Interactions of caseins with phenolic acids found in chocolate

To investigate the interactions between caseins and phenolic acids, such as the ones present in chocolate, casein was incubated with protocatechuic acid or p-coumaric acid at 55 °C. In addition, casein was isolated from chocolate and the phenolic compounds within these caseins were quantified. Electrophoresis results revealed that casein–phenolic interactions were induced by incubation; minor aggregation of casein subunits was observed after incubation of casein with protocatechuic acid. Minor aggregation of casein isolated from milk chocolate was also observed. In vitro hydrolysis of casein control, casein–protocatechuic acid, casein–p-coumaric acid, caseins isolated from milk chocolate and white chocolate using trypsin showed degree of hydrolysis of 19.3, 18.6, 17.7, 10.4 and 17.8% respectively. The presence of protocatechuic acid and p-coumaric acid in the model system and the presence of phenolic compounds in milk chocolate, in addition to the structural changes occurring during processing, affected the peptide profiles of casein hydrolysates.     

Production and characterization of a milk-clotting protease in the crude enzymatic extract from the newly isolated Thermomucor indicae-seudaticae N31: (Milk-clotting protease from the newly isolated Thermomucor indicae-seudaticae N31)

Microbial rennet-like milk-clotting enzymes are aspartic proteinases that catalyze milk coagulation, substituting calf rennet. Crude enzymatic extract produced by the thermophilic fungus, Thermomucor indicae-seudaticae N31, on solid state fermentation (SSF) using wheat bran, exhibited high milk-clotting activity and low proteolytic activity after 24 h of fermentation. Highest milk-clotting activity (MCA) was at pH 5.7, at 70 °C and in 0.04 M CaCl₂; it was stable in the pH range 3.5–4.5 for 24 h and up to 45 °C for 1 h. MCA was highly inhibited by pepstatin A. Hydrolytic activity profile of the crude enzymatic extract on whole bovine casein, analyzed by gel electrophoresis (Urea–PAGE) and RP-HPLC revealed low proteolytic action towards casein fractions and a peptide profile similar to the one obtained with commercial Rhizomucor miehei protease (Hannilase).     

Screening of a Bacillus subtilis strain producing both nattokinase and milk-clotting enzyme and its application in fermented milk with thrombolytic activity

Bacillus subtilis is a generally recognized as safe probiotic, which is used as a starter for natto fermentation. Natto is a functional food with antithrombus function due to nattokinase. Compared with natto, fermented milk is a more popular fermented food, which is commonly fermented by Lactobacillus bulgaricus and Streptococcus. However, there is no report on B. subtilis–fermented milk. In this study, to produce a functional fermented milk with antithrombus function, a B. subtilis strain (B. subtilis JNFE0126) that produced both nattokinase and milk-clotting enzyme was isolated from traditionally fermented natto and used as the starter for the functional fermented milk. In liquid fermentation culture, the peak values of thrombolytic activity and milk-clotting activity were 3,511 U/mL at 96 h and 874.5 Soxhlet unit/mL at 60 h, respectively. The optimal pH and temperature were pH 7.0 at 40°C for nattokinase and pH 6.5 and 55°C for milk-clotting enzyme, respectively. The thrombolytic activity in the fermented milk reached 215.1 U/mL after 8 h of fermentation. Sensory evaluation showed that the acceptance of the milk fermented by B. subtilis JNFE0126 was similar to the traditional milk fermented by L. bulgaricus and S. thermophilus. More importantly, oral intake of the fermented milk by the thrombosis-model mice prevented the development of thrombosis. Our results suggest that B. subtilis JNFE0126–fermented milk has potential as a novel, functional food in the prevention of thrombosis-related cardiovascular diseases.     

Lactose hydrolysis in milk as affected by neutralizers used for the preparation of crude β-galactosidase extracts from Lactobacillus bulgaricus 11842

Three different neutralizers (NaOH, KOH, NH₄OH) were employed for pH maintenance during the growth of Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842, used as a source of β-galactosidase extracts. The crude enzymatic extract (CEE) was obtained by bead milling of the cell paste, collected from the cultivation of the source microorganism in skim milk at 43 °C and constant pH. Lactose hydrolysis kinetics in skim milk and proteolytic activity during the hydrolysis were evaluated. The use of NH₄OH as a neutralizer resulted in significantly (P<0.05) higher enzyme activity of the CEE than that obtained using NaOH or KOH. The kinetic parameters, k_cat and K_m, of the Michaelis–Menten model were determined for lactose hydrolysis in skim milk using 1% (v/v) addition of a CEE. There was no significant (P>0.05) difference in k_cat among the different extracts, with a clear temperature dependence following Arrhenius kinetics. The rate of lactose hydrolysis was dependent on the initial enzyme activity and temperature. The highest initial rate was observed at 65 °C; however, the enzyme deactivation occurred within 1–1.5 h. The proteolytic activity determined by HPLC peptide mapping was significantly (P<0.05) higher in the moderate temperature range (20 and 37 °C) than at 7 or 55 °C. Industrial relevance: Since lactose intolerance affects a large proportion of the world's population, an economically feasible and effective process with a cheap source of β-galactosidase may have a substantial potential. The use of crude β-galactosidase extracts from Lactobacillus bulgaricus 11842 appears to be a promising approach for development of a technologically feasible process of lactose hydrolysis for food or non-food uses.     

Caseinolytic and milk-clotting activities from Moringa oleifera flowers

This work reports the detection and characterization of caseinolytic and milk-clotting activities from Moringa oleifera flowers. Proteins extracted from flowers were precipitated with 60% ammonium sulphate. Caseinolytic activity of the precipitated protein fraction (PP) was assessed using azocasein, as well as α(s)-, β- and κ-caseins as substrates. Milk-clotting activity was analysed using skim milk. The effects of heating (30-100°C) and pH (3.0-11.0) on enzyme activities were determined. Highest caseinolytic activity on azocasein was detected after previous incubation of PP at pH 4.0 and after heating at 50°C. Milk-clotting activity, detected only in the presence of CaCl(2), was highest at incubation of PP at pH 3.0 and remained stable up to 50°C. The pre-treatment of milk at 70°C resulted in highest clotting activity. Enzyme assays in presence of protease inhibitors indicated the presence of aspartic, cysteine, serine and metallo proteases. Aspartic proteases appear to be the main enzymes involved in milk-clotting activity. PP promoted extensive cleavage of κ-casein and low level of α(s)- and β-caseins hydrolysis. The milk-clotting activity indicates the application of M. oleifera flowers in dairy industry.     

Effect of hydrolysis of casein by plasmin on the heat stability of milk

This study examined the effect of hydrolysis of casein by added plasmin (6 mg L⁻¹) on the heat stability of raw, pre-heated, serum protein-free or concentrated skim milk. Plasmin activity markedly affected the heat stability–pH profile of skim milk and serum protein-free milk, apparently by altering the properties of the casein micelles. It is probable that changes in the surface charge of the micelles, as a result of the hydrolysis of caseins, contributed to this effect. Hydrolysis by plasmin reduced the zeta-potential of the casein micelles from ∼−19 to ∼−16 mV. The effect of hydrolysis of casein by plasmin on the heat stability of pre-heated milk was less pronounced, shifting the heat stability–pH profile to more alkaline values; the heat stability of concentrated milk was unaffected by plasmin. A very high (50 mg L⁻¹) level of added plasmin resulted in clearing of the skim milk; the L^* value decreased from ∼75 to ∼35 after 24 h incubation at 37 °C. Clearing was correlated with a change in casein micelle diameter from an initial value of ∼175 to ∼250 nm. It is suggested that plasmin-induced changes in zeta-potential may promote micellar aggregation or changes in micelle stucture.     

Differential behavior of Lactobacillus helveticus B1929 and ATCC 15009 on the hydrolysis and angiotensin-I-converting enzyme inhibition activity of fermented ultra-high-temperature milk and nonfat dried milk powder

Consumers' growing interest in fermented dairy foods necessitates research on a wide array of lactic acid bacterial strains to be explored and used. This study aimed to investigate the differences in the proteolytic capacity of Lactobacillus helveticus strains B1929 and ATCC 15009 on the fermentation of commercial ultra-pasteurized (UHT) skim milk and reconstituted nonfat dried milk powder (at a comparable protein concentration, 4%). The antihypertensive properties of the fermented milk, measured by angiotensin-I-converting enzyme inhibitory (ACE-I) activity, were compared. The B1929 strain lowered the pH of the milk to 4.13 ± 0.09 at 37°C after 24 h, whereas ATCC 15009 needed 48 h to drop the pH to 4.70 ± 0.18 at 37°C. Two soluble protein fractions, one (CFS1) obtained after fermentation (acidic conditions) and the other (CFS2) after the neutralization (pH 6.70) of the pellet from CFS1 separation, were analyzed for d-/l-lactic acid production, protein concentration, the degree of protein hydrolysis, and ACE-I activity. The CFS1 fractions, dominated by whey proteins, demonstrated a greater degree of protein hydrolysis (7.9%) than CFS2. On the other hand, CFS2, mainly casein proteins, showed a higher level of ACE-I activity (33.8%) than CFS1. Significant differences were also found in the d- and l-lactic acid produced by the UHT milk between the 2 strains. These results attest that milk casein proteins possessed more detectable ACE-I activity than whey fractions, even without a measurable degree of hydrolysis. Findings from this study suggest that careful consideration must be given when selecting the bacterial strain and milk substrate for fermentation.     

Investigations on biochemical properties of milk-clotting enzymes

The properties of proteolytic enzymes produces from calf maws and from an Ascomycete were studied. Both milk-clotting proteases have their optimum activity at pH 5.2 and 45 degrees C. The microbiological rennin has a second maximum activity at pH 3.5 and 55 degrees C. Temperatures above 55 degrees C cause a rapid decrease of activity. The behaviour of enzyme activity is similar with varying substrate and enzyme concentrations. However, increasing amounts of enzyme in ratio to the substrate lead to reaction rates of the calf rennin differing clearly from that of the microbiological rennet complex.     

辣木凝乳酶水解酪蛋白位点及其酪蛋白磷酸肽、酪蛋白糖巨肽

基于十二烷基硫酸钠-聚丙烯酰胺凝胶电泳,液相色谱-串联质谱法研究辣木凝乳酶对酪蛋白的酶切位点和水解特性,并分析该凝乳酶水解产生的酪蛋白糖巨肽和酪蛋白磷酸肽。结果表明,辣木凝乳酶酶切κ-酪蛋白的Arg 93-His 94位点导致凝乳,区别于其他已报道的凝乳酶,具有明显特异性。酶作用下κ-酪蛋白的米氏常数Km=0.49 mg/mL,最大反应速率Vm=45.2 U/min,有效促进酪蛋白胶束的聚集和凝乳。该酶对α-、β-、κ-酪蛋白产生不同程度的水解,对α-酪蛋白的水解速度最快,说明其适用于软质奶酪的加工。以酪蛋白为底物,通过钡-乙醇沉淀法得到酪蛋白磷酸肽（casein phosphopeptides,CPPs）,产率为15.87%,得到的CPPs可以使钙沉淀推迟23 min,有利于钙离子被肠道有效吸收。以水牛乳为底物时,乳凝固后析出的乳清中含酪蛋白糖巨肽6.61 mg/mL,糖基化程度477.527 μg/mg。研究为新型植物凝乳酶的研发,及其在奶酪、酪蛋白活性肽等产品的应用提供科学依据。     

Addition of sodium caseinate to skim milk increases nonsedimentable casein and causes significant changes in rennet-induced gelation,heat stability,and ethanol stability

The protein content of skim milk was increased from 3.3 to 4.1% (wt/wt) by the addition of a blend of skim milk powder and sodium caseinate (NaCas), in which the weight ratio of skim milk powder to NaCas was varied from 0.8:0.0 to 0.0:0.8. Addition of NaCas increased the levels of nonsedimentable casein (from ～6 to 18% of total casein) and calcium (from ～36 to 43% of total calcium) and reduced the turbidity of the fortified milk, to a degree depending on level of NaCas added. Rennet gelation was adversely affected by the addition of NaCas at 0.2% (wt/wt) and completely inhibited at NaCas ≥0.4% (wt/wt). Rennet-induced hydrolysis was not affected by added NaCas. The proportion of total casein that was nonsedimentable on centrifugation (3,000 × g, 1 h, 25°C) of the rennet-treated milk after incubation for 1 h at 31°C increased significantly on addition of NaCas at ≥0.4% (wt/wt). Heat stability in the pH range 6.7 to 7.2 and ethanol stability at pH 6.4 were enhanced by the addition of NaCas. It is suggested that the negative effect of NaCas on rennet gelation is due to the increase in nonsedimentable casein, which upon hydrolysis by chymosin forms into small nonsedimentable particles that physically come between, and impede the aggregation of, rennet-altered para-casein micelles, and thereby inhibit the development of a gel network.     

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.     

Assessment of multifunctional activity of bioactive peptides derived from fermented milk by specific Lactobacillus plantarum strains

Milk-derived bioactive peptides with a single activity (e.g., antioxidant, immunomodulatory, or antimicrobial) have been previously well documented; however, few studies describe multifunctional bioactive peptides, which may be preferred over single-activity peptides, as they can simultaneously trigger, modulate, or inhibit multiple physiological pathways. Hence, the aim of this study was to assess the anti-inflammatory, antihemolytic, antioxidant, antimutagenic, and antimicrobial activities of crude extracts (CE) and peptide fractions (<3 and 3–10 kDa) obtained from fermented milks with specific Lactobacillus plantarum strains. Overall, CE showed higher activity than both peptide fractions (<3 and 3–10 kDa) in most of the activities assessed. Furthermore, activity of <3 kDa was generally higher, or at least equal, to the 3 to 10 kDa peptide fractions. In particular, L. plantarum 55 crude extract or their fractions showed the higher anti-inflammatory (723.68–1,759.43 μg/mL of diclofenac sodium equivalents), antihemolytic (36.65–74.45% of inhibition), and antioxidant activity [282.8–362.3 µmol of Trolox (Sigma-Aldrich, St. Louis, MO) equivalents]. These results provide valuable evidence of multifunctional role of peptides derived of fermented milk by the action of specific L. plantarum strains. Thus, they may be considered for the development of biotechnological products to be used to reduce the risk of disease or to enhance a certain physiological function.     

Characterisation of proteolytic enzymes from muscle and hepatopancreas of fresh water prawn (Macrobrachium rosenbergii)

BACKGROUND: Fresh water prawn in Thailand is widely consumed due to its delicacy. During postmortem handling and storage, prawn meat becomes soft and mushy, probably as a result of indigenous proteases. Therefore, an understanding of prawn proteases associated with the degradation of muscle proteins from fresh water prawn could pave the way for prevention of such a phenomenon during extended storage. RESULTS: Proteolytic enzymes in the crude extract (CE) from muscle and hepatopancreas of fresh water prawn (Macrobrachium rosenbergii) were characterised. CE from muscle exhibited the highest hydrolytic activities towards haemoglobin at pH 5 and 50 °C, while that from hepatopancreas had the highest activity on casein at pH 7 and 60 °C. Based on inhibitor study, cysteine protease and serine protease were dominant in CE from muscle and hepatopancreas, respectively. CE from muscle rarely hydrolysed natural actomyosin (NAM), but could not degrade pepsin‐soluble collagen (PSC). Conversely, NAM and PSC were susceptible to hydrolysis by CE from hepatopancreas as evidenced by the marked decreases in band intensity. Activity staining using haemoglobin, casein and gelatin as substrates revealed that no proteolytic or gelatinolytic activity was observed in CE from prawn muscle, while CE from hepatopancreas exhibited pronounced hydrolytic activities towards all substrates. CE from muscle showed calpain and cathepsin L activities but CE from hepatopancreas mainly exhibited tryptic and chymotryptic activities. CONCLUSION: Serine proteases, mainly trypsin‐like or chymotrypsin‐like, from hepatopancreas were probably responsible for the softening of prawn meat during postmortem storage via the degradation of both muscle and connective tissues. Copyright © 2010 Society of Chemical Industry     

Low Volume Bioreactor Development in Dairy Industry Based on Encapsulated Rennin in Tubular Cellulose/Starch Gel Composite

A novel process development is described in this study, based on a low volume bioreactor containing enzyme (rennin) encapsulated in a nano/micro porous cellulose (or tubular cellulose; TC) and starch gel matrix (TC/SG composite), for use in continuous coagulation of milk in cheese production. To validate the process, experiments were carried out in 10- and 50-L bioreactors. The biocatalyst (rennin immobilized in the TC/SG composite) proved to be efficient for continuous coagulation of milk. The biocatalyst was divided in four portions (filters) evenly distributed in the bioreactors, and high flow rate of milk was applied in order to avoid curd formation inside the bioreactors. Both bioreactor systems provided two economically useful results for the dairy industries: (i) substantial increase of milk coagulation productivity, (ii) acceleration of maturation of the produced cheeses, and (iii) reusability of the biocatalyst. Based on the results, it was estimated that a bioreactor of 1 m³ containing 20 kg of the TC/SG biocatalyst with encapsulated rennin could be used to treat 12,000 L of milk daily for cheese production. Physicochemical analyses of the produced cheeses at various stages of maturation were performed as well as analysis of aroma volatile compounds by SPME GC/MS. No significant differences were found compared to cheeses prepared with the traditional method.     

Characterization of the purified actinidin as a plant coagulant of bovine milk

In this work, actinidin was characterized in view of its possible suitability as a coagulant enzyme in the manufacturing process of cheese. The results show that actinidin does exhibit milk-clotting activity, which is correlated with the enzyme concentrations. The combined use of urea and SDS–PAGE led to the conclusion that the milk clot is clearly separated from the whey proteins and corresponds to casein coagulum. Moreover, both the enzyme dependence on pH and temperature and the stability profiles are fully suitable with the chemical–physical conditions adopted during the cheese-making procedure. The analysis of the kinetic constants as well as the electrophoretic pattern of the hydrolysis products suggests that β-casein is the preferred substrate of actinidin, whereas κ-casein seems to be hydrolyzed only in a few large fragments.     

Interactions of Calcium,pH, Temperature,and Chymosin During Milk Coagulation,

Holstein milk samples with good and poor chymosin-coagulation characteristics were coagulated in the Formagraph using different combinations of five levels of chymosin, three pH, and three temperatures in the presence and absence of .02% added calcium chloride.All the main factors significantly altered both coagulation time and curd firmness. Multiple comparisons of mean coagulation times showed that lower concentrations of chymosin (.01, .02, and .03 rennin units/ml milk) were significantly different in altering coagulation time and were different from higher concentrations (.04 and .05 rennin units/ml milk). The three pH produced significantly different mean coagulation times. Addition of more than .02 rennin units/ml milk was not necessary for adequate curd firmness in 30 min after chymosin addition where the pH of the milk was 6.4 or lower. Addition of .02% calcium chloride to milk was not necessary for adequate curd firmness 30 min after chymosin addition if other milk coagulation factors were adequately adjusted (pH ≤ 6.4; chymosin concentration = .02 rennin unit/ml milk; temperature = 37°C).     

Assessment of the production of Bacillus cereus protease and its effect on the quality of ultra-high temperature-sterilized whole milk

Bacillus cereus is one of the most important spoilage microorganisms in milk. The heat-resistant protease produced is the main factor that causes rotten, bitter off-flavors and age gelation during the shelf-life of milk. In this study, 55 strains of B. cereus were evaluated, of which 25 strains with protease production ability were used to investigate proteolytic activity and protease heat resistance. The results showed that B. cereus C58 had strong protease activity, and its protease also had the highest thermal stability after heat treatment of 70°C (30 min) and 100°C (10 min). The protease was identified as protease HhoA, with a molecular mass of 43.907 kDa. The protease activity of B. cereus C58 in UHT-sterilized whole milk (UHT milk) showed an increase with the growth of bacteria, especially during the logarithmic growth phase. In addition, the UHT milk incubated with protease from B. cereus C58 at 28°C (24 h) and 10°C (6 d) were used to evaluate the effects of protease on the quality of UHT milk, including protein hydrolysis and physical stability. The results showed that the hydrolysis of casein was κ-CN, β-CN, and α_S-CN successively, whereas whey protein was not hydrolyzed. The degree of protein hydrolysis, viscosity, and particle size of the UHT milk increased. The changes in protein and fat contents indicated that fat globules floated at 28°C and settled at 10°C, respectively. Meanwhile, confocal laser scanning microscopy images revealed that the protease caused the stability of UHT milk to decrease, thus forming age gelation.     

Culture Filtrates from a Fermented Rice Product (Lao-Chao) and Their Effects on Milk Curd Firmness

To develop a new Oriental-style dairy product, the characteristics of culture filtrate from lao-chao with Rhizopus oryzae, used as a milk-clotting agent, and factors (heat-treatments, calcium, sucrose, and curdling temperature) affecting curd firmness were determined. The optimal conditions for proteolytic activity were around 40°C and pH 3. No activity was detected over the range of pH 8 to 11. After high-heat treatment (121°C, 15 min) on skim milk, no clotting was observed. Sucrose resulted in the retardation of milk-clotting. Ca⁺⁺ could be used to increase curd firmness which also increased from 29. 2–2.8g to 80. 3–4.7g when the curdling temperature was increased from 25°C to 45°C.     

Effects of added plasmin on the formation and rheological properties of rennet-induced skim milk gels

Elevated plasmin enzyme activity has been suggested as a likely cause of impaired functional properties that occur in milk from cows either in their late-lactational period or that are experiencing mastitis. However, there are conflicting reports on the impact of plasmin on rennet coagulation properties of milk. The effects of added plasmin on the rheological properties, at small and large deformation, of rennet-induced gels were investigated. The microstructure of rennet-induced gels was studied, using confocal scanning laser microscopy. Porcine plasmin was added to reconstituted milk, and samples were incubated at 37 degrees C for between 0.5 to 8 h. The hydrolysis reaction was terminated using soybean trypsin inhibitor. The extent of degradation of caseins was determined with SDS-PAGE. The extent of breakdown of alpha(s)- and beta-caseins increased with incubation time with plasmin. Storage modulus of rennet gels decreased linearly with increasing degradation of caseins. There was an increase in the loss tangent parameter of the gels with increasing casein degradation, indicating a more liquid-like gel character. Gelation time decreased until approximately 3 h of incubation with plasmin (when the amounts of intact alpha(s)- and beta-caseins were approximately 46 and 50%, respectively); thereafter, gelation time increased considerably. Yield stress of rennet-induced gels decreased with increasing casein breakdown. When the level of casein hydrolysis was high (<40% of intact caseins), the microstructure of rennet-induced gels was drastically altered. Even when there were low levels of casein hydrolysis, the rheological properties of rennet gels were altered, which could have negative impacts on cheese yield and texture.     

Insights into milk-clotting activity of latex peptidases from Calotropis procera and Cryptostegia grandiflora

Latex fractions from Calotropis procera, Cryptostegia grandiflora, Plumeria rubra, and Himatanthus drasticus were assayed in order to prospect for new plant peptidases with milk-clotting activities, for use as rennet alternatives. Only C. procera and C. grandiflora latex fractions exhibited proteolytic and milk-clotting activities, which were not affected by high concentrations of NaCl and CaCl₂. However, pre-incubation of both samples at 75 °C for 10 min eliminated completely their activities. Both proteolytic fractions were able to hydrolyze k-casein and to produce peptides of 16 kDa, a similar SDS-PAGE profile to commercial chymosin. RP-HPLC and mass spectrometry analyses of the k-casein peptides showed that the peptidases from C. procera or C. grandiflora hydrolyzed k-casein similar to commercial chymosin. The cheeses made with both latex peptidases exhibited yields, dry masses, and soluble proteins similar to cheeses prepared with commercial chymosin. In conclusion, C. procera and C. grandiflora latex peptidases with the ability to coagulate milk can be used as alternatives to commercial animal chymosin in the cheese manufacturing process.