Milk Clotting Enzyme Activity Research Articles

Cell immobilization for enhanced milk clotting enzyme production from Bacillus amyloliquefacien and cheese quality

BackgroundMilk clotting enzymes, essential for milk coagulation in cheese production, are obtained from the stomach of young ruminants, an expensive and limited source. This study was accomplished by finding a suitable alternative. Bacterial isolates recovered from honey were screened for milk clotting enzyme activity. and further, by immobilization of the microorganisms to enhance stability and facilitate their repeated use.ResultThe most effective enzyme was produced by a microbe identified as Bacillus amyloliquefaciens based on 16 S rRNA sequencing. The cells were encapsulated in Ca2+ alginate beads. These beads retained complete enzyme production after being used five times. Glucose and Soybean were selected as the most favorable carbon and nitrogen sources, respectively. The optimum temperature for activity was 35 ℃ for both free and immobilized cells but as the temperature was increased to 55 °C and above, the encapsulated form retained more activity than the free cells. The pH optimum shifted from 6.5 to 7 for the free cells to 7–7.5 for the immobilized cells. The immobilization process decreased the activation energy for enzyme production and activity, prolonged the enzyme half-life, and increased the deactivation energy. Enzyme produced by immobilized cells generated a more compact cheese.ConclusionsThe finding of this study was to identify a less expensive source of milk-clotting enzymes and confirm the success of cell immobilization in improving cell rigidity and stability. Also, immobilization of this B. amyloliquefaciens strain offers an enzyme source of value for industrial production of cheese.

Purification and Characterization of Milk Clotting Enzyme from Edible Mushroom (Pleurotus florida)

Oyster mushroom (Pleurotus florida 14 MICC) is one of the most widely cultivated edible fungi in the world. Milk clotting enzyme (MCE) derived from the mold was developed as a calf rennet alternative; thus, it was purified and characterized, and its effect on different milk species was investigated. The highest MCE activity (75.49 SU/ml) was observed in mushroom fruit bodies dissolved in 0.2 M sodium acetate pH 5.0; as well as the highest total MCE activity (367.85 SU) was recorded at 20% of ammonium sulfate with a specific activity of 343.79 SU mg-1 protein while size exclusion column chromatography on Sephadex G-100 purified MCE 3.46-fold with 17.96% yield. Also, it could be capable of coagulating different milk species. Mushroom MCE exhibited their optimal activity at pH 5.0 for crude extract (CE) while at pH 6.0 for partial purified (PP) and purified (P) MCE fractions; as well as at 55 °C for CE and PP MCE fractions while 50 °C for P MCE. CaCl2 concentration (0.01%) recorded the maximal activity for CE while (0.04%) and (0.02%) for PP and P fractions, respectively. It could be concluded that MCE from Oyster mushroom may be a good candidate as a calf rennet substitute in cheese production.

Optimasi Waktu Inkubasi Lactobacillus rhamnosus SKG 34 Dalam Produksi Enzim Penggumpal Susu


 
 
 
 
 
 This study aims to determine the effect of incubation time on milk clotting enzyme (MCE) activity produced by L. rhamnosus SKG 34 and determine the optimum incubation time to produce the highest its activity. This study used a randomized block design (RBD) with incubation time as a treatment consisting of 8 levels, that were 6 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 42 hours, and 48 hours. Data were analyzed with Variance Analysis (ANOVA) than followed by Duncan Multiple Range Test (DMRT). The analyzed were repeated 3 times resulting in 24 experimental units. The results showed that the incubation time significantly affected protease activity, MCE activity, specific protease activity and ratio of MCE to protease but did not affect the total LAB. The optimum incubation time of L. rhamnosus SKG 34 is 12 hours with total LAB 1.83 x 109 CFU/ml and protease activity 180.67 U/ml, MCE activity 595.06 SU, protease spesific activity 73.149 U/mg and ratio of MCE to protease 3.29 SU/U.
 Keywords : incubation time, Enzyme protease, Lactobacillus rhamnosus SKG 34, Milk clotting enzyme.
 
 
 
 
 

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.

PEMANFAATAN MILK CLOTTING ENZYME DARI Lactobacillus casei D11 UNTUK PEMBUATAN KEJU MOZZARELLA

Milk Clotting Enzyme (MCE) is an active agent for cheese making which may be produced by Lactic acid bacteria (LAB). MCE activity differs according to the LAB strains used. Lactobacillus casei D11 could produced MCE when it is grown in MRS broth medium. In this study, MCE of L. casei D11 with the addition of rennet is used and optimized for the production of mozzarella cheese using Response Surface Method (RSM) with Central Composite Design (CCD). The organoleptic properties were determined by hedonics test involving 30 respondents and analyzed statistically which was followed by a Duncan's test. Furthermore, a proximate analysis of mozzarella cheese was conducted. Our results show that the MCE activity produced by L. casei D11 was 8.471 Soxhlet Unit with protease activity of 3.28 U/mL. The ANOVA results showed that the concentration of MCE significantly influence the production of curd. The optimum concentration of MCE and rennet for the production of curd suited for the production of mozzarella cheese were 20 and 0.002%, respectively, with a maximum predicted curd yield of 14.996 % (g/100 mL milk) which is increased by 13.9% as compared to the curd yield before optimization . The statistical analysis on taste, color, flavor, and cheese texture by respondents shows that mozzarella cheese made by a combination of 15% of MCE and 0 . 00 079 and 0.0015% of rennet, were organoleptically superior to the commercial mozzarella used in this experiment. The proximate analysis shows that mozzarella produced has a moisture content of 33.34%(w/w), 3.48% ash, 30.44% fat, 25.12% protein, 7.53% carbohydrate and energy of 404 kkal/100g.

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.

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.

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.

Influence of residual milk-clotting enzyme on alpha(s1) casein hydrolysis during ripening of Reggianito Argentino cheese.

Milk-clotting enzyme is considered largely denatured after the cooking step in hard cheeses. Nevertheless, typical hydrolysis products derived from rennet action on alpha(s1)-casein have been detected during the ripening of hard cheeses. The aim of the present work was to investigate the influence of residual milk-clotting enzyme on alpha(s1)-casein hydrolysis in Reggianito cheeses. For that purpose, we studied the influence of cooking temperature (45, 52, and 60 degrees C) on milk-clotting enzyme residual activity and alpha(s1)-casein hydrolysis during ripening. Milk-clotting enzyme residual activity in cheeses was assessed using a chromatographic method, and the hydrolysis of alpha(s1)-casein was determined by electrophoresis and high performance liquid chromatography. Milk-clotting enzyme activity was very low or undetectable in 60 degrees C- and 52 degrees C-cooked cheeses at the beginning of the ripening, but it increased afterwards, particularly in 52 degrees C-cooked cheeses. Cheese curds that were cooked at 45 degrees C had higher initial milk clotting activity, but also in this case, there was a later increase. Hydrolysis of alpha(s1)-casein was detected early in cheeses made at 45 degrees C, and later in those made at higher temperatures. The peptide alpha(s1)-I was not detected in 60 degrees C-cooked cheeses. The results suggest that residual milk-clotting enzyme can contribute to proteolysis during ripening of hard cheeses, because it probably renatures partially after the cooking step. Consequently, the production of peptides derived from alpha(s1)-casein in hard cheeses may be at least, partially due to this proteolytic agent.

An Evaluation With Piglets of Bovine Milk, Hydrolyzed Bovine Milk, and Isolated Soybean Proteins Included in Infant Milk Formulas. II. Stomach-Emptying Rate and the Postprandial Change in Gastric pH and Milk-Clotting Enzyme Activity

The piglet was used as a model for the human infant to determine the effect of the protein source of a milk formula on stomach-emptying rate and the postprandial changes in gastric pH and milk-clotting enzyme activity. Three liquid milk formulas containing either intact bovine milk protein, hydrolyzed bovine milk protein, or isolated soybean protein as the sole source of dietary protein were given to five 37-day-old piglets that had undergone surgery for the preparation of a simple gastric fistula. At set times after commencement of a meal, the stomach contents were removed via a gastric cannula and weighed; then pH and milk-clotting enzyme activity were determined. The isolated soybean protein-based formula tended to empty from the stomach more slowly than did the bovine milk-based formulas. Maximum difference (p less than 0.05) was found at 3 h postfeeding, where 30% of the ingested soybean formula dry matter remained in the stomach; comparable values for the intact bovine milk-based formula and hydrolysate were 22 and 12%, respectively. The type of protein did not significantly (p greater than 0.05) affect the postprandial change in gastric pH or gastric milk-clotting enzyme activity. Gastric pH increased sharply after feeding, from pH 1.5 at 0 h to pH 5.2 at 1 h, and then fell gradually over the following 4 h to pH 1.84.

Production of fungal rennet byMucor miehei using solid state fermentation

Investigations have been carried out on the production of fungal rennet using a thermophilic strain ofMucor miehei under solid state fermentation conditions. A high milk clotting enzyme activity (58000 Soxhlet units/g) was achieved when optimum conditions were used. Further, a high ratio of 6.6:1 between milk clotting and proteolytic activities for this enzyme was obtained. Cheese prepared using this enzyme was also found to be acceptable in organoleptic quality. Large scale production of the enzyme in trays using the optimum conditions gave milk-clotting enzyme activities comparable to those in flask experiments.

Evaluation of Formagraph for Comparing Rennet Solutions1,2

A new instrument for measuring milk clotting, the Formagraph, was evaluated. Measurement is movement of small pendulums immersed in linearly oscillating samples of coagulating milk. Pendulum movements are recorded on photographic paper, producing a diagram of firmness versus time. The instrument can be used for measuring milk-clotting enzyme activity in comparison to a standard, and in constrast to the rolling bottle method, it does not require continual observation. Coagulation times measured by the Formagraph are longer than those measured by the rolling bottle method, but both methods give linear standard curves between .2 and 1.0 rennet units/ml enzyme concentrations.

Distribution of Milk Clotting Enzymes Between Curd and Whey and Their Survival During Cheddar Cheese Making1,4

A linear diffusion test capable of measuring milk clotting enzyme activity at concentrations of 1×10−4 to 1×10−1 rennin units per ml is described. The distribution of rennet activity between curd and whey in freshly coagulated milk was measured at 72% in the whey and 31% in the curd at pH 6.6. At pH 5.2, the whey contained 17% and the curd 86%. Total recovery of activity was 102±5%. Distribution of milk clotting enzymes from Mucor pusillus var. Lindt and Mucor miehei between curd and whey in freshly coagulated milk was independent of pH with approximately 83% in the whey and 17% in the curd. Porcine pepsin was unstable, precluding accurate assessment of enzyme distribution. During the manufacture of Cheddar cheese approximately 35% of the rennet activity was destroyed up to the time the whey was drained, and 6% remained in the cheese following pressing. Neither of the microbial enzymes lost activity during cheese making. Most of the activity remained in the whey while only 2 to 3% was detected in the cheese after pressing.