PROCESS FOR PRODUCING AN UNDERNATURED WHEY PROTEIN CONCENTRATE

Abstract: A process for producing an undernatured whey protein concentrate involving microfiltration of skim raw milk to provide bacterial reduction followed by microfiltration to separate the casein and ultrafiltration to give a whey protein concentrate having a serum albumin content of about 10 % or more.

Description: PR O CESS FOR PRODUCING AN UNDENATURED WHEY PROTEIN CONCENTRATE Related Applications This application is a continuation-in-part of U.S. Serial No. 929,347 filed August 13, 1992, which was a division and continuation-in-part of U.S. Serial No. 417,246 filed October 4, 1989 which was a continuation-in-part of U.S. Serial No. 289,971 filed December 23, 1988, which was a continuation-in-part of U.S. Serial No. 188,271 filed April 28, 1988 and is a continuation-in-part of U.S. Serial No. 866,756 filed April 10, 1992 which is a continuation-in-part of U.S. Serial No. 563,794 filed May 3, 1990 which in turn is a continuation-in-part of said U.S. Serial No. 289,971. The contents of said applications are hereby incorporated by reference in their entirety. Background of the invention As early as 1982 Bounous et al (1) showed that dietary whey protein concentrate (W.P.C.) 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. 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 pasteurization of milk the thermosensitive proteins serum albumin and immunoglobulin were partially heat denatured and hence precipitated in the curd. Said related applications describe experiments where W.P.C. 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 and immunoglobin. It was found that the presence in the serum albumin (B.S.A.) of 6 glutamylcysteine (Glu-Cys) group/molecule (substrate for glutathione (G.S.H.) synthesis) and the specific intramolecular diεulfide bond related to the undenatured conformation of the molecule, were a key factor in the G.S.H. promoting activity of W.P.C. Enhancement of G.S.H. levels in tissues are believed to represent the common denominator underlying the beneficial effect of W.P.C. Dietary W.P.C. produced with low level pasteurization improves systemic humoral immune response, increases the resistance of target cells against the carcinogenic effect of chemical carcinogens such as dimethylhydrazine, improves resistance to pneumococcal infection, and provides a sustained increase of tissue glutathione. The extent of denaturation produced during whey concentrate production is normally assessed by the loss of solubility at pH 4.6. Clearly the two most important factors of protein denaturation are temperature and pH whereas lower pH values usually enhance the denaturing effect of high temperature. This is why throughout our entire procedure, we have maintained a rather high pH ()6) thanks to a reduction of pH lowering bacterial contamination through microfiltration with 1.4 μ pores. With a denaturation temperature of 64°C. at pH 6, serum albumin appears to be the most easily denatured serum protein since its denaturation is not as reversible as that of alpha-lactalbumin (4) . Even a temperature as low as 55°C. causes unfolding of the B.S.A. molecule over a period of time. A pH of at least about 6 is therefore preferably maintained throughout the process. The thermosensitivity of the immunoglobulins is very similar to that of BSA (2.4). Although the amino acid sequence of most immunoglobulins in cow's milk is not known, we know that these large molecules are very rich in Cysteine hence they may contain Glu-Cys groups. Therefore they might exert upon digestion and absorption an activity similar to that of BSA. The major drawback with low temperature milk pasteurization are: a) Some degree of heat denaturation, hence precipitation of the thermosensitive proteins; b) The possibility of insufficient bacterial reduction; c) The difficulty in maintaining the delicate balance between adequate pasteurization and the avoidance of protein denaturation. This is likely to cause problems of control on a factory scale; d) The cost of low temperature pasteurization. The object of this invention is to provide an improved process for preparing a whey protein concentrate that has adequate bacterial reduction without protein denaturation. It is a particular object to provide a process that will achieve a whey protein concentrate having a serum albumin of at least 10% and adequate bacterial reduction. This approximately 10% 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) . We propose to utilize techniques of microfiltration to achieve the objectives of this invention. Microfiltration using ceramic membranes is a membrane based procedure which allows the separation of particles ranging in size between 0.1 and 10 microns. Microfiltration with. membranes having a 1.4 micron porus size has been recently utilized to remove bacteria and other particles from milk in order to obtain a commercially "sterile" milk (5) . With this method a 2 to 3 Log reduction of bacterial count in milk was achieved; hence a bactericidal effect greater than what is obtained by traditional milk pasteurization (72-78 β C / 15 sec: 98% bacterial reduction) (6) . The industrial methods of separation of the caseins are based on the destabilization of these proteins either by lowering the pH of milk to the isoelectric point (pH 4.6) at 20 β 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 because pasteurization at low pH of the whey-concentrate would entail a substantial denaturation of the proteins. The second procedure is less economic because the caseins recovered are less functional. The casein in raw milk occurs in the form of a colloidal dispersion. The particles of this dispersion range from 20 to 200 n in diameter and are generally referred to as casein micelles. A recent study (7) has shown that a microfiltration membrane containing pores with average diameter of 0.2 micron will selectively retain from skim milk the casein micelles. However this study concentrated on the separation of casein. The milk that was used was skim milk that had been pasteurized at a temperature of 72°C. which would have resulted in denaturation. Although the study does not indicate the serum albumin content of the permeate, it is certain that it would be a low value. The Invention In accordance with this invention, a process is provided for producing an undenatured serum albumin content of at least about 10% comprising the following steps, each of which is conducted at a temperature not greater than about 50°C (< 50°): 1) Microfiltration of skim raw milk that has been skimmed at a temperature not in excess of about 50°C. to provide a first permeate having a bacterial count at least as good as the standard applicable to pasteurized milk; 2) Microfiltration of the first permeate to separate most of the casein as a retentate from a second permeate; 3) Ultrafiltration of the second permeate to give a whey protein concentrate having a serum albumin content of at least 10%. Brief Description of drawings 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 μg of protein. Electrophoresis was performed at 200 volts for 70 minutes. Extent of protein denaturation by the process was determined in triplicate by the nitrogen solubility index (NSI) at pH 4.6 and 3000 g. (AOCS 1985 ref.8) The method utilized in these experiments differs from that described in Reference 2 and represent 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 the AOCS method (9) . 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 esophiles) 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. Referring now to Figure 1 of the drawings, both the microfiltration of the raw milk- and the microfiltration to separate the casein were carried out on a microfiltration system with controlled transmembrane pressure as set up by Alfa-Laval filtration system Denmark (MFS-7 system with 1.4 sqm membrane surface) . Ceramic membranes were obtained from Membralox (pore size 0.1 and 1.4 micron). Table 1 below shows the operating conditions during microfiltration of skim raw bovine milk using a ceramic membrane, as previously described having a pore size of 1.4 microns. TABLE 1 Operating conditions during microfiltration for bacterial count reduction (1.4 ). TMP = transmembrane pressure CF = concentration factor Raw milk should be used that has been skimmed at a temperature not in excess of about 50°C. The best temperature for skimming to a level of 0.05% of fat is 45°C to 50°C. The temperature used during microfiltration of the raw milk should be in the range about 40°C to 50°C. As previously noted denaturation of serum albumin does not begin to occur until about 55°C. Table 2 shows the microbial counts of the milk following microfiltration. We have noted a 2-3 log reduction in bacterial counts in accordance with the literature (5) . This confirms the efficacy of the method. As expected, the lipids are in the retentate. The protein content of the permeate is only marginally different from that of the retentate and there is no difference between permeate and retentate in the type of proteins as measured by electrophoresis. Hence no selective retention of proteins is noted over a 1.4μ membrane. As indicated in Figure 1 microfiltration is followed by diafiltration which involves adding distilled water to the retentate. 1 volume of retentate to 1 volume of water is used and this procedure is performed two or three times. TABLE 2 . 'Reduction in log from raw milk to permeate. The microbial counts of the permeate 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 fermented. The second stage involved the separation of the casein micelles with a 0.1 μ membrane. The parameters are illustrated in Table 3. It should be noted that these values may vary throughout the procedure depending on conditions such as clogging of membrane pores, increased viscosity of the retentate etc. The results of the process are given in Table 4. Bovine serum albumin passes ' through the membrane. The protein composition of the final product in powder form after concentration by ultrafiltration and lyophylization (Table 4) meets the requirements previously identified by us as essential for the development of immunoenhancing activity and tissue GSH promotion: serum albumin concentration around 10% and minimal degree of denaturation. In Table 5 and Table 6 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. The temperature and other conditions during the second stage are as shown in Table 3. TABLE 3 Operating conditions during microfiltration for milk fractionation (0.lμ) . TMP = transmembrane pressure CF = concentration factor DF = diafiltration The retentate from the second stage was subjected to ultrafiltration on a pilot scale using a Romicon cartridge (1.5 εqm) with a cut off of 50,000 Dalton to provide a powder. The temperature during ultrafiltration was <50°C. Table 4 shows the results obtained by the process. TABLE 4 Composition of milk retentate and permeate (0,lμ). Example l *NSI = Nitrogen solubility index. 2 β-lg = beta-lactoglobulin 3 α-la = alpha-lactalbumin MF = microfiltration BSA - bovine serum albumin IMMU - Immunoglobulin TABLE 5 PRODUCT BOVINE SERUM ALBUMIN in % of Total Whey Protein Promod 4 ± 1 Alacen 855 4 ± 1 Lacprodan-80 4.8 ± 2 Sapro 4.8 ± 0.1 Savorpro-75 4 ± 1 Bioisolate 5 ± 1 Promix 4.3 ± 1 Mean ± SD From De Wit J.N. et al Neth. Milk Dairy J. 37 (1983) pp. 37-49 The process of this invention therefore provides a practical procedure for making undenatured whey protein concentrate. Furthermore it has the advantage of permitting separate recovery of the casein. This is a useful by-product that contributes to the practical economies of the process. REFERENCES (which are incorporated by reference in their entirety) 1. Bounous G., Kongshavn P.A.C. "Influence of Dietary Proteins on the Immune System of Mice". J.Nutr. 112, 1747, 1982. 2. Bounous G. , Gold P. "The Biological Activity of Undenatured Dietary Whey Proteins: Role of Glutathione" Clin.Invest. Med. 14: 296-309, 1991. 3. Bounous G., 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 5. Olesen, N. and F. Jensen, 1989. "Microfiltration. The Influence of Operation Parameters on the Process". Michwissenschaft 44(8), p. 476, 1989. 6. Pedersen P.J., in "New Applications of Membrane Process" Publ. by I.D.F. chapt. 4, p.33, 1991. 7. Fauquant, J.;, Maubois, J.L.; Pierre, A. "Microfiltration du lait sur Membrane Minerale" Tech.Lait 1028, 21-23, 1988. 8. AOAC 1980, "Official Methods of Analysis" 13 Edition Association of Official Analytical Chemists, Washington, D.C. 9. AOCA 1985, "Official and Tentative Methods of the American Oil Chemist Society, Official Methods", Ball-65, Revised Edition.

Claims: CLAIMS 1. A process for producing an undenatured whey protein concentrate having a serum albumin content of about 10% or more comprising the following steps each of which is conducted at a temperature not greater than about 50°C: i) microfiltration of skim raw milk that has been skimmed at a temperature not in excess of about 50°C. to provide a first permeate having a bacterial count at least as good as the standard applicable to pasteurized milk; ii) microfiltration of the first permeate to separate most of the casein as a retentate from a second permeate; iii) ultrafiltration of the second permeate to give a whey protein concentrate having a serum albumin content of about 10% or more. 2. A process as in claim 1, in which the skim raw milk is bovine and the serum albumin is bovine serum albumin. 3. A process as in claim 1, in which the microfiltration of the skim raw milk is with a ceramic membrane having a pore size of about 1.4 microns. 4. A process as in claim 3, in which the temperature of microfiltration of the skim raw milk is in the range 45-50°c 5. A process as in claim 1, in which the microfiltration to separate casein is with a ceramic membrane having a pore size of about 0.1 microns. 6. A process as in claim 1, in which a pH of at least about 6 is maintained during the steps of microfiltration and ultrafiltration. 7. A process as in claim 1, in which said first permeate is subjected to diafiltration. 8. An undenatured whey protein concentrate having a serum albumin content of at least about 10%, whenever prepared by the process of any one of claims 1 to 7. 9. A whey protein concentrate of claim 8 containing substantially all of the heat labile whey proteins from the milk.

Inventors: AUROUZE BERNARD, BOUNOUS GUSTAVO, TURGEON SYLVIE, AUROUZE, BERNARD, BOUNOUS, GUSTAVO, TURGEON, SYLVIE
Applicants: IMMUNOTEC RES CORP LTD, IMMUNOTEC RESEARCH CORPORATION LTD.
Application year: 1993
Main classification: A23C9
IPC codes: A23C9, A23J1