AU677230B2 - A process for producing beta-casein enriched products - Google Patents

Description

OPI DATE 12/04/94 APPLN. ID 49873/93 1111 iIlll111I!I1111111111!li! AOJP DATE 07/07/94 PCT NUMBER PCT/NZ93/00086 1111111I111111 11111 IIIIIIIIII11111111 l1ii1i111111111111 AU9349873 (21) International Application Number: PCT'NZ93/00086 (74) Agen~ts: BEN NETT, Michael, R. et al.. A. Park Son, 6th Floor, Huddart Parker Building. Post office Square.

(22) International Filing Date: 22 September 1993 (22.09.93) P.O. Box 949, Wellington 6015 (NZ).

Priorih. data: (81) Designated States: AT. AU, BB, BG, BR, BY, CA, CH.

244445 22 September 1992 (22.09.92) NZ CZ, DE, DK, ES, Fl, GB, HU, JP, KP, KR, KZ, LK, LU, LV, MG, MN, MIW, NL, NO, NZ, PL, PT, RO, RU, SD, SE, SK, UA, US, UZ, VN, European patent (71 Aplicnt~fo al dzaad Sa~s~e~ UJ. EW EA(AT, BE, CH, DE, DK. ES, FR, GB, GR, IE, IT, LU, L"ANI) DAIRY1- RESEARGH &{hEtftJr MC, NL, PT, SE), OAPI patent (BF, BJ, CF, CO, CI, -~ai F~n R~dF~th~re rP~lh~,u N 1 d~JOI CM, GA, GN, NIL, MR, NE, SN, TD, TO).

(72) Inventors; and Published Inventors/Applicants (for US only) RAM, Satyendra [NZ/ W~ith international search repor, NZ]; LOH, Dan, Wee [NZ/SG]; LOVE, Donald, Craig I,; [NZ/NZ]; ELSTON, Peter, Dudley [NZ/NZ]; New Zea- 67 land Dairy Research Institute, Dairy Farm Road, Fitz-6 7 herbert, Palmerston North 5301 (NZ).

7;<e 7-e e 0 (54)Title: A PROCESS FOR PRODUCING BETA-CASEIN ENRICHED PRODUCTS (57) Abstract The invention relates to a process of extracting a 0-casein enriched product and a P-casein depleted product from a casein solids feedstock. The casein feedstock is slurried and cooled to a temperature range of -10 'C to 14 *C until the desired amount or P-casein has been dissolved and it is then separated from solid P-casein depleted product. For rennet casein feedstock the pHor the slurry is maintained at pH 5.0 8.0 while ror other casein feedstock a pH of 3.5 to 8.0 may be used. The 0-casein enriched product has a number of non-rood and food uses including as an additive to infant formulations. The (-casein depleted product can be used for most of the same purposes as the casein feedstock.

WO 94/06306 PCr/NZ93/00086 2 Isolation of individual casein proteins (that is, the casein fractions) from mammalian milk is a well known art. A number of laboratory based methods have been published in which one or several of five different technologies have been used: differential solubility, liquid/liquid extraction, electrophoresis, membrane techniques, and chromatographic separations. Details of such technologies may be found in reference 7.

Japanese patent specification 54-095768 describes a process for the indirect preparation of an enriched 6-casein fraction by removal of most of the a, and K-casein fractions. The process uses a caseinate solution as the starting material. The process uses a differential precipitation procedure.

Japanese patent specifications 59-091848 and 59-091849 address the total fractionation of whole casein. In the first specification, the preparation of a fraction containing a and 6-casein is described. The isolation of the 6-casein from the a,/6-casein fraction is the subject of the second patent. The isolation of the 6-casein involves calcium precipitation of the cta-casein and separation by centrifugation.

Japanese patent specifications 47 034936 and 55 16616 address the manufacture of human milk substitutes by altering the composition and/or character of the caseins in bovine milk. Specification 47 034936 discloses three methods for the isolation of 6-casein enriched fractions. Each method is dependent on the removal of ao-caseins. Specification 16616 discloses a process for isolating 6-casein from desalted skim milk or a sodium caseinate solution. The process utilises a calcium precipitation procedure.

French Patent Specification 2,592,769 describes the production of 6-casein enriched fraction and a 6-casein depleted fraction by either the treatment of milk with a calcium complexing agent or the treatment of a caseinate solution with an agent which polymerises all the casein, adjusting the temperature to 0 to 7 0 C and subjecting the product to microfiltration using an inorganic membrane and tangential flow. The microfiltrate material is enriched in 6-casein while the retentate material is depleted.

Microfiltrate flow rates at such cold temperatures are very low making it difficult to justify such a process at an industrial level.

WO 92/00017 describes a process for extracting 6-casein from a rennet casein suspension or solution. The process involves acidifying the suspension or solution to a pH of about 4 to 5 and cooling the solution to -2 to 10°C. Two phases are formed; a liquid phase containing 6-casein and a solid phase which is depleted of substantially all of its 6-casein.

This solid phase no longer resembles the original rennet casein. The two phases are

I

WO 94/06306 PCT/NZ93/00086 4 c) separating said slurry into two phases to obtain a solid phase which is 6-casein depleted and a liquid phase which is 6-casein enriched.

Preferably said casein feedstock is a wet casein curd or a milk protein powder containing casein.

More preferably said casein feedstock is mineral acid or lactic acid casein, milk protein isolate (TMP

T

M base), rennet casein, milk protein co-precipitate, casein co-precipitate, whole casein precipitated by calcium or other casein curd which contains 6-casein.

Preferably said casein feedstock is a curd or powder of rennet casein.

Preferably said casein is fresh wet curd having a moisture content of between 45 and w/w.

Preferably said fresh wet curd is subjected to wet milling to reduce the curd particle sizes to 1 mm diameter or less.

Alternatively said casein feedstock is a curd or powder of acid casein.

Preferably the concentration of casein in said slurry is 1-25% w/w.

More preferably the concentration of casein in said slurry is 6-15% w/w.

Preferably in step said slurry is held at a pH of between 6.6 and 7.2 when rennet casein is used.

Preferably said slurry is held for between 0.1 and 30 hours at -10°C to 14°C.

More preferably said slurry is held for between 4 and 30 hours at 1 to 8 0

C.

More preferably said slurry is held for between 10 and 16 hours.

More preferably said slurry is held between 2 and 6°C.

Preferably said feedstock is casein curd and said process includes the preliminary step of extracting said casein curd from milk.

6 Preferably an aqueous solution of sodium chloride is added in a concentration of up to 4.0% w/w is added to said slurry of step a), Preferably step (iii) comprises isolating said p-casein curd by decanter separation, filter press, membrane filtration, clarifiers, screens or the like.

In one alternative said step of isolating p-casein from said liquid phase from step c) above comprises subjecting said liquid phase to a concentrating step followed by spray drying.

Preferably said concentrating step comprises evaporation, ion exchange or membrane filtration of said liquid phase with pH adjustment when required.

Preferably said process includes the further step of drying said p-casein depleted solid phase.

In a further aspect the present invention may broadly be said to consist in a p-casein enriched product when produced by the above identified process.

Preferably said p-casein enriched product contains 40 to 95% p-casein.

In a further aspect the present invention may broadly be said to consist in a p-casein depleted casein product when produced by the above identified process.

In a still further aspect the present invention may broadly be said to consist in a food, pharmaceutical, plastics or other non-food product including said p-casein enriched product and/or said p-casein depleted product.

S 20 Preferably said product is an infant food product.

The invention consists in the foregoing and also envisages constructions of which the following gives examples.

Bi a t I 4: B 3 I [N:\LIBFF]00391:mcn WO 94/06306 PCr/1VZ93/00086 8 Figure 9 represents the urea-PAGE analysis of the products from example VII.

The lanes are: 1 and 2 casein coprecipitate used as the starting material for the extraction; 3 6-casein enriched product from the water extraction; 4 water extracted casein coprecipitate; 5 6-casein enriched product obtained from the saline extraction; 6 salt extracted casein coprecipitate; 7, 8, and 9 were the as-, 6- and K-casein standards.

Figure 10 represents the urea-PAGE analysis of the products from example VIII.

The lanes are 1 and 2 standard New Zealand sulphuric acid casein (Alacid 750) used for the extraction; 3, 4, 5, 6, 7 and 8 are the extracts recovered from the 0.25%. and 4.0% w/v salt extractions (respectively); 9 6-casein standard.

Figure 11 represents the urea-PAGE analysis of products from example IX. The lanes are: 1 a mixture of a s- and 6-casein standards; 2 and 3 pre-extraction acid casein; 4 and 5 extracted acid casein; 6 and 7 6-casein enriched product.

Figure 12 represents the urea-PAGE analysis of the products from example X. The lanes are: 1 and 2 water extracted New Zealand rennet casein Alaren T M 771; 3 6-casein enriched product from the water extraction of the same rennet casein; 4 the rennet casein sample used for the experiment; 5 6-casein enriched product obtained from the saline extraction; 6 salt extracted rennet casein sample; 7, 8, and 9 were the 6- and K-casein standards.

MODES OF CARRYING OUT THE INVENTION Rennet casein curd may be prepared from mammalian milk, preferably cows milk, using known methods (such as those .described by Weal and Southward, 1974; L L Muller, 1971; Southward and Walker, 1980; Southward and Walker, 1982; Southward, 1985; Mulvihill, 1989). A preferred method is that described in Weal and Southward, 1974 and Southward and Walker, 1980.

In the process of the invention as shown in figure 1 fresh washed, dewatered casein curd prepared from skim milk by a known method, which typically contains 40 to 75% (w/w) moisture is added to water to form a slurry. Optionally, the curd may be wet milled to reduce curd particle sizes to 1 mm or less. The concentration of curd in the slurry is within the range 5-25% w/w, preferably between 6 and 15% w/w. Alternatively, acid casein curds, the milk protein isolate (TMP m base) curd or the milk protein WO 94/06306 PCr/~NZ9/0008 of the extract produces an end product which is a micellar (calcium phosphate) proteinate or a mineral caseinate form eg. ammonium, sodium, potassium, calcium, magnesium or an alkaline earth, and monovalent or divalent or any mixture thereof. In addition, the 6-casein fraction can be dephosphorylated by known methods at this stage.

In addition hydrolysis may be used to produce modified forms or peptides of 6-casein.

Alternatively, the 6-casein extract is heated to a temperature of 2 to 65°C, preferably to 45 0 C for 0.1 minute to 4 hours, preferably 0.2 to 0.5 minutes. The 6-casein enriched curd obtained may be separated from solution by decanter separation, a filter press, membrane filtration, clarifiers and/or the use of a screen or any other suitable method.

In another alternative illustrated in figure 1 the 6-extract can be concentrated by, for example, evaporation, ion exchange or membrane filtration, followed by spray drying to obtain the 6-casein enriched product.

The 6-casein enriched curd and 6-casein depleted casein curd obtained by the process of the invention may be dried by any suitable method, for example freeze drying, ring drying, roller drying and fluid bed drying for the extracted rennet casein and the acid form of 6-casein enriched product. The proteinate or micellar forms of the 6-casein enriched product may be dried additionally by spray drying.

Using this process it is possible to extract 6-casein enriched product in an amount between 0.1 and 10%, typically 3 to 6% w/w of the total casein present in the milk or casein curd used as the feed material. The purity of the 6-casein enriched product is between 40% to 95% preferably between 60 to The 6-casein depleted casein product remaining is between 90 to 99% preferably 94 to 98% of the total casein present in the casein feedstock used as the starting material. The 6-casein depleted product was, very similar in its properties, including chemical composition, as the casein starting material. Known industrial uses for casein are discussed in reference Example I Laboratory scale demonst:ation of the feasibility of producing6-casein enriched product from rennet casein curd Fresh skim milk (2.5 L) was warmed to 30 0 C and calf rennet was added (0.22ml/L of milk). After 30 minutes at 30°C the rennet casein coagulum which formed was heated WO 94/06306 PCF/NZ93/00086 12 enriched product recovered from the aqueous 6-extract was shown to contain essentially 6-casein (Figure Densitometric analysis of the urea-PAGE gel revealed the 6-casein enriched product sample to be of a purity of approximately 80% (Figure 4).

Example III Pilot plant production of 6-casein enriched product Referring again to figure 1, approximately 900 L of fresh skim milk was set with calf rennet (200 ml rennet) at 27°C. After heating the coagulum was separated by screen dewheying. The dewheyed curd was washed in the standard way followed by decanter dewatering.

The dewatered rennet casein curd (66.8 kg) combined with chilled water (3 0 C) was subjected to wet milling to generate curd particles of 1mm or less in diameter before being placed in a silo. The mixture was continuously stirred and held overnight (19 h).

The slurry mixture was then pumped to a decanter for separation of the 6-casein depleted casein curd solids from the 6-extract. The casein curd solids recovered were dried in the usual way (21 kg of 6-casein depleted product was obtained).

The 6-extract recovered from the cold separation step (680 L) was placed in a silo and acidified to pH 4.7. The acidified mixture was then heated to 36-38 0 C prior to separation in the decanter for recovery of 6-casein enriched curd (2.49 kg) and ring drying (1 kg dried 6-casein enriched product in the form of a powder recovered).

The gross chemical composition of the pre-extraction rennet casein, the 6-casein depleted casein product and the 6-product are given in Table 1 below. The results show that the rennet casein composition is essentially unchanged from that of the pre-extraction rennet casein in respect to ash, calcium, lactose, moisture and total nitrogen (TN) contents after the extraction.

Urea-PAGE analysis (Figure 5) showed that the 6-extract contained 6-casein as the major component and that other casein fractions were not present.

TABLE I The gross chemical composition of the rennet caseins and 6-product of example III.

WO 94/06306 P(7Ir/NZ93/00086 14 6-casein enriched curd was dried in a ring drier. 13.0 Kg of dried 6-casein enriched product was obtained.

Example V Extraction '-casein enriched product from Standard New Zealand lactic acid casein Samples (1.00 0.05) g of New Zealand lactic casein (Alacid 720) were suspended in 10 mL of water containing between 0 and 4 w/w sodium chloride. The slurries were mixed by inversion for 20 hours at 3.7 0 C and then centrifuged at 13,000g to separate the extract from the solids. Total protein in the extract was estimated by determining the absorbance at 280 nm and using 6-casein as the reference protein for calibration. The results showed that the extracts obtained from the 0.25%, 2.0% and 4.0% w/v salt extractions contained 10 mg, 32 mg, 50 mg, 59 mg, 71 mg and 62 mg protein, respectively. When the extracts were analyzed by urea-PAGE (Figure 7) all the extracts were shown to contain 6-casein as the predominant protein.

Densitometric analysis of the urea-PAGE gel showed that the 6-casein in the extracts ranged in purity between 51% and 76% depending on the level of salt added for the extraction.

Example VI Extraction of 6-casein enriched product from whole casein precipitated with calcium Skim milk was warmed to 55°C and calcium was added to 3% w/v to precipitate the caseins. The casein precipitate was washed to remove whey proteins and dried in the usual way.

Samples of the dried casein (1.00 0.05 g) were suspended in water (final volume mL) containing the same range of sodium chloride concentrations described in Example WO 94/06306 PCF/NZ93/00008 16 Extraction of 6-casein enriched product from whole sulphuric acid casein Samples (1.00 0.05) g of New Zealand sulphuric acid casein (Alacid 750) were suspended in 10 mL of water containing between 0 and 4 w/w sodium chloride as described for example V above. The slurries were mixed by inversion for 22 hours at 3.7 0.5)°C and then centrifuged at 13,000g to separate the extract from the solids.

Total protein in the extract was estimated by determining the absorbance at 280 nm as described before (see example The results showed that the extracts obtained from the extractions with 0.25%, 2.0% and 4.0% w/v salt contained 10 mg, 32 mg, 56 mg, 80 mg, 88 mg and 66 mg protein, respectively. Urea-PAGE analysis (Figure 10) showed that all the extracts contained 6-casein as the predominant protein.

Densitometric analysis of the urea-PAGE gel showed that the 6-casein in the extracts ranged in purity between 57% and 86% depending on the level of salt added for the extraction.

Example IX Extraction of 6-casein enriched product from fresh acid casein curd Fresh decanter dewatered sulphuric acid casein curd was prepared as follows: 800 L of pasteurised skim milk was cooled to 21 0 C and 1.0 N sulphuric acid was injected in-line to obtain a pH of 4.5 to 4.6. .The coagulum was then heated to 49 0 C for cooking followed by standard acidulation and screen dewheying procedures. The dewheyed curd solids were subjected to the usual washes and dewatered in a decanter. (Approximately 43.03 kg of fresh acid casein curd was recovered).

The fresh acid casein curd was agitated in 480 L of water at 3.5 0 C for 191/2 hours. The curd solids were separated from the aqueous 6-casein extract (550 L) by the use of a decanter. The acid casein curd was dried in the usual way.

WO 94/06306 PCr/NZ9300086 18 A sample of New Zealand rennet casein (Alaren T M 771) (700 g) was slurried in 4.00 kg cold water. A second sample (700 g) was suspended in 4.02 kg cold water which contained 80.4 g of sodium chloride (saline extraction). Both slurries were agitated for 17 hours at 2.4 0 C after which the solids were separated from the extract by filtering the slurry (sequentially) through a 125 micron and a 25 micron GAF bag. The extracted casein solids were freeze dried. The soluble extracts were acidified with 0.05 M sulphuric acid to pH 4.65 and heated to 40 0 C under agitation. The 6-casein curd solids which formed were recovered by filtering through the 25 micron GAF bag and then freeze dried (recovering 58.9 g and 13.24 g 6-casein enriched product from the water and the saline extractions, respectively). When the dried products were analysed by urea-PAGE (Figure 12) the results showed that the protein recovered from the water and saline extracts were enriched 1.7 and 1.2 fold respectively in 6-casein.

ADVANTAGES

At least in the preferred form the invention has the following advantages: 1. The process may be readily introduced into the standard method for the commercial manufacture of casein.

2. The process results in the production of two potentially useful fractions. Because the process only strips a small fraction of the 6-casein present, the6-casein depleted casein is a useful product in addition to the 6-casein product. Thus, there is substantially reduced waste.

3. The process produces 6-casein product of high quality on an industrial scale in economically viable quantities.

WO 94/06306 PCT/NZ93/00086 7. Proteins of Milk. R McK Whitney. In Fundamentals of Dairy Chemistry, third edition, chapter 3, 81-170, by N P Wong, R P Jenness, M Keeney and E M Marth (Eds).

Van Nostrand Reinhold, New York.

Claims (16)

1. 7. A process according to claim 6 wherein said fresh wet curd is subjected to wet milling to reduce the curd particle sizes to 1mm diameter or less.
2. 8. A process according to any one of the preceding claims wherein the concentration of casein in said slurry is 1-25% w/w.
3. 9. A process according to claim 8 wherein the concentration of casein in said slurry is 6-15% w/w.
4. 10. A process according to any one of claims 4, 6 and 7 wherein the casein feedstock is rennet casein and in step said slurry is held at a pH of between 6.6 and 7.2.
5. 11. A process according to any one of claims 5, 6 and 7 wherein the casein feedstock is other than rennet casein and in step said slurry is held at a pH of between 4.5 and
6. 12. A process according to any one of the preceding claims wherein said slurry is held for between 0.1 and 30 hours at -10°C to 14 0 C.
7. 13. A process according to claim 12 wherein said slurry is held for between 4 and hours at 1 to 8°C.
8. 14. A process according to claim 13 wherein said slurry is held for between 10 and 16 hours. A process according to any one of claims 12 to 14 wherein said slurry is held between 2 and 6°C. WO 94/06306 PP/NZ93/00086 24
9. 24. A process according to claim 23 wherein when said casein feedstock is mineral acid or lactic acid casein, milk protein isolate, casein precipitated from milk by addition of calcium ions, or milk protein co-precipitate, said 6-casein enriched liquid phase is concentrated prior to said step i). A process according to claim 23 wherein said preconcentrating step comprises evaporation, ion excnange or membrane filtration of said liquid phase. A process according to any one of claims 23 to 25 wherein said step comprises adjusting the pH of said liquid phase to between 4.5 and 5.2.
10. 27. A process according to any one of claims 23 to 26 wherein said step (ii) comprises heating said liquid phase to approximately 2 to 65 0 C for a period of between 0.6 seconds to 4 hours.
11. 28. A process according to claim 27 wherein said step (ii) comprises heating said liquid phase.to 25 to
12. 29. A process according to claim 27 or 28 wherein said liquid phase is heated for 0.2 to 90 minutes. A process according to any.one of claims 23 to 29 wherein said step (iii) comprises isolating said 6-casein enriched product by decanter separation, filter press, membrane filtration, clarifiers, screens or the like.
13. 31. A process according to any one of the preceding claims which includes the additional step of adding an aqueous solution of sodium chloride in a concentration of up to 4.0% w/w to said slurry of step a). 26
14. 41. A process for extracting a p-casein enriched product and a p-casein depleted product from casein feedstock including milk protein co-precipitate substantially as hereinbefore described with reference to any one of the Examples.
15. 42. A p-casein enriched product when produced by the process of claim 41.
16. 43. A p-casein depicted product when produced by the process of claim 41. Dated 6 January, 1997 New Zealand Dairy Board Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON I I I I I i I e fit* 'V ii w N:\LBFFl00443:MCN