GB2387099A - Method for producing milk with an enhanced content of naturally expressed melatonin - Google Patents

Abstract

A method for producing milk with a concentration of naturally expressed melatonin in the range of from 15 to 30 pg/ml, involving the selection of appropriate lactating mammals. The mammals are maintained under conditions of controlled light intensity and duration, defining a dark period and a light period. At least two milkings are carried out in a 24 hour period, with one of said milkings being carried out at the end of the dark period. Separation is maintained between milk collected during this 'dark' milking and milk collected during the other milking(s). The milk collected during the 'dark' milking is then selected for processing and packaging.

Description

Method for Producing Milk with an Enhanced Content of Naturaliv Expressed

Melatonin This invention relates to a method for producing milk having an 5 enhanced content of naturally expressed melatonin. In particular it relates to a method for producing cows' milk having an enhanced concentration of naturally expressed melatonin, which may find use as a so-called Functional food" product.

Melatonin (N-acetyl-methoxytryptamine) is a naturally occurring 10 hormone, which in recent years has gained popularity as a proposed remedy for insomnia and jet-lag, amongst other things. It is produced in varying levels in the pineal glands of most mammals. Secretion of melatonin into the bloodstream of the mammal is accelerated by the onset of darkness, and this enhanced melatonin production is thought to induce sleep. Conversely, 1s melatonin production decreases when the eye of the mammal detects light.

Melatonin is present naturally in the milk of lactating mammals, such as the dairy cow. Recent studies have shown that the level of naturally expressed melatonin in milk varies according to the time of day at which the milk is expressed, and this is thought to be related to the natural daily cycle 20 of melatonin secretion in the pineal gland. The level of melatonin in milk has also been shown to fluctuate according to variations in the length of daily light and dark periods throughout the year.

Until now this was thought to be no more than an interesting observation. However, it has now been realised that the level of naturally 25 expressed melatonin in milk can be manipulated, and reliably reproduced by

controlling the lighting conditions to which the lactating mammal is exposed.

The peak time for melatonin expression into the milk may thus be identified, and milk collected at this time kept separate from milk collected at other times during a controlled 24-hour cycle.

5 Furthermore, it is now believed that melatonin can be effective in assisting sleep in humans, even at levels well below what would be considered a medicinal or therapeutic dose. The theory behind this is that even a relatively small level of melatonin ingested by a person acts as a replacement therapy and may encourage the secretion of natural melatonin to in that person's own pineal gland, thus helping to induce sleep.

The present invention stems from the realizations that the level of naturally expressed melatonin in milk can be enhanced and reliably reproduced, and that such melatonin enriched milk can assist in encouraging sleep in a person who consumes that milk. The present invention is also t5 intended to provide a practical, efficient and reproducible method of obtaining such enriched milk.

According to the present invention, there is provided a method of producing milk having a concentration of naturally expressed melatonin in the range of from 15 to 30 pg/ml, which method comprises the steps of: 20 - selecting appropriate lactating mammals; - maintaining said mammals under conditions of controlled light intensity and duration, defining a dark period and a light period; - carrying out at least two milkings in a 24 hour period, one of said milkings being a dark milking carried out substantially at the end of the dark 25 period;

- 3 - maintaining separation between milk collected during the dark milking and milk collected during the other milkings; and - selecting the milk collected during the dark milking for processing and packaging.

5 Preferably, three milkings are carried out in a 24 hour period, one of said milkings being a dark milking carried out substantially at the end of the dark period. It is also preferred that the selected mammals are fed an increased daily dry matter intake.

The method of the present invention is equally applicable to any 10 lactating mammal that naturally expresses melatonin in its milk. However:,as cows are the primary source of milk for human consumption, it is highly preferred that the lactating mammals selected are cows. Further selection criteria may be used to choose animals with desirable milk production characteristics. For example, the selection of lactating mammals may be carried out on the basis of the age of the mammals, their breed, their stage of lactation, their history of lactation, their rate of melatonin production, or their milk yield.

The control of the light conditions of the mammals is essential, as the reproducibility of the concentration achieved in the milk is dependent on this.

20 Defining an average 8 to 10 hour dark period throughout the year contributes to consistent reproducible concentrations of melatonin. It has been found by experimentation that the light intensity during the dark period should preferably not exceed 50 lux, and that the average light intensity during the light period should preferably be at least 1000 lux.

- 4 At certain times of year natural light conditions will match the light conditions needed for the method according to the present invention, but at other times it will be necessary either to limit the availability of natural light or to supplement it with artificial light. When a natural light intensity remains below 1000 lux after the end of the dark period, artificial lights may be provided to supplement or replace that light. These can also be used to supplement the natural light before the end of the light period. At other times of year the night may be shorter than the required dark period, and therefore the light intensity during parts of the dark period may be too high.

10 Consequently,: the animals may be kept in a housing or loafing facility during the dark period, and shattering or screening may be employed to limit the light within that housing facility. It is preferred that the mammals are housed within a facility that is provided both with artificial lights and with shutters or screens to control the lighting conditions therein, thereby maintaining the 15 mammals in the correct light intensity for the correct periods.

Dairy mammals such as cows are routinely housed in structures such as cow houses or sheds, and allowed out to graze in pastures dependent, amongst other factors, on the time of year. Such existing housing may be adapted to permit the control of the light intensity therein by the provision of 20 lights and shutters or screened windows as appropriate. Mammals housed in such controlled conditions could still be allowed out to pasture, but the control of such release would need to ensure that the ambient light conditions in the pasture matched those of the appropriate dark/light period necessary for the method according to the present invention. Animals may 2s be housed for the duration of the dark period, or simply for those parts

thereof in which the ambient light exceeds the appropriate level. For example when operating under certified organic standards it is necessary to permit animals access to pasture between milkings. Therefore the animals might need to be housed at the beginning and end of the dark period, but 5 could be given free to access pasture during the hours of natural darkness.

It is highly preferred that the dark period is approximately 8 to 10 hours in length, with the light period being 14 to 16 hours. Studies have been made into the duration of the light and dark periods and they have shown that these optimise the reproducibility of the melatonin concentration lo in the milk. The dark period can of course run at any stage, but it is preferred: that it coincides as much as possible with the natural day/night cycle.

Therefore it is preferred that the dark period runs from approximately 9 pm to 5 am, as this most closely coincides with the appropriate natural light/dark cycles (when annual sunrises are averaged) and with conventional milking l5 routines.

It is highly preferred that the, or one of the, other milking(s) of the mammals is carried out at or shortly before the end of the light period. In this way milk produced in the dark, and containing melatonin is not diluted by secretion into a pre-existing pool of milk (in the udder) with low or no 20 melatonin content. If only one non-dark milking is carried out, this may conveniently be carried out at approximately 7 pm, with the dark milking carried out at approximately 5 am. At least one further milking may be carried out during the light period, making a total of three milkings in a 24 hour period. In such circumstances it is particularly convenient to carry out

- 6 the dark milking at 5am and the two other milkings at 1pm and 9pm respectively. To ensure that milk collected during the dark milking is not diluted by milk at other collection times, or indeed by milk from nonselected mammals, 5 it is essential that separation of such Dark milk is maintained throughout collection and onward processing. Therefore it is preferred that the milk collected during the dark milking is directed to a dark milk storage tank, and milk collected during the other milkings is instead directed to a normal milk storage tank.

10 Milk produced by the method according to the present invention is intended for human consumption and most particularly as a so-called "functional food". The properties of the milk depend upon the careful control of the melatonin concentration in the milk and the present invention allows the milk to be farmed in a practical commercial way which gives reproducible 15 results. Therefore it is desirable to monitor the concentration of melatonin in the milk during the further processing thereof.

According to the present invention there is also provided milk or milk products which are packaged for retail sale or distribution having a concentration of natural melatonin in the range of 15 to 30 pg/ml, and To produced by a method as described above.

In order that it may be better understood, but by way of example only, the present invention will now be described in more detail, with reference to the following examples.

EXAMPLE 1a

An example of how a dairy farm is set up to operate according to the present invention is discussed below, and it entails adopting a procedure along the following lines. The cows are housed in a cow house, which is 5 provided with suitable lights, shutters and light screens, but they also have controlled access to external pastures. The following simplified standard operating procedure describes those features of the process that are relevant to the present invention. Other standard actions carried out on dairy farms may have been omitted for clarity.

10 Cow Selection As - A group of cows are selected from the herd on the basis of their stage of lactation, their milk yield and their melatonin productivity. - These selected cows (hereinafter referred to as "night milk 15 cows") are kept at least partially separate from the remainder of the herd (other cows").

Milking Times - The night milk cows are milked 3 times a day at 5 am, 1 pm and 9pm. 20 - Any other cows are milked twice daily at 6 am and 4 pm Feeding - Night milk cows are fed a variable dry matter intake (DMI) and their body condition is monitored to ensure no loss of condition.

- Other cows are fed DMI of 21 kg per day.

- 8 Lighting and Housing Night milk cows are kept in conditions suited to ensuring a light level of not more than 50 lux between 9 pm and 5 am. During the summer months this requires them to be housed or in a 5 controlled loafing area during the hours between the 9 pm and 5 am milkings when the ambient light exceeds 50 lux..

- Lux level readings are taken at 9 pm and 5 am to monitor this.

- The light readings of the other cows are not taken.

- Standard day length information from the Royal Observatory 10 indicates that housing during some: part of the time between the 9 pm and 5 am GMT milkings will be necessary between 21st May and 22nd August.

The other cows are walked out to pasture as normal for the whole period.

Daily Routine At 5 am turn all lights on in the yard and cow house Take and record light meter readings Feed night milk cows Prepare collecting yard to receive night milk cows 20 - Collect night milk cows Scrape and re-bed cubicles Dampen milking parlour surfaces Set up parlour to receive night milk cows Change milk filter stock s - Ensure water is flushed from milk pipes

- 9 - - Check storage tanks are in cool mode - Direct pipes to night milk storage tank - Milk the night milk cows following a standard milking procedure - Feed other cows 5 - Collect other cows - Change milk feed pipe to normal milk storage tank(s) - Milk other cows - Return all cows to pasture or cubicles as appropriate - Wash parlour and milking equipment 10 - Perform 1 pm milking of night milk cows and ensure milk goes to normal milk storage tanks - Perform 4 pm milking of other cows and ensure milk goes to normal milk storage tanks - Feed cows as appropriate 15 - Perform 9 pm milking of night milk cows and ensure milk goes to normal milk storage tanks - Turn light off - Take and record light meter readings Ensure light intensity for night milk cows remains at the 20 appropriate level during the dark period between 9 pm and 5 am. (Use low intensity red lights if occasional lighting is required.)

- 10 EXAMPLE 1b

In an alternative embodiment, only two milkings of the night milk cows (one Dark" and one "non-dark") are carried out in a 24 hour period.

The operating procedure is the same as for example 1a above, except 5 that: - The night milk cows are milked only twice a day, at 5 am and 9 pm, there is no 1pm milking; - Night milk cows are kept in conditions suited to ensuring a light level of not more than 50 lux between 9 pm and 5 am, and during 10 the summer months are housed between these times for any period in which the ambient light intensity exceeds 50 lux; and - Lux levels are taken at 9 pm and 5 am to monitor this.

EXAMPLE 2

15 The previous examples indicate how the present invention can be carried out on a dairy farm that has been suitably modified. During the work carried out leading to the discovery of the present invention, tests were carried out to analyse the melatonin in milk produced by the cows.

20 General Points All cattle milked at dawn were acclimatized for two days prior to the commencement of the trial to obviate any effect of this novel milking time.

All samples taken were frozen within 30 minutes and maintained at -20 C until their melatonin content was assayed by radioimmunoassay.

The effect of time of day on meiatonin content of bovine milk and day to day reproducibility. Protocol Forty cattle were each milked before dawn, estimated time 03.30, with 5 the milking parlour and yard artificially lit to a minimum level considered necessary to comply with Health and Safety considerations. Samples were also taken from the same forty cattle during the afternoon milking on the same day.

This procedure was repeated three times on alternate days (Monday, 10 Wednesday and Friday). rt: The melatonin levels from these samples are shown in Table 1 and Figures 1 and 2.

Table 1. - Effect of time of day on melatonin levels (pn/ml) in bovine milk.

Day 1 Day 2 Day 3 am pm am pm am pm Average 18.3 5.8 18.1 3.7 19.1 3.9 SD 4.7 3.1 5.1 2.3 5.5 2.4

c.v. (%) 25.5 54.3 28.5 61.7 28.7 60.3 Statistical analysis by Single Factor Anova shows a significant difference between night time melatonin content of Morning" and Afternoon milk of p<0.001.

- 12 Statistical analysis by Single Factor Anova shows a significant difference between night time melatonin levels at a level of p<O.1.

Figure 1. - Effect of time of day on melatonin levels (Pu/ml) in bovine milk.

35 - _

3o-- <,, 25 - ii 0320- - c 15 I: 5-. O- ' ' ' 1 ' I ' I. .. ..

0 2 4 6 8 10 12 14 16

5 Clock time The effect of time within and between lactations on melatonin content of bovine milk Protocol 10 50ml milk samples were collected before dawn and at the afternoon milking from four groups, each of ten cattle, as detailed below.

Group Lactation Time within lactation A 1st Start B 1 st End 15 C 5th Start 1:) 5th End

- 13 The milk melatonin levels from these samples are shown in Table 2 and Figure 2.

Table 2. The effect of lactation on melatonin levels (pn/ml) in bovine milk Group Lactation Phase Morning" milk "Afternoon milk" Mean SD Mean SD A 1 st Start 18.4 5.0 5.8 2.0 B 1st End 19.0 4.6 7.3 4.0 C 5th Start 18. 4 5.6 7.3 3.1 D 5th End 16.4 3.3 5.3 1.8 Finure 2. The effect of lactation on melatonin levels (pn/ml) in bovine milk 35 - __

- !: o 0 1 2 3 4 5 6 7 8

Bovine milk melatonin levels (pg/ml) 1 Start of 1st lactation - am 2 End of 1st lactation - am lo 3 Start of 5th lactation - am 4 End of 5th lactation - am 5 Start of 1st lactation - pm

- 14 6 End of 1st lactation - pm 7 Start of 5th lactation - pm 8 End of 5th lactation - pm 5 Statistical analysis by Single Factor Anova failed to show any significant differences in milk melatonin levels for either morning" or "afternoon" milk samples associated with start or end lactation period or early or late lactation.

10 The effect of exposure to a period of light between dawn and milking A group of ten cows at the start of their first lactation were milked at their normal morning milking time, between 0600 and 0700. At this time of year, the cows would have been exposed to approximately 2 hours light between dawn and milking.

Is The milk melatonin levels in this group were 13.1+ 5.7pg/ml which when compared with the levels obtained from milk collected before dawn (18.4 + 4.9pg/ml) and tested statistically using Single Factor Anova produced a difference significant at p<O.05. The results are shown in Figure 3.

- 15 Figure 3. - Bovine milk melatonin levels (pq/ml! before and approximately 2 hours after dawn.

15- O- 1 1

0 1 2 14. Group 1 Milked before dawn Group 2 Milked approximately 2 hours after dawn Conclusions

1. Bovine milk contains higher levels of melatonin in the morning than in the afternoon/evening.

to 2. Levels of melatonin in bovine milk obtained from the same animal are reproducible from day to day. However, considerable inter-individual variation is indicated by the coefficients of variation of between 25 and 30% for nighttime milk melatonin levels.

Of 40 cows assessed, 16 (40%) had night time melatonin levels of 20pg/ml or higher and a further 14 (35%) had levels between 15 and 20pg/ml.

- 16 3. Bovine milk melatonin levels do not vary significantly between the start and end of a lactation period or between the 1st and 5th lactation although there was a trend towards lower levels at the end of the 5th lactation. 5 4. Exposure to natural light for approximately 2 hours after dawn reduces melatonin levels in the milk. It is therefore necessary to milk before dawn or within a short time after dawn to maximise melatonin levels.

5. The option exists of darkening the cowshed on summer mornings, but in order not to compromise fertility, extra light would be needed in the 10 evening so as not to lengthen the dark phase.

Methodolonv Melatonin Extraction procedure 1. Thaw milk samples and shake on a mechanical shaker for 5 minutes I5 to disperse the fat content; 2. Dispense 2ml aliquots of milk into glass tubes; 3. Add 10ml chloroform, Analar grade and shake gently for 10 minutes.

Vigorously shaking at this point will result in the production of an emulsion; 20 4. Centrifuge at 3000rpm for 10 minutes; 5. Remove the upper, aqueous phase by aspiration; 6. Tip the lower, organic phase into a clean dry glass tube ensuring that no residual aqueous phase is transferred; 7. Evaporate the organic phase to dryness at 40 C under a flow of 25 nitrogen;

- 17 8. Reconstitute residue with 2ml tricine assay buffer; 9. Subject 2 x 0.5ml aliquots to analysis by radioimmunoassay as detailed in Assay Protocol.

5 Assay Protocol 1. Pipette standards and buffer to form standard curve; 2. Add 500,u1 saliva samples to assay tubes; 3. Add 1001 of diluted antiserum to all tubes except totals and NSBs (non specific binding). Vortex and incubate at room temperature for 10 30 minutes.: 4. Add 1001 4251-melatonin and vortex. Incubate at 4 C for 15 - 18 hours. (If a solid phase separation is used this incubation time can be decreased to 4 hours at room temperature) EITHER:

15 5. Solid phase separation system. Add 1001 solid phase double antibody and incubate for 1 hour at room temperature with intermittent mixing. Add 1 ml Brij / saline wash and centrifuge at room temperature at 1 50Og for 10 minutes.

OR 20 5. Liquid phase separation system. Add 100111 diluted double antibody, 1001 diluted normal rabbit serum and 500 Ill 6% PEG solution. Mix and incubate at 4 C for 4 hours. Centrifuge at 300Og for 20 minutes.

6. Decant over a mesh and discard supernatant. This must be done immediately after centrifugation. (An aluminium or Teflon (RTM) 25 covered mesh is placed over the rack of tubes, and the whole carefully

- 18 inverted over a sink to remove supernatant. The whole assembly is then blotted with absorbent paper to remove final drips before righting the rack).

7. Count the pellet in an appropriate gamma radiation counter.

5 Determine the melatonin concentration from the dose response curve.

Reanents All water used is freshly double glass distilled water (DGDW).

Buffer: tricine (Sigma Ltd product no. TQ377) is made up at 0.1M, pH 8.0 10 with 0.9% NaCI and 0.2% gelatin. Heat to 50 C for 30 minutes to dissolve the gelatin.

17.99 tricine 9.Og NaCI) to 11 with DGDW 2.0g gelatin) 5 Bring to pH 8.0 with 1 M NaOH Buffer is stored at 4 C and made up fresh weekly.

Antiserum: rabbit anti-melatonin antiserum (code no. R/R/19540-16876) is supplied freeze dried.

AB/R/03 - 1331 Of a 1/10 dilution (sufficient for 150 assay tubes).

20 The contents of the vial should be reconstituted with 2ml DGDW and 50111 aliquots stored at -20 C. The working dilution sufficient for 100 assay tubes is prepared by diluting a 50,u1 aliquot for 1 Oml with assay buffer.

AB/R/031 - 50,ul of a 1/100 dilution (sufficient for 150 assay tubes).

The contents of the vial should be reconstituted with 15ml assay buffer and 25 are sufficient to add 100,1 to each of 150 assay tubes.

- 19 These working dilutions were prepared fresh daily. Antiserum dilution curves were performed as appropriate.

251-melatonin tracer: this tracer is available from Amersham International Double antibody separation system 5 a) solid phase separation system i) A second antibody raised against rabbit IgG and linked to a suitable solid phase.

ii) Brij / saline wash solution: dissolve 9g NaCI in 998ml DGDW and add 2ml Brij 35.

10 b) liquid phase separation system i) normal rabbit serum diluted 1:200 in assay buffer ii) donkey anti-rabbit IgG suitably diluted in assay buffer iii) 6% polyethylene glycol 6000 SPEW in DGDW.

Standards: melatonin (Sigma Ltd product no. M5250) stock solution is made 15 up at 1mg/ml by dissolving 10mg melatonin in 0.5ml absolute ethanol and adjusting the volume to 10ml with DGDW. This solution is stable for at least a year at 4 C. The working standard is freshly prepared daily from this ethanolic stock as follows: 100111 (1mg/ml) to 100ml in DGDW = 1pg/ml 20 500111 (1,ug/ml) to 50ml in DGDW = 10ng/ml 501 (10ng/ml) to 2. 5ml in assay buffer = 0.2ng/ml Further dilutions with assay buffer provide a standard curve as follows:

- 20 MT standard Assay buffer MT pa / tube MT pu / ml 0.2nq/ml _ 0 500 0 0

5 495 1 2

10 490 2 4

25 475 5 10

50 450 10 20

125 375 25 50

250 250 50 100

500 100 200

The standards are treated in exactly the same way as the milk samples in the assay.

Claims (15)

-21 CLAI MS

1. A method of producing milk having a concentration of naturally expressed melatonin in the range of from 15 to 30 pg/ml, which method comprises the steps of: - selecting appropriate lactating mammals; maintaining said mammals under conditions of controlled light intensity and duration, defining a dark period and a light period; - carrying out at least two milkings in a 24 hour period, one of said 10 milkings being a dark milking carried out substantially at the end of the dark period; maintaining separation between milk collected during the dark milking and milk collected during the other milking(s); and - selecting the milk collected during the dark milking for processing 5 and packaging.

2. A method as claimed in claim 1, wherein three milkings are carried out in a 24 hour period.

3. A method as claimed in claim 1 or claim 2, wherein the lactating mammals are cows.

20

4. A method as claimed in any of the preceding claims, wherein the light intensity during the dark period does not exceed 50 lux.

5. A method as claimed in any of the preceding claims, wherein the average light intensity during the light period is 1000 lux.

- 22

6. A method as claimed in any of the preceding claims, wherein the lighting conditions are controlled with the assistance of artificial lights and shutters.

7. A method as claimed in any of the preceding claims, wherein the dark 5 period is approximately 8 to 10 hours in length.

8. A method as claimed in 7, wherein the dark period runs from approximately 9 pm to 5 am.

9. A method as claimed in any of the preceding claims, wherein two non dark milkings are carried out, one 8 to 10 hours after the end of the dark to milking, the other at the end of the light period.

10. A method as claimed in any of claims 7 to 9, wherein the dark milking is carried out at 5am and the two other milkings are carried out at 1pm and 9pm.

11. A method as claimed in any of the preceding claims, wherein the milk 75 collected during the dark milking is directed to a dark milk tank, and milk collected during the other milkings is directed to a normal milk tank.

12. A method as claimed in any of the preceding claims, wherein the processing of the dark milk includes monitoring the concentration of melatonin in the milk.

20

13. A method as claimed in any of the preceding claims, wherein the selection of lactating mammals is carried out on the basis of at least one of the age of the mammals, their stage of lactation, their milk yield and their natural level of melatonin expression.

14. A method of producing milk as claimed in claim 1 and substantially as 25 herein described with reference to the accompanying examples.

- 23

15. Milk or milk products packaged for retail sale or distribution having a concentration of natural melatonin in the range of 15 to 30 pg/ml, and produced by a method according to any of the preceding claims.