NZ550859A - Production of commercial quantities of lactoferrin-enriched milk where milking is re-established after an extended period of non-milking - Google Patents

Abstract

Provided is a method of producing a lactoferrin-enriched milk product comprising the steps 1) providing milk by ceasing to milk an ungulate for more than 24 hours and then collecting milk for up to 7 days and 2) processing the milk sample to produce a milk product. Further provided is a milk product produced by the said method.

Description

55 0 8 5 9 NEW ZEALAND PATENTS ACT, 1953 No: Date: COMPLETE SPECIFICATION ENRICHED MILK PRODUCTS We, FONl'ERRA CO-OPERATIVE GROUP LIMITED, 9 Princes Street, Auckland, New Zealand, do hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: (followed by page la) INTELLECTUAL PROPERTY OFFICE OF N.Z. 2? OCT 2006 received la ENRICHED MILK PRODUCTS 55 0 8 5 9 Field of the Invention The present invention relates to a method for producing lactoferrin-enriched milk products, and to milk products produced by the methods.

Background Art Lactoferrin is a component of milk whey, valuable for many biological properties, including those as an iron-binding, bacteriostatic and anti-bacterial protein. Commonly, lactoferrin concentrations are high in colostrum (Nonnecke & Smith, 1984, J Dairy Science 62, 2863-72). In particular, the lactoferrin content of mammary secretion is markedly increased during mammary involution when expression of the lactoferrin gene is initiated in milk-engorged secretory alveoli (Molenaar el al, 1996 J Dairy Research 60:1-11).

The process of mammary gland involution occurs during the transition from a lactating to a non-lactating state. This transition phase begins after cessation of milk removal and results in changes in mammary secretion composition. Secretion volume declines during involution, as does the concentration of most milk-specific components. Lactoferrin, hydrolytic enzymes, immunoglobulins, and serum-derived components increase in concentration in the secretions during involution (Hurley WL 1989, J Dairy Sci. 17,6:1637-46).

It has also been shown that once-daily milking results in an increase in the concentration of lactoferrin, however lactoferrin concentration slowly declined following the resumption of twice-daily milking (Farr et al,, 2002 Proceedings of the New Zealand Society of Animal Production 62: 225-236).

It would be beneficial to establish a milking regime which would result in a significant increase in lactoferrin yield, that is a regime in which increased lactoferrin concentration is achieved whilst milk yield is maintained. Milk products derived from such milk and/or lactoferrin isolated from the milk would be useful due to the beneficial properties of lactoferrin discussed above. 2 It is an object of the invention to provide a method for the production of lactoferrin-enriched milk products and/or at least to provide the public with a useful choice.

Summary of the Invention The applicants have shown, for the first time, that it is possible to re-establish milking after an extended period of non-milking, and at the same time to maintain a several-fold elevation in the concentration of lactoferrin in the milk for a significant period after milk production is re-established. Using the procedure established by the applicants, 10 commercial quantities of lactoferrin-enriched milk can be harvested over a period of a few days and can be processed to produce a lactoferrin enriched milk product.

In one aspect the invention provides a method of producing a lactoferrin-enriched milk product comprising the steps of: 3) providing a milk sample prepared by a) ceasing to milk a ungulate for a period of greater than 24 hours; b) collecting milk from the ungulate within a period of up to 7 days after completion of a); and 2) processing the milk sample to produce a milk product.

In one embodiment the method comprises the steps of: a) ceasing to milk an ungulate for a period of greater than 24 hours; b) collecting milk from the ungulate within a period of up to 7 days after completion of a); and c) processing the milk collected to produce a milk product.

Preferably the ungulate is a cow or a goat. More preferably the ungulate is a cow.

In one embodiment the cow is in late lactation (within 90 days of the next calving).

In a further embodiment the period in a) is at least 2 days, preferably at least 3 days, more preferably at least 4 days, more preferably at least 5 days, most preferably at least 6 days. 3 In a further embodiment prior to step a) the ungulate is subjected to once daily milking for a period of at least 2 days.

In a further embodiment the period in b) is less than 7 days, alternatively less than 6 days, 5 alternatively less than 5 days, alternatively less than 4 days.

In a preferred embodiment the ungulate is milked twice daily during the period in b) Preferably the level of lactoferrin in the milk collected is at least 400 mg/1, more preferably 10 at least 600 mg/1, more preferably at least 800 mg/l, more preferably at least 1000 mg/1, more preferably at least 1500 mg/1, more preferably at least 2000 mg/1, more preferably at least 2500 mg/1, more preferably at least 3000 mg/1, more preferably at least 3500 mg/1, more preferably at least 4000 mg/1, more preferably at least 4500 mg/1, more preferably at least 5000 mg/1, most preferably at least 5000 mg/1.

To obtain lactoferrin concentrations greater than 1500 mg/1 it is generally necessary to select high lactoferrin producing ungulates.

In a further embodiment the ungulate used in the method produces milk with a lower 20 concentration of beta-lactoglobulin than does an average ungulate of the same breed.

Preferably the ungulate is a cow identified by the BB beta-lactoglobulin genotype (Hill, JP 1993. Journal of dairy science 76, 281-286) or other genetic or laboratory tests. Use of such cows in the method would enable production of whey protein concentrate (WPC) containing an even greater proportion of lactoferrin.

The milk product may be a milk powder including whole milk powder (WMP) and skimmed milk powder (SMP), a milk protein concentrate (MPC) or a whey protein concentrate (WPC).

The milk product may be a food product. | INTELLECTUAL HKUKcnl i I The milk product may be a nutritional supplement.

Alternatively the milk product may be lactoferrin isolated from the milk OFFICE OF N.z. 11, MAR 2007 eceived 4 The milk product may also be a milk product to which the isolated lactoferrin is added.

In a further aspect the invention provides a milk product produced by the method of the 5 invention.

In a further aspect the invention provides a nutritional supplement containing a milk product of the invention.

In a further aspect the invention provides a food product containing a milk product of the invention.

Detailed Description of the Invention 15 Definitions The term "once daily milking" means milking the ungulate once within a twenty-four hour period.

The term "twice daily milking" means milking the ungulate twice within a twenty-four period.

The term "milk protein concentrate" or "MFC" means a powder formed from the concentration of milk, usually comprising 55-85% protein and manufactured via filtration, 25 evaporation, and drying.

The term "whey protein concentrate" or "WPC" means a powder formed from the concentration of whey proteins, comprising at least 50% protein and manufactured from whey via filtration, evaporation, and drying.

The term "food product" as used herein includes solid foods, liquid beverages and other edible materials regardless of their specific form.

The term "comprising" as used in this specification and claims means "consisting at least in part of'; that is to say when interpreting statements in this specification and claims which include "comprising", the features prefaced by this term in each statement all need to be present but other features can also be present. Related terms such as "comprise" and 5 "comprised" are to be interpreted in similar manner.

In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, 10 reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

The Applicants have discovered that following a period of non-milking of greater than 24 15 hours followed by a resumption of milking, the concentration of lactoferrin in milk collected is significantly increased and surprisingly that the increased concentration is maintained while milk production recovers for a period of at least four days resulting in an increased yield of lactoferrin in milk collected during this period.

The invention provides methods for producing lactoferrin enriched milk products, milk, milk powder, WMP, SMP, MPC and WPC nutritional supplements and food products.

Methods for preparing milk powder, WMP, SMP, MPC, WPC are known to those skilled in the art (Encyclopaedia of Dairy Sciences 2003, Eds Roginski, H., Fuquay, J.W. Fox, 25 P.F. and Academic Press and references cited therein). Further, it is well known that, to avoid losses of lactoferrin during manufacture of milk powders, it is necessary to carefully regulate temperature during spray drying and also the pH and ionic strength of the milk or milk fraction. It is known to those skilled in the art that to avoid denaturation of lactoferrin, steps requiring heat treatment should preferably be performed with the 30 lactoferrin in a low ionic strength solution. The relationship between the temperature and ionic strength is discussed in European Patent Application 0364912 which is incorporated herein by reference. Low ionic strength may be effected by way of dialysis, ultrafiltration, 6 diafiltration, resuspension of dried material in a suitable solution, or other methods known to those skilled in the art.

In one embodiment, the processing of the milk or milk sample is processing to isolate 5 lactoferrin. Preferably lactoferrin is isolated by ion exchange chromatography, preferably cation exchange. Preferably ion exchange is carried out at neutral pH. Preferably ion exchange is carried out using column chromatography. In one embodiment a membrane adsorber, that immobilises the exchange functional groups on a microporous membrane, is used for ion exchange. Preferably lactoferrin is extracted from whey using a strongly 10 acidic membrane adsorber.

Examples of methods for isolating lactoferrin from milk are found in New Zealand Patent Numbers 221082, 227097, 249235, in US Patent numbers 4,791,193 and 5,596, 082, and in Mitchell et al. 1994. Extraction of lactoperoxidase and lactoferrin from cheese whey using 15 membrane cation exchangers. Page 89 in IDF Seminar: Indigenous Antimicrobial Agents of Milk — Recent Developments Int. Dairy Fed., Brussels, Belgium, all of which are incorporated herein by reference.

Methods for pasteurising lactoferrin containing solutions as described in European Patent 20 Application 0364912 which is also incorporated herein by reference.

Methods for incorporating milk powder, WMP, SMP, MPC, WPC or lactoferrin into nutritional supplements and food products are known to those skilled in the art.

Preferred food products include dairy products such as milk drinks (including treated milks such as skimmed milk and consumer products such as flavoured milks), fermented milk products (including yoghurts and cheeses), and milk preparations (including infant milk formulas and products made using milk powders or milk components as ingredients).

Brief Description of the Drawings Figure 1 shows change in milk yield during resumption of milking after a 2-day (♦) or a 4-day (■) milking interval in cows (n=10 per group) at the end of lactation. Pre-treatment 7 yields are shown at day -4. Error bars are standard error of mean. Horizontal bars indicate duration of non-milking period.

Figure 2 shows change in milk lactoferrin concentration during resumption of milking after 5 a 2-day (♦) or a 4-day (a) milking interval in cows (n=l 0 per group) at the end of lactation. Pre-treatment concentrations are shown at day -4.

Figure 3 shows change in milk lactoferrin yield (g/d) during resumption of milking after a 2-day (♦) or a 4-day (a) milking interval in cows (n=10 per group) at the end of lactation. 10 Pre-treatment yields are shown at day -4. Note sustained, elevated yields 2-4 days after milking was resumed.

Figure 4 shows cumulative milk yield and lactoferrin yield over 4-days after resumption of milking after a 2-day (♦) or a 4-day (a) milking interval in cows (n=l 0 per group) at the 15 end of lactation.

Figure 5 shows lactoferrin concentrations, n=6 per group (mg/ml) in milk at each milking after resumption of milking following 2, 4 or 7 days without milking in mid-lactation.

Data at milking -1 show the pre-treatment milk lactoferrin concentrations and at milking 0 20 at the first milking after the non-milking period. Milking 6 was a composite am + pm sample, as was milking 7. Hence the data presented covers a total of 10 milkings post-treatment.

Figure 6 shows changes in milk yield (kg/day) following cessation of milking in mid-25 lactation. First point at day -1 is the yield during twice-daily milking. The second point at time 0 is the accumulated volume of secretion during the non-milking period (2,4 or 7 days -see legend). Thereafter, yields are for 24 h periods. Average yields are shown for 12 cows per group.

Figure 7 shows daily lactoferrin yields, n=6 per group (g/d). Day -1 represents daily yield before treatment and day 0 is the yield at the first milking after 2, 4 or 7 days without milking in mid-lactation.

Examples 8 The invention will now be illustrated with reference to the following non-limiting examples.

Example 1: Milking regime resulting in increased yield of lactoferrin from cows in late lactation.

Twenty cows, grazing good quality pasture, in late-lactation, free of mastitis and with an average milk yield about 9 kg/d during twice-daily milking, were not milked for 3 10 consecutive milkings (n=l 0) following a morning milking (2-day interval) or for 7 consecutive milkings (n=10) following a morning milking (4-day interval). Milking was then resumed and continued twice daily for a further 5 days. Milk yield was measured at each milking and milk lactoferrin concentrations measured using an ELISA kit ( Bethyl Laboratories, Montgomery, Texas, USA ) specific for bovine lactoferrin.

Milk yields and lactoferrin concentrations in milk from the experimental cows are shown in Figures 1-3. Milk yield at the resumption of milking was the accumulation of secretion during the extended interval and as such was close to the normal twice-daily yield. However, secretion rate was suppressed by the lack of milking such that milk secretion 20 over the first 24 hours after resumption of milking was less than 30% of previous yields on twice-daily milking (Figure 1). Yield recovered to close to previous levels over the next 3 days, recovery being somewhat slower in the 4-day group.

Milk lactoferrin concentrations differed between the 2-day and 4-day treatment groups in 25 the first milk after resumption of milking, but after 48 hours, concentrations of lactoferrin in milk of both groups was elevated about four-fold compared to pre-treatment and this increase was sustained for a further 48 hours, at least (Figure 2).

Daily yield (g/day) of lactoferrin was elevated in the 4-day interval group at the 30 resumption of milking but declined in both groups over the first 24 hours only to rebound to a daily secretion rate 2-3 fold greater than pre-treatment values 2-4 days after the resumption of milking and through the time period when recovery of milk yield was maximal (Figure 3). 9 Data in Figure 4 show that both treatments result in accumulated secretion of about 30 litres of milk containing 30-35g of lactoferrin in the four day period following resumption of milking.

Example 2: Milking regime resulting in increased yield of lactoferrin from cows in mid-lactation Methods This trial was undertaken in Taranaki, New Zealand in December 2004. Thirty-six cows of mixed age and breed (Jersey, Friesian) and approximately 16 weeks of lactation (July/August 2004 calving) and early (<2months) pregnancy were selected for the trial. Milk from these cows tested baeteriologically negative approximately 5 days before trial began. All selected cows were producing > 8kg milk/day at the herd test on December 15 3rd/4th. Animals grazed ryegrass/white clover pasture as their sole diet. Sufficient pasture was offered to maximise feed intake.

Cows were allocated into 3 groups of 12 (balanced for age breed and milk yield). The cows were milked twice daily, then were not milked for 3 consecutive milkings (n=12) following 20 a morning milking (2-day interval) or for 7 consecutive milkings (n=12) following a morning milking (4-day interval) or for 13 consecutive milkings (n=12) following a morning milking (7-day interval). The last milking for each group was on the morning of 6th December, 9th December and 11th December for 7-day, 4-day and 2-day groups respectively. They were then re-milked (twice daily) and recovery of milk yield and 25 composition monitored for the next 5 days (or longer for some parameters).

Milk samples Milk samples were collected at the pre-trial herd test and at every milking following the 30 resumption of milking for 3 days. Thereafter, composite samples of am and pm milk were taken for a further 2 days.

Milk analyses Lactoferrin concentrations were determined in the milk samples using a lactoferrin EL1SA kit (Bethyl Laboratories, Montgomery, Texas, USA). Milks from 6 cows in each group were analysed.

Results All groups showed elevation of milk lactoferrin concentration for at least 7 milkings (3-4 days) after the restart of twice-daily milking following cessation of milking for 2, 4 or 7 days (Figure 5). Maximal concentrations were obtained at around milking 3 following the 10 restart, representing a 4-5 fold increase in lactoferrin concentration yield. Elevated lactoferrin concentrations were still apparent at milking 7.

Milk yield recovered rapidly with the resumption of milking and was fully recovered by day 4 in the 2 and 4 day groups (Figure 6). Milk yield recovery was slower in the 7-day 15 group but yield was close to the 2 and 4-day groups by day 7 following the resumption of milking.

Calculation of lactoferrin yields indicate that daily production of lactoferrin was stimulated 2-4 fold by the non-milking treatment especially in the 2 and 4-day groups. (Figure 7).

Example 3: Purification of Lactoferrin from the lactoferrin-enriched milk Lactoferrin can be purified, from milk produced by the method of the invention, using the method of US 5,596,082.

Milk produced by the method of the invention is used to produce a clari fied cheese whey having a solids content of 5.6% and a pH of 6.6, by cross-flow micro filtration 1.4 .mu.m (Alfa Laval), A column having a diameter of 1.6 cm is packed with 20 ml of the strong cation exchanger S Sepharose Big Beads (Pharmacia). The bed height is 10 cm. After equilibration of the resin with 0.025M phosphate buffer pH 6.5, the clarified cheese whey is pumped through the column at a rate of 150 or 200 bed volumes/hour 11 After washing of the column with buffer, LF is eluted in with 0.8M NaCl in phosphate buffer.

The eluates may be freeze dried.

Example 4: Purification of Lactoferrin from lactoferrin-enriched milk at pilot production scale A 10 cm column of a pilot production chromatography system (Bioprocess System Pharmacia) is packed with SP Sepharose Big Beads (Pharmacia). The diameter of the column is 10 cm, the bed height is 11 cm and the bed volume is 0.864 1.

After equilibration of the resin at room temperature with 0.025M phosphate buffer pFI 6.5, 15 clarified cheese whey, prepared as described in Example 3, is pumped through the column at a rate of flow of 180 litre/hour.

After washing of the column with buffer solution, lactoferrin is eluted with l.OMNaCl in phosphate buffer. The liquid flow rate during washing and elution was 8.6 litre/hour (10 20 bed volumes/hour).

The eluates may be freeze dried.

Example 5: Pasteurization of lactoferrin Freeze-dried lactoferrin-enriched milk powder, produced from milk produced by the method of the invention, can be pasteurised using the method of EP 0344912. 100 g of a freeze-dried bovine lactoferrin powder is dissolved into 1 litre of deionized water. This aqueous lactoferrin solution is placed in a vessel with a lid, and is heated in hot water at 85°C for 30 minutes. After the heating, the aqueous lactoferrin solution is immediately cooled to room temperature with running water. 12 Example 6: Large scale purification and pasteurisation of lactoferrin Ten tonnes of raw skim milk, obtained by standard procedures from milk produced by the 5 method of the invention, is passed through a carrier comprising chitosan beads to which sulphuric ester is attached. Bovine lactoferrin which is absorbed onto the beads, washed then eluted with a 1 M saline solution. The eluate is desalted/concentrated through an ultrafilter (DDS 20), so that 20 litres of a lactoferrin solution are obtained. The solution is then pasteurized by heating at a temperature of 95°C for 4 seconds by the use of a plate 10 type heat-exchanger, and then cooled to TC.

The above Examples illustrate practice of the invention. It will be appreciated by those skilled in the art that numerous variations and modifications may be made without 15 departing from the spirit and scope of the invention. 13

Claims (29)

WHAT WE CLAIM IS:

1. A method of producing a lactoferrin-enriched milk product comprising the steps of: 1) providing a milk sample prepared by a) ceasing to milk an ungulate for a period of greater than 24 hours; b) collecting milk from the ungulate within a period of up to 7 days after completion of a); and 2) processing the milk sample to produce a milk product.

2. The method of claim 1 comprises the steps of: a) ceasing to milk an ungulate for a period of greater than 24 hours; b) collecting milk from the ungulate within a period of up to 7 days after completion of a); and c) processing the milk collected to produce a milk product.

3. The method of claim 1 or claim 2 wherein the ungulate is in late lactation (within 90 days of the next calving). ' '

4. The method of any preceding claim in which the period in a) is at least 2 days.

5. The method of any preceding claim in which the period in a) is at least 3 days.

6. The method of any preceding claim in which the period in a) is at least 4 d lys. .w

7. The method of any preceding claim in which the period in a) is at least ^iays.

8. The method of any preceding claim in which the period in a) is at least § days.

9. The method of any preceding claim in which the period in a) is at least 7 days. j

10. The method of any preceding claim in which the pcriod in a) is at least 8 days. 14

11. The method of any preceding claim in which, prior to step a), the ungulate is subjected to once daily milking for a period of at least 2 days.

12. The method of any preceding claim in which the period in b) is less than 7 days. 5

13. The method of any preceding claim in which the period in b) is less than 6 days.

14. The method of any preceding claim in which the period in b) is less than 5 days. 10

15. The method of any preceding claim in which the period in b) is less than 4 days.

16. The method of any preceding claim in which the ungulate is milked twice daily during the period in b). 15

17. The method of any preceding claim in which the level of lactoferrin in the milk collected is at least 400 mg/1.

18. The method of any preceding claim in which the ungulate used in the method produces milk with a lower concentration of beta-lactoglobulin than does an average ungulate of the 20 same breed.

19. The method of claim 18 in which a ungulate is a cow identified by the BB beta-lactoglobulin genotype. 25

20. The method of any one of claims 1 to 19 in which the milk product is selected from a group consisting of a milk powder (including whole milk powder [WMP] and skimmed milk powder [SMP]), a milk protein concentrate (MPC) and a whey protein concentrate (WPC). 30

21. The method of any one of claims 1 to 19 in which the milk product is a food product.

22. The method of claim 21 in which the food product is selected from a group consisting of milk drinks (including treated milks such as skimmed milk and consumer products such as flavoured milks), fermented milk products (including yoghurts and cheeses), and milk INTELLECTUAL PROPERTY OFFICE OF IM.Z. 11, MAR 2007 RECEIVED. 15 55 08 59 preparations (including infant milk formulas and products made using milk powders or milk components as ingredients).

23. The method any one of claims 1 to 19 in which the milk product is a nutritional 5 supplement.

24. The method of any one of claims 1 to 19 in which the milk product is lactoferrin isolated from the milk. 10

25. The method of any preceding claim in which the milk product is a milk product to which the lactoferrin of claim 24 is added.

26. The method of any preceding claim in which the ungulate is a goat or a cow. 15

27. The method of claim 25 in which the ungulate is a cow.

28. A milk product produced by the method of any preceding claim.

29. The method of any one of claims 1 to 19 and 24 substantially as herein described with 20 reference to any example thereof. By the authorised agent* AJ PARK Per INTELLECTUAL PROPERTY OFFICE OF N.Z, 2 7 OCT 2006 RECE VED