Commercial Rennet Research Articles

Gastric Lipase Characterization and Utilization in Cheese Manufacture

Gastric lipase has been demonstrated in some rennet paste preparations but not in commercial rennet extracts. Lamb gastric lipase had a pH optimum from 7.0 to 7.6 and was most stable at pH 7.0. It hydrolyzed tributyrin most rapidly at 45C and β-naphthyl laurate very slowly. Lamb gastric lipase hydrolyzed mono- and dibutyrins more rapidly than lamb pregastric esterase. It hydrolyzed milk fat very slowly but may have a role in cheese flavor development. Cheddar and Provolone cheeses were organoleptically preferred when gastric lipase preparations were included in their manufacture.

X-ray diffraction studies of rennin crystals.

Rennin, purified from commercial rennet powder by Foltmann’s method (Foltmann 1959) and precipitated from solution by sodium chloride, crystallizes in the form of rectangular blocks, often nearly equidimensional but sometimes elongated, usually along the direction of highest refractive index. X-ray diffraction photographs (figure 1 is an example) show that the symmetry is orthorhombic and the lattice body-centred. Essential information is given in table 1.

Activation of Prorennin by Pepsin1,2

The ability of pepsin to catalyze the activation of prorennin was demonstrated when purified prorennin solutions buffered at pH 6.0 and 5.5 were activated by pepsin at 25C with little interference from autocatalytic activation by rennin. Addition of m NaCl retarded activation of prorennin by pepsin at pH 6.0 and 5.5, but the reaction was still faster than in rennin-catalyzed activation mixtures containing the same concentration of salt. Pepsin-catalyzed activation of prorennin at pH 6.0 appeared to follow zero-order kinetics during the early stages of reaction, followed by a marked change to a reduced rate of activation. However, the kinetics of activation may have been complicated by lack of pepsin stability at pH 6.0.Pepsin was more proteolytic than rennin on casein substrates at all pH values from 2.0 to 6.0. The general proteolytic activities of rennin and pepsin as distinguished from their milk-clotting activities may be related to their respective abilities to activate prorennin. Pepsin was able to stimulate activation of crude rennet extracts at pH 5.5, where rennin has good stability. Use of pepsin in commercial rennet activation could reduce or eliminate activation losses.

Degradation of Casein Fractions by Rennet Extract

The proteolytic activity of commercial rennet on purified fractions of αs- and β-casein was studied. Products of proteolysis, soluble in 5% trichloroacetic acid, were examined by gel-filtration and by absorbancy at 280mμ. Residual casein fractions were determined by polyacrylamide-gel electrophoresis.Rennet enzymes appear to preferentially degrade αs-casein. β-casein evidently is more resistant to rennet proteolysis than αs-casein. These findings confirm results of our studies with whole casein and suggest that successful rennet substitutes need to have the delicate differential proteolytie characteristics of rennet.

Comparison of the Proteolysis Produced by Rennet Extract and the Pepsin Preparation Metroclot during Ripening of Cheddar Cheese

Summary This investigation determined the differences in proteolysis produced by commercial rennet extract and a pepsin preparation during the ripening of Cheddar cheese. Preliminary experiments with the two enzymatic agents were made in fresh pasteurized milk. The bacterial factor was eliminated by the addition of toluene. The general course of protein degradation, as measured by nonprotein nitrogen production, was found to be higher when rennet extract was used. When the pH of milk was adjusted to four different levels from pH 6.5 to 5.3, both rennet extract and pepsin had maximum proteolysis at pH. 6.3, with rennet extract liberating more nonprotein nitrogen than pepsin. The proteolysis produced by rennet extract in Cheddar cheese during ripening was higher than that produced by pepsin as measured by the nonprotein nitrogen. However, there was almost no variation in the amino acid pattern of Cheddar cheese of the same age, regardless of clotting agent used. The concentrations of the amino acids were measurably different, but no qualitative differences were observed. Organoleptic evaluation of the cheese revealed that both cheeses were of good quality. However, the texture and body of the cheese made with pepsin was slightly curdy for a longer period during ripening. Bitter flavor did not develop during the ripening period in any lots of cheese.

Viscometric measurements of the activities of commercial rennets using sodium caseinate as substrate

SummaryThe considerable falls in viscosity of sodium caseinate solutions under the action of rennet are shown to be due to changes in volume and shape (or either alone) of loosely coiled long chain molecules, resulting from the discharge of ionization (‘Third-type electro-viscous effects’). This reduction may be as well effected by small amounts of NaCl or by the separated decomposition products of the rennin-caseinate reaction. Even at the highest practicable caseinate concentrations (9–10% by weight) the solutions are truly fluid over the range of shear-rate of the Ostwald viscometers usedThe initial rate of fall of viscosity is found to be proportional to the maximum (initial) specific viscosity, leading to an equation Xk1 = 1/τ where X the proportional viscosity loss, varies with the source of caseinate but is constant for all normal rennets; k1, the first-order reaction constant, varies with the rennet but is independent of the source of casein over a wide range; and τ, which has the dimensions of time, varies with both caseinate and rennet.The parameter k1 may be found either from the exponential viscosity fall or calculated from the asymptotic initial slope and has been shown to serve as a useful index of rennet activity. This method of rennet testing does not depend on the keeping qualities of rennins, rennets, milks or caseinates. Details of the preparation of caseinates and of the methods for practical testing of commercial rennets are given.

ミルクおよびミルクゲルのレオロジー

By the use of the coaxial cylinder rheometer, the viscoelastic properties of reconstituted skim milk and renneted milk gel have been measured under various temperatures, concentration of skim milk and rennet.The aqueous solutions of skim milk in the concentration range from 35 to 50 weight percent show a remarkable non-Newtonian behavior over a frequency range between 10-4 and 10-1cps. The viscosity at the zero frequency, therefore, could not be measured. According to Ferry's reduction method, however, the master curves for the reduced viscosity and rigidity have been obtained for both the temperature and concentration relationships. The activation energy calculated from the linear relation between log aT and 1/T is 93kcal/mol.As skim milk coagulates by adding commercial rennet, the viscoelasticity of renneted milk gel increases at the lapse of setting time. The velocity of gelation varies with the concentration of skim milk, the temperature of setting and the concentration of added rennet. The velocity constant K of gelation was defined as the reciprocal of the setting time when the value of viscosity and rigidity reaches the half value of maximum respectively. It has been found that log K increases linearly with temperature above about 25°C, below which the enzyme action of rennet is inactive. The activation energy for gelation is about 59kcal/mol.The relation between K and the concentration of rennet c, is represented by an equation K=K∞(1-e-kc), where K∞ is the maximum velocity constant and k is a constant. The gelation of milk seems to be caused primarily by the adsorption of rennet to casein molecules which is followed by the denaturation.

Crystalline Rennin in the Manufacture of Cheddar Cheese1,2

Summary Unsuccessful attempts to produce good Cheddar cheese with crystalline rennin led other workers to suggest the cheese-making value of substances other than rennin in rennet extract. Eight three-vat lots of Cheddar cheese were made to compare the effects of crystalline rennin, commercial rennet, and the supernatant resulting from the salt precipitation of about 80% of the clotting activity from rennet extract. The activity of each rennin preparation was measured in a reconstituted substrate (60g. nonfat dry milk in 500ml. 0.01 M CaCl 2 ). Clotting solutions containing equal substrate clotting activities were added to each vat. The cheese, cured at 50° F., was of good quality, and no significant differences were apparent between treatments when graded organoleptically at 2, 10, and 34 wk. Small but consistent differences in soluble nitrogen content were noted. Cheese made with crystalline rennin yielded the highest soluble nitrogen, and that made with the supernatant the lowest. Good-quality Cheddar cheese was produced with crystalline rennin. Other substances in rennet extract were found to be nonessential to the cheese-making process.

An invariant casein substrate for rennet standardization, derivable from diverse sources of cows milk.

WHEN rennin or commercial rennets (impure rennins) cause milk to coagulate, it is well known that the primary enzyme action consists in decomposing a part only of the casein, known as ϰ-casein1. Later, either through the activity of the products of decomposition or through loss of protective action, the casein aggregates and finally a gel (curd) is formed.

A viscometric study of the breakdown of casein in milk by rennin and rennet

SummaryIn the later stages of the reaction between rennin and casein drastic viscometric methods are undesirable, but in the first stages there is a fall in viscosity which may be satisfactorily measured in an Ostwald viscometer. Using fat-free milk, the viscosity at first falls linearly with time. At low rennet concentrations (Ce) this may be said to constitute a zero-order reaction (constantk0). At higher rennet concentrations and after longer times, the reaction passes to first order (constantk1). After very long times it doubtless becomes more complex.The values ofk0are proportional, over a considerable range, to the milk concentration (Cm), those ofk1being independent ofCm.For pure rennink0andk1are proportional toCe, but for commercial rennets they vary as a power (N) ofCeand the value ofNappears to measure the rennin purity. The potentialities of the method for assessing rennet activities are also discussed.If the reduced viscosity (specific viscosity÷Cm) of fat-free milks is plotted againstCm, good straight lines are obtained which may be extrapolated to zero concentration to give a reliable value of intrinsic viscosity. The intrinsic viscosity falls progressively during the protein breakdown process but the slope of the curves (‘second order term’) remains unchanged.

634. The determination of the coagulating power of commercial rennet extracts using an automatic tester

1. A method for preparing rennet reference standards by freeze-drying is described.2. A substrate has also been developed using ordinary spray dried separated milk powder which will give reproducible results under specified conditions. These include:(i) A constant temperature (37° C.) for the duration of the test.(ii) The exclusion of added calcium ions until actual testing begins.(iii) The equilibration of the milk at 37° C. for 2 hr. before use.3. The construction and use of an automatic apparatus for recording rennet coagulation time is also described.4. The instrument error of a single observation is about 1·0%.5. With liquid rennet extracts, the total experimental error, based on one sample of three replicates, is 2% or less: with two samples, it is 1% or less.6. With freeze-dried rennet preparations, the experimental error with two ampoules, sampled thrice with three replicates per sample, was ±2·.0%. With three ampoules it was ±1% or less.

牛乳凝固酵素に関する研究

A colorimetric micromethod for estimating little proteolytic action (pepsin action) contained in milk coagulating enzyme preparations is presented in this paper. The principle of the method consists of cansing tyrosine to react, formed by proteolytic action from casein (substrate), with mercuric sulfate and nitrous acid, estimating colorimetrically the reacting colored substance by a photometer and representing the proteolytic action with γ units of tyrosine from 1mg of enzyme preparations. By this method, commercial rennets, rennet powders prepared by salting out method, papain and some pepsin preparations were compared with one another in their proteolytic action.

A simple method for preparing crystalline rennin.

The preparation of crystalline rennin previously described (1) was somewhat long and inconvenient, but the knowledge gained about the properties of the crystals suggested that a simpler method might be successful. Since then Møgensen (2) has shown that the mere precipitation of commercial rennet with saturated sodium chloride gives a product with proteolytic properties resembling those of the crystalline material, and with enhanced milk-clotting activity per unit of dry weight. Some years previously, Hankinson (3) had also achieved considerable purification by these means. The results of an attempt to discover a simple method of crystallization are described below.

The preparation of crystalline rennin and a study of some of its properties

Crystalline rennin enzyme has been prepared using a commercial cheese rennet as the starting material. The over-all yield is approximately 5%. The crystalline enzyme protein has been subjected to electrophoretic analysis, revealing the presence of three components. The major component (75%) contained both milk-clotting and proteolytic activities. The minor components did not clot milk. The ultraviolet absorption spectrum of the protein, also studied, indicated the presence of tyrosine in the molecule. Crystalline and crude rennins are affected in the same way by changes in pH; temperature; and concentration of sodium ion, calcium ion, and substrate. Of these factors, pH is quantitatively the most important. Crystalline rennin exhibits a pH optimum for proteolysis against hemoglobin at pH 3.9. Some pepsin was detected in commercial rennet, which has a plateau of maximum proteolytic action between pH 3.5 and 4. In the presence of 1% sodium chloride, the pH-activity curves for both crude and purified preparations lose their skewed nature and become dome-shaped, with optima near pH 3. The proteolytic action of commercial and crystalline rennins is not affected by the presence of H 2S. Activation energies for the proteolytic action of rennin are small (3500–3900 and 2600 cal./mole in the absence and presence of salt, respectively). This is in contrast to an apparent activation energy of 12,300 cal./mole for milk clotting by the same enzyme. The above results are discussed in the light of the various theories of the enzyme-catalyzed coagulation of milk.

Paper chromatography of casein and rennin.

Purified rennins of very high activity were prepared. The amino acid com position of the hydrolyzates of these preparations was studied by paper chromato graphy. Several fractions were obtained from the purified rennins by paper chromatography showing strong enzyme activity which was comparable to the density of their color development with a benzidine reagent. Whether these fractions represent distinct molecular species is not known as yet. The active enzyme was separated from the inert material of a crude commercial rennet powder by chromatography. The action of rennin on milk and casein was also studied by chromatographic techniques. It would appear that rennin causes an unfolding and breaking of the alpha casein molecule with the appearance of several large polypeptides. No free amino acids were detected after rennin action on casein.

Purification of Rennin from Commercial Rennin Extract: Properties of Purified Product

Summary A stable purified rennin sol was prepared from a commercial rennet extract by means of an isoelectric precipitation procedure. One part of dry rennin coagulated 72,300,000 parts of fresh raw skim milk at pH 5.75-5.80 (obtained by added CaCl 2 ) in 10 minutes at 40° C, which was 4.55 times that of the dry organic matter of the original extract. The peptic activity, measured by a method which estimates quantitatively 10μg. crystalline pepsin, was only 6.25 per cent of the original dry organic mixture. The rennin was therefore 99.77 per cent pure from the standpoint of peptic activity. The purified rennin exhibited the properties of a globulin. Its isoelectric point in 16.7 per cent NaCl solution was pH 4.5. The rennin sol, freed from NaCl by dialysis, showed a progressive increase in negative zeta potential from pH 6.0 to 7.5, and at pH 6.5 showed about the same negative zeta potential as calcium caseinate particles. Rennin and calcium casemate, therefore, do not exhibit opposite electropotentials at the pH of milk.

Effect of Heat and pH on the Inactivation of Rennin in Whey

Summarry The zone of maximum stability of the milk-coagulating power of commercial rennet extract, when heated in whey to 49° C. or above, centers at pH 4.0. The pH-inactivation curves at the temperatures of 49° to 70° C. are nearly symmetrical both above and below pH 4.0. In the regions where alterations in pH accelerate inactivation, an alteration of one pH unit includes approximately the complete inactivation effect. No peptic activity could be demonstrated, using the carmine-fibrin test, in solutions in which the rennin activity had been destroyed. The data indicate that normal amounts of rennet can be inactivated by holding at 50° C. for 14 minutes at pH 6.8 to 7.0.

161. Studies in Cheddar cheese. VI. The degradation of milk proteins by lactic acid bacteria isolated from cheese, alone, with sterile rennet and with whole rennet

It has been shown by experiments in milk that the enzymes of commercial rennet in conjunction with the lactic acid bacteria occurring in Cheddar cheese can bring about protein breakdown similar in extent to that found in the ripe cheese as far as the non-protein nitrogen is concerned. The amino nitrogen produced is, however, much less than in cheese. This may be ascribed to the higher pH of cheese as compared with that of the milk cultures, since acidity adversely affects the peptidases present. Attention is drawn to the differences between the conditions.

Rennet Test for the Detection of Mastitis

Summary A new test with fresh commercial fluid rennet for the detection of mastitis is described. It is believed that this method of testing, because of its simplicity and cheapness, may have practical use. Comparisons of the results secured by the rennet method with those secured by other methods are presented.