GB2024828A - Process for decreasing the thermal stability of microbial rennet - Google Patents

Abstract

An aqueous solution of Mucor microbial rennet is contacted with hydrogen peroxide, preferably at a temperature of 4 to 30 DEG C and pH 4 to 8 thereby to decrease the thermal stability of the said rennet. The rennet product which, can serve as a replacement for calf rennet in traditional cheese-making processes, is thermally unstable at the temperature at which cheese-makers whey is pasteurised.

Description

SPECIFICATION Process for decreasing the thermal stability of microbial rennet Background and prior art For centuries, calf rennet has been used as a milk coagulant in the production of all varieties of cheese. In fact, cheeses made with this enzyme set the standards for flavor, appearance and texture with which cheeses made from other coagulants are compared. Recently, dramatic increases in world cheese production and decreases in calf rennet supplies have stimulated the use of alternative milk coagulant enzymes.

Among the available enzymes for this purpose, the microbial rennets are favored because they can be mass produced and offer a variety of properties permitting selection of those most suitable in cheese production. Unfortunately, the increased thermal stability, especially when compared to calf rennet, of microbial rennets, such as the Mucor microbial rennets, has been considered to be a less desirable property of these enzymes.

In the cheese-making process, the whey which separates from the rennet coagulated curd is collected as a valuable source of whey proteins. Normally, the collected whey is pasteurized, at a temperature from about 600C to 71 C, for a sufficient time to destroy any microorganisms and to thermally inactivate at least about 80 to 90% of any residual rennet coagulant. Residual levels of coagulant enzyme retaining much more than about 20% of their original milk clotting activities after normal pasteurization have been found to undesirably hydrolyze the valuable whey proteins and restrict the use of the whey as an ingredient in various food products.The normal pasteurizing conditions which are sufficiently mild to leave the whey proteins unaffected but sufficiently severe to thermally inactivate calf rennet are much siower to inactivate microbial rennet coagulants to the desired level. This problern is further complicated by the fact that more microbial rennet is partitioned into the whey than is calf rennet. As a result there has been a continuing effort to improve the thermal lability of microbial rennets.

Several microbiological approaches to obtain more heat-labile microbial coagulants have been tried. These approaches include comprehensive screens for new microorganisms and mutations of known rennet-producing microorganisms. To date, neither approach has been successful in obtaining an acceptable coagulant with the desired thermal properties.

Chemical approaches to modify various properties of enzymes have also been tried but, in general, are severely limited by a concomitant loss in the enzyme's milk-clotting activity. Recent studies, however, to determine the structural and functional determinants of one Mucor microbial rennet indicate that some chemical modification of microbial rennet may not result in the total loss of milk coagulant activity. Such modifications include nitration (Biochemica et Biophysica Acta, 37t, 368 [1974]), carbamylation (ibid., 271, 93 [1972]) and periodate-induced deglycosylation (ibid., 328, 52 [1 973]) of the glycoenzyme from Mucor microbial rennet Of these modifications, there is some suggestion that carbamylation and periodate-induced deglycosylation may impart some increased thermal lability.Unfortunately these chemical approaches have not been completely successful in producing a microbial rennet with the desired degree of thermal lability.

SUMMARY OF THE INVENTION It is therefore highly desirable to produce a microbial rennet with desirable milk coagulant properties but with substantially increased thermal labile properties.

In accordance with the present invention, a process is provided for decreasing the thermal stability of Mucor microbial rennet by contacting an aqueous solution of the microbial rennet with hydrogen peroxide under conditions effective to substantially decrease the enzymes' thermal stability.

DESCRIPTION OF THE INVENTION The thermal stable microbial rennets which are most effectively treated by this process are prepared from Mucor microorganisms by well-known procedures and are commercially available in aqueous solution. Such rennets include those produced from Mucor miehei, Mucor pusillus and the like.

The native coagulant enzymes from these sources are known to be more heat stable than calf rennet.

This process may be applied to pure, partially pure or even very crude fermentation liquors of the microbial rennet.

The hydrogen peroxide used in this invention is commercially available or can be produced in situ by familiar means. For example, inorganic and inorganic peroxides are known to produce hydrogen peroxide on contact with water. Any suitable hydrogen peroxide-producing means which does not adversely inactivate the treated rennet may be selected, including the use of sodium peroxide, calcium peroxide, benzoyl hydroperoxide, a mixture of cumene hydroperoxide and peroxidase, urea hydrogen peroxide and the like. The inorganic peroxides are preferred.

In the practice of this invention, an aqueous solution of the thermal stable microbial rennet mentioned above is contacted with the hydrogen peroxide under conditions effective to substantially decrease its thermal stability (i.e. increase thermal lability), preferably to at least 20 percent of its original stability expressed in terms of its residual milk clotting activity after thermal treatment and most preferably to at least about the thermal stability of calf rennet. The thus treated solution of microbial rennet can be used as is, concentrated or further purified as may be required by the particular cheesemaking process.

Those skilled in the art will appreciate that the above process can be practiced as a batch or continuous process and that the conditions of contact with hydrogen peroxide, i.e. concentration of materials, pH, temperature and contact time, will vary widely depending upon the thermal stability of the native microbial rennet, the type of process and the equipment selected. Such conditions, however, should be selected which do not adversely affect the original milk clotting activity of the microbial rennet.

The concentration of microbial rennet in the aqueous solution is most conveniently expressed in terms of milk clotting enzymatic activity, i.e. Soxhlet Units (SU) per ml, as determined by the procedure described in J. of Diary Science, Vol. 54, No. 2, 1 59-1 67 (1 971) which is incorporated herein by reference. The milk clotting activity is determined on a 10% solution of skim milk at pH 6.45 to 6.50 containing 0.01 MCaCl2. Five milliliters of milk are incubated at 37 OC + 0.5 with 0.5 ml of enzyme solution. The time necessary for the appearance of the milk clot is measured. One Soxhlet Unit is defined as that enzyme activity which clots one (1) ml of a milk substrate in 40 minutes under the conditions of the assay procedure.In practice, the milk clotting activity of the aqueous solution of microbial rennet will preferably vary from about 10,000 SU/ml to about 100,000 SU/ml.

The amounts of hydrogen peroxide used to treat the aqueous solution of microbial rennet will depend upon the enzyme's activity, original heat stability and purity. The concentration of hydrogen peroxide in the aqueous solution varies between about 1% and 25% hydrogen peroxide on a volume/volume (V/V) basis based on the original volume of the aqueous solution. Preferably, the amount is between 3% and 1 0% (V/V) based on the original volume. At concentrations much above about 25% (V/V), the benefit becomes asymptotical and at concentrations much below about 1% (V/V) the reaction rate becomes unduly long.

The temperature for hydrogen peroxide contact varies between about 40C and 30"C. Preferably the temperature is between about 40C and 200 C. At temperatures much above 300C the enzyme undesirably loses activity and at temperatures much below about 40C the reaction rates become unduly long.

The pH for the hydrogen peroxide treatment will preferably range between about pH 4.0 and 8.0.

At pH values much above about pH 8.0, the enzyme is undesirably inactivated and at pH values much below pH 4.0 the solubility of the enzyme diminishes and the reaction time increases.

The contact time is a function of the particular contact conditions selected but should be sufficient to substantially decrease the microbial rennet's thermal stability to a desired level.

Under the preferred conditions, contact times between about 1 5 and 72 hours have been used most successfully.

In a preferred mode of this invention, the hydrogen peroxide treated solution is further treated to substantially destroy an residual or excess hydrogen peroxide in the solution using any convenient means which does not inactivate the microbial rennet. Residual hydrogen peroxide may be destroyed with the use of a material selected from the group consisting of catalase such as beef liver catalase, peroxidase such as horseradish peroxidase, ascorbic acid and the like. These hydrogen peroxide destroying means can be effectively used under the prevailing conditions used for the hydrogen peroxide treatment. In practice, the amount of hydrogen peroxide remaining in the solution after treatment is determined by any convenient detection means as is well known in the cheese industry such as sodium iodide titration.A suitable amount of the hydrogen peroxide destroying means is then added to the microbial rennet solution and residual amounts of the hydrogen peroxide are monitored with the detection means until the hydrogen peroxide is substantially destroyed. Beef liver catalase is a preferred means and is effective to substantially decrease the hydrogen peroxide to the desired level under the preferred conditions above in a period from about 1 to 3 hours.

The thermal stability of the hydrogen peroxide treated microbial rennet is determined under conditions which are normally used to pasteurize cheese whey solutions. The foilowing model is useful in reproducing these pasteurization conditions. A one (1 ) ml aliquot of the rennet-containing solution is diluted to 100 ml with a sodium phosphate buffer (pH 5.5 to 6.0). Aliquots (2 to 3 ml) of this diluted solution are then placed in sealed glass tubes and heated in a water bath to a temperature from 600C to 71 OC for periods of time from 0 to 20 minutes. The heated samples are cooled and then assayed for their milk clotting activities.Under these model pasteurization conditions, the residual activity of the rennet found in cheese whey is decreased after the thermal treatment to a desired residual milk clotting activity level at least below about 20 percent of the original milk clotting activity before the thermal treatment Using the preferred conditions of this invention, the hydrogen peroxide treated microbial rennets are found to be as thermally labile as calf rennet when subjected to the same thermal conditions.

The microbial rennet produced according to this process is useful in all traditional cheese making processes which now use microbial rennet as a milk coagulant. Since the novel hydrogen peroxide treated microbial rennet is dramatically less thermal stable than the untreated microbial rennet, it can serve as a reasonable replacement for even calf rennet. Used in these cheese making processes, the treated microbial rennet of this invention is desirably inactivated by the normal conditions used to pasteurize the cheese whey and the resulting whey can be fully utilized for its valuable whey protein.

The following examples are illustrative of the inventive concept but are not intended to limit the scope thereof.

EXAMPLE 1 This example illustrates a typical treatment of various Mucor microbial rennets using hydrogen peroxide.

Aqueous fermentation broths of microbial rennet from Mucor miehei (pH 4.5) containing 49,800 SU/ml and Mucorpuslllus (pH 5.5) containing 49,800 SU/ml were obtained from commercial'sources.

Samples (1 000 ml) of the aqueous broths were cooled to 40C and the pH was adjusted to pH 97.0 by the addition of 20% (W/V) aqueous sodium hydroxide. To each sample there was then added 100 ml of 30% (V/V) hydrogen peroxide (analyticai reagent grade). This corresponds to a concentration of about 3% (V/V) hydrogen peroxide based on the original volume of the aqueous broth. These samples were allowed to stand for 24 hours at 40 C in contact with the hydrogen peroxide. Beef liver catalase [0.3 ml, 100 Keil Units (KU) per 1 ml; 1 KU decomposes 1.0 g. of H202/1 0 min. at 250C at pH 7.0] was then added to the solutions to destroy any excess hydrogen peroxide and the mixture was allowed to stand (about 2 to 3 hours) at room temperature until a negative peroxide test was obtained.The pH values of the solutions were back adjusted to their original pH values with 1 2N HCI and concentrated to their original volume of 1000 ml by flash evaporation.

The milk clotting activity of the microbial rennet was determined on 10% solutions of skim milk containing 0.01 M CaCI2 adjusted to about pH 6.5 with lactic acid. Five mis of milk were incubated at 370C with 0.5 ml of sample of the enzyme solution. The time necessary for the appearance of the clot was measured. Enzyme activity was expressed in terms of Soxhlet clotting units (SU). The hydrogen peroxide treatment of Mucor miehei microbial rennet resulted in the total retention of milk clotting activity while the hydrogen peroxide treatment ofMucorpuslllus microbial rennet resulted in the retention of 80 to 90% of the milk clotting activity.

To determine the thermal stability of the hydrogen peroxide treated enzymes, aliquots (1 ml) of the native and treated rennets were diluted to 100 ml with 0.2 M sodium phosphate buffer (pH 5.5).

Aliquots (2 ml) of these diluted solutions, representing an excess amount of rennet present in cheese whey, were placed in screw top test tubes and heated in a water bath at 600 to 700C for various times.

After the heat treatment, the samples were cooled by immersion in an ice-water bath and were assayed for their residual milk clotting (MC) activities by the method described above. The results of these studies are given in Table A below.

TABLE A Pasteurization % Residual MC Conditions Activity After Sample T"C Time (Min) Pasteurization M. pusillus (native) 60 0 100 4 95 8 90 12 89 16 87 20 82 M. pusillus (H2O2 treated) 60 0 100 4 34 8 18 12 11 16 6.9 20 - M. miehei (native) 70 0 100 4 94 8 79 12 72 16 66 20 61 M. meihei (H2O2 treated) 70 0 100 4 19 8 5.2 12 2.0 16 - 20 - The above data clearly illustrate that the hydrogen peroxide treatment is effective to dramatically reduce the heat stability of the enzyme as expressed in terms of its milk clotting activity and as compared with the native (untreated) enzyme.

EXAMPLE 2 This example illustrates the effect of pH on the milk clotting activity and thermal stability of microbial rennet treated by thins process.

Ten ml aliquots of an aqueous broth of microbial rennet from Mucor miehei (pH 4.5) containing 49,800 SU/ml were adjusted to various pH values between pH 4.5 and 7.5 using 20% (W/V) aqueous sodium hydroxide. Each sample was treated with 0.6 ml of 30% (V/V) hydrogen peroxide (corresponding to 1.8% (V/V) H202 based on the original volume). One untreated sample served as a control. All samples were stored at 4.0for 24 hours and then treated with 5 ml of 20% (W/V) aqueous ascorbic acid (to decompose any H202) ahd stored at 40C for 48 hours. Each mixture was diluted 100 fold with 0.2 M sodium phosphate buffer (pH 5.5). The milk clotting (MC) activities were determined using the procedure described in example 1. These results are given in Table B below.

TABLE B Treatment pH % of Original MC Activity 4.5 (control) 100 4.5 94 5.1 89 5.8 92 6.3 94 6.9 97 7.5 100 . .

The data in Table B clearly demonstrates that the instant process does not adversely affect the milk clotting activity of the treated microbial rennet even over the broad pH range from 4.5 to 7.5.

The thermal stability of the treated microbial rennet was determined using the pasteurization procedure described in Example 1. The milk clotting activities were measured after pasteurization and reported as a percentage of the original activity before pasteurization. The data is reported in Table C below.

TABLE C Pasteurization Time % Residual MC Treatment pH Minutes at 700C Activity 4.5 (control) 0 100 4 93 8 -84 12 72 4.5 0 100 4 39 8 12.4 12 5.6 6.3 0 100 4 37.6 8 13 12 4.4 7.5 0 100 4 10.7 8 2.6 12 The data in Table C illustrates that the samples treated with hydrogen peroxide over the pH range from 4.5 to 7.5 are dramatically more heat labile than the untreated control.

EXAMPLE 3 This example illustrates the effects of contact time on the milk clotting activity and thermal stability of microbial rennet treated by this process, A 200 ml portion of an aqueous solution of Mucor miehei microbial rennet containing 49,800 SU/ml was adjusted to pH 7.0 with 20% (W/V) aqueous sodium hydroxide cooled to 40C and 20 ml of 30% (V/V) hydrogen peroxide (corresponding to 3% (V/V) H202 based on the original volume) was added to the solution. This mixture was maintained at 4 C for several days during which time aliquots (1 ml) were removed, then treated with beef liver catalase (0.01 ml, 100 KU/ml) and further diluted 100 fold with 0.2 M sodium phosphate buffer (pH 5.5). The milk clotting (MC) activities were determined using the procedure described in Example 1. These results are given in Table D below.

TABLE D Contact Time (Hours) % of Original MC Activity 0 100 17 103 26 108 41 105 50 113 65 101 73 113 136 111 The data in Table D demonstrates that this process improves rather than adversely affects the milk clotting activity of the treated microbial rennet even over extended contact times.

The thermal stability of selected samples of the treated microbial rennet was determined using the pasteurization procedure described in Example 1. The milk clotting (MC) activities were measured after pasteurization and reported as a percentage of the original MC activity in the sample before pasteurization. The data is reported in Table E below.

TABLE E Contact Time Pasteurization Time % Residual MC Hours Minutes at 70"C Activity 0 o 100 4 94 8 79 12 72 26 0 100 4 19 8 5.2 12 2 73 0 100 4 9.6 8 3.2 12 136 0 100 4 6.6 8 1.6 12 The data in Table E shows that the samples treated with hydrogen peroxide over a broad range of contact times are dramatically more heat labile than an untreated control.

EXAMPLE 4 This example illustrates the effects of hydrogen peroxide concentration on the milk clotting activity -and thermal stability of microbial rennet treated by this process.

A 250 ml portion of an aqueous solution of Mucormieheimicrobial rennet containing 99,600 Su/ml was adjusted to pH 7.0 with 20% (W/V) aqueous sodium hydroxide. Ten ml aliquots were removed and various amounts of hydrogen peroxide (20% V/V) were added ranging from 3% (V/V) to 9% (V/V) H207 based on the original volume. Each sample was then stored at 40C for 77 hours.

Sufficient beef liver catalase was added (0.1 ml, 100 KU/ml) to destroy the hydrogen peroxide in the sample. Each sample was then diluted 100 fold with 0.2 M phosphate buffer (pH 5.5). The milk clotting (MC) activities were determined using the procedure described in Example 1. These results are reported in Table F below.

TABLE F H202 Concentration % V/V % of Original MC Activity 0 100 3.0 91 4.2 91 5.4 96 6.0 97 7.2 97 8.4 93 9.0 93 The data in Table F shows that this process does not adversely affect the milk clotting activity of the treated microbial rennet even over a range of 2 2 concentrations.

The thermal stability of selected samples of the treated samples was determined using the pasteurization procedure (at 650 C) described in Example 1. The milk clotting activities were measured after pasteurization and reported as a percentage of the original MC activity in the sample before pasteurization. The data is reported in Table G below.

TABLE G H202 Concentration Pasteurization Time % Residual MC % V/V Minutes at 650C Activity 0 0 100 8 100 3 0 100 8 21 6 0 100 8 10 9 0 100 8 4 The data ip Table G shows that the samples treated with hydrogen peroxide over a range of H202 concentrations are dramatically more heat labile than an untreated control.

EXAMPLE 5 This example illustrates the use of hydrogen peroxide produced in situ on the thermal stability of microbial rennet treated by this process.

Purified microbial rennet (500 mg) from Mucor miehei was dissolved in 100 ml of 1.0 N sodium acetate buffer (pH 4.5). This aqueous solution of the enzyme contained about 9,960 SiJ/ml. A ten ml aliquot of this solution was removed and 500 mg of calcium peroxide was added thereto. This amount of calcium peroxide on contact with water present was sufficient to produce hydrogen peroxide in situ equivalent to about 2.35% H202 (V/V) based on the original volume. The sample was stored at about 20"C for about 15 to 18 hours at pH 4.5. To destroy excess hydrogen peroxide, beef liver catalase (0.1 ml; 100 KU/ml) was then added.The sample was diluted 20 fold with 0.2 M sodium phosphate buffer (pH 5.5) and the thermal stabilities of the treated sample and untreated control were determined using the pasteurization procedure (at 600C) described in Example 1. The milk clotting (MC) activities were measured before and after pasteurization and values reported as a percentage of the original MC activity. The data is reported in Table H below.

TABLE H CaO2 % H 202 Pasteurization Time (Min) % Residual MC (mg) (V/V) at 60"C Activity 0 0 0 100 8 97 16 97 20 94 500 2.35 0 100 8 37 16 23 20 21 The data in Table H clearly shows that hydrogen peroxide produced in situ by this process is effective to dramatically reduce the thermal stability of the treated microbial rennet compared to the untreated control.

EXAMPLE 6 This example illustrates the use of the microbial rennet treated by this process to produce cheese.

Batches of cheddar cheese were prepared, using a conventiqnal procedure, from 196 kg of pasteurized whole milk and 1 O/o commercial starter culture. A control vat was coagulated with 22.5 ml of untreated Mucor miehei microbial rennet containing about 93,000 SU/ml. A test vat was coagulated with 26.5 ml of Mucormiehei microbial rennet treated as described in Example 4 with 3% (V/V) hydrogen peroxide containing about 79,000 Su/ml. The respective times for milk clot formation were measured and were determined to be equivalent. After cheese manufacture, the whey liquids were collected, adjusted to pH 6.1 with sodium hydroxide and analyzed for residual enzyme activity before and after flash pasteurization. The enzyme activities are compared in Table I below as a percentage of the original MC activities in the whey.

TABLE I % of Original MC Sample Pasteurization Conditions Activity in Whey Untreated Control None 100 630C for 20 sec 99 63iC for 30 sec 100 H202 Treated None 100 63"C for 5 sec 30 63"C for 10 sec 30 630C for 15 sec 26 630C for 20 sec 23 The above data clearly shows that the H202 treated microbial rennet is as effective as the native microbial rennet to produce cheese but the H202 treated rennet is much more heat labile than the native rennet.

The cheeses prepared above were aged at 70C for 30 days and compared with one another by a trained sensory panel. The cheese made with H202 treated rennet was well accepted and found to be desirably similar in flavor and texture to the cheese made with untreated native rennet.

EXAMPLE 7 This example compares the heat stabilities of calf rennet and microbial rennet treated by this process.

Mucormieheimicrobial rennet containing 99,600 SU/ml was treated with 3% (v/v) H202 as described in Example 4 and its thermal stability was compared with the native microbial rennet before treatment and with calf rennet containing about 49,800 SU/ml using the pasteurization procedure (at 600 C) described in Example 1. The milk clotting (MC) activities were measured after pasteurization and reported as a percentage of the original MC activity in the sample before pasteurization. The data is reported in Table J below.

TABLE J Pasteurization Time % Residual MC Sample Minutes at 60 C Activity Microbial rennet 0 100 (untreated) 8 98 20 98 Microbial rennet 0 100 (H202 treated) 8 60 20 38 Calf rennet 0 100 8 72 20 55 The data in Table J clearly shows that the instant process is effective to reduce the heat stability of microbial rennet to about the heat stability of calf rennet.

Claims (23)

1. A process for decreasing the thermal stability of Mucor microbial rennet which comprises contacting an aqueous solution of the microbial rennet with hydrogen peroxide under conditions effective to substantially decrease the microbial rennet's thermal stability.

2. A process according to claim 1 wherein the microbial rennet is produced from Mucor miehei.

3. A process according to claim 1 wherein the microbial rennet is produced from Mucor pusillus.

4. A process according to claim 1, 2 or 3, wherein the thermal stability of the microbial rennet expressed in terms of residual milk clotting activity after thermal treatment is decreased to at least below about 20 percent of the original activity.

5. A process according to claim 1,2 or 3, wherein the thermal stability of the microbial rennet is decreased to about the thermal stability of calf rennet when subjected to the same thermal conditions.

6. A process according to any preceding claim wherein the microbial rennet is present in the aqueous solution at a concentration expressed in terms of milk clotting activity values ranging from about 10,000 to 100,000 Soxhlet Units per milliliter.

7. A process according to any preceding claim wherein the concentration of hydrogen peroxide is between 1% and 25% on a volume/volume basis based upon the original volume of the aqueous solution.

8. A process according to claim 7 wherein the hydrogen peroxide concentration is between 3% and 10% on a volume/volume basis, based upon the original volume of the aqueous solution.

9. A process according to any preceding claim wherein the hydrogen peroxide contact is carried out at a temperature between 40C and 300 C.

1 0. A process according to any preceding claim wherein the hydrogen peroxide contact is carried out at a pH between 4.0 and 8.0.

11. A process according to any one of claims 1 to 6 wherein the concentration of hydrogen peroxide is between 1% and 25% on a volume/volume basis based upon the original volume of the aqueous solution, and the hydrogen peroxide contact is carried out at a temperature between 40C and 300C and pH of between 4.0 and 8.0 for between 1 5 and 72 hours.

12. A process according to any preceding claim wherein the hydrogen peroxide is produced in situ.

13. A process according to claim 12 wherein the hydrogen peroxide is produced in situ by adding sodium peroxide, benzoyl hydrqperoxide, urea hydrogen peroxide or a mixture of cumeme hydroperoxide and peroxidase to the aqueous solution.

14. A process according to claim 12 wherein the hydrogen peroxide is produced in situ by adding calcium peroxide to the aqueous solution.

1 5. A process according to any preceding claim wherein residual hydrogen peroxide in the solution is substantially destroyed.

16. A process according to claim 1 5 wherein residual hydrogen peroxide is substantially destroyed by adding catalase, peroxidase or ascorbic acid to the aqueous solution.

17. A process according to claim 1 6 wherein beef liver catalase or horseradish peroxidase is added to the aqueous solution.

18. A process according to claim 1, substantially as described in any one experiment of the Examples.

19. AMucor microbial rennet produced by a process claimed in any preceding claim.

20. A process for making cheese which comprises subjecting milk to the clotting action of a Mucor microbial rennet claimed in claim 19, separating curd from whey, and pasteurising the whey.

21. A process according to claim 20 wherein the pasteurising temperature is from 60 to 71 OC.

22. A whey product produced by a process claimed in claim 20 or 21.

23. A cheeseproduct produced by a process claimed in claim 20 or 21.