein, (NH4)2SO4, (NH4)3C6H5O7, and NH4NO3 were added into PDA medium, respectively. Fermentation and MCA assay were carried out following 1.3.5(1). (4) Effect of concentration of liquid PDA on the activity of milk-clotting enzyme: PDA medium was concentrated to 2 times, 3 times, and 4 times by Rotary Evaporator; Fermentation and MCA assay were carried out following 1.3.5(1).
2 2.1
Results and discussion
Isolation and purification of microorganisms in glutinous rice wine Four bacteria and fungi were screened out from glutinous rice wine (Fig.1). It can be judged from the colony and morphology of microorganisms that the four strains included one mold, one yeast, and two bacteria. In this study, the microorganisms were isolated from glutinous rice wine, which was fermented, and thus fewer microorganisms were obtained compared with that isolated from the starter. Since in the starter, the microorganisms balanced themselves by rivalizing the environment and space for living, and since the starter was produced in open system, some adventitious microorganism were easy to intrude. During fermenting, the growth of adventitious microorganism can be inhibited by preponderant microorganism; therefore, isolating microorganism from glutinous rice wine can minish the work and find the milk-clotting enzyme producing strain quickly. 2.2 Confirmation of milk-clotting enzyme producing strain 2.2.1 Microorganisms counting and MCA assay during fermentation Microorganisms counting and MCA assay are carried out following 1.3.4(1), and the results are shown in Fig.2.
Qiaoling Cheng et al. / Chinese Journal of Biotechnology, 2008, 24(6): 999–1003
Fig. 1 Morphology of microorganisms isolated from glutinous rice wine
Fig. 2
Relation between the change of bacteria, yeast, mold
counts and milk-clotting activity during fermentation of glutinous rice wine
As seen in Fig.2, there were more bacteria in the initial fermentation period than in the later period. It was possibly because along with the fermentation time increasing, the system acidity increased gradually, which was not suitable for the bacteria to grow; also, with the increase of yeast and mold quantity, the habitat of bacteria was destroyed unceasingly. The mold quantity achieved a peak after 48 h and started to drop, and the yeast quantity also increased until fermentation for 60 h, and then maintained stably. This was mainly because the mold can hydrolyze starch into monosaccharide, which can accelerate yeast to propagate. However, alcohol, one of the yeast metabolites, restrained the propagating of mold, and therefore, the mold quantity started to drop when the yeast increased to a certain level. It is noteworthy that the change tendency of the mold quantity and MCA of glutinous rice wine maintained consistently. After fermenting for 48 h, the mold quantity and MCA of glutinous rice wine achieved their peaks
synchronously. From this result, mold was thought to be the most probable milk-clotting enzyme producing strain by and large. 2.2.2 Confirmation of milk-clotting enzyme producing strain by casein plate method According to 1.3.4(2), four strains were inoculated to the casein plate; two bacteria and the yeast could grow on the plates, but no precipitation circle appeared. Around the mold, a white precipitation circle with diameter of about 1 cm was observed. This result further confirmed that mold was the milk-clotting enzyme producing strain in glutinous rice wine. 2.3 Optimization of fermentation conditions for milkclotting enzyme production The MCA of enzyme produced by microorganism is related with the way and the conditions of fermentation besides being related with the microorganism itself. Generally speaking, the liquid in-depth fermentation is simple to operate, and the automation and control sterile process is easy to realize. This study optimized the fermentation conditions for milk-clotting enzyme production, and enhanced the MCA to a certain extent. 2.3.1 Effect of different carbon source on the activity of milk-clotting enzyme produced by mold: Experiments were carried out according to 1.3.5(1), and results are shown in Fig. 3. As seen in Fig.3, the results indicate that adding glucose into PDA fluid had a very clear function to increase MCA whereas adding glutinous rice powder and soluble starch into PDA fluid resulted in the decrease in MCA slightly. Supplemented with 3% glucose, the enzyme activity was 137% that of the control. This indicated that adding glucose as carbon source could promote mold propagating, and was advantageous for increasing MCA. Therefore, glucose was selected as the additional carbon source. 2.3.2 Effect of different concentration of glucose in PDA on the activity of milk-clotting enzyme: According to 1.3.5(2), the effect of different concentrations of glucose in PDA on the activity of milk-clotting enzyme was investigated, and the results are shown in Fig. 4.
Fig. 3
Effect of different carbon source on activity of milk-clotting enzyme produced by mold
1: blank; 2: glucose; 3: glutinous rice power; 4: soluble starch
Qiaoling Cheng et al. / Chinese Journal of Biotechnology, 2008, 24(6): 999–1003
From Fig.4 it suggested that when 5% glucose was added in PDA, the MCA was maximal. As the glucose concentration continued to increase, the mold was eugonic, but the MCA dropped gradually. This was possibly because the nutrition needed for mold propagating was different from that of enzyme production. The suitable nutritive proportion was the essential condition for mold to produce enzyme. 5% of glucose was chosen for the best concentration. 2.3.3 Effect of different nitrogen source on the activity of milk-clotting enzyme produced by mold: Different nitrogen sources were tested according to 1.3.5(3), and the results obtained are shown in Fig.5. The result shown in Fig.5 indicated that the nutrition of PDA fluid was rich and its nitrogen source could meet the needs of mold. Additional nitrogen source had a certain function to decrease MCA, and inorganic nitrogen source showed stronger inhibitory action than organic nitrogen source. This was mainly related to the nutrient needs, which were provided for enzyme production. Our results show that there was no need to add additional nitrogen source. 2.3.4 Effect of different concentration of liquid PDA on the activity of milk-clotting enzyme: The result shown in Fig.4 indicated that the concentration of glucose in PDA had a very clear influence on MCA. Thus, we tested the effect of other nutrition ingredient in PDA fluid on MCA produced by mold. Experiments were carried out according to 1.3.5(4) and the results are shown in Fig.6.
Fig. 4 Effect of different concentration of glucose in PDA on activity of milk-clotting enzyme
Fig. 6 Effect of different concentration of liquid PDA on activity of milk-clotting enzyme
As seen in Fig. 6, two times of PDA fluid was more advantageous for mold to produce milk-clotting enzyme compared with original liquid PDA. With the increase of PDA concentration, although the mold grew well, the MCA dropped. This was possibly because the nutrition that was provided for mold propagating was different from that for enzyme production. Two times of PDA fluid was the most suitable PDA concentration.
3
Conclusions
This study has isolated milk-clotting enzyme producing strain from glutinous rice wine and has studied the fermentation condition. Four bacteria and fungus were screened out by gradient dilution. The relations between the change of bacteria, yeast, mold counts, and milk-clotting activity during the fermentation of glutinous rice wine were investigated It was proved that mold played the most important role in the production of milk-clotting enzyme. This was further confirmed by the casein plate method. The optimization of the fermentation conditions revealed that two times concentrated potato medium supplemented with 5% glucose without additional nitrogen was better for production of the enzyme. The relative enzyme activity was increased to 144% under the conditions established. Further identification of the mold and optimization of its fermentation process conditions were carried out by our team.
REFERENCES [1] Dao RN. New dairy product of home market-cheese. China Dairy Cattle, 2000, 2: 4748. [2] Zhang FX. Studies on extraction technology of calf rennet. Journal of Northwest Sci-Tech University of Agriculture and Forestry, 2002, 30(1): 2426. Fig. 5
Effect of different nitrogen source on activity of milk-clotting enzyme produced by mold
1: blank; 2: tryptone; 3: animal protein hydrolysate; 4: soybean protein; 5: (NH4)2SO4; 6: (NH4)3C6H5O7; 7: NH4NO3
[3] Meng GZ. Progress in research of milk clotting enzymes. Micorbiology, 1987, 14(2): 3537. [4] Zhou JQ, Lin QL, Zhao MM. Studies on the production of chymosin by microorganisms. China Food Addtives, 2004,
Qiaoling Cheng et al. / Chinese Journal of Biotechnology, 2008, 24(6): 999–1003
2: 69. [5] Liu ZM, Luo CY. Studies on milk-clotting mechanism of
[7] Arimk K, Iwasake K. Milk-clotting enzyme from microorganisms part I screening test and the identification of the
glutinous rice wine. Food Science, 2000, 1(21): 1316.
potent fungus. Agric Biol Chem, 1967, 31(5): 540545.
[6] Du LX. Protocols for Industrial Microbiology. Tianjin:
[8] Zhang JZ. Microbiology Chemotaxonomy. Beijing: China
Tianjin Science and Technology Press, 1992, 8788.
Light Industry Press, 1996, 154156.