Research Article Received: 10 February 2014
Revised: 10 June 2014
Accepted article published: 9 October 2014
Published online in Wiley Online Library: 20 November 2014
(wileyonlinelibrary.com) DOI 10.1002/jsfa.6947
Influence of stage of lactation and year season on composition of mares’ colostrum and milk and method and time of storage on vitamin C content in mares’ milk a* Grazyna b ̇ ̇ Maria Markiewicz-Keszycka, ¸ Czyzak-Runowska, d Jacek Wójtowski,b Artur Jó´zwik,c Radosław Pankiewicz,d Bogusława Łeska, ¸ c Nina Strzałkowska,c Joanna Marchewkae ̇ Józef Krzyzewski, and Emilia Bagnickac
Abstract BACKGROUND: Mares’ milk is becoming increasingly popular in Western Europe. This study was thus aimed at investigating the impact of stage of lactation and season on chemical composition, somatic cell count and some physicochemical parameters of mares’ colostrum and milk, and at developing a method for the determination of vitamin C (ascorbic acid) in mares’ milk and to determine its content in fresh and stored milk. RESULTS: The analysis conducted showed an effect of the stage of lactation on contents of selected chemical components and physicochemical parameters of mares’ milk. In successive lactation periods levels of fat, cholesterol, energy value, citric acid and titratable acidity decreased, whereas levels of lactose and vitamin C, as well as the freezing point, increased. Analysis showed that milk produced in autumn (September, October, November) had a higher freezing point and lower concentrations of total solids, protein, fat, cholesterol, citric acid and energy value in comparison to milk produced in summer (June, July, August). Mares’ milk was characterised by low somatic cell count throughout lactation. In terms of vitamin C stability the most advantageous method of milk storage was 6-month storage of lyophilised milk. CONCLUSION: In general, the results confirmed that mares’ milk is a raw material with a unique chemical composition different from that produced by other farm animals. © 2014 Society of Chemical Industry Keywords: mares’ milk; chemical composition; stage of lactation; seasonal variation; method of storage
INTRODUCTION
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∗
Correspondence to: Maria Markiewicz-Ke¸ szycka, School of Agriculture and Food Science, University College Dublin, Belfield, Dublin 4, Ireland. E-mail: [email protected]
a School of Agriculture and Food Science, University College Dublin, Dublin 4, Ireland b Department of Small Mammals Breeding and Raw Materials of Animal Origin, Poznan University of Life Sciences, 62-002, Suchy Las, Poland c Department of Animal Sciences, Institute of Genetics and Animal Breeding of the Polish Academy of Sciences, 05-552, Magdalenka, Poland d Faculty of Chemistry, Adam Mickiewicz University, 61-614, Pozna´n, Poland e Animal Production Department, Neiker-Tecnalia, Arkaute Agrifood Campus, E-01080, Vitoria-Gasteiz, Spain
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Mares’ milk is characterised by unique chemical composition as compared to the milk of other farm animal species. It is comprised of approximately 1% fat, 2% protein and 7% lactose, and is a source of bioactive components such as lysozyme, lactoferrin, immunoglobulin, long-chain n-3 fatty acids and fat-soluble vitamins.1 – 11 Despite its potential, mares’ milk is a niche raw material, difficult to obtain for large-scale production. One of the factors limiting mares’ milk production is the seasonality of reproduction in mares, and for this reason its preservation and storage are required. Owing to its content of biologically active substances, the preservation methods most frequently applied for this raw material include freezing and freeze drying, i.e. lyophilisation.12,13 It is commonly known that freezing and lyophilisation are optimal methods of food preservation, causing minimal changes to the original properties. Freezing is the oldest and most readily available method of raw material storage. For this reason mares’ milk is most frequently
stored in frozen form, although it may be troublesome from a practical point of view, especially with respect to its further distribution.14 Milk lyophilisation may prove to be a much easier
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method applicable to extended milk storage. Lyophilisation guarantees complete freedom in the management of the raw material, including its storage. This process involves the removal of water from frozen material by ice sublimation, i.e. its direct transition to the vapour state, bypassing the liquid state. The obtained product is characterised by high quality, with a minimised risk of adverse reactions connected with oxidation or microbial activity. This process makes it possible to maintain much higher product quality in comparison to other drying methods; however, its high cost needs to be stressed here.13 Quality and stability of mares’ milk lyophilisate are determined by its content of water and its activity.15 There is no information in the available scientific literature on changes in the contents of biologically active components, particularly vitamin C, during milk storage. The available literature contains no information on properties of mares’ milk subjected to freezing and lyophilisation. It results only from a study by Gruda and Postolski16 concerning cows’ milk where milk freezing is rarely used, since this process has a negative effect on its quality. The main problem is connected with low stability of casein, which, after only 3 weeks of storage at a temperature range from −8 to −12 ∘ C, forms a flaky precipitate. Owing to the seasonality of reproduction in mares, their milk is subjected to significant variations related to the season (probably the feeding system and lactation stage) and thus the milking season has a significant effect on milk composition.4 – 6,17 This issue is not as visible in milk of dairy cows, which undergoes only slight seasonal changes, while cows calve all year round. There are no reports available in the scientific literature on the effect of year season on the nutritive value and somatic cell count (SCC) in mares’ milk. There is also a very limited number of studies on chemical composition of mares’ milk throughout 180 days of lactation.5,7,17,18 There is even less information on SCC, vitamin C, cholesterol content and physicochemical parameters of mares’ milk at different stages of lactation.9,18 – 22 The vast majority of information concerns the composition of mares’ milk in the period of the most intensive growth of foals.1,2,7,23 There is a very limited number of studies conducted after the third month of lactation, even though, due to the rearing of the foal, milk for human consumption can only be collected beginning in the eighth week of lactation at the earliest.24 Studies concerning the nutritional value of mares’ colostrum are also scarce. In the last 20 years very few original research studies assessing chemical composition and physicochemical parameters of mares’ colostrum, have been published.1,2,4,5,19,23,25 Cited information collected from literature sources shows that the results of studies on the chemical composition, vitamin C content as well as SCC of mares’ colostrum and milk are scarce and often differ between each other, thus indicating the need for further experiments on that subject. Available literature sources also lack information on periodic changes in chemical composition and physicochemical parameters of mares’ milk. Therefore, the aim of the current study was to investigate the effect of the stage of lactation and year season (summer vs. autumn) on fat, protein, lactose, total solids (TS), cholesterol, vitamin C, citric acid concentration and some physicochemical parameters such as density, titratable acidity (TA) and freezing point (FPD), as well as on SCC in mares’ milk. Moreover, the mentioned parameters were evaluated in mares’ colostrum. Another aim of the study was to develop a method for the determination of vitamin C (ascorbic acid) in mares’ milk and to determine its content in fresh and stored milk.
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MATERIAL AND METHODS Animals, feeding and milk sample collection The experiment was carried out on milk obtained from 16 mares: eight Thoroughbred mares and eight Polish Half Bred mares, which foaled during the period from January to May in one of the horse breeding stations in the Opolszczyzna region (southwestern Poland). Polish Half Bred breed is one of the most popular breeds in Poland. It was created strictly as a sport horse based on different horse breeds without clear pedigree structure and regulations.26 All Polish Half Bred mares used in this study were related to Thoroughbred mares in the first, second or third generation. The average body weight (BW) of Thoroughbred mares was 496 kg and of Polish Half Bred mares was 544 kg. The age of experimental animals ranged from 5 to 16 years. All the experimental animals were reared under identical environmental conditions and were in a similar body condition, according to the stud’s internal evaluation criteria. Diet of mares in early lactation (1–3 month of lactation) contained 45% forage and 55% concentrate mix, while diet of mares in late lactation (4–6 months of lactation) consisted of 60% forage and 40% concentrate mix. The concentrate mix consisted of 66% oats and 34% commercial blend of grain for breeding mares. The rations were calculated to meet the daily nutrient requirements of the lactating mares of 500 kg mature weight, according to the Polish Nutrient Requirement Standards for Horses,27 which are as follows: for mares in 1–3 months of lactation – 12.5 kg dry matter, 130 MJ digestible energy, 1130 g digestible crude protein, 50 g Ca, 34 g P, 50 g NaCl and 90 mg carotene; and for mares in 4–6 month of lactation – 11 kg dry matter, 111 MJ digestible energy, 840 g digestible crude protein, 41 g Ca, 27 g P, 50 g NaCl and 80 mg carotene. From the beginning of May until the end of October, mares and foals spent 8–10 h on a good-quality pasture (approximately 1 ha per mare–foal pair), whereas at night dams with their offspring were kept in pens of 16 m2 . In the summer, pasture supplied most of the roughage, in spring and autumn it was supplemented with hay, and in winter hay was used in place of pasture. Mares were hand-milked without oxytocin injection. Prior to every milking forestripping, udder massage and teat cleaning were performed. Mares were always milked at the same time of the day, between 08:00 and 09:00 p.m., 60 min after separating foals from their mothers. During milking, foals remained in visual and tactile contact with their mothers. Milk was completely removed from both udder halves. The minimum quantity of milked milk from mares was obtained at the beginning of lactation (∼300 mL per milking) and maximum at the end of lactation (∼1500 mL per milking). Representative samples of colostrum and milk for laboratory analyses (∼100 mL) were collected from each mare into sterile plastic containers. Colostrum samples were collected twice: up to 4 h after foaling and 8–12 h after foaling. Milk was collected four times: at day 7, day 28, day 90 and day 180 of lactation. Samples in which analysis of basic chemical composition and SCC were performed, had been fixed immediately after milking with a Broad Spectrum Microtabs II preservative and cooled to a temperature of 3–6 ∘ C. The remaining milk and colostrum were frozen (−20 ∘ C). In those samples, shortly after freezing analysis of cholesterol and vitamin C concentrations was conducted. Storage conditions Analysis was conducted on a total of 30 milk samples coming from 10 Coldblood mares of the Polish Coldblood horse breed. Milk was collected individually from each mare, three times at weekly intervals, in the autumn season.
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Influence of stage of lactation and season on composition of mares’ colostrum and milk Collected milk was cooled immediately and transported under cold-storage conditions at 4 ∘ C to a laboratory to determine content of vitamin C as well as basic composition in the fresh milk. Afterwards, half of the milk samples were frozen and stored at −30 ∘ C in 50 mL test tubes. In turn, samples to be lyophilised were frozen in a shell freezer (Labconco, Kansas City, MO, USA) at −40 ∘ C, also in 50 mL test tubes half-filled with milk. Frozen samples were lyophilised in a Labconco FreeZone apparatus, at a collector temperature of −50 ∘ C and lyophilisation capacity of 10 kg water/24 h drying in the frozen form at a pressure below 0.100 mBa. Lyophilised samples were stored at room temperature. Analysis was performed on fresh and frozen as well as lyophilised milk after 6 and 12 months of milk storage. Frozen samples were thawed at a temperature of 4 ∘ C, while lyophilised samples were rehydrated to original mass. Experimental procedures Fat, protein, lactose, TS and citric acid content as well as density, TA and FPD were determined by automated infrared analysis with a Milkoscan FT2 instrument (Foss Electric, Hillerød, Denmark). The samples were heated to 40 ∘ C in a water bath. Based on the results of chemical composition the energy value of 1 kg milk was calculated using gross physiological energy indexes according to Rubner, as described by Barłowska.28 The hygienic status of the milk was based on SCC determined using an IBCm instrument (Bentley, MN, USA). The instrument applies laser-based flow cytometry, with ethidium bromide used to stain DNA in somatic cells. The laser-based counting section uses fluorescence characteristics of the dye to count the cells one by one. Cholesterol was assayed after a prior extraction and foaming of milk fat according to Fletouris et al.,29 and quantitative analysis was performed by colorimetry according to Searcy and Berquist.30 Analytical standard was supplied by Alpha Diagnostics (Cholesterol Standard, 30 mL, C6509STD). Vitamin C was determined using a modified method described by Omaye et al.31 for the determination of ascorbic acid in plasma, animal tissues and urine. Analytical standards were supplied by Sigma-Aldrich (L-ascorbic acid BioXtra, ≥99.0%, crystalline,
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A5960). To remove proteins from the analysed milk solution, 800 μL of 10% trichloroacetic acid (TCA; Aldrich) solution in distilled water was added to 800 μL mares’ milk homogenate. The samples (in test tubes) were centrifuged. After centrifugation the following reagents were added in sequence to 500 μL of the supernatant: 1000 μL of 10% TCA, 200 μL orthophosphoric acid (Aldrich) (100 μL concentrated acid + 100 μL distilled water), 200 μL 2,2′ -bipyridyl (bipy) solution in 70% alcohol (POCH) and 100 μL aqueous solution of 3% iron(III) chloride (Aldrich). After addition of each reagent the samples were mixed immediately. The prepared samples were allowed to stand at 37 ∘ C for 60 min while the ferrous-2,2′ - bipyridyl red-orange complex was being prepared. The analysed samples were than centrifuged and read at 525 nm using a spectrophotometer (model LabdaBio-20; PerkinElmer, Waltham, MA, USA), and vitamin C content in the sample was calculated. Blank sample was 1500 μL 5% TCA + other used reagents. The main process, i.e. reduction of the ferric ion Fe3+ to the ferrous ion Fe2+ by ascorbic acid, depends on the concentration of vitamin C. The amount of vitamin C was determined by formation of the [Fe(bipy)3]2 + red-orange complex: [ ]2+ bipy + ox.vit.C Fe3+ + vit.C (e− ) −−−−→ Fe (bipy)3 The reduction of iron ions and formation of this coloured complex is possible as a result of the addition of orthophosphoric acid (pH about 1–2), which stops other reducing processes in tested milk. Statistical analysis In order to assess the significance of the effect of stage of lactation and season results were processed by the PROC MIXED procedure with Tukey–Kramer’s post hoc comparison test for SAS (version 9.2, 2011). Normality tests were used to establish whether the investigated traits were well modelled by a normal distribution. The preliminary analyses revealed that there were no differences between both mare breeds for all studied parameters; thus this effect was not included in the final statistical model. In the model
Table 1. LSM and their SE for chemical composition, somatic cell count and physicochemical properties of mares’ colostrum and milk during lactation Time after foaling Item
0–4 h
8–12 h
7 DIM
28 DIM
90 DIM
Fat (g kg−1 ) Protein (g kg−1 ) Lactose (g kg−1 ) TS (g kg−1 ) Cholesterol (g kg−1 ) Vitamin C (g kg−1 ) Citric acid (g kg−1 ) Energy (MJ kg−1 ) Density (g kg−1 ) TA (∘ SH) Freezing point (−∘ C) SCS, Ln SCC (×103 cells mL−1 )
21.6ABa 128.1A 31.9Aa 192.9A 0.22A 0.06A 2.3A 3.79A 1,056A 12.55A 0.583A 5.54A 342.00
23.9A 66.5B 39.1Ab 143.1B 0.22A 0.03B 2.3A 2.85B 1,039Ba 9.32B 0.580A 4.96A 263.00
17.0B 29.2C 58.5Bc 114.4C 0.09B 0.07A 1.8B 2.23C 1,031BC 5.72Ca 0.574A 3.37B 37.00
15.9B 22.5C 63.7CBC 112.2C 0.08B 0.08A 1.7B 2.16CDa 1,030BCb 4.44C 0.565A 3.05B 23.00
15.1Bb 18.3C 65.5BCd 109.0C 0.07BCa 0.10C 1.4BC 2.09CD 1,029BCb 3.54C 0.535B 3.00B 21.00
180 DIM 4.3C 16.7C 66.3C 97.4C 0.03Cb 0.12C 1.3C 1.64Db 1,028C 2.92Cb 0.506C 2.90B 20.00
SE 1.4 6.4 1.59 5.85 0.008 0.04 0.08 0.12 1.96 0.43 0.005 0.18 –
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Means within a row with different letters differ: P ≤ 0.01 (upper case); P ≤ 0.05 (lower case). MJ, megajoule; TA, titratable acidity, measured in Soxhlet–Henkel degrees (∘ SH); SCS, somatic cell score; SCC somatic cell count; For statistical analysis SCC was transformed to natural logarithm values (Ln); DIM, days in milk.
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the following effects were included: fixed effect of the type of milk (colostrum, milk); fixed effect of stage of lactation; fixed effect of year season; and random error. In order to assess the significance of the effect of the method and time of milk storage on vitamin C content, two-way analysis of variance was conducted (GLM-SAS procedure v.9.2 2012). In the model the following effects were included: the fixed effect of milk storage method and the fixed effect of milk storage time and random error. A detailed comparison of object means was performed using Tukey’s test.
successive analysed lactation periods SCC in milk decreased slightly, but there were no significant differences between stages of lactation. A decrease in SCC during lactation was also reported by other authors.17,19,20,35 Centoducati et al.17 and Cagalj et al.,35 who obtained milk from mares following similar methodology to ours and reported similar SCC in mares’ milk to those obtained in the current study. In our earlier study we found lower SCC levels in machine-milked late-lactation mares’ milk, at the level of 3 × 103 mL−1 .10 However, most authors who did not mention whether the udder was completely empty or not recorded higher SCC in mares’ milk in comparison to the presented results. Pecka et al.23 indicated that SCC in colostrum collected up to 2 h postpartum were over twofold higher than in colostrum included in this experiment. The above-mentioned authors also recorded a more than tenfold higher SCC in milk in comparison to the results collected in this study.23 In the experiment conducted by Danków et al.19 the mean SCC recorded between days 15 and 150 of lactation was 46 × 103 mL−1 – 95% higher than that found in this study. Kulisa et al.20 recorded, in turn, a considerable reduction of SCC from 111 × 103 mL−1 in the second to third month of lactation to 16 × 103 mL−1 in the fourth and seventh months of lactation. Probably among the factors influencing SCC level in mares’ milk are milking technique (machine vs. hand milking), the level of udder emptying and efficiency of milking. To our knowledge there are no recent scientific and technical reports on analytical, sanitary, productive and technological aspects of SCC level in mares’ milk. Numerous studies, mostly on cows’ milk, showed that an increase SCC level is related to intramammary infection.36 For this reason the SCC in cows’ milk has been considered as the index of glandular irritation in the mammary gland.37 However, changes in SCC, especially in goats’ milk, can also be associated with non-infectious factors such as breed, parity, stage of lactation, type of birth, oestrus, diurnal, monthly and seasonal variations.36 Bagnicka et al.38 showed that the presence of bacterial pathogens in goats’ milk led to an increase in the total SCC. However, their study revealed that bacterial pathogens were present in 20% of milk samples with low SCC. Therefore, SCC cannot be the only decisive indicator of bacterial infection of the mammary gland in goats. There are important differences between species in number and pattern of somatic cells according to physiology status of the mammary gland. In the healthy mammary gland of cows, the predominant cell types are leucocytes, in goat they are leucocytes and epithelial cells and in women they are epithelial cells.39 The increase in SCC in milk from infected animals is accompanied by a change in the pattern of cell populations.40 In healthy cows leucocytes contribute to the defence against invading pathogens, while the immune system in mares may differ from that of cows or goats and may relate to the high concentration of lysozyme, lactoferrin and immunoglobulin. Further studies are needed to establish a pattern of mares’ milk somatic cells, and to determine whether pathological and non-pathological factors influence SCC level in mares’ milk. The determination of colostrum density is an indirect method used to assess its quality, including the level of immunoglobulins.24 In this study the density of mares’ colostrum was higher than that of milk (1.047 vs. 1.029 g mL−1 ). The specific gravity of colostrum collected up to 4 h after foaling was higher than that from 8–12 h post foaling (P ≤ 0.01; Table 1). Pecka et al.23
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Effect of stage of lactation Chemical composition of colostrum differed from that assayed for milk (Table 1). Tested colostrum, in comparison to milk, was characterized by higher content of fat, cholesterol, protein, TS and citric acid, and lower content of lactose and vitamin C. Presented results concerning nutritive value of mares’ colostrum and milk were similar to those recorded by other authors and indicate that the composition of mares’ milk changes in the course of lactation.3,6,17,18,32 In colostrum collected up to 4 h postpartum the energy value as well as concentration of vitamin C, protein and TS were higher than in colostrum collected from the same mares between 8 and 12 h post foaling (P ≤ 0.01; Table 1). The rapid drop in protein level in mares’ colostrum has been confirmed by the results of other authors. Salimei et al.8 and Pecka et al.23 determined the chemical composition of colostrum collected up to 2 h after foaling; the authors reported higher protein content than the level detected in this study. Salimei et al.,8 after a repeated colostrum analysis at 6 h post foaling, recorded a considerable decrease in protein content, to 49 g kg−1 . Protein content of mares’ milk was stable. Its concentration in milk decreased in successive stages of lactation but there were no significant differences between retrievals. The results of this study indicate that mares’ milk contains twice as much protein as human milk and, on average, 60% less protein than cows’ milk. Mares’ milk is characterized by very low energy value, which is related to low fat content. Fat concentration in human and cows’ milk is 30–50 g kg−1 .33,34 Mean fat content in mares’ milk was lower than that in colostrum (12.8 vs. 22.0 g kg−1 ). Milk from the first half of lactation (7, 28 and 90 days) was characterized by a higher content of fat than that collected towards the end of lactation (P ≤ 0.01; Table 1), which was confirmed in studies conducted by other authors.4,6,17,32 A very low fat level (below 6 g kg−1 ) in the sixth month of lactation was also reported in other studies.10,18,32 In the experiments of Pikul et al.5 and Pikul and Wójtowski,4 conducted on mares in the fifth and sixth months of lactation, fat content was higher (over 10 g kg−1 ) than that in this study. Mean lactose concentration in the colostrum of mares was lower than in milk (35.6 vs. 63.5 g kg−1 milk). Lactose concentration in milk increased in successive stages of lactation and at day 7 of lactation it was lower than at days 90 and 180 of lactation (P ≤ 0.05; P ≤ 0.01; Table 1). Similar upward trends in the course of 180-day lactation were also recorded by Santos and Silvestre,6 Summer et al.32 and Centoducati et al.17 The high content of lactose (60–70 g kg−1 ) in mares’ and human milk distinguish them from cows’ milk (50 g kg−1 ). SCC decreased in mares’ colostrum and milk during the progress of lactation (Table 1). Mean SCC in colostrum was over tenfold higher than in milk (301.57 vs. 25.73 × 103 cells mL−1 ). In the
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J Sci Food Agric 2015; 95: 2279–2286
Influence of stage of lactation and season on composition of mares’ colostrum and milk
J Sci Food Agric 2015; 95: 2279–2286
Human milk is a richer source of vitamin C than cows’ milk and its content also changes with the stage of lactation and is greater in colostrum than in milk.49 – 51 Effect of season Climatic conditions Winter months (February, March) had a mean temperature of 4.97 ∘ C during the day and −1.27 ∘ C during the night; spring time (April and May) was characterized by mean temperatures of 19.67 and 6.46 ∘ C during the day and night, respectively. In the summer (June, July and August) the average temperature was 23.47 ∘ C during the day and 11.69 ∘ C during the night. In turn, in the autumn (September, October, November), during the day the average temperature was 13.68 ∘ C and during the night it was 5.38 ∘ C. It has been already shown that cows’ milk composition varies with season, stage of lactation, health status of the cow, milking interval, feeding, genetic factors and other day-to-day variations.52 Studies on the effects of feeding, breed, stage of lactation or genetic variation on mares’ milk composition are scarce.4,5,53,54 To the best of our knowledge, the effect of season on mares’ milk composition has been not assayed and requires further research. Table 2 presents the average values, range and seasonal variation for the composition and some physical properties of mares’ milk collected in summer and autumn. No effect of the season of the year was found on nutrient content or values of physicochemical parameters of colostrum produced by mares foaling in winter (February, March) or in spring (April, May). The averages for fat, protein, lactose, dry matter, SCC and energy value in the spring season were 2.23%, 9.67%, 3.73%, 16.88%, 300 × 103 cells mL−1 and 3.27 MJ kg−1 respectively; and in the winter season were 2.41%, 9.29%, 3.37%, 16.30%, 263 × 103 cells mL−1 and 3.14 MJ kg−1 , respectively. The density was the same for the colostrum collected in spring and in winter, with a mean value of 1.046 g mL−1 . Titratable acidity was 11.54 ∘ SH and 10.49 ∘ SH, while FPD was −0.585 and −0.577 ∘ C in winter and spring, respectively. There was a slight variation in the level of citric acid, which ranged from 0.21% in winter to 0.25% in spring; however, this difference was not significant. Variation in the basic chemical components and cytology parameter of milk from summer (June, July, August) and autumn (September, October, November) was manifested mainly in the fluctuations in fat and cholesterol content, as well as protein, citric acid and FPD of mares’ milk. Milk collected in summer had a significantly higher fat and cholesterol content than in autumn (P ≤ 0.01). Compared with fat, the protein and citric acid concentrations showed less variability (P ≤ 0.05). Milk collected in summer was characterized by higher content of protein and citric acid than that collected in autumn (P ≤ 0.05; Table 2). The average concentrations of vitamin C and SCC were similar for milk collected in summer and in autumn and there were no specific significant seasonal variations. Generally, the level of lactose, which is known to be one of the least variable milk components in cows’ milk, fluctuated over the study, but no significant seasonal differences were observed. Milk produced in summer had lower a FPD value than that collected in autumn. This variation could be attributed to the higher total solids level in summer versus autumn milk (P ≤ 0.01; Table 2). The FPD displays a relatively narrow range in cows’ milk, because of the osmotic balance between milk and blood.52 Bjerg et al.55 suggested that variations in cows’ milk freezing point may be due to the increased water intake due to the increased temperature and sunshine hours in summer. The lower FPD of mares’ milk is probably related to the concentration of water-soluble substances such as lactose, citrates and phosphates.24,35 Cagaj et al.35 suggested
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recorded a higher colostrum density, reaching 1.070 g kg−1 . In turn, Yao et al.25 determined the specific gravity of colostrum to be 1.080 g kg−1 in the second hour post foaling and at 1.030 g kg−1 in the 24th hour postpartum. The presented results indicate differences in mean TA between colostrum and milk (10.89 vs. 4.15 ∘ SH). Milk from the end of lactation was characterized by lower TA than that collected at the beginning of lactation (P ≤ 0.05; Table 1). Recorded results were similar to those reported by other authors18,23,32 and confirmed our earlier results.10 The presented data indicate that mares’ milk is characterized by lower TA than cows’ milk. According to Barłowska,28 TA of cows’ milk fluctuates around 7 ∘ SH and remains stable throughout lactation. Mares’ milk, in comparison to cows’ milk, is characterized by a low level of TS, and thus a lower concentration of nutrients, including casein, which is a factor responsible for TA.10 In the second half of lactation, when the level of TS in mares’ milk is less than 10.0 g kg−1 , the difference between TA of cows’ and mares’ milk is the most visible. Citric acid content in mares’ colostrum was higher than in milk (on average, 2.3 vs. 1.5 g kg−1 ). With the progress of lactation its concentration in milk decreased, which was confirmed statistically (P ≤ 0.01; Table 1). Original research papers concerning content of citric acid in milk are scarce.10,24,41 Citric acid contained in the milk salts binds calcium and magnesium in the form of citrates and increases its thermal stability.24 Moreover, it is a substrate for certain lactic bacteria species, using it to form aroma substances. Citrates have also been ascribed antiscorbutic properties and a capacity to promote calcium absorption by the mucosa in the alimentary tract in the presence of vitamin D, which might influence the biological value of milk.42 Mares’ milk contained small amounts of cholesterol. Colostrum contained over three times more cholesterol than milk (0.22 vs. 0.07 g kg−1 ). Cholesterol content in milk collected at day 180 of lactation was lower than in previously collected samples (P ≤ 0.01; Table 1). Cholesterol content determined in this study was similar to the level reported by Marconi and Panfili.3 A downward trend for cholesterol content during lactation was also shown by Pikul and Wójtowski,4 who investigated the fatty acid profile and cholesterol content in mares’ milk at the different stages of lactation. Cholesterol content determined by those authors in colostrum (0.20 g kg−1 ) and milk (0.06 g kg−1 ) was very similar to that obtained in this study. Mean cholesterol content in mares’ milk analysed in this study was lower than in human or cows’ milk. Its concentration in human milk ranges from 0.10 to 0.20 g kg−1 .34,43 At 3.5% fat-corrected, cows’ milk cholesterol content is 0.14 g kg−1 , but depending on fat content it may vary from 0.08 to 0.20 g kg−1 .44 – 46 Concentration of vitamin C in colostrum was lower than in milk (0.05 vs. 0.09 g kg−1 ). Colostrum collected up to the fourth h postpartum contained over twofold higher content of vitamin C than that collected between the 8th and 12th hour postpartum (P ≤ 0.01; Table 1). Content of vitamin C in milk was higher at days 90 and 180 than at the beginning (days 7 and 28) of lactation (P ≤ 0.01; Table 1). Cows’ milk contains ∼0.02 g vitamin C kg−1 , which provides antioxidant protection to this raw material.47 Vitamin C concentration in cows’ milk also depends on the stage of lactation, but changes in its concentration differ from that in the analysed mares’ milk. Hidiroglou et al.48 observed that the concentration of vitamin C in cows’ milk decreased with the progress of lactation.
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Table 2. Chemical composition, SCC and physicochemical parameters of mares’ milk collected in autumn and summer Season of milk collectiona Summer Item
LSM
Fat (g kg−1 ) Protein (g kg−1 ) Lactose (g kg−1 ) TS (g kg−1 ) Cholesterol (g kg−1 ) Vitamin C (g kg−1 ) Citric acid (g kg−1 ) Energy (MJ kg−1 ) Density (g kg−1 ) TA (∘ SH) Freezing point (∘ C) SCS (Ln) SCC (×103 cells mL−1 )
11.9A 17.8a 65.6 105.5A 0.06A 0.10 1.4a 1.95A 1,029 3.51 0.527A 2.90 19.00
Minimum 2.0 15.5 60.4 91.1 0.03 0.05 1.1 1.43 1,026 2.67 0.405 – 9.00
Autumn Maximum 21.1 20.5 69.9 117.0 0.09 0.15 1.7 2.35 1,030 4.87 0.737 – 31.00
LSM
Minimum
4.1B 16.7b 66.6 97.3B 0.03B 0.11 1.3b 1.64B 1,028 2.80 0.506B 2.89 21.00
Maximum
1.1 14.6 5.61 85.9 0.02 0.09 1.0 1.34 1,022 1.14 0.409 – 7.00
8.3 18.9 7.04 103.6 0.06 0.13 1.6 1.84 1,030 4.29 0.716 – 46.00
SE
P-value
1.16 0.34 0.87 1.61 0.003 0.005 0.04 0.054 0.0004 0.25 0.005 0.12 –
0.002 0.03 0.435 0.0013 < 0.0001 0.170 0.047 0.0004 0.255 0.057 0.0098 0.959 –
a Summer: June, July, August; autumn: September, October, November. Means within a row with different letters differ: P ≤ 0.01 (upper case); P ≤ 0.05 (lower case). MJ, megajoule; TA, titratable acidity, measured in Soxhlet–Henkel degrees (∘ SH); SCS, somatic cell score; SCC somatic cell count; For statistical analysis SCC was transformed to natural logarithm values (Ln).
that determining the limits for freezing point of mares’ milk is particularly significant, taking into consideration its high price. Density was constant throughout the summer and autumn seasons. Titratable acidity displayed some fluctuations but no significant seasonal variation was found. There are no reports in the available literature on the effect of season of the year on the nutritive value or the cytology parameter in mares’ milk. The differences between the results obtained in this study are difficult to interpret. One may assume that they were caused by differences in the chemical composition of diets offered to mares. The quality of grass and of oats could be different in June and September. This hypothesis must be confirmed by nutritional studies taking into account detailed analysis of pasture and feed samples during different seasons. A more detailed study of the effect of nutritive and non-nutritive factors like breed and age of mares are needed. Our results clearly show that the effect of season of the year on the chemical composition of mares’ milk cannot be considered without paying attention to the fact that it is related to the stage of lactation. Because the collection of mares’ milk, in contrast to cows’ milk, is seasonal in character, strongly determined by the season of the year, milk collected in summer will be obtained from the mares being in the earlier stage of lactation than milk harvested in autumn. This will result in higher fat content in summer milk in comparison to low-fat milk collected in autumn. This may serve as valuable information for potential producers and consumers.
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Effect of method and time of storage on vitamin C concentration The basic chemical composition of the fresh milk used for this part of the experiment was: 3.79 g kg−1 fat, 15.98 g kg−1 protein, 70.75 g kg−1 lactose and 93.06 g kg−1 total solids. Mean content of vitamin C in milk was 0.13 g kg−1 and it was slightly lower than in the study by Marconi and Panfili,3 who recorded 0.15 g kg−1 , but higher than in the warm-blooded mares’ milk examined in this study.
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Table 3. Vitamin C content (g kg−1 ) in mares’ milk depending on method and time of storage
Method of storage Fresh Frozen Lyophilisate
Time of storage (months)
6 12 6 12
LSM 0.134A 0.120BC 0.104D 0.125AB 0.115C
SD 0.0122 0.0102 0.0146 0.0142 0.0171
Means within a column with different letters differ (P ≤ 0.01).
The duration of milk storage, irrespective of its preservation method (freezing, lyophilisation), had an effect on the concentration of vitamin C (P ≤ 0.01; Table 3). At 12-month storage the losses were over twofold greater (18%) than in the case of 6-month storage (8%). In turn, no differences were found (P > 0.05) when comparing content of vitamin C in frozen and lyophilised milk after 6 months of storage. Only 12-month storage resulted in a reduction of its concentration. Losses greater by ∼10% were recorded in frozen milk in relation to lyophilised milk (P ≤ 0.01). Moreover, in frozen milk after 6 months of storage, losses of the vitamin amounted to 10%, while after 12 months it was 22% in relation to fresh milk and in lyophilised milk it was 6% and 14%, respectively. A review of the literature shows that most studies on the effect of milk storage on vitamin C content concern human milk.51,56,57 Investigations conducted by Romeu-Nadal et al.50 indicate that in the period of 12 months the concentration of vitamin C in human milk freeze-stored at a temperature of −20 ∘ C decreased by 24%, while in that stored at −80 ∘ C it decreased by 12%. In turn, a study conducted by Marconi and Panfili3 showed that the preservation of milk by drying had a considerable effect on the
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Influence of stage of lactation and season on composition of mares’ colostrum and milk reduction of concentration, not only for vitamin C but also vitamins A and E as well as cholesterol.
CONCLUSIONS Analyses showed that the stage of lactation is a major factor significantly influencing the chemical composition of mares’ milk. Moreover, it was shown that milk collected in autumn differs from that produced in summer, first in the lower content of fat and cholesterol. In terms of vitamin C stability, lyophilisation and 6-month storage of milk proved to be the most advantageous.
ACKNOWLEDGMENTS The authors wish to thank the Moszna Horse Stud for cooperation during the experiment. We wish to acknowledge the help provided by Billy Carey in revising the English.
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39 Boutinaud M, Rulquin H, Keisler DH, Djiane J and Jammes H, Use of somatic cells from goat milk for dynamic studies of gene expression in the mammary gland. J Anim Sci 80:1258–1269 (2002). 40 Lindmark-Månsson H, Bränning C, Aldén G and Paulsson M, Relationship between somatic cell count, individual leukocyte population and milk components in bovine udder quarter milk. Int Dairy J 16:717–727 (2006). ̇ ´ 41 Strzałkowska N, Jó´zwik A, Bagnicka E, Krzyzewski J, Horbanczuk K, Pyzel B et al, The concentration of free fatty acids in goat milk as related to the stage of lactation, age and somatic cell count. Anim Sci Pap Rep 28:389–395 (2009). 42 Budsławski J, Outline of Dairy Chemistry (in Polish). PWRiL, Warsaw (1971). 43 Koletzko B, Rodriguez-Palmero M, Demmelmair H, Fidler N, Jensen R and Sauerwald T, Physiological aspects of human milk lipids. Early Hum Dev 65 3–18 (2001). 44 Focant M, Mignolet E, Marique M, Clabots F, Breyne T, Dalemands D et al, The effect of vitamin E supplementation of cow diets containing rapeseed and linseed on the prevention of milk fat oxidation. J Dairy Sci 81:1095–1101 (1998). 45 Manzi P and Pizzoferrato L, Cholesterol and antioxidant vitamins in fat fraction of whole and skimmed dairy products. Food Bioprocess Technol 3:234–238 (2008). ̇ ´ 46 Strzałkowska N, Jó´zwik A, Bagnicka E, Krzyzewski J and Horbanczuk JO, Studies upon genetic and environmental factors affecting the cholesterol content of cow milk. II. Effect of silage type offered. Anim Sci Pap Rep 27:199–206 (2009). 47 Morrissey P and Hill T, Fat-soluble vitamins and vitamin C in milk and milk products. Adv Dairy Chem 3:527–589 (2009). 48 Hidiroglou M, Ivan M and Batra T, Concentrations of vitamin C in plasma and milk of dairy cattle. Ann Zootech (Paris) 44:399–402 (1995).
49 Tawfeek H, Muhyaddin O, al-Sanwi H and al-Baety N, Effect of maternal dietary vitamin C intake on the level of vitamin C in breast milk among nursing mothers in Baghdad, Iraq Food Nutr Bull 23:244–247 (2002). 50 Romeu-Nadal M, Castellote A and López-Sabater M, Effect of cold storage on vitamins C and E and fatty acids in human milk. Food Chem 106:65–70 (2008). 51 Moltó-Puigmartí C, Permanyer M, Castellote A and López-Sabater M, Effects of pasteurisation and high-pressure processing on vitamin C, tocopherols and fatty acids in mature human milk. Food Chem 124:697–702 (2011). 52 Chen B, Lewis MJ and Grandison AS, Effect of seasonal variation on the composition and properties of raw milk destined for processing in the UK. Food Chem 158:216–223 (2014). 53 Doreau M, Boulot S, Bauchart D, Barlet JP and Martin-Rosset W, Voluntary intake, milk production and plasma metabolites in nursing mares fed two different diets. J Nutr 122:992–999 (1992). ´ ´ 54 Pietrzak-Fie´cko R, Tomczynski R, Swistowska A, Borejszo Z, Kokoszko ´ E and Smoczynska K, Effect of mare’s breed on the fatty acid composition of milk fat. Czech J Anim Sci 54:403–407 (2009). 55 Bjerg M, Rasmussen MD and Nielsen MO, Changes in freezing point of blood and milk during dehydration and rehydration in lactating cows. J Dairy Sci 88:3174–3185 (2005). 56 Spitzer J and Beuttner A, Characterisation of off-odor development in human milk during storage at −18 degrees C. Food Chem 120:240–246 (2010). 57 Sandgruber S, Much D, Amann-Gassner U, Hauner H and Buettner A, Sensory and molecular characterisation of the protective effect of storage at −80 ∘ C on the odour profiles of human milk. Food Chem 130:236–242 (2012).
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Revised: 10 June 2014
Accepted article published: 9 October 2014
Published online in Wiley Online Library: 20 November 2014
(wileyonlinelibrary.com) DOI 10.1002/jsfa.6947
Influence of stage of lactation and year season on composition of mares’ colostrum and milk and method and time of storage on vitamin C content in mares’ milk a* Grazyna b ̇ ̇ Maria Markiewicz-Keszycka, ¸ Czyzak-Runowska, d Jacek Wójtowski,b Artur Jó´zwik,c Radosław Pankiewicz,d Bogusława Łeska, ¸ c Nina Strzałkowska,c Joanna Marchewkae ̇ Józef Krzyzewski, and Emilia Bagnickac
Abstract BACKGROUND: Mares’ milk is becoming increasingly popular in Western Europe. This study was thus aimed at investigating the impact of stage of lactation and season on chemical composition, somatic cell count and some physicochemical parameters of mares’ colostrum and milk, and at developing a method for the determination of vitamin C (ascorbic acid) in mares’ milk and to determine its content in fresh and stored milk. RESULTS: The analysis conducted showed an effect of the stage of lactation on contents of selected chemical components and physicochemical parameters of mares’ milk. In successive lactation periods levels of fat, cholesterol, energy value, citric acid and titratable acidity decreased, whereas levels of lactose and vitamin C, as well as the freezing point, increased. Analysis showed that milk produced in autumn (September, October, November) had a higher freezing point and lower concentrations of total solids, protein, fat, cholesterol, citric acid and energy value in comparison to milk produced in summer (June, July, August). Mares’ milk was characterised by low somatic cell count throughout lactation. In terms of vitamin C stability the most advantageous method of milk storage was 6-month storage of lyophilised milk. CONCLUSION: In general, the results confirmed that mares’ milk is a raw material with a unique chemical composition different from that produced by other farm animals. © 2014 Society of Chemical Industry Keywords: mares’ milk; chemical composition; stage of lactation; seasonal variation; method of storage
INTRODUCTION
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∗
Correspondence to: Maria Markiewicz-Ke¸ szycka, School of Agriculture and Food Science, University College Dublin, Belfield, Dublin 4, Ireland. E-mail: [email protected]
a School of Agriculture and Food Science, University College Dublin, Dublin 4, Ireland b Department of Small Mammals Breeding and Raw Materials of Animal Origin, Poznan University of Life Sciences, 62-002, Suchy Las, Poland c Department of Animal Sciences, Institute of Genetics and Animal Breeding of the Polish Academy of Sciences, 05-552, Magdalenka, Poland d Faculty of Chemistry, Adam Mickiewicz University, 61-614, Pozna´n, Poland e Animal Production Department, Neiker-Tecnalia, Arkaute Agrifood Campus, E-01080, Vitoria-Gasteiz, Spain
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Mares’ milk is characterised by unique chemical composition as compared to the milk of other farm animal species. It is comprised of approximately 1% fat, 2% protein and 7% lactose, and is a source of bioactive components such as lysozyme, lactoferrin, immunoglobulin, long-chain n-3 fatty acids and fat-soluble vitamins.1 – 11 Despite its potential, mares’ milk is a niche raw material, difficult to obtain for large-scale production. One of the factors limiting mares’ milk production is the seasonality of reproduction in mares, and for this reason its preservation and storage are required. Owing to its content of biologically active substances, the preservation methods most frequently applied for this raw material include freezing and freeze drying, i.e. lyophilisation.12,13 It is commonly known that freezing and lyophilisation are optimal methods of food preservation, causing minimal changes to the original properties. Freezing is the oldest and most readily available method of raw material storage. For this reason mares’ milk is most frequently
stored in frozen form, although it may be troublesome from a practical point of view, especially with respect to its further distribution.14 Milk lyophilisation may prove to be a much easier
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method applicable to extended milk storage. Lyophilisation guarantees complete freedom in the management of the raw material, including its storage. This process involves the removal of water from frozen material by ice sublimation, i.e. its direct transition to the vapour state, bypassing the liquid state. The obtained product is characterised by high quality, with a minimised risk of adverse reactions connected with oxidation or microbial activity. This process makes it possible to maintain much higher product quality in comparison to other drying methods; however, its high cost needs to be stressed here.13 Quality and stability of mares’ milk lyophilisate are determined by its content of water and its activity.15 There is no information in the available scientific literature on changes in the contents of biologically active components, particularly vitamin C, during milk storage. The available literature contains no information on properties of mares’ milk subjected to freezing and lyophilisation. It results only from a study by Gruda and Postolski16 concerning cows’ milk where milk freezing is rarely used, since this process has a negative effect on its quality. The main problem is connected with low stability of casein, which, after only 3 weeks of storage at a temperature range from −8 to −12 ∘ C, forms a flaky precipitate. Owing to the seasonality of reproduction in mares, their milk is subjected to significant variations related to the season (probably the feeding system and lactation stage) and thus the milking season has a significant effect on milk composition.4 – 6,17 This issue is not as visible in milk of dairy cows, which undergoes only slight seasonal changes, while cows calve all year round. There are no reports available in the scientific literature on the effect of year season on the nutritive value and somatic cell count (SCC) in mares’ milk. There is also a very limited number of studies on chemical composition of mares’ milk throughout 180 days of lactation.5,7,17,18 There is even less information on SCC, vitamin C, cholesterol content and physicochemical parameters of mares’ milk at different stages of lactation.9,18 – 22 The vast majority of information concerns the composition of mares’ milk in the period of the most intensive growth of foals.1,2,7,23 There is a very limited number of studies conducted after the third month of lactation, even though, due to the rearing of the foal, milk for human consumption can only be collected beginning in the eighth week of lactation at the earliest.24 Studies concerning the nutritional value of mares’ colostrum are also scarce. In the last 20 years very few original research studies assessing chemical composition and physicochemical parameters of mares’ colostrum, have been published.1,2,4,5,19,23,25 Cited information collected from literature sources shows that the results of studies on the chemical composition, vitamin C content as well as SCC of mares’ colostrum and milk are scarce and often differ between each other, thus indicating the need for further experiments on that subject. Available literature sources also lack information on periodic changes in chemical composition and physicochemical parameters of mares’ milk. Therefore, the aim of the current study was to investigate the effect of the stage of lactation and year season (summer vs. autumn) on fat, protein, lactose, total solids (TS), cholesterol, vitamin C, citric acid concentration and some physicochemical parameters such as density, titratable acidity (TA) and freezing point (FPD), as well as on SCC in mares’ milk. Moreover, the mentioned parameters were evaluated in mares’ colostrum. Another aim of the study was to develop a method for the determination of vitamin C (ascorbic acid) in mares’ milk and to determine its content in fresh and stored milk.
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MATERIAL AND METHODS Animals, feeding and milk sample collection The experiment was carried out on milk obtained from 16 mares: eight Thoroughbred mares and eight Polish Half Bred mares, which foaled during the period from January to May in one of the horse breeding stations in the Opolszczyzna region (southwestern Poland). Polish Half Bred breed is one of the most popular breeds in Poland. It was created strictly as a sport horse based on different horse breeds without clear pedigree structure and regulations.26 All Polish Half Bred mares used in this study were related to Thoroughbred mares in the first, second or third generation. The average body weight (BW) of Thoroughbred mares was 496 kg and of Polish Half Bred mares was 544 kg. The age of experimental animals ranged from 5 to 16 years. All the experimental animals were reared under identical environmental conditions and were in a similar body condition, according to the stud’s internal evaluation criteria. Diet of mares in early lactation (1–3 month of lactation) contained 45% forage and 55% concentrate mix, while diet of mares in late lactation (4–6 months of lactation) consisted of 60% forage and 40% concentrate mix. The concentrate mix consisted of 66% oats and 34% commercial blend of grain for breeding mares. The rations were calculated to meet the daily nutrient requirements of the lactating mares of 500 kg mature weight, according to the Polish Nutrient Requirement Standards for Horses,27 which are as follows: for mares in 1–3 months of lactation – 12.5 kg dry matter, 130 MJ digestible energy, 1130 g digestible crude protein, 50 g Ca, 34 g P, 50 g NaCl and 90 mg carotene; and for mares in 4–6 month of lactation – 11 kg dry matter, 111 MJ digestible energy, 840 g digestible crude protein, 41 g Ca, 27 g P, 50 g NaCl and 80 mg carotene. From the beginning of May until the end of October, mares and foals spent 8–10 h on a good-quality pasture (approximately 1 ha per mare–foal pair), whereas at night dams with their offspring were kept in pens of 16 m2 . In the summer, pasture supplied most of the roughage, in spring and autumn it was supplemented with hay, and in winter hay was used in place of pasture. Mares were hand-milked without oxytocin injection. Prior to every milking forestripping, udder massage and teat cleaning were performed. Mares were always milked at the same time of the day, between 08:00 and 09:00 p.m., 60 min after separating foals from their mothers. During milking, foals remained in visual and tactile contact with their mothers. Milk was completely removed from both udder halves. The minimum quantity of milked milk from mares was obtained at the beginning of lactation (∼300 mL per milking) and maximum at the end of lactation (∼1500 mL per milking). Representative samples of colostrum and milk for laboratory analyses (∼100 mL) were collected from each mare into sterile plastic containers. Colostrum samples were collected twice: up to 4 h after foaling and 8–12 h after foaling. Milk was collected four times: at day 7, day 28, day 90 and day 180 of lactation. Samples in which analysis of basic chemical composition and SCC were performed, had been fixed immediately after milking with a Broad Spectrum Microtabs II preservative and cooled to a temperature of 3–6 ∘ C. The remaining milk and colostrum were frozen (−20 ∘ C). In those samples, shortly after freezing analysis of cholesterol and vitamin C concentrations was conducted. Storage conditions Analysis was conducted on a total of 30 milk samples coming from 10 Coldblood mares of the Polish Coldblood horse breed. Milk was collected individually from each mare, three times at weekly intervals, in the autumn season.
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Influence of stage of lactation and season on composition of mares’ colostrum and milk Collected milk was cooled immediately and transported under cold-storage conditions at 4 ∘ C to a laboratory to determine content of vitamin C as well as basic composition in the fresh milk. Afterwards, half of the milk samples were frozen and stored at −30 ∘ C in 50 mL test tubes. In turn, samples to be lyophilised were frozen in a shell freezer (Labconco, Kansas City, MO, USA) at −40 ∘ C, also in 50 mL test tubes half-filled with milk. Frozen samples were lyophilised in a Labconco FreeZone apparatus, at a collector temperature of −50 ∘ C and lyophilisation capacity of 10 kg water/24 h drying in the frozen form at a pressure below 0.100 mBa. Lyophilised samples were stored at room temperature. Analysis was performed on fresh and frozen as well as lyophilised milk after 6 and 12 months of milk storage. Frozen samples were thawed at a temperature of 4 ∘ C, while lyophilised samples were rehydrated to original mass. Experimental procedures Fat, protein, lactose, TS and citric acid content as well as density, TA and FPD were determined by automated infrared analysis with a Milkoscan FT2 instrument (Foss Electric, Hillerød, Denmark). The samples were heated to 40 ∘ C in a water bath. Based on the results of chemical composition the energy value of 1 kg milk was calculated using gross physiological energy indexes according to Rubner, as described by Barłowska.28 The hygienic status of the milk was based on SCC determined using an IBCm instrument (Bentley, MN, USA). The instrument applies laser-based flow cytometry, with ethidium bromide used to stain DNA in somatic cells. The laser-based counting section uses fluorescence characteristics of the dye to count the cells one by one. Cholesterol was assayed after a prior extraction and foaming of milk fat according to Fletouris et al.,29 and quantitative analysis was performed by colorimetry according to Searcy and Berquist.30 Analytical standard was supplied by Alpha Diagnostics (Cholesterol Standard, 30 mL, C6509STD). Vitamin C was determined using a modified method described by Omaye et al.31 for the determination of ascorbic acid in plasma, animal tissues and urine. Analytical standards were supplied by Sigma-Aldrich (L-ascorbic acid BioXtra, ≥99.0%, crystalline,
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A5960). To remove proteins from the analysed milk solution, 800 μL of 10% trichloroacetic acid (TCA; Aldrich) solution in distilled water was added to 800 μL mares’ milk homogenate. The samples (in test tubes) were centrifuged. After centrifugation the following reagents were added in sequence to 500 μL of the supernatant: 1000 μL of 10% TCA, 200 μL orthophosphoric acid (Aldrich) (100 μL concentrated acid + 100 μL distilled water), 200 μL 2,2′ -bipyridyl (bipy) solution in 70% alcohol (POCH) and 100 μL aqueous solution of 3% iron(III) chloride (Aldrich). After addition of each reagent the samples were mixed immediately. The prepared samples were allowed to stand at 37 ∘ C for 60 min while the ferrous-2,2′ - bipyridyl red-orange complex was being prepared. The analysed samples were than centrifuged and read at 525 nm using a spectrophotometer (model LabdaBio-20; PerkinElmer, Waltham, MA, USA), and vitamin C content in the sample was calculated. Blank sample was 1500 μL 5% TCA + other used reagents. The main process, i.e. reduction of the ferric ion Fe3+ to the ferrous ion Fe2+ by ascorbic acid, depends on the concentration of vitamin C. The amount of vitamin C was determined by formation of the [Fe(bipy)3]2 + red-orange complex: [ ]2+ bipy + ox.vit.C Fe3+ + vit.C (e− ) −−−−→ Fe (bipy)3 The reduction of iron ions and formation of this coloured complex is possible as a result of the addition of orthophosphoric acid (pH about 1–2), which stops other reducing processes in tested milk. Statistical analysis In order to assess the significance of the effect of stage of lactation and season results were processed by the PROC MIXED procedure with Tukey–Kramer’s post hoc comparison test for SAS (version 9.2, 2011). Normality tests were used to establish whether the investigated traits were well modelled by a normal distribution. The preliminary analyses revealed that there were no differences between both mare breeds for all studied parameters; thus this effect was not included in the final statistical model. In the model
Table 1. LSM and their SE for chemical composition, somatic cell count and physicochemical properties of mares’ colostrum and milk during lactation Time after foaling Item
0–4 h
8–12 h
7 DIM
28 DIM
90 DIM
Fat (g kg−1 ) Protein (g kg−1 ) Lactose (g kg−1 ) TS (g kg−1 ) Cholesterol (g kg−1 ) Vitamin C (g kg−1 ) Citric acid (g kg−1 ) Energy (MJ kg−1 ) Density (g kg−1 ) TA (∘ SH) Freezing point (−∘ C) SCS, Ln SCC (×103 cells mL−1 )
21.6ABa 128.1A 31.9Aa 192.9A 0.22A 0.06A 2.3A 3.79A 1,056A 12.55A 0.583A 5.54A 342.00
23.9A 66.5B 39.1Ab 143.1B 0.22A 0.03B 2.3A 2.85B 1,039Ba 9.32B 0.580A 4.96A 263.00
17.0B 29.2C 58.5Bc 114.4C 0.09B 0.07A 1.8B 2.23C 1,031BC 5.72Ca 0.574A 3.37B 37.00
15.9B 22.5C 63.7CBC 112.2C 0.08B 0.08A 1.7B 2.16CDa 1,030BCb 4.44C 0.565A 3.05B 23.00
15.1Bb 18.3C 65.5BCd 109.0C 0.07BCa 0.10C 1.4BC 2.09CD 1,029BCb 3.54C 0.535B 3.00B 21.00
180 DIM 4.3C 16.7C 66.3C 97.4C 0.03Cb 0.12C 1.3C 1.64Db 1,028C 2.92Cb 0.506C 2.90B 20.00
SE 1.4 6.4 1.59 5.85 0.008 0.04 0.08 0.12 1.96 0.43 0.005 0.18 –
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Means within a row with different letters differ: P ≤ 0.01 (upper case); P ≤ 0.05 (lower case). MJ, megajoule; TA, titratable acidity, measured in Soxhlet–Henkel degrees (∘ SH); SCS, somatic cell score; SCC somatic cell count; For statistical analysis SCC was transformed to natural logarithm values (Ln); DIM, days in milk.
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the following effects were included: fixed effect of the type of milk (colostrum, milk); fixed effect of stage of lactation; fixed effect of year season; and random error. In order to assess the significance of the effect of the method and time of milk storage on vitamin C content, two-way analysis of variance was conducted (GLM-SAS procedure v.9.2 2012). In the model the following effects were included: the fixed effect of milk storage method and the fixed effect of milk storage time and random error. A detailed comparison of object means was performed using Tukey’s test.
successive analysed lactation periods SCC in milk decreased slightly, but there were no significant differences between stages of lactation. A decrease in SCC during lactation was also reported by other authors.17,19,20,35 Centoducati et al.17 and Cagalj et al.,35 who obtained milk from mares following similar methodology to ours and reported similar SCC in mares’ milk to those obtained in the current study. In our earlier study we found lower SCC levels in machine-milked late-lactation mares’ milk, at the level of 3 × 103 mL−1 .10 However, most authors who did not mention whether the udder was completely empty or not recorded higher SCC in mares’ milk in comparison to the presented results. Pecka et al.23 indicated that SCC in colostrum collected up to 2 h postpartum were over twofold higher than in colostrum included in this experiment. The above-mentioned authors also recorded a more than tenfold higher SCC in milk in comparison to the results collected in this study.23 In the experiment conducted by Danków et al.19 the mean SCC recorded between days 15 and 150 of lactation was 46 × 103 mL−1 – 95% higher than that found in this study. Kulisa et al.20 recorded, in turn, a considerable reduction of SCC from 111 × 103 mL−1 in the second to third month of lactation to 16 × 103 mL−1 in the fourth and seventh months of lactation. Probably among the factors influencing SCC level in mares’ milk are milking technique (machine vs. hand milking), the level of udder emptying and efficiency of milking. To our knowledge there are no recent scientific and technical reports on analytical, sanitary, productive and technological aspects of SCC level in mares’ milk. Numerous studies, mostly on cows’ milk, showed that an increase SCC level is related to intramammary infection.36 For this reason the SCC in cows’ milk has been considered as the index of glandular irritation in the mammary gland.37 However, changes in SCC, especially in goats’ milk, can also be associated with non-infectious factors such as breed, parity, stage of lactation, type of birth, oestrus, diurnal, monthly and seasonal variations.36 Bagnicka et al.38 showed that the presence of bacterial pathogens in goats’ milk led to an increase in the total SCC. However, their study revealed that bacterial pathogens were present in 20% of milk samples with low SCC. Therefore, SCC cannot be the only decisive indicator of bacterial infection of the mammary gland in goats. There are important differences between species in number and pattern of somatic cells according to physiology status of the mammary gland. In the healthy mammary gland of cows, the predominant cell types are leucocytes, in goat they are leucocytes and epithelial cells and in women they are epithelial cells.39 The increase in SCC in milk from infected animals is accompanied by a change in the pattern of cell populations.40 In healthy cows leucocytes contribute to the defence against invading pathogens, while the immune system in mares may differ from that of cows or goats and may relate to the high concentration of lysozyme, lactoferrin and immunoglobulin. Further studies are needed to establish a pattern of mares’ milk somatic cells, and to determine whether pathological and non-pathological factors influence SCC level in mares’ milk. The determination of colostrum density is an indirect method used to assess its quality, including the level of immunoglobulins.24 In this study the density of mares’ colostrum was higher than that of milk (1.047 vs. 1.029 g mL−1 ). The specific gravity of colostrum collected up to 4 h after foaling was higher than that from 8–12 h post foaling (P ≤ 0.01; Table 1). Pecka et al.23
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Effect of stage of lactation Chemical composition of colostrum differed from that assayed for milk (Table 1). Tested colostrum, in comparison to milk, was characterized by higher content of fat, cholesterol, protein, TS and citric acid, and lower content of lactose and vitamin C. Presented results concerning nutritive value of mares’ colostrum and milk were similar to those recorded by other authors and indicate that the composition of mares’ milk changes in the course of lactation.3,6,17,18,32 In colostrum collected up to 4 h postpartum the energy value as well as concentration of vitamin C, protein and TS were higher than in colostrum collected from the same mares between 8 and 12 h post foaling (P ≤ 0.01; Table 1). The rapid drop in protein level in mares’ colostrum has been confirmed by the results of other authors. Salimei et al.8 and Pecka et al.23 determined the chemical composition of colostrum collected up to 2 h after foaling; the authors reported higher protein content than the level detected in this study. Salimei et al.,8 after a repeated colostrum analysis at 6 h post foaling, recorded a considerable decrease in protein content, to 49 g kg−1 . Protein content of mares’ milk was stable. Its concentration in milk decreased in successive stages of lactation but there were no significant differences between retrievals. The results of this study indicate that mares’ milk contains twice as much protein as human milk and, on average, 60% less protein than cows’ milk. Mares’ milk is characterized by very low energy value, which is related to low fat content. Fat concentration in human and cows’ milk is 30–50 g kg−1 .33,34 Mean fat content in mares’ milk was lower than that in colostrum (12.8 vs. 22.0 g kg−1 ). Milk from the first half of lactation (7, 28 and 90 days) was characterized by a higher content of fat than that collected towards the end of lactation (P ≤ 0.01; Table 1), which was confirmed in studies conducted by other authors.4,6,17,32 A very low fat level (below 6 g kg−1 ) in the sixth month of lactation was also reported in other studies.10,18,32 In the experiments of Pikul et al.5 and Pikul and Wójtowski,4 conducted on mares in the fifth and sixth months of lactation, fat content was higher (over 10 g kg−1 ) than that in this study. Mean lactose concentration in the colostrum of mares was lower than in milk (35.6 vs. 63.5 g kg−1 milk). Lactose concentration in milk increased in successive stages of lactation and at day 7 of lactation it was lower than at days 90 and 180 of lactation (P ≤ 0.05; P ≤ 0.01; Table 1). Similar upward trends in the course of 180-day lactation were also recorded by Santos and Silvestre,6 Summer et al.32 and Centoducati et al.17 The high content of lactose (60–70 g kg−1 ) in mares’ and human milk distinguish them from cows’ milk (50 g kg−1 ). SCC decreased in mares’ colostrum and milk during the progress of lactation (Table 1). Mean SCC in colostrum was over tenfold higher than in milk (301.57 vs. 25.73 × 103 cells mL−1 ). In the
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© 2014 Society of Chemical Industry
J Sci Food Agric 2015; 95: 2279–2286
Influence of stage of lactation and season on composition of mares’ colostrum and milk
J Sci Food Agric 2015; 95: 2279–2286
Human milk is a richer source of vitamin C than cows’ milk and its content also changes with the stage of lactation and is greater in colostrum than in milk.49 – 51 Effect of season Climatic conditions Winter months (February, March) had a mean temperature of 4.97 ∘ C during the day and −1.27 ∘ C during the night; spring time (April and May) was characterized by mean temperatures of 19.67 and 6.46 ∘ C during the day and night, respectively. In the summer (June, July and August) the average temperature was 23.47 ∘ C during the day and 11.69 ∘ C during the night. In turn, in the autumn (September, October, November), during the day the average temperature was 13.68 ∘ C and during the night it was 5.38 ∘ C. It has been already shown that cows’ milk composition varies with season, stage of lactation, health status of the cow, milking interval, feeding, genetic factors and other day-to-day variations.52 Studies on the effects of feeding, breed, stage of lactation or genetic variation on mares’ milk composition are scarce.4,5,53,54 To the best of our knowledge, the effect of season on mares’ milk composition has been not assayed and requires further research. Table 2 presents the average values, range and seasonal variation for the composition and some physical properties of mares’ milk collected in summer and autumn. No effect of the season of the year was found on nutrient content or values of physicochemical parameters of colostrum produced by mares foaling in winter (February, March) or in spring (April, May). The averages for fat, protein, lactose, dry matter, SCC and energy value in the spring season were 2.23%, 9.67%, 3.73%, 16.88%, 300 × 103 cells mL−1 and 3.27 MJ kg−1 respectively; and in the winter season were 2.41%, 9.29%, 3.37%, 16.30%, 263 × 103 cells mL−1 and 3.14 MJ kg−1 , respectively. The density was the same for the colostrum collected in spring and in winter, with a mean value of 1.046 g mL−1 . Titratable acidity was 11.54 ∘ SH and 10.49 ∘ SH, while FPD was −0.585 and −0.577 ∘ C in winter and spring, respectively. There was a slight variation in the level of citric acid, which ranged from 0.21% in winter to 0.25% in spring; however, this difference was not significant. Variation in the basic chemical components and cytology parameter of milk from summer (June, July, August) and autumn (September, October, November) was manifested mainly in the fluctuations in fat and cholesterol content, as well as protein, citric acid and FPD of mares’ milk. Milk collected in summer had a significantly higher fat and cholesterol content than in autumn (P ≤ 0.01). Compared with fat, the protein and citric acid concentrations showed less variability (P ≤ 0.05). Milk collected in summer was characterized by higher content of protein and citric acid than that collected in autumn (P ≤ 0.05; Table 2). The average concentrations of vitamin C and SCC were similar for milk collected in summer and in autumn and there were no specific significant seasonal variations. Generally, the level of lactose, which is known to be one of the least variable milk components in cows’ milk, fluctuated over the study, but no significant seasonal differences were observed. Milk produced in summer had lower a FPD value than that collected in autumn. This variation could be attributed to the higher total solids level in summer versus autumn milk (P ≤ 0.01; Table 2). The FPD displays a relatively narrow range in cows’ milk, because of the osmotic balance between milk and blood.52 Bjerg et al.55 suggested that variations in cows’ milk freezing point may be due to the increased water intake due to the increased temperature and sunshine hours in summer. The lower FPD of mares’ milk is probably related to the concentration of water-soluble substances such as lactose, citrates and phosphates.24,35 Cagaj et al.35 suggested
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recorded a higher colostrum density, reaching 1.070 g kg−1 . In turn, Yao et al.25 determined the specific gravity of colostrum to be 1.080 g kg−1 in the second hour post foaling and at 1.030 g kg−1 in the 24th hour postpartum. The presented results indicate differences in mean TA between colostrum and milk (10.89 vs. 4.15 ∘ SH). Milk from the end of lactation was characterized by lower TA than that collected at the beginning of lactation (P ≤ 0.05; Table 1). Recorded results were similar to those reported by other authors18,23,32 and confirmed our earlier results.10 The presented data indicate that mares’ milk is characterized by lower TA than cows’ milk. According to Barłowska,28 TA of cows’ milk fluctuates around 7 ∘ SH and remains stable throughout lactation. Mares’ milk, in comparison to cows’ milk, is characterized by a low level of TS, and thus a lower concentration of nutrients, including casein, which is a factor responsible for TA.10 In the second half of lactation, when the level of TS in mares’ milk is less than 10.0 g kg−1 , the difference between TA of cows’ and mares’ milk is the most visible. Citric acid content in mares’ colostrum was higher than in milk (on average, 2.3 vs. 1.5 g kg−1 ). With the progress of lactation its concentration in milk decreased, which was confirmed statistically (P ≤ 0.01; Table 1). Original research papers concerning content of citric acid in milk are scarce.10,24,41 Citric acid contained in the milk salts binds calcium and magnesium in the form of citrates and increases its thermal stability.24 Moreover, it is a substrate for certain lactic bacteria species, using it to form aroma substances. Citrates have also been ascribed antiscorbutic properties and a capacity to promote calcium absorption by the mucosa in the alimentary tract in the presence of vitamin D, which might influence the biological value of milk.42 Mares’ milk contained small amounts of cholesterol. Colostrum contained over three times more cholesterol than milk (0.22 vs. 0.07 g kg−1 ). Cholesterol content in milk collected at day 180 of lactation was lower than in previously collected samples (P ≤ 0.01; Table 1). Cholesterol content determined in this study was similar to the level reported by Marconi and Panfili.3 A downward trend for cholesterol content during lactation was also shown by Pikul and Wójtowski,4 who investigated the fatty acid profile and cholesterol content in mares’ milk at the different stages of lactation. Cholesterol content determined by those authors in colostrum (0.20 g kg−1 ) and milk (0.06 g kg−1 ) was very similar to that obtained in this study. Mean cholesterol content in mares’ milk analysed in this study was lower than in human or cows’ milk. Its concentration in human milk ranges from 0.10 to 0.20 g kg−1 .34,43 At 3.5% fat-corrected, cows’ milk cholesterol content is 0.14 g kg−1 , but depending on fat content it may vary from 0.08 to 0.20 g kg−1 .44 – 46 Concentration of vitamin C in colostrum was lower than in milk (0.05 vs. 0.09 g kg−1 ). Colostrum collected up to the fourth h postpartum contained over twofold higher content of vitamin C than that collected between the 8th and 12th hour postpartum (P ≤ 0.01; Table 1). Content of vitamin C in milk was higher at days 90 and 180 than at the beginning (days 7 and 28) of lactation (P ≤ 0.01; Table 1). Cows’ milk contains ∼0.02 g vitamin C kg−1 , which provides antioxidant protection to this raw material.47 Vitamin C concentration in cows’ milk also depends on the stage of lactation, but changes in its concentration differ from that in the analysed mares’ milk. Hidiroglou et al.48 observed that the concentration of vitamin C in cows’ milk decreased with the progress of lactation.
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Table 2. Chemical composition, SCC and physicochemical parameters of mares’ milk collected in autumn and summer Season of milk collectiona Summer Item
LSM
Fat (g kg−1 ) Protein (g kg−1 ) Lactose (g kg−1 ) TS (g kg−1 ) Cholesterol (g kg−1 ) Vitamin C (g kg−1 ) Citric acid (g kg−1 ) Energy (MJ kg−1 ) Density (g kg−1 ) TA (∘ SH) Freezing point (∘ C) SCS (Ln) SCC (×103 cells mL−1 )
11.9A 17.8a 65.6 105.5A 0.06A 0.10 1.4a 1.95A 1,029 3.51 0.527A 2.90 19.00
Minimum 2.0 15.5 60.4 91.1 0.03 0.05 1.1 1.43 1,026 2.67 0.405 – 9.00
Autumn Maximum 21.1 20.5 69.9 117.0 0.09 0.15 1.7 2.35 1,030 4.87 0.737 – 31.00
LSM
Minimum
4.1B 16.7b 66.6 97.3B 0.03B 0.11 1.3b 1.64B 1,028 2.80 0.506B 2.89 21.00
Maximum
1.1 14.6 5.61 85.9 0.02 0.09 1.0 1.34 1,022 1.14 0.409 – 7.00
8.3 18.9 7.04 103.6 0.06 0.13 1.6 1.84 1,030 4.29 0.716 – 46.00
SE
P-value
1.16 0.34 0.87 1.61 0.003 0.005 0.04 0.054 0.0004 0.25 0.005 0.12 –
0.002 0.03 0.435 0.0013 < 0.0001 0.170 0.047 0.0004 0.255 0.057 0.0098 0.959 –
a Summer: June, July, August; autumn: September, October, November. Means within a row with different letters differ: P ≤ 0.01 (upper case); P ≤ 0.05 (lower case). MJ, megajoule; TA, titratable acidity, measured in Soxhlet–Henkel degrees (∘ SH); SCS, somatic cell score; SCC somatic cell count; For statistical analysis SCC was transformed to natural logarithm values (Ln).
that determining the limits for freezing point of mares’ milk is particularly significant, taking into consideration its high price. Density was constant throughout the summer and autumn seasons. Titratable acidity displayed some fluctuations but no significant seasonal variation was found. There are no reports in the available literature on the effect of season of the year on the nutritive value or the cytology parameter in mares’ milk. The differences between the results obtained in this study are difficult to interpret. One may assume that they were caused by differences in the chemical composition of diets offered to mares. The quality of grass and of oats could be different in June and September. This hypothesis must be confirmed by nutritional studies taking into account detailed analysis of pasture and feed samples during different seasons. A more detailed study of the effect of nutritive and non-nutritive factors like breed and age of mares are needed. Our results clearly show that the effect of season of the year on the chemical composition of mares’ milk cannot be considered without paying attention to the fact that it is related to the stage of lactation. Because the collection of mares’ milk, in contrast to cows’ milk, is seasonal in character, strongly determined by the season of the year, milk collected in summer will be obtained from the mares being in the earlier stage of lactation than milk harvested in autumn. This will result in higher fat content in summer milk in comparison to low-fat milk collected in autumn. This may serve as valuable information for potential producers and consumers.
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Effect of method and time of storage on vitamin C concentration The basic chemical composition of the fresh milk used for this part of the experiment was: 3.79 g kg−1 fat, 15.98 g kg−1 protein, 70.75 g kg−1 lactose and 93.06 g kg−1 total solids. Mean content of vitamin C in milk was 0.13 g kg−1 and it was slightly lower than in the study by Marconi and Panfili,3 who recorded 0.15 g kg−1 , but higher than in the warm-blooded mares’ milk examined in this study.
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Table 3. Vitamin C content (g kg−1 ) in mares’ milk depending on method and time of storage
Method of storage Fresh Frozen Lyophilisate
Time of storage (months)
6 12 6 12
LSM 0.134A 0.120BC 0.104D 0.125AB 0.115C
SD 0.0122 0.0102 0.0146 0.0142 0.0171
Means within a column with different letters differ (P ≤ 0.01).
The duration of milk storage, irrespective of its preservation method (freezing, lyophilisation), had an effect on the concentration of vitamin C (P ≤ 0.01; Table 3). At 12-month storage the losses were over twofold greater (18%) than in the case of 6-month storage (8%). In turn, no differences were found (P > 0.05) when comparing content of vitamin C in frozen and lyophilised milk after 6 months of storage. Only 12-month storage resulted in a reduction of its concentration. Losses greater by ∼10% were recorded in frozen milk in relation to lyophilised milk (P ≤ 0.01). Moreover, in frozen milk after 6 months of storage, losses of the vitamin amounted to 10%, while after 12 months it was 22% in relation to fresh milk and in lyophilised milk it was 6% and 14%, respectively. A review of the literature shows that most studies on the effect of milk storage on vitamin C content concern human milk.51,56,57 Investigations conducted by Romeu-Nadal et al.50 indicate that in the period of 12 months the concentration of vitamin C in human milk freeze-stored at a temperature of −20 ∘ C decreased by 24%, while in that stored at −80 ∘ C it decreased by 12%. In turn, a study conducted by Marconi and Panfili3 showed that the preservation of milk by drying had a considerable effect on the
© 2014 Society of Chemical Industry
J Sci Food Agric 2015; 95: 2279–2286
Influence of stage of lactation and season on composition of mares’ colostrum and milk reduction of concentration, not only for vitamin C but also vitamins A and E as well as cholesterol.
CONCLUSIONS Analyses showed that the stage of lactation is a major factor significantly influencing the chemical composition of mares’ milk. Moreover, it was shown that milk collected in autumn differs from that produced in summer, first in the lower content of fat and cholesterol. In terms of vitamin C stability, lyophilisation and 6-month storage of milk proved to be the most advantageous.
ACKNOWLEDGMENTS The authors wish to thank the Moszna Horse Stud for cooperation during the experiment. We wish to acknowledge the help provided by Billy Carey in revising the English.
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J Sci Food Agric 2015; 95: 2279–2286
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