Lactoferrin, lysozyme and immunoglobulin G content in milk of four breeds of cows managed under intensive production system

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1 Polish Journal of Veterinary Sciences Vol. 13, No. 2 (2010), Original article, lysozyme and immunoglobulin G content in milk of four breeds of cows managed under intensive production system J. Król 1, Z. Litwińczuk 2, A. Brodziak 1, J. Barłowska 1 1 Department of Commodity Science and Processing of Animal Raw Materials, University of Life Sciences, Akademicka 13, Lublin, Poland 2 Department of Cattle Breeding, University of Life Sciences, Akademicka 13, Lublin, Poland Abstract The aim of the study was to analyze chosen antibacterial protein content, i.e. immunoglobulin G, lactoferrin and lysozyme in bovine milk obtained from cows of four breeds raised and maintained under intensive production technology, that is in the free stall system, and fed total mixed ration diet (TMR system). The studies were conducted on milk from four breeds of dairy cows maintained in Poland, i.e. Polish Holstein-Friesian Black-White and Red-White variety, Jersey and Simental. Milk samples were collected solely from healthy cows, having performed the TOK test with Mastirapid. Further analyses were made only on the milk samples with SCC determined under 2.0 x 10 5 cells/ml, i.e. 423 samples. Each sample was examined for lactoferrin and lysozyme determination using reversed-phase high-performance liquid chromatography (RP-HPLC) with UV-VIS detector. The immunoglobulin G (IgG) levels were established by the aid of radial immunodiffusion technique with Bovine IgG LL tests (The Binding Site, Birmingham, UK). It should be stated that a breed of cow had a significant effect on a content of the antimicrobial proteins analyzed in bovine milk evaluated. Milk obtained from Simental and Jersey cows prove to be their excellent source, i.e. lactoferrin ( and mg/l), lysozyme (9.84 and μg/l) and immunoglobulin G (579.9 and mg/l). The highest content of these protein was stated in milk of multiparous cows at the late stage of lactation. Key words: cows milk, antimicrobial proteins, intensive production system Introduction The last decades have been marked with growing sale of products that boost immunity levels of purchasers. Obviously, it is conditioned on increasing consumers concern for health benefits and immune system improvement. Regular administration of food supplements can translate to the reduction of viral and bacterial infection incidence and thus, enhancement of customers immunity defense mechanisms. Such products are based on milk and colostrum, especially bovine immune proteins isolated from them (Gapper et al. 2007, Król et al. 2008). These proteins are particularly adapted to fortify baby nutritional formulas, dietetic foods, high-protein preparations for convalescents and sportsmen. Besides, they are used Correspondence to: J. Król, jolanta.krol@up.lublin.pl

2 358 J. Król et al. in pharmacology and cosmetology (Mehra et al. 2006, Gapper et al. 2007). A critical group of proteins showing the antibacterial activity prove to be immunoglobulins that have important roles in specific humoral immunity and destroy foreign antigens (Korhonen 2004, Gapper et al. 2007). and lactoferrin also contribute significantly to organism protection. They constitute one of the most vital component of non-specific immunity mechanisms (Struff and Sprotte 2008). An antibacterial protein content in a diet is one of the factors that condition eliciting the appropriate immune response of organism. The research objective was to analyze antibacterial protein content, i.e. immunoglobulin G, lactoferrin and lysozyme in bovine milk obtained from cows of four breeds raised and maintained under intensive production technology, that is in the free stall system and fed total mixed ration diet (TMR system). Materials and Methods The studies were conducted on milk from four breeds of dairy cows maintained in Poland, i.e. Polish Holstein-Friesian Black-White and Red-White variety, Jersey and Simental. Cows were managed in the intensive system, i.e. housed in free stall dairy barns and fed according to TMR system. A cow feed ration included bulky feeds (maize silage, hay silage, hay), concentrates and mineral-vitamin mixtures. The feeds were mixed at proper ratios tailored to a nutritional group in a feed carrier and provided to the animals. Only for Jersey breed cows, separated and regulated feeding with concentrates in the feed station was implemented. In all the farms, milking operation was performed twice a day in the milking parlors. Milk samples were collected from evening milking solely from healthy cows, having performed the TOK test with Mastirapid. The cooled samples were transported to the laboratory to measure the somatic cell counts (SCC) on Somacount 150 (Bentley Instruments). Further analyses were made only on the milk samples with SCC determined under 2.0 x 10 5 cells/ml. A total of 423 milk samples were analyzed, i.e. 154 milk samples from Polish Holstein Friesian Black-White variety, 108 from Polish Holstein-Friesian Red-White variety, 85 from Simental breed and 76 from Jersey. Each sample was examined for lactoferrin and lysozyme determination using reversed-phase high-performance liquid chromatography (RP-HPLC) with UV-VIS detector, acetonitrile water eluent mixtures as mobile phase and 250 x 4.6mm NUCLEOSIL C18 (Varian) column. Calibration of the chromatographic system for lactoferrin and lysozyme determination was carried out by the calibration curve and external standard method. For this purpose, each protein was calibrated individually by injection (20 μl) solutions of the standards (lactoferrin from bovine milk and egg white lysozyme Sigma, Germany). All chemicals were of HPLC analytical grade. Solutions of lactoferrin and lysozyme standard with a concentration ranging, respectively from 0 to 200 mg/l and from 0 to 20 μg/l, were prepared to create the calibration curves. The limits of quantification LOQ (for lactoferrin 40 mg/l and for lysozyme 2.8 μg/l) and detection LOD (for lactoferrin 8.7 mg/l and lysozyme 0.9 μg/l) were determined. The immunoglobulin G (IgG) levels were established by the aid of radial immunodiffusion technique with Bovine IgG LL tests (The Binding Site, Birmingham, UK). Control samples, provided in the IgG LL set, were used before the test samples. To evaluate the impact of the subsequent lactation and its stage on a content of the antibacterial proteins analyzed, there were formed 4 age classes including cows at I, II, III and IV lactation periods and 3 lactation stages, that is until 120 day, from 121 up to 200 day and day. The results obtained were analyzed statistically by StatSoft Inc. STATISTI- CA ver. 6 program, based on single factor analysis of variance. Significance of differences was determined with L Fisher s test. Results Milk obtained from Simental breed cows showed the highest content of IgG (579.9 mg/l) and lactoferrin ( mg/l). While the lowest amount of proteins was recorded in milk of Polish Holstein-Friesian Black-White and Red-White variety. The differences in each protein concentrations (in favor of Simentalers) were as follows: IgG and mg/l; lactoferrin and mg/l. Milk from Jersey cows was characterized by a relatively high level of the studied proteins with the highest lysozyme concentration (13.02 μg/l). The poorest source of antimicrobial protein appeared to be milk obtained from primiparous cows. However, immunoglobulin and lysozyme content progressed successively with the lactations, with peaks reported in IV lactation. Whereas, the highest lactoferrin concentration was found in milk from cows at II and IV lactation, and mg/l, respectively. An antibacterial protein level underwent some significant changes throughout the lactation period. Milk gained during the first lactation months had the lowest lactoferrin and lysozyme content. Significantly more protein qualities were determined in the late stage of lactation, i.e. by mg/l (P 0.01) and 3.12 μg/l (P 0.05). Milk obtained at this stage of lactation

3 , lysozyme and immunoglobulin G Table 1. Effect of breed on antimicrobial protein content in bovine milk. Breed Polish Holstein- Polish Holstein- Friesian Black-White Friesian Red-White n = 154 n = 108 Simental n=85 Jersey n= A A B AB A A AB B A AB C BC 29.1 A, B, C differences significant at P 0.01 Table 2. Effect of subsequent lactation on antimicrobial protein content in bovine milk. Subsequent lactation I II III IV n = 162 n = 128 n = 136 n = A a A B ab AB AB b AB B b B 25.9 a, b differences significant at P 0.05; A, B differences significant at P 0.01 Table 3. Effect of lactation stage on antimicrobial protein content in bovine milk. Lactation stage < 120 days days days n = 168 n = 106 n = A a a AB ab a B b b 23.6 a, b differences significant at P 0.05; A, B differences significant at P 0.01 also showed the highest level of immunoglobulin G. During the first stages of lactation, this protein content was significantly lower (P 0.05), i.e. from 70.6 mg/l (early stage) up to 78.4 mg/l (middle stage). Discussion Most of available research reports pertaining to the problem of antimicrobial proteins concern colostrum, in particular its immunoglobulin content. Both plasma and bovine milk contain three classes of immunoglobulins, i.e. G, A and M, with predominance of immunoglobulin IgG, approximately 80% (Korhonen 2004, Gapper et al. 2007). However, Newstead (1976) who determined IgG concentration in milk from Jersey and Friesian cows, found a substantially lower level of this protein (compared to the present research) in milk from the Jersey breed animals 0.32 g/l. While milk gained from the Friesian cows contained 0.46 g/l of IgG which is comparable to the results obtained in the present study for milk from the Polish Holstein-Friesian cows. Krukowski et al. (2006) determined IgG content in milk from Black-White variety cows with 50-75% Holstein-Friesian genes. IgG concentration in these cows milk, from the

4 360 J. Król et al. healthy quarters, was 628 mg/l. In the present research, a lower IgG content has been found. As regards milk from the Polish Holstein-Friesian Black and White variety cows, differences in this protein concentration reached over 200 mg/l. According to El-loly and Farrag (2007) and Smithers (2008), sweet whey derived from the manufacture of cheese proves to be the rich source of immunoglobulins. The studies of Levieux and Ollier (1999) revealed that milk gained from Holstein-Friesian cows contained 0.47 mg/ml of IgG at average, while colostrum 59.8 mg/ml. The primiparous cows were shown to produce significantly less IgG as compared to cows at 2-4 lactation (P 0.05) and older (P 0.01). These findings have been confirmed in the present investigations. The lowest IgG concentration was found in the first lactation (454.8 mg/l). In the subsequent lactations, IgG content progressed successively and the significant differences occurred in IV lactation. The research results reported by Guliński et al. (2006) collaborate a higher immunoglobulin content (determined by Colostrodoser) in colostrum produced by multiparous cows. Mian-bin and Yin-jun (2009) studying colostrum obtained from cows managed at a farm in China (Hangzhou) found 25 mg/ml of immunoglobulin G and 1.02 mg/ml of lactoferrin, which content increased with the subsequent lactation. Concentration of IgG investigated by Caffin et al. (1983) was also noted to change, alike the present study, during the lactation course. In the early (30 days) and middle stage of lactation (150 days), there were recorded comparable values, that is 0.37 and 0.38 mg/ml. A significantly higher IgG content was found in milk gained in the late lactation (270 days) 0.60 mg/l. Another important ingredient of milk whey is also lactoferrin, which mean level is 0.1 g/l. Antimicrobial activity of this protein consists in its binding with iron ions that inhibits bacteria growth (Indyk and Filonzi 2005, Rilay et al. 2008). A high lactoferrin concentration was noted by Król et al. (2007) in milk obtained from Polish Red cows ( mg/l) and Whitebacks ( mg/l). Whereas Lindmark- -M4nsson et al. (2003) reported 90 mg/l ( mg/l) of this protein determined in raw milk from Swedish farms and this content appeared to be comparable to that noted in the present investigations. A substantially lower lactoferrin level ( mg/l) in milk from Black-White variety cows was determined by Reklewska et al. (2003). Tsuji et al. (2003) analyzed lactoferrin concentration in colostrum obtained from four breeds of cows in Japan. A higher content of this protein was established in the colostrum produced by Holstein-Friesian and Jersey cows ( mg/ml) as compared to Japanese Brown and Japanese Black ones ( mg/ml). These authors also indicated significant differences in lactoferrin levels in the successive lactations; the primiparous cows were observed to produce significantly less lactoferrin compared to the multiparous. Wielgosz-Groth et al. (2009) found elevated lactoferrin concentration in milk of Jersey breed cows during lactation. Its level, determined by sandwich Elisa, ranged from μg/ml in the first lactation month up to ,g/ml in the tenth one. The changes in this protein content throughout the lactation were also noted by Hiss et al. (2008), who studied caprine milk. A lactoferrin level maintained between 10 and 28 μg/ml until 32 lactation week, then it successively progressed to reach over 100 μg/ml in 44 lactation week., an enzymatic protein (hydrolase group) functions in the synergism with immunoglobulins and lactoferrin showing antibacterial activity against a number of bacteria (Pellergino and Tirelli 2000). According to the Carlsson et al. (1989) investigations, lysozyme concentration ranged from up to 2.74 μg/ml subject to somatic cell count and its level increased with SCC growth. Elegamy et al. (1996) found two times less lysozyme (70 μg/ml) in bovine milk as against camel s milk (150 μg/l). The research result closest to the present study findings have been obtained by Reklewska et al. (2003), who evaluated milk from Black-White cows. It contained μg/l of lysozyme, depending on a nutritional system. In conclusion, it should be stated that a breed of cow had a significant effect on a content of the antimicrobial proteins in bovine milk evaluated. Their excellent source prove to be milk obtained from multiparous cows of Simental and Jersey breed at the late stage of lactation. References Caffin JP, Poutrel B, Rainard P (1983) Physiological and pathological factors influencing bovine immunoglobulin G1 concentration in milk. 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