Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4732—Casein
• • 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
  • A23J1/202—Casein or caseinates
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• This invention relates to a method of producing a 6-casein enriched product and a 6-casein depleted product from casein curd or powder and to products produced by the method.
• Casein may be obtained from mammalian milk by known techniques.
• acid casein is obtained by acidifying milk with mineral or organic acids.
• Rennet casein is obtained by precipitation of the casein micelles of mammalian milk after hydrolysis of ⁇ -casein with calf rennet or equivalent enzymes of bacterial, fungal or plant origin. Methods of commercial production of caseins are described in references 1 to 4 and 6.
• 6-casein One of the known fractions of casein is 6-casein.
• 6-casein has many properties which make it a commercially desirable product to obtain.
• Known properties include:
• 6-casein is a phosphorylated protein which, upon digestion in the human gut, can release peptides which exert specific physiological effects. These include peptides which have opioid activity. These properties allow 6-casein to be used in both non-food and food products such as in low allergenic foods, low lactose dairy drinks, and pharmaceutical products. 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 s and ⁇ -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.
• the preparation of a fraction containing ⁇ s - and 6-casein is described.
• the isolation of the 6-casein from the ⁇ s /6 -casein fraction is the subject of the second patent.
• the isolation of the 6-casein involves calcium precipitation of the ⁇ .-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 ⁇ .-caseins.
• Specification 55 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°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 physically separated.
• the process of the present invention unlike the process of WO 92/00017, strips only a small proportion of the total 6-casein from the casein curd.
• United States Patent 5,169,606 described a process of cooling bovine milk to a temperature sufficient to dissociate 6-casein to equilibrium and filtering the cooled milk through a microfilter to produce a humanized bovine milk product having an enriched 6-casein content.
• the specification gives no indication as to what is done with the " retentate by product of the microfiltration. Further, it is an object of this invention to produce a product containing a mixture of whey proteins and 6-casein.
• the Japanese Patent Specification 5,146,258 (91-337,701) describes a process for the isolation of 6-casein using a combination of precipitation and membrane technologies.
• a three stage procedure is used for isolating 6-casein.
• Skim milk is first acidified to a pH of 3.5 or less and cooled.
• the pH is readjusted to 4.2 -4.4 to effect the precipitation of the ⁇ --casein fraction.
• a subsequent membrane filtration step in the cold (third stage) is used to effect the separation of 6-casein.
• the remaining (6-casein depleted) casein is substantially altered in character and its commercial usefulness is no longer preserved.
• the present invention may be said broadly to consist in a process for extracting a 6-casein enriched product and a 6-casein depleted product from a casein feedstock including milk protein co-precipitate, said process comprising the steps of: a) forming a slurry of said casein feedstock and cold water, b) holding said slurry at a cold temperature and a pH between 5.0 and 8.0 for a rennet casein feedstock or a pH between 3.5 and 8.0 for other casein feedstock for a suitable period of time to effect extraction of 6-casein, and 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.
• casein feedstock is a wet casein curd or a milk protein powder contaimng casein.
• casein feedstock is mineral acid or lactic acid casein, milk protein isolate (TMPTM base), rennet casein, milk protein co-precipitate, casein co-precipitate, whole casein precipitated by calcium or other casein curd which contains 6-casein.
• said casein feedstock is a curd or powder of rennet casein.
• said casein is fresh wet curd having a moisture content of between 45 and 75% w/w.
• said fresh wet curd is subjected to wet milling to reduce the curd particle sizes to 1 mm diameter or less.
• casein feedstock is a curd or powder of acid casein.
• the concentration of casein in said slurry is 1-25% w/w.
• the concentration of casein in said slurry is 6-15% w/w.
• step (b) 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°C.
• More preferably said slurry is held for between 10 and 16 hours.
• said slurry is held between 2 and 6°C.
• said feedstock is casein curd and said process includes the preliminary step of extracting said casein curd from milk.
• said process is conducted as a batch process.
• said 6-casein depleted solid phase from step c) is subjected to a second extraction comprising said steps a) to c).
• said 6-casein depleted solid phase from step c) of said second extraction is subjected to a third extraction comprising said steps a) to c).
• Preferably said process includes the further step of isolating 6-casein enriched product from said liquid phase.
• Preferably said process includes the further step of drying said isolated 6-casein enriched product.
• said step of isolating the said 6-casein enriched product comprises steps of: i) where necessary, acidifying said separated liquid phase, ii) heating said liquid phase to effect precipitation of a 6-casein curd, and iii) isolating said 6-casein curd.
• said casein feedstock is mineral acid or lactic acid casein, milk protein isolate (TMPTM base), 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).
• Preferably said preconcentrating step comprises evaporation, ion exchange or membrane filtration of said liquid phase.
• step (i) comprises adjusting the pH of said liquid phase to between 4.5 and 5.2.
• step (ii) comprises heating said liquid phase to approximately 2 to 65°C for a period of between 0.6 seconds to 4 hours.
• step (ii) comprises heating said liquid phase to 25 to 45°C.
• liquid phase is heated for 0.2 to 90 minutes.
• 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).
• step (iii) comprises isolating said 6-casein curd by decanter separation, filter press, membrane filtration, clarifiers, screens or the like.
• step of isolating 6-casein from said liquid phase from step c) above comprises subjecting said liquid phase to a concentrating step followed by spray drying.
• 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 6-casein depleted solid phase.
• the present invention may broadly be said to consist in a 6-casein enriched product when produced by the above identified process.
• said 6-casein enriched product contains 40 to 95% 6-casein.
• the present invention may broadly be said to consist in a 6-casein depleted casein product when produced by the above identified process.
• the present invention may broadly be said to consist in a food, pharmaceutical, plastics or other non-food product including said 6-casein enriched product and/or said 6-casein depleted product.
• said product is an infant food product.
• This invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, and any or all combinations of any two or more of said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which this invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.
• Figure 1 is a schematic representation of the process of the invention including the optional preliminary step of forming a casein curd.
• Figure 2 represents urea-PAGE analysis of samples from the investigation described in Example I. All samples were analysed in duplicate. The lanes are: 1
• Figure 3 represents the urea-PAGE analysis of the dry-products obtained in example II.
• Lane 1 a mixture of ⁇ s - and 6-casein standards; 2 and 3 pre-extraction rennet curd; 4 and 56-casein depleted casein curd; 6 and 76-casein enriched product.
• Figure 4 represents a densitometer trace of a 6-casein enriched product sample after electrophoresis on the urea-PAGE gel. (Refer lane 7 on Figure 3).
• Figure 5 represents the urea-PAGE analysis of samples obtained from example III.
• Figure 6 is a schematic diagram of the process of the large scale pilot plant extraction of 6-casein enriched product described in example IN.
• Figure 7 represents the urea-PAGE analysis of the products from example V.
• Figure 8 represents the urea-PAGE analysis of the products from example VI.
• Figure 9 represents the urea-PAGE analysis of the products from example VII.
• Figure 10 represents the urea-PAGE analysis of the products from example VIII.
• Figure 11 represents the urea-PAGE analysis of products from example IX.
• Figure 12 represents the urea-PAGE analysis of the products from example X.
• 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.
• 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.
• 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.
• acid casein curds, the milk protein isolate (TMPTM base) curd or the milk protein co-precipitate may be used in the process of the invention instead of rennet casein curd with appropriate adjustment of the reaction conditions.
• the water to which the rennet casein curd is added is at a temperature of between 1 and 30°C, preferably between 2 and 5°C. Alternatively, the water may be added at ambient temperature and then cooled to the desired temperature.
• the rennet casein curd slurry is held for 3 to 30 hours, preferably for 12 to 16 hours at 1 to 12°C, preferably 2 to 5°C. It is preferred that the slurry is gently agitated during this cold extraction. In a commercial process it is envisaged that the curd may be extracted either as detailed above or for periods between 0.1 minute and 16 hours at the given or lower temperatures.
• the pH of a slurry of rennet casein is adjusted to be above pH 5.0 during the cold extraction, preferably between pH 6.8 and 7.2 which is the natural pH of the system.
• the pH is adjusted to 3.0 to 8.0.
• a soluble 6-casein enriched fraction and a solid 6-casein depleted curd fraction are obtained in either case.
• the curd solids and soluble 6-casein fraction may be separated using a screen, decanter, clarifier, filter press, membrane filtration, centrifugation or any other suitable method.
• An aqueous 6-extract and a 6-casein depleted rennet casein curd solid fraction are obtained.
• the 6-casein depleted rennet casein curd fraction recovered by decanter centrifugation (or any other suitable method) is dried in the usual way.
• the 6-casein may be recovered from the aqueous 6-casein extract by acidifying the 6-extract after warming.
• the pH is adjusted to 4.5 - 5.2, most preferably 4.6 - 4.9.
• the pH may be adjusted as the 6-extract comes off the mechanical centrifuge or alternatively within a holding vat.
• the pH is adjusted by the addition of an organic or inorganic acid or a mixture thereof.
• Suitable organic acids include but are not limited to acetic acid, lactic acid, citric acid, tartaric acid or a mixture of any two or more of these.
• Suitable inorganic acids include but are not limited to hydrochloric acid, sulphuric acid, phosphoric acid or mixtures thereof.
• the 6-casein Prior to acidification the 6-casein may be concentrated or desalted by any combination of by evaporation, membrane filtration or the like.
• the mineral composition of the extract can be altered by ultrafiltration and/or diafiltration of the extract.
• the diafiltered mineral reduced or mineral adjusted product can in addition, or alternatively, be recovered by spray drying.
• Altering the mineral composition 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.
• the 6-casein fraction can be dephosphorylated by known methods at this stage.
• hydrolysis may be used to produce modified forms or peptides of 6-casein.
• the 6-casein extract is heated to a temperature of 2 to 65°C, preferably 25 to 45°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.
• 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.
• 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 85%.
• 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 5.
• the fresh dewatered curd was slurried with 480L of water and chilled to 2.7°C. The slurry was held under gentle agitation at this temperature for 23 hours. The slurry was then pumped to a screen to separate the curd from the aqueous 6-extract.
• the 6-extract so recovered (approximately 400 L) was acidified manually to pH 4.4 with dilute sulphuric acid and heated to 55°C.
• the 6-casein solids which formed as the acid curd were collected (manually) by filtration through 50 ⁇ m GAF bags.
• the 6-casein enriched curd so recovered was freeze dried to obtain 0.59 kg of 6-casein enriched product.
• the dewatered rennet casein curd (66.8 kg) combined with chilled water (3°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°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.
• fresh skim milk (13850 L) was treated with calf rennet and processed under standard conditions for the manufacture of casein curd.
• Wet casein curd (881 kg) was conveyed via a holding tank to a colloid mill where the curd was wet milled to particles of approximately 1.02 mm diameter. Approximately 3200 L of chilled water was used to complete the wet milling of the curd.
• the milled rennet casein curd slurry was pumped to a silo (at approximately 6°C) and cold water was added (final volume of 11430 L approx). The slurry was held cold and agitated gently for the next 17 h (end temperature was 3.6°C).
• the 6-casein depleted casein curd solids were recovered by a decanter and dried in the usual way.
• the liquid 6-casein enriched extract at 30°C from the decanter was acidified (in-line) with 4% w/w H 2 S0 4 en route to a second decanter for the recovery of the 6-casein enriched product.
• the end pH to which the extract was acidified was 4.7.
• the resulting 6-casein enriched curd was dried in a ring drier. 13.0 Kg of dried 6-casein enriched product was obtained.
• 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.
• TMPTM 1000 base New Zealand milk protein isolate
• 8.07 kg water which contained 106 g of sodium chloride (saline extraction).
• Both slurries were agitated with the aid of an overhead stirrer at 2.4°C for 18 hours after which the solids were separated from the extract by filtering the slurry sequentially through 125 micron and 25 micron GAF bags.
• the extracted casein solids were freeze dried (recovered 1.74 kg and 1.49 kg for the coprecipitate samples extracted with water and the saline solution, respectively).
• the soluble extracts (4.228 kg and 3.49 kg from the samples extracted with water and the saline solution, respectively) were heated to (and held at) 60°C under agitation for between 30 and 90 min.
• the 6-casein enriched curd solids which formed were recovered by filtering through the 25 micron GAF bag.
• the 6-casein emiched curd solids were freeze dried (3.24 g and 100.3 g 6-casein emiched product were obtained from the water and the saline extractions, respectively).
• urea-PAGE Figure 9 the results showed that the products recovered from the water and saline extracts were emiched 1.54 and 1.23 fold respectively in 6-casein.
• Fresh decanter dewatered sulphuric acid casein curd was prepared as follows: 800 L of pasteurised skim milk was cooled to 21°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°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 5 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°C for 19V2 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. 0
• the 6-casein extract (550 L) recovered from the cold separation step was heated in-line by direct steam injection and fed to the decanter at 38°C for the recovery of the 6-casein curd.
• the wet 6-casein curd was dried.
• the dry 6-casein product recovered weighed 0.225 kg.
• 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).
• 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.

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• 1993
  • 1993-09-22 AU AU49873/93A patent/AU677230B2/en not_active Ceased
  • 1993-09-22 WO PCT/NZ1993/000086 patent/WO1994006306A1/en not_active Application Discontinuation
  • 1993-09-22 EP EP93919728A patent/EP0661927A4/en not_active Withdrawn
  • 1993-09-22 JP JP6507997A patent/JPH08501688A/ja active Pending

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