CA2165937A1 - Process for producing an undenatured whey protein concentrate - Google Patents
Process for producing an undenatured whey protein concentrateInfo
- Publication number
- CA2165937A1 CA2165937A1 CA 2165937 CA2165937A CA2165937A1 CA 2165937 A1 CA2165937 A1 CA 2165937A1 CA 2165937 CA2165937 CA 2165937 CA 2165937 A CA2165937 A CA 2165937A CA 2165937 A1 CA2165937 A1 CA 2165937A1
- Authority
- CA
- Canada
- Prior art keywords
- whey
- milk
- temperature
- protein
- drying
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/14—Milk preparations; Milk powder or milk powder preparations in which the chemical composition of the milk is modified by non-chemical treatment
- A23C9/142—Milk preparations; Milk powder or milk powder preparations in which the chemical composition of the milk is modified by non-chemical treatment by dialysis, reverse osmosis or ultrafiltration
- A23C9/1425—Milk preparations; Milk powder or milk powder preparations in which the chemical composition of the milk is modified by non-chemical treatment by dialysis, reverse osmosis or ultrafiltration by ultrafiltration, microfiltration or diafiltration of whey, e.g. treatment of the UF permeate
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/14—Milk preparations; Milk powder or milk powder preparations in which the chemical composition of the milk is modified by non-chemical treatment
- A23C9/142—Milk preparations; Milk powder or milk powder preparations in which the chemical composition of the milk is modified by non-chemical treatment by dialysis, reverse osmosis or ultrafiltration
- A23C9/1422—Milk preparations; Milk powder or milk powder preparations in which the chemical composition of the milk is modified by non-chemical treatment by dialysis, reverse osmosis or ultrafiltration by ultrafiltration, microfiltration or diafiltration of milk, e.g. for separating protein and lactose; Treatment of the UF permeate
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
- A23J1/20—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from milk, e.g. casein; from whey
- A23J1/205—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from milk, e.g. casein; from whey from whey, e.g. lactalbumine
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C2210/00—Physical treatment of dairy products
- A23C2210/20—Treatment using membranes, including sterile filtration
- A23C2210/208—Removal of bacteria by membrane filtration; Sterile filtration of milk products
Abstract
A process for producing an undenatured whey protein concentrate as a by-product in making cheese to give a whey protein concentrate having serum albumin content of about 9.5% or more involving microfiltration, ultrafiltration, control of temperature and other factors.
Description
A PROCESS l~)R PRODUCING AN UNDI~NATUR13~D W~ih;Y PROI~
CONCENTRATE
Related Applications This appli~tio,n is a co~ t;on-in-part of U.S. Serial No 08/315,904 filed September 30, 1994 and a co~ t;on-in-part of U.S. Serial No.
08/175,637, filed Dec~ll,ber 30, 1993, which are con~ ;ons-in-part of U.S.
Serial No. 07/989,186 filed Dece,.,ber 11, 1992, which is a col-t;..u~l;on-in-part of u.S. serial No. 07/929,347 filed August 13, 1992. The cont~llLs of said related applications are hereby incol~ldled by reference in their enl~ely.
Background of the Invention As early as 1982 Bounous et al (1) showed that dietary whey protein concPn~lP~ (W.P.C.) i.l.pr~ved the active systemic hllmoral imm~ne response in a .. ~.. ~1, as measured by sheep red blood cell injection~. It was however found that the use of high te~ dlur~ teuri7~tion of milk in 1988 and subsequent years following a Salmonello~i~ epidemic in Europe gready reduced the effectiveness of commercially available whey protein concP~ es in improving the immune response.
In said related applications the discov~l~ that unden~tllred whey protein concentrate had an - enh~nce~ immllnological effect was presP,nt~d. It was furthermore ~Ypl~ined that in the conventional high l~nl~ tule p~te,uri7~tion ofmiLt~ the thermosensitive proteins serum albumin and laclof~ were partially heat denatured and hence preci~ led in the curd. Said related applications describe eXperim~nt~ where W.P.C. was ~l~P~ ed using the lowest level of heat treatment of miLk compatible with safety standards, so as to obtain a whey protein distribution having a relatively high content of the thermolabile serum albumin, It was found that the presence in the serum albumin (B.S.A.) of 6 glutamylcyst(e)ine (Glu-Cys) group/molecule (substrate for glutathione (G.S.H.) synthesis) and the specific intramolecular disulfide bond related to the - - lmdPnqtllred co~fo~tion of the mol~clllP, were a key fætor in the G.S.H.
promoting .ctivity of W.P.C. The term cyst(e)ine implies the presence of a fiAe bond (cystine), . nd ~e ~s~ . reduction to ~ c. Fnhq~cpment of G.S.H. levels in tissues are believed to ~ the common d~lo...;n~or S underlying the benPfi<~iql effect.
Dietary W.P.C. produced with low level p ~ ;on im~ s ~;,t~mic humoral immllnP ~.s~nse, increases the re~i~tqn~ of target cells against the carcinogenic effect of chPmi~-q1 carr-inogpnc such s dh--elhylhy~ t,;n~ n~es re-~ict~n~e to pnellmococc~1 infection, and provides a mod~r~t~ but sllst~in~
increase of tissue g1tlt~thion~
The extent of de~l.-.~ n produced during whey c4ncpi~L~te production is norm~11y ~cs~-~sed by the loss of so1-1bi1ity at pH 4.6. Clearly the two most?olL~u~l factors of protein denAI~ on are ten~pe~dlule and pH whereas lower pH values usually enh~nc~ the ~ ;n~ effect of high temperature. This is why throughout our entire ploce-lule, we have .. ~ ~ a rather high Ph (6) thanks to a reduction of pH lowering b~ t~-n~1 c~nt~ ion through microfi1t~tion with 1.4 pores. With a dena~ul~lion len~ ldlulc; of 64C. at pH
6, serum albumin d~J~)ea[S to be the most easily de,~alult;d serum protein since its den~tl1ration is not as reversible as that of alpha-lactalbumin (4). Even a temp~ldlule as low as 55C. causes unfolding of the B.S.A. molecule over a period of time. A pH of at least about 6 is th~l~ro,e preferably ...~il;t~ined throughout ~e process.
The object of this invention is to provide an improved process for prepa~ g a whey protein concen~ldt~ that has adequate b~ctP,ri~1 reduction without protein ~n~ tion. It is a particular object to provide a process that will achieve a whey protein cQnc~ntl dte having a serum albumin of at least 9.5 %
and adequate b~ct~Pri~1 reduction. This appl.~ill,ately 9.5% level of serum albumin was found in our studies to be important for t,he achievement of sustained increase of tissue glutathione for the proy~lies such as improved systemic humoral immune response described above (References 2 and 3).
We propose to utilize techniques of microfiltration to achieve t,he objectives of this invention. Microfiltration using ceramic membranes is a m~mhrstne based procedure which allows the s~qpStr,ttinn of particles rztn~in~ in size belween 0.1 and 10 microns.
Micr )filtr~tinn with me...bli.r.Ps having a 1.4 micron porus si~ has been recel~tly utilized to l~.llove b~r~ and other particles from milk in order to obt~in a co~ e~ially "sterile~ milk (5). With this me~hoA a 2 to 3 Log reduction of bS~ctçtiS~l count in milk was achieved; hence a bstcteri~Asll effect greater than what is ol~tained by traditional milk ~u~ l;on (72-78C / 15 sec.: 98% bSlrtçri~l reduction) (6). The inAustrisll methods of sb~ on of the caseins are based on the destabilization of these proteins either by lowering the pH of milk to the ~ ~tric point (pH 4.6) at 20C or by e~ alic (rennet) hydrolysis of the Kappa casein which stabilizes the mirP11~, The first procedureis not suitable for the ,eco~ of native whey pç~eins because pA~te~ri7~til~n at low pH of the whey-conre~-t~te would entail a subst~ullial denSIt~lr~tion of theproteins.
Brief Description of Drawing Figure 1 is a srhemsltic ~ep,~ ~ti~lion of the process of this invention for producing a whey protein concPI~ te with immllnoenhslnrin~ ~,ro~ .ies.
Figure 2 illu~L~dl~s the gluPthione promoting activity of said whey protein concP-~L~i1lp- to which we have applied the trSl~ems~rk Tmmlln-x~l, in an in vitro assay as described below. In this assay, the activity of said product is cGlllpa~ed with a control milk protein (casein i.e. Ca c~Pin~te) and another commercial whey protein conrPnt~tp with low serum albumin content (Alacen 855, mentioned in Table 5).
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 (T ~emmli- buffer system) after the samples were reduced with 10% 2-mercaptoethanol. Samples were applied so that each slot received 10-20 microgrammes of protein. Electrophoresis was performed at 200 volts for 216~937 70 I.lh.~ s. The results were also co~ ....P~l by .,hr~.l"atog.a~hy.
E~stent of protein d~ ;on by the process was de ~ i nP~ in trjr~ tP, by the ~ g~ s ~luki1ity index ~NSI) at pH 4.6 and 3000 g. (AOAC 1985 ref.
6). The mPshod ntili7ed in these ~?e~;...~nt~ differs from ~at describe~d in S Reference 2 and ~ ls a more ~ccl~r~tP~ r~flection of the unden~h~red state of protein.
Protein content (N X 6.38) and total lipids of ~mp1~s were ~el~
in dupli~ vely by the standard method of Kjeldahl and the method of Mojonnier.
~ istl~e cont~nt was det~-rmined in dupli~te by the AOAC method C7)-Total coli~olllls count was det~ ined following incub~tion at 37C for 18 hours in brilli~nt green using the most probable number method. Total b~cteri~
count (aerobic mesophiles) was ~let~.rmin~ follorving incub~tion at 32C for 48 hours in PCA m~ium. Both methods are a~pn,ved by the Tnt~rn~tic)nal Dairy Fede~tion and ~m~oric~n Public Health ~ tion Lactose was measured by the enzymatic method.
Glut~thione de~ .n~;Qn Tn~rellular gl-.~thione was measured by erkylllalic assay using ni~in~mi-le-diphosphate, dithio-n,~bellzene and gluhtl~ionto. reductase. Briefly, MT-4 human lymphocyte T cells were halv~sled after 72 hours of incubation at 37 C under 5 % CO2 using standard methods of tissue culture in the presence of varying concentration of WPC (0.01 to 1 mg/m1). The cells were then lysed with ice-cold water and 6% sulfosalicylic acid, centrifuged at 600g for 10 min, and sUpern~t~nt~ collected and proc~s~ . S~t~ophotometric analysis was pelrolllled for 2 min at 412 nanometres (nm) and results were expressed in nmoleof GSH /107 cells.
Dr. S. Baruchel is Associate Professor of Pediatric and Oncology at McGill University in Montreal, C~n~1a. He has developed in his laboratory at -Montreal Children Hospital Research Tnstitute this in vitro assay (8,9) which is utilized in collaboration with us in evaluating the GSH promoting (potency) activity of each WPC production batch.
216~937 The Invention In accol~lce with an aspect of this invention, a pr~)CeSS iS provided for ploducillg an l)n~en~tured whey protein con~P~ tP, having a serum albumin content of about 9.5% or more as a byproduct of a process for m~king cheese S comrri~ing As ill~ ~ in Figure 1 in step 1, cold stand~ ;on of the fat contPnt in milk is err~ at a ~.~ not greater than about 4C. By this approach we reduce the Oulgl'OWl}l of b~teri~. Fur~Prmore this method reduces the breakdown of fat globules which would lead to further reactions involving enzymes and b~rP-fi~l metabolism, This involves s1~immin~ the milk to a desired fat content. The usual practice has been to use a tel"~ ur~ in the range of 50C to 65C as this is themost effi~ient range for slrimming to a level of 0.05% of fat. However, we prefer in accof~ance with this invention to ...~ . the te~ e at a lower level not greater than about 4C. This low ~ during the fat removal process is nP~s~.~ to avoid n~ tion of lipids which would then tend to continue over time during storage and produce r~n~ idity ~iberation of fatty acids) which could in turn damage the conform~tion without ~ffecting nutritional P.ffi~iPncy of the labile proteins.
In the eY~mples described in this applic~ti~ln standar 1i7~tion of milk was carried out with a cold sep~ Qr (Alfa Laval, CMRPX 714-HGV) coupled with an automatic standardizer (Alfa Laval, Alfast Model 110). All standardization steps were carried out at 4C. (Figure 1).
In next stPp shown in Figure 1, the skim raw milk obtained after step 1, is microfiltred to provide a first pP.rmP~tP- having a bacterial count at least as good as the standard applicable to p~te~lri7~1 milc.
The microfiltration of the skim milk obtained in step 1 was carried out on a microfiltration system with controlled t~n~mPmhrane pressure as set up by Alfa-Laval filtration system De-nm~rk (MFS-7 system with 1.4 sq m membrane surface). Ceramic membranes were obtained from Membralox (pore size 1.4 micron).
Table 1 below shows the operating conditions during microfiltration of `' - 2165g~7 - skim raw bovine milk using a oe~miC me~ dne, as previously df~lib~d having a pore size of 1.4 microns.
S T~ c 500 Tr~ncmP.. h.i~nc P~ ur~ IP) 0,5 bar Tal~genlialvelocity >6.6m/s ~oncent~~ n Factor (CF) 20 Flux 950 Llh/m2 Raw milk should be used that has been ~l~immf~d at a ~ .e not in excess of about 4C.
The le~ J~e used during microfiltr~tion of the raw milk should be in the range of about 40C to 45C. As previously noted ~i~ifi~-~nt ~lçn~ .tion of serum albumin does not begin to occur until about 55C.
Table 2 shows the m-icrobial counts of the milk following microfil~r~tion.
We have noted a 2-3 log red~cti< n in b~t`t~-ri~l counts in aco~f~lce with the l; If.. ~ . C (5). ~is c"n r....,~ the efficacy of the method. As ~ec~d, the lipids are in the l~P;n~;1le The protein content of the Ferme~ is only ~I~argillally dirr~r~lt from that of the re~ P and there is no di~rerellce between permeate and rt;le~ le in the type of proteins as measured by electrophoresis. Hence no selective retention of proteins is noted over a 1.4 micron membrane.
` 21~937 Microbial counts (per/g) expressed in log E;~peri- Raw M;lk R.~P~ IP. Perm~t~ Reduction ment of total number Total Coli- T C T C b~ tPri~l ~I~ forms counts (C) 4.40 -- 6.78 -- 2.37 -- 2.03 2 6.054.25 6.644.23 3.070.48 2.98 - ~ 3 3.931.80 4.661.29 1.76 0 2.17 4 5.723.08 6.333.18 3.35 0.3 2.37 5.493.67 6.32 -- 2.53 -- 2.96 6 5.033.08 7.20 -- 2.89 -- 2.14 7 6.034.60 7.51 -- 4.032.41 2.00 8 6.905.32 8.457.51 4.272.48 2.63 9 6.524.00 7.805.78 3.761.80 2.76 ~eduction in log from raw milk to permeate.
The microbial counts of the perm~te compare favourably wi~ standards applicable to conventional p~teun7~tion.
20The next step, step 3 according to Figure 1, is p~teun7~tion of the microfiltered milk. The leml}e,~lu,c; used in this invention is above 72C up to about ~0 seconds.
The following step 4 involves cooling of the microfiltered miLlc. In accordance with the examples of this invention the temperature is accordingly 25immediately reduced, such as by flash cooling to a temperature of about 30C
~165 937 - prior to cheese m~king At this prç~imin~ry stage of cheese m~kin~, c~lrinm ch1- ri~e is usually added to milk to ~ a~ the ~ nP-~S of the curd during the m~mlf~tllre of cheese. The net result is an increase in cheese yield due to better p c~ip;~lionand less losses of cheese curd particles in the whey.
Based on the report of .Shim~d~ & ]U~ h;~ (1981) (10) dP~ n of BSA is enh~nr~d by c~lrillm ions. Thcl~fo~ we avoid the addition of c~1cil-m ions COl ~ to normal pr~ctice~ in cheese m~mlf~cture.
Additives of this sort are avoided during produc-ti~ n of cheese if the whey gPnPr~tPd is being used for this invention. Yield can also be increased in conventional pr~ctice by the addition of miL deliv~livt;s (casein, whey protein)to milk being transformed into cheese. ThiS procedure is avoided because it influPnres the quality of the protein contPnt of the whey. The various f~ti~
found in normal whey will be mo~ifi~ reslllting in a whey protein cOI rentr~tP~
having less un-lçn~t lred bovine seNm albumin per unit of total protein. The pH
is to be ...~i~.LqinP~l at not less than 6 at this chilling stage and until col ~entr~tion of the whey.
~Ppining follows in step S and this involves the ~litit)n of cultures to milk about 24 hours before starting cheese m~king. In the examples the only additives are the usual lactic cultures.
The next step in m~nllf~ctllring cheese is curd making, illustrated in step 6, Figure 1, which includes the following stages namely:
(a) Rpnnp-tting (b) Coagulum cutting (c) Stirring (d) ~P~ting (cooking) (e) Post stirring Following post stirring the curd is prep,essed and separated from the whey.
Conventional additives are used for rennPttin~. The making and subsequent handling of the cheese are generally represented in Figure 1 at step 7. This step will depend on the type of cheese. They therefore will not further be described.
" ~165~7 g What is i~ ?o~ l however for the pu~poses of this invention is to avoid any additives or lr~l~ that will de1PtP~iously affect the whey. Thus the ~du~;on of P.mmPnt~l cheese l~uir~s a ~-~ G of more than 50C.
Raising the curd to this lGlll~GldlurG before ~...tiol- of the whey and .--~ ingsuch lGlll~)GldlUrt~S wouldaffecttheserumalbumincontPnt Itis highly desirable to avoid any cheese making steps that involve a te~ lJI G in e~cess of 40C .
No ~lition~l whey is collP~hd for the ~Ul~oS~s of this invention during .~haril-g and final pressing of the curd as the pH by then of any whey ~cc with the curd would be below pH 6.
The whey obtained in step 6 should be chilled to about 4C. as soon as it is sep~ e~ from the curd, and this is illllstr~tP~ on Figure 1 as step 8. By this procedure, the metabolism of the lactic acid b~^tP-ti~ is reduced and acidity does not increase. No additives (such as H2O2) should be used as ~ntih~t~ti~l agents.Instead we use low ~---~ e inhih-~ition of b~ tPri~l metabolism. The pH of the whey should never he below pH 6 before it is co~
The whey that has been collPct~d is first ce~ iruged to take out excess fat (Alfa-Laval M\H~PX-214TGV) that was present during cheese production, chilled to 4C. and stored till llltr~filtr~tion (U.F.) is carried out.
During next step 9, the l~lnp~lalure is then raised at 40C. for the ultr~filtr~tion using, for eY~mple, Romicon cartridge (2.3 sq.m.) with a cut offof 50,000 dalton. During U.F. the l~lP~ tP is s~n~ d to ~ filtr~tion by adding distilled water so as to reduce the lactose level in the dry matter to less than 10% of total solids. 1 volume of lel~ to 1 volume of water is used and this procedure is p~lrolllled two or three times. The relPnl~te following completion of U.F. has a total solids of 19-20%. The conditions of ~llt~filtr~tion are set forth below in Table 3.
" ~165937 .
-TEMPERATURE approx. 40C
TRANSMFMR~RANE 1.0 BAR
PRESSURE ~I~IP) TANGENTIAL VELOCITY TANGENTIAL VELOCITY
>7mls FACTOR (CF) FLUX 24.5 1/m2/h The membrane flux increases 2 to 2.5 % per degree centigr~le, giving a similar increase in capacity of a m~m1f~rtl1nng plant. This means that withoutspecial reasons for opPr~til)n~ at low te~ ~, it is an advantage in con~e~-l;on~1 practice to operate at as high a t l"~ dlure as possible.
The le",~eldluf~ utilized in most other commercial methods during this procedure is 50C. This level of l~ f~-~ilit~tP~ a higher flux through the membranes hence more relent;11P~ pr~duction per unit of time and per unit of membrane. In our mPthod, the draw-back of less production per unit of membrane is co~.~n.~te~ by increasing the membrane surface.
The objective of not exc~ing 40C. is obt~uned throughout the system by fine tuning the points of input and output in the system so as to avoid a heat pro~ucing unbalance between the two.
Next, the ~e",~e,dlure is lowered to 4C. in step l0 and kept at that ~ ?erdlule till freeze drying or spray drying is started. The microbial counts of the retPntate compare favourably with standards applicable to conventional p~ ion.
These standards differ in each jurisdiction. As an example, the Province of Quebec, ~n~ , requires that total bacteria count (aerobic mesophiles (32C) be m~in~ined 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 l0.
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 ~165g37 -- p~t~-JI~ ;,~1 or fPrm~Pn~
ConrPntr~tion to produce a dry product by lyophili7~tion (free~e drying) is p~rul,l,ed in step 11 at l --..~ '--.es under 0C for 15 to 18 hours. This does not denature a ~i~nifir~nt p~ lion of the thPrm~ hile ~l~leins.
S Table 4 ill.. `il.~f~ the co",~osilion of whey protein concpllt~ e powder obtained using the prinripl~s described above.
Certain factors cannot be controlled during normal production of whey concPnl.~, powders. Se~con~l v~ri~tinn of milk co"-p(j;,iLion and b~ctP-ri~l metabolism that occurs in milk at each step of the cheese making process will bemainly respolsible for the dirrel~nces obs~.ved in the composition of the cQ~ l.dLo. However, the prdrfire of the prinriplP~s of this invention may be eY~cte~ to produce a ~n~i~t~ntly high level of th~rmol~hilP proteins such as serum albumin and to avoid ~ulJst~ lial loss of the glul~u"ylcysteine group conl~inillg protein, together with bactP-n~l counts subs~.lL;~lly lower than that obtained with the method disclosed in the PCT/CA93/00518 (Bounous et al.) and in the United States Applir~ti~ n SN. 08/175,637 (Lange et al.), both ~Csign~ toTmmlmotec Research Col~ldLion Ltd., which is the ~ignPe of the present applir~tit-n Clearly the co.~.hin~ n of microfiltr~tion as in the first part of the method disclosed in PCT/CA93/00518 (Bounous et al.) and p~tPuri7~tion low temperature lenient procedures as disclosed in United States Application SN.
08/175,637 (Lange et al.) greatly reduced the b~r-tP-ri~l content of the end ., product. With this method of production, it is possible to obtain desired b~rtp-ri~l results with less stringent conditions than are disclosed in these prior applications. This is i~ >ol~nt for large scale productions on infl~lstri~l scale.
If fur~er b~rtpri~l redllction is desired a second p~tpllri7~tion or microfiltration procedure can be applied to the whey between steps 8 and 9, and namely prior to ultrafiltration. Further evidence of the leniency of this methodof production is the relatively high level of la~;lof~llin ~able 4) in colllp~ison to commercial WPC. This i"l~oll~lt protein is similar to serum albumin in that it contains 4 Glu-Cys peptides (Reference 11) and it is ~imil~rly thermolabile.
The analysis of laclorellin as reported in Table 4 were ~ roillled by Drs. G.
Regester and G. Smithers at the CSIRO Dairy ~ ch Laboratory in Victoria, str~ , and will likely be publi~h~d soon in the Journal of Nutrition.
COMPOSlTION OF WHEY PROTEIN CONCENTRATE POWDER
S EXAMPLE EXAMPLE EXAMPLE
PROTEIN(%) 78.04 77.00 77.08 ~-Lactalbumin(%) 22.00 æ.80 22.20 B-Lactoglobulin(%) 57.80 56.30 54.45 Serum Albumin (%) 11.7 11.06 10.85 Lactoferrin (%) 0.67 - - 0.70 0.69 Tmmllnoglobulin (%) 8.39 9.20 11.10 FAT (%) 3. 4. 3.5 LACTOSE (%) 9.40 9.20 9.4 MOISTURE (%) maximum 4. 4. 4.
Ash 3 3 3 MlCROBIOLOGY
Salmonella (/lOOg) No growth No growth No growth Coli (/lOOg) <5 <5 <5 Staphylococcus (/g) < 5 < 5 < 5 Total count (/g) < 1000 < 1000 < 500 NITROGEN SOLUBILlTY 99% 98.7% 99%
INDEX
The protein composition and solubility of the final product in powder form after concentration by Ill~filtr~tion and lyophili7~tion CIable 4) me,ets the requirements previously i-lentifi~d herein as es.sPnti~l for the development of immunoenh~n~.ing activity and tissue GSH promotion: serum albumin concentration around 9.5 % or more and minim~l degre,e of den~ tion. In Table S and Table 6 are ~resen~d for co~ a,ison the concentrations of serum albumin - in current ccilll"~ ially available W.P.C.'s and the nill~gel~ sol~bility inde~
d~ ned by De Wit in some W.P.C. products, as shown in Table 6. Table S is derived from Bounous et al. (2) W~Y PROTEIN CONCENTRATE BOVINE SERUM ALBUMIN
in % of Total Whey Protein Promod ~I~ 4+ 1 Alacen 855 4+ 1 Lacprodan-80 4.8+2 Sapro 4+0.1 Savorpro-75 4+1 Bioisolate 5 + 1 Promix 4.3 + 1 WHEY PROTEIN CONCENIRATE ¦ NSI AT Ph 4.6 Normal UF WPC 83 %
Neutral UF-DF-WPC 78%
Acid UF-DF-WPC 42%
De-fatted UF WPC 91%
Sphèrosil 'OMA' WPC ~I~I) 79%
Sphèrosil 'S' WPC ~) 35%
Vistec WPC ~I~I) 35%
Demin. delact. WPC 72%
From De Wit J.N. et al. Neth. Milk Dairy J. 37 (1983) pp. 37-49 Table 7 r~,esellts analysis results of an un~en~ red whey protein ~16~937 -- con~n~ d generally in accol~lce with the present invention made by ~e Japan Gove~ s.l Food analysis centre.
. - - 15--lTEMS RESULTS MEI EIOD
Water 4.4% Air Oven Method Protein 83.1% KiP~ hl Method (N x 6.38) Lipid 6.2% Roese~ot~ieb Method l~oi~ re 2.2% ~r~ition at 550 Carbohydrate 4.1% lO~(water + protein + lipid +
moisture) Energy 423kacVlOOg protein x 4.22 + lipid x 9.16 +
carbohydrate x 3.87 As Absent ( < O. lppm) Silver Diethyl~lithioc~rbamate Specl,ophotometric Method Pb Absent (<0.05ppm) Atomic Absorption Spectrophotometric Method Cd Absent (<O.Olppm) Atomic Absorption Specllopllotometric Method Total Hg Absent (<O.Olppm) Cold Vapor Atomic Absorption Spectrophotometric Method Sn Absent ( < lppm) Polalog~l)hy Method Cu 1.52ppm Atomic Absorption S~illopllotometric Method 3s Total Aerobic 8.7x103/g Standard Agar Plating Method ~ teri7~
Coliforms Absent/2.22g BGLB Broth Inoculating Method Surface Spread Plating Method S. Aureus Absent/O.Olg Fnri~hmPnt Culture Method Salmonella Absent/25g Fn~i-hment Culture Method Pseudomonas Absent/O. lg ~165~37 The J~plnPse Go~e- .. Pnt Food analysis centre also c4n~uct~ a co...~ Qn of the serum albumin content of the lm-lçnslu.ed whey protein cQnc~ e ~ner~lly made in acco~lce with this invention with three best commercial whey protein C~ PJ~ available on the m~rl~P.t The serum S albumin c4nPnt for the product of this invention was found to be 11.7 as c~...l~ed with 7.7, 7.7 and 5.5 ~s~;livGl~ for the co~,v~.l;~n~l products.
The ~xess of this invention therefore provides a pp~tir~l p~1ulè for m~king lln~P~ .~d whey protein con~ e. Furthermore, it has the advantage of using a by product of cheese pr~uction which is a high pollut~nt It is a promising prophylactic and thGl~peulic approach which utiliæs what was until now a continlling fin~nr;~l problem for the dairy industry which is responsible for the disposal of this major water polll~t~nt In conrlllQion~ it is the objective of this invention to plGSGl~/G intact the confo....~;on of the labile whey proteins in the W.P.C. This objective of leniency is obtained through several inter-dependent steps involving microfiltr~ti- n, lGlllpel~lule~ ions col tPnt, ultPfiltr~tinn flwc and drying technique.
216~37 REFERENCES
(which are inco~po,dled by reference in their e~ ~y) 1. Bounous G., Knn~hAvn P.A.C. "Tnfluent,~e of Dietary Proteins on the Tmm~ne System of Mice" J. Nutr. 112, 1747, 1982.
2. Bounous G., Gold P. "The biol~i~Al Activity of Un~lenAtured Dietary Whey Proteins: Role of ~3h~tAthione" Clin.Invest.Med. 14: 29~309, 1991.
CONCENTRATE
Related Applications This appli~tio,n is a co~ t;on-in-part of U.S. Serial No 08/315,904 filed September 30, 1994 and a co~ t;on-in-part of U.S. Serial No.
08/175,637, filed Dec~ll,ber 30, 1993, which are con~ ;ons-in-part of U.S.
Serial No. 07/989,186 filed Dece,.,ber 11, 1992, which is a col-t;..u~l;on-in-part of u.S. serial No. 07/929,347 filed August 13, 1992. The cont~llLs of said related applications are hereby incol~ldled by reference in their enl~ely.
Background of the Invention As early as 1982 Bounous et al (1) showed that dietary whey protein concPn~lP~ (W.P.C.) i.l.pr~ved the active systemic hllmoral imm~ne response in a .. ~.. ~1, as measured by sheep red blood cell injection~. It was however found that the use of high te~ dlur~ teuri7~tion of milk in 1988 and subsequent years following a Salmonello~i~ epidemic in Europe gready reduced the effectiveness of commercially available whey protein concP~ es in improving the immune response.
In said related applications the discov~l~ that unden~tllred whey protein concentrate had an - enh~nce~ immllnological effect was presP,nt~d. It was furthermore ~Ypl~ined that in the conventional high l~nl~ tule p~te,uri7~tion ofmiLt~ the thermosensitive proteins serum albumin and laclof~ were partially heat denatured and hence preci~ led in the curd. Said related applications describe eXperim~nt~ where W.P.C. was ~l~P~ ed using the lowest level of heat treatment of miLk compatible with safety standards, so as to obtain a whey protein distribution having a relatively high content of the thermolabile serum albumin, It was found that the presence in the serum albumin (B.S.A.) of 6 glutamylcyst(e)ine (Glu-Cys) group/molecule (substrate for glutathione (G.S.H.) synthesis) and the specific intramolecular disulfide bond related to the - - lmdPnqtllred co~fo~tion of the mol~clllP, were a key fætor in the G.S.H.
promoting .ctivity of W.P.C. The term cyst(e)ine implies the presence of a fiAe bond (cystine), . nd ~e ~s~ . reduction to ~ c. Fnhq~cpment of G.S.H. levels in tissues are believed to ~ the common d~lo...;n~or S underlying the benPfi<~iql effect.
Dietary W.P.C. produced with low level p ~ ;on im~ s ~;,t~mic humoral immllnP ~.s~nse, increases the re~i~tqn~ of target cells against the carcinogenic effect of chPmi~-q1 carr-inogpnc such s dh--elhylhy~ t,;n~ n~es re-~ict~n~e to pnellmococc~1 infection, and provides a mod~r~t~ but sllst~in~
increase of tissue g1tlt~thion~
The extent of de~l.-.~ n produced during whey c4ncpi~L~te production is norm~11y ~cs~-~sed by the loss of so1-1bi1ity at pH 4.6. Clearly the two most?olL~u~l factors of protein denAI~ on are ten~pe~dlule and pH whereas lower pH values usually enh~nc~ the ~ ;n~ effect of high temperature. This is why throughout our entire ploce-lule, we have .. ~ ~ a rather high Ph (6) thanks to a reduction of pH lowering b~ t~-n~1 c~nt~ ion through microfi1t~tion with 1.4 pores. With a dena~ul~lion len~ ldlulc; of 64C. at pH
6, serum albumin d~J~)ea[S to be the most easily de,~alult;d serum protein since its den~tl1ration is not as reversible as that of alpha-lactalbumin (4). Even a temp~ldlule as low as 55C. causes unfolding of the B.S.A. molecule over a period of time. A pH of at least about 6 is th~l~ro,e preferably ...~il;t~ined throughout ~e process.
The object of this invention is to provide an improved process for prepa~ g a whey protein concen~ldt~ that has adequate b~ctP,ri~1 reduction without protein ~n~ tion. It is a particular object to provide a process that will achieve a whey protein cQnc~ntl dte having a serum albumin of at least 9.5 %
and adequate b~ct~Pri~1 reduction. This appl.~ill,ately 9.5% level of serum albumin was found in our studies to be important for t,he achievement of sustained increase of tissue glutathione for the proy~lies such as improved systemic humoral immune response described above (References 2 and 3).
We propose to utilize techniques of microfiltration to achieve t,he objectives of this invention. Microfiltration using ceramic membranes is a m~mhrstne based procedure which allows the s~qpStr,ttinn of particles rztn~in~ in size belween 0.1 and 10 microns.
Micr )filtr~tinn with me...bli.r.Ps having a 1.4 micron porus si~ has been recel~tly utilized to l~.llove b~r~ and other particles from milk in order to obt~in a co~ e~ially "sterile~ milk (5). With this me~hoA a 2 to 3 Log reduction of bS~ctçtiS~l count in milk was achieved; hence a bstcteri~Asll effect greater than what is ol~tained by traditional milk ~u~ l;on (72-78C / 15 sec.: 98% bSlrtçri~l reduction) (6). The inAustrisll methods of sb~ on of the caseins are based on the destabilization of these proteins either by lowering the pH of milk to the ~ ~tric point (pH 4.6) at 20C or by e~ alic (rennet) hydrolysis of the Kappa casein which stabilizes the mirP11~, The first procedureis not suitable for the ,eco~ of native whey pç~eins because pA~te~ri7~til~n at low pH of the whey-conre~-t~te would entail a subst~ullial denSIt~lr~tion of theproteins.
Brief Description of Drawing Figure 1 is a srhemsltic ~ep,~ ~ti~lion of the process of this invention for producing a whey protein concPI~ te with immllnoenhslnrin~ ~,ro~ .ies.
Figure 2 illu~L~dl~s the gluPthione promoting activity of said whey protein concP-~L~i1lp- to which we have applied the trSl~ems~rk Tmmlln-x~l, in an in vitro assay as described below. In this assay, the activity of said product is cGlllpa~ed with a control milk protein (casein i.e. Ca c~Pin~te) and another commercial whey protein conrPnt~tp with low serum albumin content (Alacen 855, mentioned in Table 5).
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 (T ~emmli- buffer system) after the samples were reduced with 10% 2-mercaptoethanol. Samples were applied so that each slot received 10-20 microgrammes of protein. Electrophoresis was performed at 200 volts for 216~937 70 I.lh.~ s. The results were also co~ ....P~l by .,hr~.l"atog.a~hy.
E~stent of protein d~ ;on by the process was de ~ i nP~ in trjr~ tP, by the ~ g~ s ~luki1ity index ~NSI) at pH 4.6 and 3000 g. (AOAC 1985 ref.
6). The mPshod ntili7ed in these ~?e~;...~nt~ differs from ~at describe~d in S Reference 2 and ~ ls a more ~ccl~r~tP~ r~flection of the unden~h~red state of protein.
Protein content (N X 6.38) and total lipids of ~mp1~s were ~el~
in dupli~ vely by the standard method of Kjeldahl and the method of Mojonnier.
~ istl~e cont~nt was det~-rmined in dupli~te by the AOAC method C7)-Total coli~olllls count was det~ ined following incub~tion at 37C for 18 hours in brilli~nt green using the most probable number method. Total b~cteri~
count (aerobic mesophiles) was ~let~.rmin~ follorving incub~tion at 32C for 48 hours in PCA m~ium. Both methods are a~pn,ved by the Tnt~rn~tic)nal Dairy Fede~tion and ~m~oric~n Public Health ~ tion Lactose was measured by the enzymatic method.
Glut~thione de~ .n~;Qn Tn~rellular gl-.~thione was measured by erkylllalic assay using ni~in~mi-le-diphosphate, dithio-n,~bellzene and gluhtl~ionto. reductase. Briefly, MT-4 human lymphocyte T cells were halv~sled after 72 hours of incubation at 37 C under 5 % CO2 using standard methods of tissue culture in the presence of varying concentration of WPC (0.01 to 1 mg/m1). The cells were then lysed with ice-cold water and 6% sulfosalicylic acid, centrifuged at 600g for 10 min, and sUpern~t~nt~ collected and proc~s~ . S~t~ophotometric analysis was pelrolllled for 2 min at 412 nanometres (nm) and results were expressed in nmoleof GSH /107 cells.
Dr. S. Baruchel is Associate Professor of Pediatric and Oncology at McGill University in Montreal, C~n~1a. He has developed in his laboratory at -Montreal Children Hospital Research Tnstitute this in vitro assay (8,9) which is utilized in collaboration with us in evaluating the GSH promoting (potency) activity of each WPC production batch.
216~937 The Invention In accol~lce with an aspect of this invention, a pr~)CeSS iS provided for ploducillg an l)n~en~tured whey protein con~P~ tP, having a serum albumin content of about 9.5% or more as a byproduct of a process for m~king cheese S comrri~ing As ill~ ~ in Figure 1 in step 1, cold stand~ ;on of the fat contPnt in milk is err~ at a ~.~ not greater than about 4C. By this approach we reduce the Oulgl'OWl}l of b~teri~. Fur~Prmore this method reduces the breakdown of fat globules which would lead to further reactions involving enzymes and b~rP-fi~l metabolism, This involves s1~immin~ the milk to a desired fat content. The usual practice has been to use a tel"~ ur~ in the range of 50C to 65C as this is themost effi~ient range for slrimming to a level of 0.05% of fat. However, we prefer in accof~ance with this invention to ...~ . the te~ e at a lower level not greater than about 4C. This low ~ during the fat removal process is nP~s~.~ to avoid n~ tion of lipids which would then tend to continue over time during storage and produce r~n~ idity ~iberation of fatty acids) which could in turn damage the conform~tion without ~ffecting nutritional P.ffi~iPncy of the labile proteins.
In the eY~mples described in this applic~ti~ln standar 1i7~tion of milk was carried out with a cold sep~ Qr (Alfa Laval, CMRPX 714-HGV) coupled with an automatic standardizer (Alfa Laval, Alfast Model 110). All standardization steps were carried out at 4C. (Figure 1).
In next stPp shown in Figure 1, the skim raw milk obtained after step 1, is microfiltred to provide a first pP.rmP~tP- having a bacterial count at least as good as the standard applicable to p~te~lri7~1 milc.
The microfiltration of the skim milk obtained in step 1 was carried out on a microfiltration system with controlled t~n~mPmhrane pressure as set up by Alfa-Laval filtration system De-nm~rk (MFS-7 system with 1.4 sq m membrane surface). Ceramic membranes were obtained from Membralox (pore size 1.4 micron).
Table 1 below shows the operating conditions during microfiltration of `' - 2165g~7 - skim raw bovine milk using a oe~miC me~ dne, as previously df~lib~d having a pore size of 1.4 microns.
S T~ c 500 Tr~ncmP.. h.i~nc P~ ur~ IP) 0,5 bar Tal~genlialvelocity >6.6m/s ~oncent~~ n Factor (CF) 20 Flux 950 Llh/m2 Raw milk should be used that has been ~l~immf~d at a ~ .e not in excess of about 4C.
The le~ J~e used during microfiltr~tion of the raw milk should be in the range of about 40C to 45C. As previously noted ~i~ifi~-~nt ~lçn~ .tion of serum albumin does not begin to occur until about 55C.
Table 2 shows the m-icrobial counts of the milk following microfil~r~tion.
We have noted a 2-3 log red~cti< n in b~t`t~-ri~l counts in aco~f~lce with the l; If.. ~ . C (5). ~is c"n r....,~ the efficacy of the method. As ~ec~d, the lipids are in the l~P;n~;1le The protein content of the Ferme~ is only ~I~argillally dirr~r~lt from that of the re~ P and there is no di~rerellce between permeate and rt;le~ le in the type of proteins as measured by electrophoresis. Hence no selective retention of proteins is noted over a 1.4 micron membrane.
` 21~937 Microbial counts (per/g) expressed in log E;~peri- Raw M;lk R.~P~ IP. Perm~t~ Reduction ment of total number Total Coli- T C T C b~ tPri~l ~I~ forms counts (C) 4.40 -- 6.78 -- 2.37 -- 2.03 2 6.054.25 6.644.23 3.070.48 2.98 - ~ 3 3.931.80 4.661.29 1.76 0 2.17 4 5.723.08 6.333.18 3.35 0.3 2.37 5.493.67 6.32 -- 2.53 -- 2.96 6 5.033.08 7.20 -- 2.89 -- 2.14 7 6.034.60 7.51 -- 4.032.41 2.00 8 6.905.32 8.457.51 4.272.48 2.63 9 6.524.00 7.805.78 3.761.80 2.76 ~eduction in log from raw milk to permeate.
The microbial counts of the perm~te compare favourably wi~ standards applicable to conventional p~teun7~tion.
20The next step, step 3 according to Figure 1, is p~teun7~tion of the microfiltered milk. The leml}e,~lu,c; used in this invention is above 72C up to about ~0 seconds.
The following step 4 involves cooling of the microfiltered miLlc. In accordance with the examples of this invention the temperature is accordingly 25immediately reduced, such as by flash cooling to a temperature of about 30C
~165 937 - prior to cheese m~king At this prç~imin~ry stage of cheese m~kin~, c~lrinm ch1- ri~e is usually added to milk to ~ a~ the ~ nP-~S of the curd during the m~mlf~tllre of cheese. The net result is an increase in cheese yield due to better p c~ip;~lionand less losses of cheese curd particles in the whey.
Based on the report of .Shim~d~ & ]U~ h;~ (1981) (10) dP~ n of BSA is enh~nr~d by c~lrillm ions. Thcl~fo~ we avoid the addition of c~1cil-m ions COl ~ to normal pr~ctice~ in cheese m~mlf~cture.
Additives of this sort are avoided during produc-ti~ n of cheese if the whey gPnPr~tPd is being used for this invention. Yield can also be increased in conventional pr~ctice by the addition of miL deliv~livt;s (casein, whey protein)to milk being transformed into cheese. ThiS procedure is avoided because it influPnres the quality of the protein contPnt of the whey. The various f~ti~
found in normal whey will be mo~ifi~ reslllting in a whey protein cOI rentr~tP~
having less un-lçn~t lred bovine seNm albumin per unit of total protein. The pH
is to be ...~i~.LqinP~l at not less than 6 at this chilling stage and until col ~entr~tion of the whey.
~Ppining follows in step S and this involves the ~litit)n of cultures to milk about 24 hours before starting cheese m~king. In the examples the only additives are the usual lactic cultures.
The next step in m~nllf~ctllring cheese is curd making, illustrated in step 6, Figure 1, which includes the following stages namely:
(a) Rpnnp-tting (b) Coagulum cutting (c) Stirring (d) ~P~ting (cooking) (e) Post stirring Following post stirring the curd is prep,essed and separated from the whey.
Conventional additives are used for rennPttin~. The making and subsequent handling of the cheese are generally represented in Figure 1 at step 7. This step will depend on the type of cheese. They therefore will not further be described.
" ~165~7 g What is i~ ?o~ l however for the pu~poses of this invention is to avoid any additives or lr~l~ that will de1PtP~iously affect the whey. Thus the ~du~;on of P.mmPnt~l cheese l~uir~s a ~-~ G of more than 50C.
Raising the curd to this lGlll~GldlurG before ~...tiol- of the whey and .--~ ingsuch lGlll~)GldlUrt~S wouldaffecttheserumalbumincontPnt Itis highly desirable to avoid any cheese making steps that involve a te~ lJI G in e~cess of 40C .
No ~lition~l whey is collP~hd for the ~Ul~oS~s of this invention during .~haril-g and final pressing of the curd as the pH by then of any whey ~cc with the curd would be below pH 6.
The whey obtained in step 6 should be chilled to about 4C. as soon as it is sep~ e~ from the curd, and this is illllstr~tP~ on Figure 1 as step 8. By this procedure, the metabolism of the lactic acid b~^tP-ti~ is reduced and acidity does not increase. No additives (such as H2O2) should be used as ~ntih~t~ti~l agents.Instead we use low ~---~ e inhih-~ition of b~ tPri~l metabolism. The pH of the whey should never he below pH 6 before it is co~
The whey that has been collPct~d is first ce~ iruged to take out excess fat (Alfa-Laval M\H~PX-214TGV) that was present during cheese production, chilled to 4C. and stored till llltr~filtr~tion (U.F.) is carried out.
During next step 9, the l~lnp~lalure is then raised at 40C. for the ultr~filtr~tion using, for eY~mple, Romicon cartridge (2.3 sq.m.) with a cut offof 50,000 dalton. During U.F. the l~lP~ tP is s~n~ d to ~ filtr~tion by adding distilled water so as to reduce the lactose level in the dry matter to less than 10% of total solids. 1 volume of lel~ to 1 volume of water is used and this procedure is p~lrolllled two or three times. The relPnl~te following completion of U.F. has a total solids of 19-20%. The conditions of ~llt~filtr~tion are set forth below in Table 3.
" ~165937 .
-TEMPERATURE approx. 40C
TRANSMFMR~RANE 1.0 BAR
PRESSURE ~I~IP) TANGENTIAL VELOCITY TANGENTIAL VELOCITY
>7mls FACTOR (CF) FLUX 24.5 1/m2/h The membrane flux increases 2 to 2.5 % per degree centigr~le, giving a similar increase in capacity of a m~m1f~rtl1nng plant. This means that withoutspecial reasons for opPr~til)n~ at low te~ ~, it is an advantage in con~e~-l;on~1 practice to operate at as high a t l"~ dlure as possible.
The le",~eldluf~ utilized in most other commercial methods during this procedure is 50C. This level of l~ f~-~ilit~tP~ a higher flux through the membranes hence more relent;11P~ pr~duction per unit of time and per unit of membrane. In our mPthod, the draw-back of less production per unit of membrane is co~.~n.~te~ by increasing the membrane surface.
The objective of not exc~ing 40C. is obt~uned throughout the system by fine tuning the points of input and output in the system so as to avoid a heat pro~ucing unbalance between the two.
Next, the ~e",~e,dlure is lowered to 4C. in step l0 and kept at that ~ ?erdlule till freeze drying or spray drying is started. The microbial counts of the retPntate compare favourably with standards applicable to conventional p~ ion.
These standards differ in each jurisdiction. As an example, the Province of Quebec, ~n~ , requires that total bacteria count (aerobic mesophiles (32C) be m~in~ined 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 l0.
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 ~165g37 -- p~t~-JI~ ;,~1 or fPrm~Pn~
ConrPntr~tion to produce a dry product by lyophili7~tion (free~e drying) is p~rul,l,ed in step 11 at l --..~ '--.es under 0C for 15 to 18 hours. This does not denature a ~i~nifir~nt p~ lion of the thPrm~ hile ~l~leins.
S Table 4 ill.. `il.~f~ the co",~osilion of whey protein concpllt~ e powder obtained using the prinripl~s described above.
Certain factors cannot be controlled during normal production of whey concPnl.~, powders. Se~con~l v~ri~tinn of milk co"-p(j;,iLion and b~ctP-ri~l metabolism that occurs in milk at each step of the cheese making process will bemainly respolsible for the dirrel~nces obs~.ved in the composition of the cQ~ l.dLo. However, the prdrfire of the prinriplP~s of this invention may be eY~cte~ to produce a ~n~i~t~ntly high level of th~rmol~hilP proteins such as serum albumin and to avoid ~ulJst~ lial loss of the glul~u"ylcysteine group conl~inillg protein, together with bactP-n~l counts subs~.lL;~lly lower than that obtained with the method disclosed in the PCT/CA93/00518 (Bounous et al.) and in the United States Applir~ti~ n SN. 08/175,637 (Lange et al.), both ~Csign~ toTmmlmotec Research Col~ldLion Ltd., which is the ~ignPe of the present applir~tit-n Clearly the co.~.hin~ n of microfiltr~tion as in the first part of the method disclosed in PCT/CA93/00518 (Bounous et al.) and p~tPuri7~tion low temperature lenient procedures as disclosed in United States Application SN.
08/175,637 (Lange et al.) greatly reduced the b~r-tP-ri~l content of the end ., product. With this method of production, it is possible to obtain desired b~rtp-ri~l results with less stringent conditions than are disclosed in these prior applications. This is i~ >ol~nt for large scale productions on infl~lstri~l scale.
If fur~er b~rtpri~l redllction is desired a second p~tpllri7~tion or microfiltration procedure can be applied to the whey between steps 8 and 9, and namely prior to ultrafiltration. Further evidence of the leniency of this methodof production is the relatively high level of la~;lof~llin ~able 4) in colllp~ison to commercial WPC. This i"l~oll~lt protein is similar to serum albumin in that it contains 4 Glu-Cys peptides (Reference 11) and it is ~imil~rly thermolabile.
The analysis of laclorellin as reported in Table 4 were ~ roillled by Drs. G.
Regester and G. Smithers at the CSIRO Dairy ~ ch Laboratory in Victoria, str~ , and will likely be publi~h~d soon in the Journal of Nutrition.
COMPOSlTION OF WHEY PROTEIN CONCENTRATE POWDER
S EXAMPLE EXAMPLE EXAMPLE
PROTEIN(%) 78.04 77.00 77.08 ~-Lactalbumin(%) 22.00 æ.80 22.20 B-Lactoglobulin(%) 57.80 56.30 54.45 Serum Albumin (%) 11.7 11.06 10.85 Lactoferrin (%) 0.67 - - 0.70 0.69 Tmmllnoglobulin (%) 8.39 9.20 11.10 FAT (%) 3. 4. 3.5 LACTOSE (%) 9.40 9.20 9.4 MOISTURE (%) maximum 4. 4. 4.
Ash 3 3 3 MlCROBIOLOGY
Salmonella (/lOOg) No growth No growth No growth Coli (/lOOg) <5 <5 <5 Staphylococcus (/g) < 5 < 5 < 5 Total count (/g) < 1000 < 1000 < 500 NITROGEN SOLUBILlTY 99% 98.7% 99%
INDEX
The protein composition and solubility of the final product in powder form after concentration by Ill~filtr~tion and lyophili7~tion CIable 4) me,ets the requirements previously i-lentifi~d herein as es.sPnti~l for the development of immunoenh~n~.ing activity and tissue GSH promotion: serum albumin concentration around 9.5 % or more and minim~l degre,e of den~ tion. In Table S and Table 6 are ~resen~d for co~ a,ison the concentrations of serum albumin - in current ccilll"~ ially available W.P.C.'s and the nill~gel~ sol~bility inde~
d~ ned by De Wit in some W.P.C. products, as shown in Table 6. Table S is derived from Bounous et al. (2) W~Y PROTEIN CONCENTRATE BOVINE SERUM ALBUMIN
in % of Total Whey Protein Promod ~I~ 4+ 1 Alacen 855 4+ 1 Lacprodan-80 4.8+2 Sapro 4+0.1 Savorpro-75 4+1 Bioisolate 5 + 1 Promix 4.3 + 1 WHEY PROTEIN CONCENIRATE ¦ NSI AT Ph 4.6 Normal UF WPC 83 %
Neutral UF-DF-WPC 78%
Acid UF-DF-WPC 42%
De-fatted UF WPC 91%
Sphèrosil 'OMA' WPC ~I~I) 79%
Sphèrosil 'S' WPC ~) 35%
Vistec WPC ~I~I) 35%
Demin. delact. WPC 72%
From De Wit J.N. et al. Neth. Milk Dairy J. 37 (1983) pp. 37-49 Table 7 r~,esellts analysis results of an un~en~ red whey protein ~16~937 -- con~n~ d generally in accol~lce with the present invention made by ~e Japan Gove~ s.l Food analysis centre.
. - - 15--lTEMS RESULTS MEI EIOD
Water 4.4% Air Oven Method Protein 83.1% KiP~ hl Method (N x 6.38) Lipid 6.2% Roese~ot~ieb Method l~oi~ re 2.2% ~r~ition at 550 Carbohydrate 4.1% lO~(water + protein + lipid +
moisture) Energy 423kacVlOOg protein x 4.22 + lipid x 9.16 +
carbohydrate x 3.87 As Absent ( < O. lppm) Silver Diethyl~lithioc~rbamate Specl,ophotometric Method Pb Absent (<0.05ppm) Atomic Absorption Spectrophotometric Method Cd Absent (<O.Olppm) Atomic Absorption Specllopllotometric Method Total Hg Absent (<O.Olppm) Cold Vapor Atomic Absorption Spectrophotometric Method Sn Absent ( < lppm) Polalog~l)hy Method Cu 1.52ppm Atomic Absorption S~illopllotometric Method 3s Total Aerobic 8.7x103/g Standard Agar Plating Method ~ teri7~
Coliforms Absent/2.22g BGLB Broth Inoculating Method Surface Spread Plating Method S. Aureus Absent/O.Olg Fnri~hmPnt Culture Method Salmonella Absent/25g Fn~i-hment Culture Method Pseudomonas Absent/O. lg ~165~37 The J~plnPse Go~e- .. Pnt Food analysis centre also c4n~uct~ a co...~ Qn of the serum albumin content of the lm-lçnslu.ed whey protein cQnc~ e ~ner~lly made in acco~lce with this invention with three best commercial whey protein C~ PJ~ available on the m~rl~P.t The serum S albumin c4nPnt for the product of this invention was found to be 11.7 as c~...l~ed with 7.7, 7.7 and 5.5 ~s~;livGl~ for the co~,v~.l;~n~l products.
The ~xess of this invention therefore provides a pp~tir~l p~1ulè for m~king lln~P~ .~d whey protein con~ e. Furthermore, it has the advantage of using a by product of cheese pr~uction which is a high pollut~nt It is a promising prophylactic and thGl~peulic approach which utiliæs what was until now a continlling fin~nr;~l problem for the dairy industry which is responsible for the disposal of this major water polll~t~nt In conrlllQion~ it is the objective of this invention to plGSGl~/G intact the confo....~;on of the labile whey proteins in the W.P.C. This objective of leniency is obtained through several inter-dependent steps involving microfiltr~ti- n, lGlllpel~lule~ ions col tPnt, ultPfiltr~tinn flwc and drying technique.
216~37 REFERENCES
(which are inco~po,dled by reference in their e~ ~y) 1. Bounous G., Knn~hAvn P.A.C. "Tnfluent,~e of Dietary Proteins on the Tmm~ne System of Mice" J. Nutr. 112, 1747, 1982.
2. Bounous G., Gold P. "The biol~i~Al Activity of Un~lenAtured Dietary Whey Proteins: Role of ~3h~tAthione" Clin.Invest.Med. 14: 29~309, 1991.
3. Bounous G., Batist. G; Gold P.: "TmmlmoçnhAncing Plop~lly of Dietary Whey Protein in Mice" Role of QllltAthione: Clin. ~vest. Med. 12: 154-61, 1989.
4. Brown R.T "Milk Coagulation and Protein Dç~ ...A~ion in 'fund~,mlontAl~
of Dairy Ch~-mi~t y"', 3rd Edition N.P, Wong (Ed) Van Nostr~n51 Reynold C. (Publ.) New York, 1988 pp. 583-607.
of Dairy Ch~-mi~t y"', 3rd Edition N.P, Wong (Ed) Van Nostr~n51 Reynold C. (Publ.) New York, 1988 pp. 583-607.
5. Fauquant, J.; Maubois, J.L.; Pierre, A. "Microfiltration du lait sur Membrane Miner-Ale" Tech .T Ait 1028, 21-23, 1988.
6. AOAC 1980, "Official Methods of Analysis" 13 FAiti(m Association of Official Analytical ChPmi~t~, Washington, D.C.
7. AOAC 1985, "Official and Tentative Methods of the ~m~ n Oil Chemi~t Society, Official Methodsn, R~ 65, Revised Edition.
8. Baruchel S., Oliver R., Wainberg M., "Anti-HIV and Antiapoptotic Activity Of The Whey Protein Concentrate: Immunocal TM. n~ 1994 9. Baruchel, S. "In vitro Modulation of MATO Breast Cancer Cells With Whey Protein Con-Pntrate ammunocal). (in preparation) 10. Shim~ K, M~t~ hit~ S. J. Agric. Food Chem. 1981, 29, 15-20.
11. Gootman et al, "Bovine Laclof~ mRNA: Sequence*, Analysis, and Expression in the M~mm~ry Gland", Biochemir~l and Biophysical Research Communit~tions, Vol 180, No. 1, 1991, pp. 75-84
Claims (16)
1. A process for producing whey protein concentrate having a serum albumin of about 9.5% or more as a by-product of a process for making cheese, comprising the steps:
(a) cold standardization of the fat content in milk, for producing skim milk, at a temperature not greater than about 4°C;
(b) spore and bacteria reduction by microfiltration of the skim milk through a 1.2 to 1.7 micron pore membrane;
(c) optional pasteurization prior to cheese making at a high temperature for a time short enough to avoid denaturing of the protein in the skim milk;
(d) chilling the skim milk preliminary to cheese production while maintaining the pH at 6 to 6.5 and without the addition of calcium ions;
(e) making curd and whey at a temperature in the range of about 30 to 45°C;
(f) separating the curd from the whey;
(g) removal of excess fat from the whey;
(h) microfiltration with a membrane having a pore size in the range 1 to 1.3 microns, or pasteurization, if needed, at a high temperature for a short time enough to avoid denaturing of the protein in the milk, to remove further bacteria from the milk and produce a commercially sterile permeate;
(i) removal of further water and small water-soluble molecules by ultrafiltration of the resulting whey at a temperature in the range of 35 to 42°C
to provide a retentate;
j) concentrating and drying said retentate under conditions of temperature and time that will not denature at least 95 % of the protein.
(a) cold standardization of the fat content in milk, for producing skim milk, at a temperature not greater than about 4°C;
(b) spore and bacteria reduction by microfiltration of the skim milk through a 1.2 to 1.7 micron pore membrane;
(c) optional pasteurization prior to cheese making at a high temperature for a time short enough to avoid denaturing of the protein in the skim milk;
(d) chilling the skim milk preliminary to cheese production while maintaining the pH at 6 to 6.5 and without the addition of calcium ions;
(e) making curd and whey at a temperature in the range of about 30 to 45°C;
(f) separating the curd from the whey;
(g) removal of excess fat from the whey;
(h) microfiltration with a membrane having a pore size in the range 1 to 1.3 microns, or pasteurization, if needed, at a high temperature for a short time enough to avoid denaturing of the protein in the milk, to remove further bacteria from the milk and produce a commercially sterile permeate;
(i) removal of further water and small water-soluble molecules by ultrafiltration of the resulting whey at a temperature in the range of 35 to 42°C
to provide a retentate;
j) concentrating and drying said retentate under conditions of temperature and time that will not denature at least 95 % of the protein.
2. A process as in claim 1 in which pasteurization is at a temperature in the range of 70 to 75°C for a predetermined time.
3. A process as in claim 2, wherein said predetermined time is between 10 and 20 seconds.
4. A process as in claim 1 in which the step of chilling the skim milk preliminary to cheese production comprises flash cooling to a temperature of approximately 30°C.
5. A process as in claim 1, in which ultrafiltration is accompanied by diafiltration to reduce the lactose level in the final dry product to less than 10%
of total solids.
of total solids.
6. A process as in claim 1, in which the retentate in step (j) is concentrated.
by lyophilization (freeze drying).
by lyophilization (freeze drying).
7. A process as in claim, 1 in which the whey for further processing is separated in a prepressing operation, prior to shaping and final pressing of thecurd.
8. A process as in claim 1, in which the retentate in step (j) is concentrated by spray drying.
9. A process as in claim 1, wherein the conditions for drying step include freeze drying at a temperature below 0° C
10. A process as in claim 9, wherein the time of freeze-drying is about 15 to 18 hours.
11. A process as in claim 9, wherein the resulting product has a moisture content of about 3 to 5 %.
12. A process as in claim 9, wherein the resulting product has a moisture content of about 4%.
13. A process as in claim 1, wherein the removal of excess fat from the whey in step (g) is carried out by centrifugation.
14. A process as in claim 1, wherein the removal of excess fat from whey in step (g) comprises cooling the whey at less than 4°C, maintaining the pH over 6 and centrifugation of whey, while no additives are supplied during this step.
15. A process as claimed in claim 1, wherein said membrane is a ceramic membrane.
16. A process for producing a substantially undenatured whey protein concentrate having a biologically effective amount of proteins containing glutamylcyst(e)ine peptides comprising cold standardization of milk, microfiltration, pasteurization, the formation of curd and whey, separation of the whey and filtering and drying the whey under conditions of time and temperature that will avoid substantial loss of said glutamylcysteine peptide containing protein.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US43785295A | 1995-05-09 | 1995-05-09 | |
US08/437,852 | 1995-05-09 |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2165937A1 true CA2165937A1 (en) | 1996-11-10 |
Family
ID=23738190
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA 2165937 Abandoned CA2165937A1 (en) | 1995-05-09 | 1995-12-21 | Process for producing an undenatured whey protein concentrate |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP0825816A1 (en) |
AU (1) | AU5641396A (en) |
BR (1) | BR9608112A (en) |
CA (1) | CA2165937A1 (en) |
WO (1) | WO1996035336A1 (en) |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
LT5362B (en) | 2005-11-02 | 2006-10-25 | Vytautas Fedaravicius | Method and device for producing casein |
US9055752B2 (en) | 2008-11-06 | 2015-06-16 | Intercontinental Great Brands Llc | Shelf-stable concentrated dairy liquids and methods of forming thereof |
UA112972C2 (en) | 2010-09-08 | 2016-11-25 | Інтерконтінентал Грейт Брендс ЛЛС | LIQUID DAIRY CONCENTRATE WITH A HIGH CONTENT OF DRY SUBSTANCES |
CA2808934C (en) | 2012-04-10 | 2019-01-15 | Kraft Foods R&D, Inc. | Process for producing cream cheese |
EP3298903B1 (en) | 2016-09-27 | 2020-11-18 | DMK Deutsches Milchkontor GmbH | Low-germ milk powders with high whey protein index (iv) |
CN112931677B (en) * | 2021-03-09 | 2024-02-06 | 江南大学 | High-activity whey protein and preparation method thereof |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CH576235A5 (en) * | 1973-08-20 | 1976-06-15 | Mueller Hans Maennedorf | Production of sterile milk protein - by multi-stage filtration |
CH627079A5 (en) * | 1977-04-15 | 1981-12-31 | Nestle Sa | Process for preparing a protein concentrate containing immunological factors of milk origin |
US5456924A (en) * | 1988-12-23 | 1995-10-10 | Immunotec Research Corporation Ltd. | Method of treatment of HIV-seropositive individuals with dietary whey proteins |
CA2151506A1 (en) * | 1992-12-11 | 1994-06-23 | Gustavo Bounous | Process for producing an undenatured whey protein concentrate |
ZA949789B (en) * | 1993-12-30 | 1995-10-25 | Immunotec Res Corp Ltd | Process for making undenatured whey protein concentrate |
-
1995
- 1995-12-21 CA CA 2165937 patent/CA2165937A1/en not_active Abandoned
-
1996
- 1996-05-08 AU AU56413/96A patent/AU5641396A/en not_active Abandoned
- 1996-05-08 WO PCT/CA1996/000293 patent/WO1996035336A1/en not_active Application Discontinuation
- 1996-05-08 EP EP96913399A patent/EP0825816A1/en not_active Withdrawn
- 1996-05-08 BR BR9608112A patent/BR9608112A/en not_active Application Discontinuation
Also Published As
Publication number | Publication date |
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WO1996035336A1 (en) | 1996-11-14 |
BR9608112A (en) | 1999-07-20 |
AU5641396A (en) | 1996-11-29 |
EP0825816A1 (en) | 1998-03-04 |
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