AU692508B2 - Process for making undenatured whey protein concentrate - Google Patents

Description

R~ III 11 I 15 io ol ooo °o o i* ft a PROCESS FOR MAKING UNDENATURED WHEY PROTEIN CONCENTRATE This invention relates to an improved process for producing a whey protein concentrate having a serum albumin content of about 9% or more.

Backzround of the Invention and ,ectives As early as 1982 Bounous et al showed that dietary whey protein concentrate (WPC) improved the active systemic humoral immune response in a mammal, as measured by sheep red blood cell injections. It was however found that the use of high temperature pasteurization of milk [in 1988 and subsequent years following a Salmonellosis epidemic in Europe] greatly reduced the effectiveness of commercially available whey protein concentrates in improving the immune response.

United States Patent 5,230,902 dated July 27, 1993 described a method of improving the humoral immune response or increasing the concentration levels of glutathione in mammals, comprising administering orally to a mammal a therapeutically or prophylactically effective amount of undenatured whey protein concentrate. U.S. Application 989,186 filed December 11, 1992 as a continuation in part of previous related U.S. applications provided an improved method for making undenatured whey protein concentrate.

In said related applications the discovery that undenatured whey protein concentrate had an enhanced immunological effect was presented. It was furthermore explained that in the conventional high temperature treatment of milk the thermosensitive protein serum albumin were partially heat denatured and hence precipitated in the curd. Said related applications describe experiments where WPC was prepared using the lowest level of heat treatment of milk compatible with safety standards, so as to obtain a whey protein distribution having a high content of the thermolabile serum albumin. It was wU a111 i/e PCT/CA94/0072(, found that the preence of the 6-glmiamylcysteiie (Glo-Cys) group/molecule in the serum albumin (BSA) and the specific intramolecular disulfide bond ofcysK; r related to the undenatured conformation of the molecule, were a key factor in the glutathione (GSH) promoting activity of WPC (enhancement of GSH levels in tissues). They are believed to represent the common denominator underlying the beneficial effect of WPC Dietary W.P.C. produced with lower levels of heat treatment improves systemic humoral immune response, increases the resistance of target cells against the effect of carcinogens including chemical carcinogens such as dimethylhydrazine, improves'resistance to pneumococcal infection, and provides a sustained increase of tissue (intracellular) glutathione.

The object of this invention is to provide an improved process for preparing a whey protein concentrate that has adequately low bacterial levels without excessive levels of protein denaturation. It is a particular object to provide a process that will achieve a whey protein concentrate having a serum albumin of about 9% and adequate bacterial reduction. This approximately 9% level of serum albumin was found in our studies to'be important for the achievement of sustained increase of tissue glutathione for the properties such as improved systemic humoral immune response described above (References 2 and 3).

It is a further object to provide a process that will achieve a whey protein concentrate having not only a high serum albumin content but also a high content of other cystine and Glu-Cys containing proteins or Glutathione homologues such as (Glu- Cys-Ala). The use of process conditions to give a high serum albumin content will also give a high content of such other Glu-Cys containing proteins.

Serum albumin is the most easily denatured serum protein since its denaturation is not as reversible as that of alpha-lactalbumin It therefore is a further object of this invention to maintain rigorous control of temperature and other conditions to minimize denaturation of serum albumin.

The casein in raw milk occurs in the form of a colloidal dispersion. The particles of this dispersion range from 20 to 200 nm (namometers) in 2 tMENDED SHEET I II I diameter and are generally referred to as casein micelles.

The industrial methods of separation of the casein are based on the destabilization of these proteins either by lowering the pH of milk to the isoelectric point (pH 4.6) at 20 0 C or by enzymatic (rennet) hydrolysis of the Kappa casein which stabilizes the micelles.

The first procedure is not suitable for the recovery of native whey proteins since low pH has been shown to denature Bovine serum albumin, apparently because of repulsion of acidic amino acids (Haurowitz, 1963) (11).

Furthermore, if excessively high temperatures are used in this procedure, there would be a considerable increase in the denaturing effect of a low pH.

The second procedure is less economically feasible because the caseins recovered are less functional for dairy industry use.

Considering those facts and other economical aspects, our objective is to develop a combination of method-steps which are compatible with cheese making. The cheese represents roughly 10% (by weight) of milk being treated and the whey represents approximately 90%, of which 0.6 to 0.7% consists of whey proteins.

A recent study has shown that a microfliltration membrane containing pores with average diameter of 0.2 micron will selectively retain 20 the casein micelles from skim milk. However, that study concentrated on the separation of caseul.

The Invention We propose to utilize low temperature lenient techniques to achieve the objectives of this invention.

In accordance with this invention a process is provided for producing an undenatured whey protein concentrate having a serum albumin content of about 9% or more as a by-product of a process for making cheese, preferably 0 cheddar cheese, comprising: cold standardization of the fat content in xnilk-at a temperature preferably not greater than about 4 0 C. By this approach we reduce the outgrowth of bacteria; furthermore this method -educes fat losses and breakdown of fat globules which would lead to further reactions involving enzymes and bacterial metabolism; pasteurisation under conditions that will avoid any substantial denaturing of the protein in the milk; chilling preliminary to cheese production preferably to a temperature of about 30 0

C;

making curd and whey at a temperature not greater than about 400C; separating the curd from the whey; removing excess fat from the whey; pasteurisation, if needed, at a high temperature for a time short enough to avoid denaturing of most of the protein in the whey; ultrafiltration at a temperature close to but not substantially in excess of 400C to provide a retentate; which may provide an end product in liquid form; where a dry powder is desired, drying said retentate, preferably by freeze drying at a temperature and for a time that will not denature the protein.

The pH is maintained at not less that 6 throughout steps 1 to 8.

:i The present invention also resides in a process for producing an undenatured whey protein concentrate, which concentrate constrains substantially all thermolabile whey proteins of the source milk with glutamylcysteine groups and disulfide bonds, in an amount effective to replenish depleted intracellular glutathione, enhance systemic humoral immune response and exert anti-cancer effects, comprising cold standardisation of milk, 25 pasteurisation, the formation of curd and whey, separation of the whey, filtering concentrating and drying the whey under conditions of time and temperature that will avoid substantial loss of said glutamylcysteine groups and at a pH of not less than 6 prior to concentration.

The invention also relates to a process wherein the whey protein concentrate comprises a substantially undenatured whey protein isolate mixture containing bovine serum albumin, a-lactalbumin and lactoferrin proteins, which MC C- VNWORDVA\ALONODELETESPECR408785.MX I I I II 4A are cystine rich, in a biologically effective amount to replenish depleted cellular glutathione (GSH), enhance the immune response and exert anti-cancer effects.

Brief description of the drawing Figure 1 is a schematic representation of the process of this invention for producing a whey protein concentrate with immunoenhancing properties.

Detailed description of the invention Material and Methods Individual whey proteins were measured by polyacrylamide gel electrophoresis. Samples of concentrated whey were applied on 16% polyacrylamide at pH 8 (Laemmli-buffer-system) after the samples were reduced with 10% 2-mercaptoethanol. Samples were applied so that each slot received 10-20 pg (micrograms) of protein. Electrophoresis was performed at 200 volts for minutes. The results were also confirmed by chromatography.

Extent of protein denaturation by the process was determined in triplicate by the nitrogen solubility index (NSI) at pH 4.6 and 3000 g.

oees

I

so **0 MC C IWWORDMARLOWODELETEDSPECRIn409795.DC 1 I I WO 95/17830 PCT/CA94/00726 (Association of Official Analytical Chemists (AOAC) 1985 ref. The method utilized in these experiments differs from that described in Reference 2 and represents a more accurate reflection of the undenatured state of protein.

Protein content (N X 6.38) and total lipids of samples were determined in duplicate respectively by the standard method of Kjeldahl and the method of Mojonnier.

Moisture content was determined in duplicate by AOAC method Total coliforms count was determined following incubation at 37°C for 18 h in brilliant green using the most probable number method. Total bacteria count (aerobic mesophiles) was determined following incubation at 32"C for 48 h in PCA medium. Both methods are approved by the International Dairy Federation and American Public Health Association.

Lactose was measured by the enzymatic method.

Standardization Referring now to Figure 1, the first step is the standardization of the milk. This involves skimming the milk to a desired fat content. This procedure is used for two reasons. The first is to attain the legal percentage of fat on a dry basis of the final cheese. The second is to achieve the best yield and quality (body and texture) of cheese.

For cheddar cheese the optimum fat to casein ratio in milk is one part fat to 0.7 part of casein (Kosikowski)(9).

The usual practice has been to use a temperature in the range of 50 0

C

to 65 0 C as this is the most efficient range for skimming to a level of 0.05% of fat. However, we prefer in accordance with this invention to maintain the temperature at a lower level, preferably not greater than about 4"C. This low temperature during the fat removal process is necessary to avoid oxidation of lipids which would then tend to continue over time during storage and produce rancidity (liberation of fatty acids) which could in turn damage the conformation without affecting nutritional efficiency of the labile proteins.

The raw milk used in the examples had the following composition: Composition I I WO 95117830 WO 95/7830 C'r/CA94/00726 protein 3.2 fat 3.65 lactose 4.7 The pH of the composition was 6.65.

In the examples described in this application standardization of milk was carried out with a cold separator (Alfa Laval, CMIRPX 714-HGV) coupled with an automatic standardizer (Alfa Laval, Alfast Model 110). All1 standardization steps were carried out at 4*C. (Figure 1) and for all examples the fat content was adjusted to 3.58% of total milk.

The fat content of 3.58% was in accordance with standardization according to the following calculation: fat %protein X1. 12 (in final product) (in raw milk) fat -3.2 X1. 12

PASTEURIZATION

The next step according to Figure 1 is pasteurization. In the examples in this application pasteurization of the milk was achieved on a heat exchanger type HTST (Alfa Laval H1-10) being set at 72.6"C with a retention time of 16 sec., followed by flash cooling to Most countries do not allow the production of dairy products without pasteurization. Different types of pasteurization can be used: HTST high temperature short time 72.8 0 C, 16 sec.

=TL low temperature long time 65.6'C, 30 mmi.

UHT ultra high temperature 1200'C for a few seconds; but the effect on denaturation of protein and especially serum albumin are mostly evident with LTLT and URT.

The introduction of high temperature pasteurization of milk was prompted by two different objectives. The first was to obtain a near sterilization of milk following the cheese related infection diseases reported during 1988 in Europe. The second objective was to improve the yield during cheese making.

In fact, cheese makers have noticed that at these temperatures, or WO 95/17830 PCT/CA94/00726 increasing by five degrees or less above pasteurizing temperature, yield could be increased in certain types of cheese.

In accordance with the teaching of this invention ultra high temperatures should be avoided, contrary to present practice. Long time exposure to a lowest temperature of the order of 65.6 0 C should also be avoided. Even a temperature as low as 55 0 C causes unfolding of the bovine serum albumin molecule over a period of time (Wong Call. 1988).

However, this invention may use high temperatures in the range 63 0 C to and preferably about 72*C for a short time up to about 20 seconds.

The conditions such as time and temperature must avoid denaturing of the protein in the milk, particularly the heat labile serum albumin, and other Glu- Cys containing proteins, however such conditions of pasteurization should be such that pathogens are killed or reduced to an acceptable level.

Cheese Production Temperature Reduction The next step according to Figure 1 is cheese production.

The preliminary step in cheese production is to reduce the temperature.

In accordance with the examples of this invention the temperature is accordingly immediately reduced, such as by flash cooling to a temperature of for cheese making.

Cheese prodoctiou-addition of cultures Since cheese making is a fermentation process, we add lactic acid cultures. Those cultures and their metabolites are mainly responsible for the acidification and repining of cheese. For cheddar cheese we use a commercial culture of mesophilic organisms which in our examples are provided by CH.

Hansen Laboratory. Suitable cultures are readily available from other sources such as Miles Laboratory. The amount of cultures that has been added was 1 of total milk being transformed in cheddar.

These cultures are added after chilling to about 30 0 C, which allows the necessary growth of mesophilic lactic acid bacteria for cheese making. The activity of these bacteria will be controlled in such a way that the pH of the milk will never drop below pH6. Since bacteria are living organisms their rate of metabolism may vary. If it is too rapid the amount of bacteria added IWO 95117830 PCT/CA94/00726 to the milk can be reduced, eg. from 1% of total milk to 0.75%. With less bacteria less acid is produced.

Another way of controlling the rate of metabolism is to reduce the fermentation temperature. A reduction of 2 0 C will normally be used for this purpose.

At this preliminary stage of cheese making calcium chloride is usually added to milk to increase the firmness of the curd during the manufacture of cheese. The net result is an increase in cheese yield due to better precipitation and less losses of cheese curd particles in the whey. However, we have found that denaturation of BSA is enhanced by the presence of calcium ions.

Therefore we avoid the addition of calcium ions, contrary to normal practice in cheese manufacture. Reference is also made to the report of Shimada Matsushita 1981 Additives of this sort are avoided during production of cheese if the whey generated is being used for this invention. Yield can also be increased in conventional practice by the addition of milk derivatives (casein, whey protein) to milk being transformed into cheese. This procedure is generally avoided by us because it influences the quality of the protein content of the whey. The various fraction found in normal whey will be modified resulting in a whey protein concentrate having less undenatured bovine serum albumin per unit of total protein.

Cheese Production fermentation The fermentation period (repining period) was 1.5 hour for our examples at a temperature of 30°C. The pH decreased from 6.65 to 6.55.

The pH drop was thus 0.1 before rennet was added.

Cheese producing Addition of rennet Rennet was added at a rate of 20 ml/100 liters of milk. The time required for curd formation was 25 minutes.

The rennet used was a pure calf rennet single strength from CH.

Hansens Laboratory. The quantity was 20 ml./100 liters of milk. The temperature is maintained at 30 0 C. The rennet was diluted in 10 times its volume with water before adding to milk. The clotting time was 25 minutes.

slI ICCI~ WO 95/17830 PCTICA94/00726 Cheese production culturing the curd The curd was then cut using 6 mm. cheese knives at a temperature of to provide 6 mm cubes and stirred for 15 minutes before it was cooked.

Cheese production cooking In our examples the cooking period lasted 75 min and continuous agitation was used. The peak temperature of 38 0 C was reached in 30 minutes (1.3 0 C per 5 minutes). After this cooking period agitation was maintained for 1 hour.

The pH'of the whey after cooking was It is highly desirable to avoid any cheese making steps that involves a temperature in excess of 40°C. In fact some cheese production such as Emmental requires a temperature of more than 50°C. Raising the temperature to this level before separation of the whey and maintaining such temperature would adversely affect the serum albumin content.

Following post stirring the curd is separated from the whey.

SEPARATION OF CURD FROM WHEY The curd should be s-parated from the whey at this level.

No additional whey is collected for the purposes of this invention during shaping and final pressing of the curd as acidification during this period would result in a pH drop below 6.

The whey should be chilled to about 4 0 C as soon as it is separated from the curd if it is to be kept for more than 1/2 hour. By this procedure, the metabolism of the lactic acid bacteria is reduced and acidity does not increase. No additives (such as H202) should be used as antibacterial agents.

Instead we use low temperature inhibition of bacterial metabolism. The pH of the whey should never be below pH=6 before it is concentrated.

Whev fat removal The whey that has been collected is first pumped at 38 0 C in a centrifugal separator to take out excess fat (Alfa-Laval MHMRPX-214TGV) that was present during cheese production. The amount of fat is reduced to a level of 0.06% in the resulting whey. The Ph in our examples was at 6.48.

The whey is then chilled to 4 0 C and stored till ultrafiltration is WO 95/17830 PCT/CA94/00726 carried out. The temperature is then raised at 40°C for the Ultrafiltration (using Romicon cartridge with a cut off of 50,000 Dalton). If necessary it can be given a second pasteurization as shown in Figure 1 under conditions similar to the pasteurization previously described.

Whe pasteurization In our examples, whey was pasteurized. This heat treatment was mainly applied to control the activity of lactic acid bacteria that was previously added to milk. It also serve to control post pasteurization contamination that could occur during cheese making.

Ultrafiltration During ultrafiltration the retentate is submitted to diafiltration by adding water so as to reduce the lactose level so that the retentate from ultrafiltration contains less than 1 of lactose. The retentate following completion of ultrafiltration has a total solids of 19-20%.

The conditions of ultrafiltration are set forth below in Table 1.

TABLE 1 TEMPERATURE 40 0

C

Transmembrane pressure (TMP) 1.0 BAR

TANGENTIAL

VELOCITY 7m/s Concentration factor (CF) 29 FLUX 24.5 l/m 2 /h The membrane flux increases 2-2'1 per degree centigrade, giving a similar increase in capacity of a plant. This means that without special reasons for operations at low temperature, it is an advantage in conventional practice to operate at as high a temperature as possible.

The temperature utilized in most other commercial methods during this procedure is 50 0 C. This level of temperature facilitates a higher flux through the membranes hence more retentate production per unit of time and per unit of membrane. In our method, the draw-back of less production per unit of membrane is compensated by increasing the membrane surface.

The objective of not exceeding 40*C is obtained throughout the system r~r* r WO 95/17830 PCT/CA94/00726 by fine tuning the points of input and output in the system si to avoid a heat producing unbalance between the two. After ultrafiltrm, n the temperature of the retentate is then lowered to 4 0 C and kept at that temperature till freeze drying is started.

The composition of the retentate is about 19-20% total solids. A typical composition is: Fat 2.09% True protein 15.91 Non protein nitrogen 0.04 Lactose 0.84 Ash 'uotal Solids 19.88% Vitamins and flavours may, if desired, be added to the retentate after ultrafiltration. The retentate may be regarded as a final product and sold in liquid form. Alternatively, the retentate can be concentrated to provide a dry product as described below.

Whey Freeze drying Concentration to produce a dry product by lyophilization (freeze drying) is performed at temperatures under OoC for 15 to 18 hours, This does not denature the thermolabile proteins.

In our examples, the retentate was subjected to a blast freeze at before entering in the freeze dryer. The temperature of the condenser were maintained at -50*C during the 17 hours of the freeze drying period.

The microbial counts of the retentate compare favourably with standards applicable to conventional pasteurization. These standards differ in each jurisdiction. As an example, the Province of Quebec, Canada, requires that total bacteria count (aerobic mesophiles (32*C) be maintained below 50,000 (log 4.69), both in the factory and in the final product in the case of powdered milk products. Coliforms are to be below 10. The Province of Quebec has a standard of a bacteria count of 25,000 (log 4.39) and a coliform count of 5 in the factory for milk products that have not been pasteurized or I IlslLI a WO 95/17830 PCT/CA94/00726 fermented.

Table 2 illustrates the composition of whey protein concentrate powder obtained using the principles described above.

Certain factors cannot be controlled during normal production of whey concentrate powders. Seasonal variation of milk composition and bacterial metabolism that occurs in milk at each step of the chees making process will be mainly responsible for the differences observed in the composition of the concentrate. However, the practice of the principles of this invention may be expected to produce a consistently high level of thermolabile proteins such as serum albumin and to avoid substantial loss of the glutamylcysteine groups in the whey proteins.

-U

WO 95/17830 Pl'CT/CA94/00726 TABLE 2 COMPOSITION OF WHEY PROTEIN CONCENTRATE POWDER Example 1 Example 2 Example 3 PROTEIN 77.5 77.04 78.08 a Lactalbumin 23.68 23.85 22.20 0 Lactoglobulin 59.90 60.37 61.40 Serum Albumin 11.11 9.67 11.35 Others 5.31 6.14 5.05 (immunoglobulin, lactoferrin, etc.) FAT 10. 9.76 8.12 LACTOSE 4.25 4.6 4.9 MOISTURE 4. 4. 4.

(maximum) OTHER NUTRIENTS (mg/g) Ca 5.06 4.78 4.84 Mg 0.66 0.65 0.66 Cu 3.05 2.48 3.05 Zn 8.76 7.12 9.35 Na 4.23 4.67 3.53 K 6.45 6.35 4.05 Cl 10.82 11.70 9.59

MICROBIOLOGY

Salmonella (/100g) No growth No growth No growth Coli (/100g) <50 <50 Staphyloccoccus <50 <50 Total count 10000 10000 10000 NITROGEN SOLUBILITY 99% 98.9% 99.5%

INDEX

13- WO 95/17830 PCT/CA94/00726 The protein composition and solubility of the final product in powder form after concentration by ultrafiltration and lyophilization (Table 2) meets the requirements previously identified by us as essential for the development of immunoenhancing activity and tissue GSH promotion: serum albumin concentration around 9% and minimal degree of denaturation. In each of the examples the serum albumin is over In Table 3 and Table 4 are presented for comparison the concentrations of serum albumin in current commercially available W.P.C.'s and the nitrogen solubility index determined by De Wit in some W.P.C. products.

TABLE 3 WHEY PROTEIN

CONCENTRATE

Promod' Alacen 855' Sapro' Bioisolate" Promix' Mean SD *trade-marks BOVINE SERUM ALBUMIN in of Total Whey Protein 4 1 4 1 4.8 2 4 +0.1 4 1 5 1 3 1 I I L I WO 95/17830 W095/7830 CTCA9110072(i TABLE 4 WHEY PROTEN

CONCENTRATE

Normal UF WPC Neutral UF-DF-WPC Acid UF-DF-WPTC De-fatted UP WPC Spherosil* 'OMIA' Spherosil* WPC Vistec* WPC Demin. de-lact. WPC From De Wit J.N. et al Neth. Milk Dairy 37 (1983) pp. 57-49 NSI at pH 4.6 83 78% 42 91 79% 35 35 72 The process of this invention therefore provides a practical procedure for making undenatured whey protein concentrate. Furthermore it has the advantage of using a by-product of cheese production which is otherwise considered to be a troublesome waste product, and a potential pollutant. it should solve what was until now a continuing financial problem for the dairy industry responsible for the disposal of this major water pollutant.

conformation of the labile whey proteins in s objective of leniency is obtained -sv intr-dependent steps involving *Trademarks

I

L I In conclusion, it is the objective of this invention to preserve intact the conformation of the labile whey proteins in the W.P.C. This objective of leniency is obtained through several inter-dependent steps involving temperature, ions content, ultrafiltration flux and drying techniques.

Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises", is not intended to exclude other additives, components, integers or steps.

**e ee•• SC CA WINWORD SIMIONSNODELESEIJ(I97LC- DOC i P WO 951.17830 PCT/CA94/00726

REFERENCES

(which are incorporated by reference in their entirety) 1. Bounous Konshavn P.A.C. "Influence of Dietary Proteins on the Immune System of Mice". J.Nutr. 112, 1747, 1982.

2. Bounous Gold P. "The Biological Activity of Undenatured Dietary Whey Proteins: Role of Glutathione" Clin.Invest.Med. 14: 296-309, 1991.

3. Bounous Batist. G; Gold P. "Immunoenhancing Property of Dietary Whey Protein in Mice: Role of Glutathione". Clin. Invest. Med. 12: 154-61, 1989.

4. Brown R.T. "Milk Coagulation and Protein Denaturation in 'Fundamentals of Dairy Chemistry'", 3rd Edition, N.P. Wong (Ed) Van Nostrand Reynold C. (Publ.) New York, 1988, pp.583-607.

Fauquant, Maubois, Pierre, A. "Microfiltration du lait sur Membrane Minerale" Tech.Lait 1028, 21-23, 1988.

6. AOAC 1980, "Official Methods of Analysis" 13 Edition Association of Official Analytical Chemists, Washington, D.C.

7. AOAC 1985, "Official and Tentative Methods of the American Oil Chemist Society, Official Methods", Ball-65, Revised Edition.

8. Shimada K, Matsushita S.J. Agric. Food Chem. 1981, 29, 15-20.

9. Kosikowski F. (1977), Cheese and Fermented milk Foods, 2d Edition, F.V. Kosikowski Associates, N.Y.

II~I -rr i it -I WO 9$/17830 PCI/CA94OO 726 Wong Jenness Kenney M; Marth E.H. 1988, Fundamentals of Dairy Chemistry, 3d Edition, Van Nostrand Reinbold, N.Y.

11. Haurowitz F. "Albumnins, globulins and other proteins" in The Chemnistry and Functions of Proteins, (Academic Press NY 1963).

Claims (19)

1. A process for producing a undenatured whey protein concentrate having a serum albumin content of about 9% or more as a by-product of a process for making cheese comprising the following steps: cold standardisation of the fat content in milk; pasteurisation prior to cheese making under conditions that will avoid any substantial denaturing of the protein in the milk; chilling preliminary to cheese production without the addition of calcium ions; making cheese curd and whey at a temperature not greater than 400C; separating the curd from the whey; removal of excess fat from the whey; pasteurisation, if needed, at a high temperature for a time short enough to avoid any substantial denaturing of the protein in the milk; ultrafiltration at a temperature close to but not substantially in 1 ,excess of 40°C to provide a retentate; the pH being maintained at not less than 6 throughout steps 1 to 8.

2. A process as in claim 1, in which the retentate is dried at a temperature 20 and time that will not denature the protein.

3. A process as in claim 1 or 2, in which cold standardisation is at a temperature of about 4 0 C.

4. A process as in any one of the preceding claims, in which pasteurisation of steps and is at temperatures of about 70°C 750C for less than 25 seconds.

5. A process as in any one of the preceding claims in which pasteurisation prior to cheese making is immediately followed by flash cooling to a temperature of approximately 300C.

6. A process as in any one of the preceding claims, in which ultrafiltration is accompanied by diafiltration to reduce the lactose level in the retentate to less than 1%. MC C.IWINWORDVARLOVO0ELETEC0JlSPEC114O9795.00C 19

7. A process as in claim 2, in which the retentate is concentrated by lyophilization.

8. A process as in claim 7, in which the whey protein concentrate has a lactose content of

9. A process as in claim 1, in which no additional whey is collected during final shaping and pressing of the curd.

A process as in claim 1, in which the retentate is freeze dried.

11. A process as in claim 1, in which the cheese curd is cheddar cheese curd.

12. A process as in claim 1, in which the addition of calcium chloride to milk is avoided.

13. A process as in claim 1, in which the retentate from ultrafiltration is a final liquid product having a solids content of about 19-20%.

14. A process for producing an undenatured whey protein concentrate, which concentrate constrains substantially all thermolabile whey proteins of the source milk with glutamylcysteine groups and disulfide bonds, in an amount effective to replenish depleted intracellular glutathione, enhance systemic humoral immune response and exert anti-cancer effects, comprising cold standardisation of milk, pasteurisation, the formation of curd and whey, separation of the whey, filtering concentrating and drying the whey under conditions of time and temperature that will avoid substantial loss of said glutamylcysteine groups and at a pH of not less than 6 prior to concentration.

15. A process as in claim 14, wherein the whey protein concentrate comprises a substantially undenatured whey protein isolate mixture containing bovine serum albumin, a-lactalbumin and lactoferrin proteins, which are cystine rich, in a S 25 biologically effective amount to replenish depleted cellular glutathione (GSH), enhance the immune response and exert anti-cancer effects.

16. A process as in claim 14 or 15 in which the addition of a calcium compound that would cause denaturation of serum albumin is avoided.

17. A process as in any one of claims 13 to 15 in which the whey is separated in a prepressing operator, prior to shaping and final pressing of the curd.

18. A process as in any one of claims 13 to 16 in which the undenatured whey Sprotein concentrate has a serum albumin concentration of at least 9%. MC CAV 1NWORD\MARLO NODELETEUfJTSPECIli 9795.OC -II I L-

19. A process as in any one of claims 1 to 17 in which the undenatured whey protein concentrate has a serum albumin content of at least A process as claimed in any one of the preceding claims substantially as herein described. DATED: 21 April, 1998 PHILLIPS ORMONDE FITZPATRICK Attorneys for: IMMUNOTEC RESEARCH CORPORATION LTD. S* S **S e s e• MC C'.IWINWnrDWARLO\NDELETE\DJPEC1M4077.0 I Ir -r le r P