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Research ArticleReceived: 5 February 2013 Revised: 4 September 2013 Accepted article published: 14 November 2013 Published online in Wiley Online Library: 12 December 2013

(wileyonlinelibrary.com) DOI 10.1002/jsfa.6480

Night milking adds value to cow’s milkMaria P Milagres,a∗ Valeria P R Minim,b Luis A Minim,b Andrea A Simiqueli,b

Liliane E S Moraesb and Hercia S D Martinoc

Abstract

BACKGROUND: Melatonin is synthesized in greater concentration at night, and it plays an important role in sleep regulation.This study aimed to evaluate the melatonin concentration in milk collected by milking during the night and evaluates its effect,with or without tryptophan supplementation, in the sleep quality of adult Wistar rats.

RESULTS: A difference (P < 0.05) was observed between the milking times, where the milk obtained at 02:00 presented a highermelatonin concentration (39.43 pg mL−1) than that acquired at 15:00 (4.03 pg mL−1). A biological assay was also performed on32 male adult Wistar rats distributed among four groups (n = 8): those receiving an AIN-93M diet (control group) and threetest groups [diets containing milk from milking at 02:00 (M2h), milking at 15:00 (M15h), and milking at 02:00 plus tryptophansupplementation (M2hT)] for 28 days. It was observed that the control group did not differ (P > 0.05) from the M15h group interms of the levels of blood melatonin and urinary sulfatoxymelatonin, but differed from groups M2h and M2hT, whereas groupM2hT presented higher blood melatonin and urinary sulfatoxymelatonin concentrations.

CONCLUSION: Combining the techniques of night milking with tryptophan supplementation resulted in production of milk thatimproves sleep quality in rats.c© 2013 Society of Chemical Industry

Keywords: melatonin; sleep; milk; tryptophan

INTRODUCTIONMelatonin is responsible for circadian regulation and the controlof sleep, encountered in the organism at highest concentrationsduring the night.1 With aging, there is a substantial reduction inmelatonin production, which may impair sleep quality among theelderly.2

Melatonin is naturally present in many foods including cherry,banana, peppermint, red wine, and milk.3 It is not considered asa food additive by the Harmonized General List of Food Additivesand their Functional Classes of the Mercosur,4 thus not permittingit to be included as food.

Cow’s milk contains roughly 5 pg mL−1 of melatonin, and studieshave focused on naturally increasing the melatonin concentrationby using different milking techniques. Valtonen et al.5 showedthat it is possible to increase the concentration of melatonin upto 56.4 pg mL−1 with changes in the photoperiod of cows; thatis, by increasing the period in which the animals remain in thedark for 17 h. Haigh6 increased the concentration of melatonin inmilk to 35 pg mL−1 by night milking, with maximum illuminationof 50 lux; Gnann7 developed a method for naturally increasingmelatonin in milk by night milking animals exposed to light atdifferent wavelengths during the day and at night.

Tryptophan is an essential amino acid, a precursor of melatoninsynthesis by the pineal gland, and acts to improve sleep andreduce stress and antioxidant effects. Tormo et al.8 and Sanchezet al.9 demonstrated that it is possible to increase the concentrationof plasma melatonin by tryptophan administration. However, nostudies in the literature have described an increase in plasmamelatonin concentration because of the addition of tryptophanto milk.

Also, no studies have been reported on the concentration ofmelatonin in the milk of cows in Brazil. However, it is known thatmelatonin concentration in milk varies with the breed, nutrition,stress level of the animals, and ambient temperature.10 Thiswork aimed to evaluate the melatonin concentration in milkcollected by night milking and to evaluate its effect, with ourwithout tryptophan supplementation, in the sleep quality of adultWistar rats

MATERIAL AND METHODSMilking of the animalsFor milking, 10 Holstein cows (age range, 3–7 years; averageweight, 580 kg; average milk production, 25 L day−1; and lactationperiod, 100–150 days after calving), located at the dairy house ofthe Federal University of Vicosa, Brazil, were randomly selected forthe study. The experiment was conducted in randomized blocks,with each cow representing one block.

∗ Correspondence to: Maria P Milagres, Department of Chemistry, State Universityof Southwest Bahia, Jequiezinho, Jequie - BA, 45206-190, Brazil. E-mail:patricia@uesb.edu.br

a Department of Chemistry, State University of Southwest Bahia, Brazil

b Department of Food Technology, Federal University of Vicosa, Av. P. H. Rolfs,Vicosa, Minas Gerais, Brazil

c Department of Nutrition and Health, Federal University of Vicosa, Vicosa, MinasGerais, Brazil

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Table 1. Composition of the cows’ diet

Component Amount (g kg−1)

Corn silage 714.2

Cornmeal 130.0

Soybean meal 136.9

Vitamin and mineral mixture 18.7

The animals’ diet was maintained as per the routine of the dairystable, with corn silage and feed supplied ad libitum (Table 1).Milking was performed at two different times: 02:00 and 15:00,one sample per animal at a time for 15 days (2–16 June 2011),totaling 300 samples. A 5-day adaptation period was kept beforesample collection to reduce the stress level from the new scheduleof milking, which could influence melatonin production. Duringthis period, the animals were milked at 02:00 and 15:00, but the milkobtained was not analyzed. Therefore, the experimental periodwas of 10 days. During the experiment, the animals were milkedat 02:00 and 15:00 and the melatonin concentration was analyzed.After milking, the samples were collected and immediately frozenat −16◦C until analyses.

Determination of melatonin concentration in the milksamplesDetermination of melatonin concentration in the milk wasperformed according to the methodology of Kollmann et al.11

Briefly, the fat was extracted from the milk samples obtained fromeach cow at each milking time by centrifugation at 3300 × g for 15min at 4◦C. Next, the defatted phase obtained after centrifugationwas diluted to 1:10 with Standard A contained in the analyticalkit (IBL International, Hamburg, Germany) and referenced as 0pg mL−1, following which the enzyme immunoassay for directdetermination and quantification of melatonin was performed(RE54041; IBL International, Hamburg, Germany).

Development of the formulations and physicochemicaland microbiological analyses.For the production of formulations M15h and M2h, milk wascollected at 15:00 and 2:00, respectively; the samples were thenskimmed to 3 g kg−1 fat, heat treated at 75◦C for 5 s, and lyophilized.The skimmed and pasteurized milk samples were weighed, placedon stainless-steel trays, and subjected to a temperature of −35◦Cfor 3 days to ensure complete freezing. Next, they were transferredto a semi-industrial lyophilizer (Terroni model 0500 LH; Sao Paulo,Brazil), whose condenser temperature varied between −50◦C and−55◦C for a period of 24 h.

For the production of M2hT, milk collected at 2:00 wasskimmed to 3 g kg−1 fat, heated to 40◦C, supplemented with2.5 g L−1 tryptophan, then subjected to heat at 75◦C for 5 s. TheM2hT formulation was then lyophilized according to the samemethodology as described for the formulations M15h and M2.

The amount of tryptophan used in the formulation M2hT wasbased on the study performed by Sanchez et al.,9 who showed thatit is possible to increase the concentration of plasma melatonin inWistar rats by administering 125 mg day−1 tryptophan.

Skimming of all milk formulations was required to not exceed thefat content in the experimental diet of Wistar rats, as establishedby AIN-93 M.12 This was followed by heat treatment of the milk at75◦C for 5 s.

Table 2. Composition of the experimental diets

Milk sample

Componenta(g kg−1) Control M15h M2h M2hT

Casein (85%) 140.0 70.0 70.0 70.0

Milk powder 0.00 274.5 274.5 274.5

Dextrinized starch 155.0 155.0 155.0 155.0

Sucrose 100.0 100.0 100.0 100.0

Soybean oil 40.0 30.0 30.0 30.0

Fiber (cellulose) 50.0 50.0 50.0 50.0

Saline mixture 35.0 35.0 35.0 35.0

Vitamin mixture 10.0 10.0 10.0 10.0

Cystine 1.8 1.8 1.8 1.8

Choline bitartrate 2.5 2.5 2.5 2.5

Starch 465.7 271.2 271.2 271.2

The control was the AIN, 93M diet.M15h, milk collected from milking performed at 15 h 00 min; M2h, milkcollected from milking performed at 2 h 00 min; M2hT, milk collectedfrom milking performed at 2 h 00 min, supplemented with tryptophan.a The amount is given as g kg−1 except for casein, which is in %.

The formulations were submitted to physicochemical andmicrobiological analyses in order to define the proximatecomposition and to verify food safety. The parameters determinedwere concentrations of fat,13 titratable acidity,14 density,13

protein,13 freezing point,14 standard plate count,14 coliform countat 35◦C XXXXX (MPN) mL−1,14 and coliform count at 45◦C MostProbable Number mL−1.14

Biological assayThe experiment was conducted after approval by the EthicsCommittee for Animal Experimentation of the Federal Universityof Minas Gerais (UFMG-CETEA), according to protocol 026/2010.

A biological assay was performed with 32 male adult Wistar rats(Rattus norvegicus var. albinus, class Rodentia) from the CentralBiotherium of the Center for Biological Sciences and Health,Federal University of Vicosa, Vicosa, Brazil. The animals werepurchased early, weaned, and maintained in individual cages in anacclimatized environment. They received commercial chow untilreaching the adult stage, that is age of 65 days and weight of300–350 g.

During the experimental phase, the animals were maintainedin individual cages in a temperature-controlled environment of22 ± 2◦C, 12-h photoperiod, and ad libitum supply of a daily dietand distilled water. They were divided into four groups of eightanimals each and fed the experimental diets for a period of 28 days(Table 2).

The effects of melatonin concentration in milk with or withouttryptophan supplementation on the sleep quality of adults Wistarrats were evaluated in four groups of animals who received thefollowing diet: AIN-93 M diet (control group) and three test groups[diets containing milk from milking at 02:00 (M2h), milking at 15:00(M15h), and milking at 02:00 plus tryptophan supplementation(M2hT) for 28 days. The composition of the diet was based on therecommendation of the American Institute of Nutrition for rodents(AIN-93 M), according to Reeves et al.12 The test groups receivedlyophilized milk formulation, which replaced 50% of proteinprovided by the AIN-93 M diet. The contents of carbohydrate andlipid provided by the lyophilized milk formulation were calculatedto balance the ingredients provided by the AIN-93 M diet (Table 2).

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Table 3. Analysis of variance of the melatonin concentration in milkfrom milking animals (randomized block design)

Source of variation Degrees of freedom Square average Pr > F

Cows 9 10.7146 0.5617

Day 9 17.7306 0.1885

Time (in hours) 1 64029.0269 < 0.0001

Cows × day 81 15.9031 0.1270

Cows × time (in hours) 9 8.9629 0.6882

Pr, probability level.

Table 4. Physicochemical characteristics of the milk samples atdifferent times for milk supplemented or not with tryptophan

Milk sample

Characteristica M15ha M2h

a M2hTa

Fat (g kg−1) 30.0 32.0 32.0

Relative density 15◦C (g mL−1) 1.03 1.03 1.03

Titratable acidity (g kg−1) 1.6 1.5 1.5

Protein (g kg−1) 31.0 32.0 32.0

Melatonin (pg mL−1) 4.20 36.72 36.23

a Means of the sample used to prepare the test diet.Abbreviations are as in the footnote to Table 2.

The ingredients were individually weighed and mixed in a quasi-industrial mixer (Lieme, Sao Paulo, Brazil) at low rotation over 15min. The diets were stored in polyethylene bags under refrigerationat 10◦C until the animals were fed.

The dietary intake of the animals was evaluated by recordingthe daily feed intake, and weight gain was obtained from thedifference between the initial and final weights of the animals. Thefeed efficiency ratio was determined from the ratio of the weightgain of the animal and the consumption of the experimental diet.

At 28 days of testing, the animals were transferred to individualmetabolic cages constructed of stainless steel in order tocollect urine over a 48-h period from each animal for analysisof 6-sulfatoxymelatonin. Analysis of 6-sulfatoxymelatonin wasperformed according to the enzyme immunoassay (RE54031; IBLInternational).

At the end of the experiment, the animals were euthanized inan atmosphere of carbon dioxide according to Resolution 714 ofthe Federal Council of Veterinary Medicine, followed by makingan incision along the abdominal and thoracic cavities for bloodcollection by cardiac puncture.

Analysis of melatonin in the blood of all animals of the fourgroups (M15h, M2h, M2hT and control) was performed using theenzyme immunoassay for quantitative determination via an invitro diagnosis of melatonin in serum and plasma (RE54021; IBLInternational).

Statistical analysisThe experimental design was performed in blocks, with fourtreatments and eight replications, so that the average initialweights of the animals were similar. The data of the initial and finalweight, weight gain and feed, milk and melatonin intake, urinarysulfatoxymelatonin levels, and melatonin concentration in plasmaby the animals were analyzed by analysis of variance (ANOVA)

Table 5. Microbial counts of the milk samples, supplemented or notwith tryptophan, at different times of milking

Milk sample

Microbial counta M15ha M2h

a M2hTa

Standard plate count (cfu mL−1) 4.7 × 103 2.0 × 103 8.7 × 103

Coliform count at 35◦C (MPN mL−1) < 0.3 < 0.3 < 0.3

Coliform count at 45◦C (MPN mL−1) < 0.3 < 0.3 < 0.3

a Means of the samples used to prepare test diet.cfu, colony-forming units; MPN, Most Probable Number. Otherabbreviations are as in the footnote to Table 2.

(α = 5%). For a significant F value, a post-hoc Tukey test was per-formed with a probability of 5% in order to compare the averagesof the four groups (diet M15h, diet M2h, diet M2hT, and control). Themean dispersion was expressed as standard deviation. The sta-tistical analysis was performed using Statistical Analysis Systems(SAS), version 9.1, licensed to the Federal University of Vicosa.

RESULTS AND DISCUSSIONEffect of milking time on the concentration of melatoninin cows’ milkTable 3 presents a summary of the ANOVA for melatoninconcentration in milk. It is observed that the interactions betweencow × day and cow × time were not significant (P > 0.05). Therewas also no significant difference in the concentration of melatoninin the milk when the variation factors assessed were cow and day.However, there was a significant difference (P < 0.05) betweenmilking times.

Although melatonin production is influenced by factors suchas genetics and psychological state, the animals presentedsimilar concentrations of melatonin production. There was asignificant difference (P < 0.05) between the milking times,where the milk obtained at 2:00 presented a higher melatonincontent (39.43 pg mL−1) as compared to that acquired at 15:00(4.03 pg mL−1). These results are consistent with those foundby Eriksson et al.,15 who observed an increase in the melatoninconcentration in milk from cows milked at night.

The proposed technique is a methodology that permits asignificant increase in the concentration of melatonin in milkwithout major investments, modifying only the time of milking,unlike in the methodologies presented by Valtonen et al.,5 Haigh6

and Gnann,7 which involve the use of artificial lighting andproduction in confinements. Because a large portion of milkproduction in Brazil occurs under semi-extensive managementconditions, the methodologies for producing milk with a highmelatonin concentration as proposed by Valtonen et al.,5 Haigh6

and Gnann7 are not viable for rural Brazilian producers dueto the large investments needed for the utilization of thesetechniques. Thus, the use of a night milking technique maybe a viable alternative for Brazilian milk producers to increasethe concentration of melatonin in milk while maintaining theeconomic and productive conditions of this sector in Brazil.

Physicochemical and microbiological compositions of themilk samplesPhysicochemical compositions of the milk samples used in theexperiment are shown in Table 4, which is in accordancewith the Technical Regulation of Milk Production, Identity and

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Table 6. Initial and final weight of the animals, weight gain and consumption of the diet, milk and melatonin of animals fed with the experimentaldiets

Weight Consumption

Diet Initial (g) Final (g) Gain (g) Diet (g) Milk (g) Melatonin (pg)

Control (Casein) 330.14a 421.43a 91.29a 947.48a 260.08a < 0.50a

M15h 337.66a 397.76b 60.10b 932.27a 255.91a 6.82b

M2h 332.16a 404.32b 72.16b 903.89a 248.12a 58.73c

M2hT 339.28a 406.83b 67.55b 915.95a 251.43a 56.93c

Results were obtained after 28 days of experimentation with rats (N = 8 animals in each group) fed the following diets: control, the AIN-93 M diet;M15h, milk collected from milking performed at 15 h 00 min; M2h, milk collected from milking performed at 2 h 00 min; M2hT, milk collected frommilking performed at 2 h 00 min, supplemented with tryptophan.The Tukey test was used to compare the experimental groups. For each evaluated characteristic, means followed by the same superscript letter inthe column are not significantly different at 5% probability.

Quality of the Ministry of Agriculture – Normative Instruction n.62/2011.16 Microbiological characterization of the samples is alsoin accordance with the referred Normative Instruction (Table 5).

Biological assayEffects of treatments on body weight and food consumptionData related to weight gain and feed consumption of the rats areshown in Table 6. Animals in the control group, which received thecasein diet, presented higher final body weight than the animals ofthe test groups (P < 0.05), although no significant difference wasfound between their feed consumption. The ability of melatonin toreduce body weight gain was also observed by Rasmussen et al.17

in experiments with middle-aged rats.

Effects of treatments on melatonin levelsAccording to Benloucif et al.,18 plasma melatonin is the moststable form of melatonin and is most highly correlated with sleeptime. Therefore, the melatonin levels were analyzed to determinethe sleep quality of adult Wistar rats. The melatonin levels inthe blood (P > 0.05) did not differ between the M15h group andthe control group. Therefore, the milk acquired at 15:00 didnot have increased blood melatonin. However, the M2h groupincreased the levels of melatonin (P < 0.05) as compared to theM15h group by 26.5%. The milking at 02:00 (M2h) increased theconcentration of melatonin in the plasma by 26.5%. The additionof tryptophan to formulation M2h increased its melatonin levels(P < 0.05) by 35.5% as compared to the M2h group (Table 7).

Increased intake of tryptophan elevated the concentration ofplasma melatonin. Other authors have also reported higher levelsof blood melatonin in rats after tryptophan ingestion. Tormo et al.8

observed increased plasma melatonin after oral administration of125 mg day−1 of tryptophan in rats. Sanches et al.9 analyzedmelatonin concentrations over a period of 24 h in rats after oraladministration of 125 mg day−1 of tryptophan and found anincrease in blood melatonin concentration in the animals. Theformulations M2h and M2hT can improve the sleep time becausethey are good sources of melatonin.

Effects of treatments on urinary sulfatoxymelatonin levelsAccording to Wulff et al.,19 collection of urine for analysis of 6-sulfatoxymelatonin is an alternative to the use of actigraphy for thecontrol of circadian rhythm. Therefore, urinary sulfatoxymelatoninlevels were analyzed to determinate the sleep quality. The milk

Table 7. Plasma melatonin levels of rats treated with milk,supplemented or not with tryptophan, at different times

Group Average melatonin concentration (pg mL−1)

Control 30.83a

M15h 35.04a

M2h 44.36b

M2hT 60.12c

Results were obtained after 28 days of experimentation with rats(N = 8 animals in each group) fed the following diets: control, theAIN-93 M diet; M15h, milk collected from milking performed at 15 h 00min; M2h, milk collected from milking performed at 2 h 00 min; M2hT,milk collected from milking performed at 2 h 00 min, supplementedwith tryptophan.The Tukey test was used to compare the experimental groups. Foreach evaluated characteristic, means followed by the same superscriptletter in the column are not significantly different at 5% probability.

Table 8. Urinary sulfatoxymelatonin levels of rats treated with milk,supplemented or not with tryptophan, at different times

Group Average urinary sulfatoxymelatonin (pg mL−1)

Control 50.22a

M15h 60.13a

M2h 78.20b

M2hT 95.76c

Results were obtained after 30 days of experimentation with rats (N = 8animals in each group) fed the following diets: control, the AIN-93 Mdiet; MS15h, milk collected from milking performed at 15 h 00 min;M2h, milk collected from milking performed at 2 h 00 min; M2hT, milkcollected from milking performed at 2 h 00 min, supplemented withtryptophan.The Tukey test was used to compare the experimental groups. Foreach evaluated characteristic, means followed by the same superscriptletter in the column are not significantly different at 5% probability.

acquired at 15:00 did not have increased urinary sulfatoxymela-tonin levels (P > 0.05). However, the M2h and M2hT groups showedincreased excretion levels of urinary sulfatoxymelatonin in 55.7and 90.68%, respectively, compared to the control group. Thus,urinary sulfatoxymelatonin is a good marker of sleep time (Table 8).

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CONCLUSIONThe night milking technique increases melatonin concentrationsin milk. Consumption of formulations using milk obtainedat night resulted in increased plasma melatonin and urinarysulfatoxymelatonin levels of rats. Milk obtained at night milkingand supplemented with tryptophan was most efficient inincreasing blood melatonin and urinary sulfatoxymelatonin levelsin Wistar rats. The combination of the night milking techniquewith tryptophan supplementation resulted in a natural productthat improves sleep quality.

ACKNOWLEDGEMENTThe authors are grateful to the Foundation for Research Supportof the State of Minas Gerais and Coordination of Improvement ofHigher Education Personnel for financial support.

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