JoVE Journal > Biochemistry
Summary
Abstract
Introduction
Protocol
Risultati
Discussion
Divulgazioni
Acknowledgements
Materials
Riferimenti
Traduzione automatica
Biochimica

Determining Total Protein and Bioactive Protein Concentrations in Bovine Colostrum

Published: December 10, 2021
doi:
10.3791/63001
, , , , , , , , , ,
1Department of Molecular Biology and Genetics,Çanakkale Onsekiz Mart University, 2Uluova Dairy Farm, 3Department of Nutrition,University of Nevada, Reno, 4Department of Food Science and Technology,University of Nebraska, Lincoln, 5Evolve BioSystems Inc.

Summary

Bovine colostrum is both a primary source of nutrients and immunological support for the newborn calf. The understanding of the level of therapeutic proteins (lactoferrin and IgG) is important for the bovine colostrum dosing and standardization for human consumption.

Abstract

Colostrum is a complex biological fluid produced by mammals immediately after parturition. It meets all the nutritional requirements for neonates as a good source of macro- and micronutrients, bioactive peptides, and growth factors. Bovine colostrum is also a potential source of nutrition and bioactive because of its rich protein content that includes immunoglobulin G (IgG) and lactoferrin. However, the level of lactoferrin and IgG in bovine colostrum changes markedly during the lactation period. Therefore, monitoring the concentration of IgG and lactoferrin for the use of bovine colostrum as a protein source is an important question to study. Methods in this article describe how to determine protein content, as well as specific concentrations of lactoferrin and IgG. These methods include the following steps: Isolation of bovine colostrum proteins, Determination of protein concentration via Bicinchoninic acid assay (BCA), Visualization of proteins via SDS-PAGE, Determination of lactoferrin, and IgG concentration using an ELISA Assay.

Introduction

Colostrum is the initial secretion of the mammary gland produced by mammals shortly after parturition. Colostrum is rich in macro- and micronutrients, antimicrobial peptides, and growth factors1,2,3,4. The composition varies gradually over time through the transition to mature milk5,6,7 but most significantly within 24 h after parturition8. The composition of colostrum is also influenced by maternal factors, including age, parity, breed, health, and nutritional status, as well as extrinsic factors, including season, premature parturition, premature lactation, colostral handling factors (pooling colostrum and storage temperature), and induction of parturition9,10,11. Compared with mature milk, colostrum contains less lactose and more fat, protein, peptides, non-protein nitrogen, ash, hormones, growth factors, cytokines, nucleotides, vitamins, and minerals12. Bovine colostrum contains a wide range of proteins, including immunoglobulins, lactoferrin, α-lactalbumin (α-LA), β-lactoglobulin (β-Lg), lactoperoxidase, and several growth factors13. The total protein concentration of bovine colostrum ranges between 11.26 mg/mL and 169.55 mg/mL14. The protein content comprises whey and casein at an average concentration of 124.00 mg/mL and 26.00 mg/mL, respectively15. The whey portion contains three major types of immunoglobulins (Igs) as IgG (85%-90%), IgM (7%), and IgA (5%)16. The major Ig in bovine colostrum is IgG, which provides passive immunity and modulates the adaptive and innate immune systems in the calf17. The initial Ig concentration of the first milking bovine colostrum can range from 20 to 200 mg/mL and decrease to around 0.4-1.0 mg/mL18. The mean IgG concentration is approximately 60 mg/mL and declines steadily to the levels below 1 mg/mL throughout the transition to mature milk19.

Another important bioactive protein in colostrum is lactoferrin, an iron-binding glycoprotein with a concentration of 1.5-5 mg/mL. Properties of lactoferrin include enhancing iron absorption as well as possessing antimicrobial activity20,21, binding lipopolysaccharide, immune-modulation, and stimulating the growth of intestinal epithelial cells and fibroblasts22. Bovine colostrum also contains α-lactalbumin and β-lactoglobulin. These proteins are sources of essential amino acids and also have bactericidal activity23,24,25. The mean α-LA and β-Lg concentrations in colostrum average 2.77 mg/mL2, and 11.5 mg/mL26, respectively. Thereafter, these concentrations decrease to 1-1.5 mg/mL27, and 4.8 mg/mL26 in mature milk. Colostrum also contains a significant amount of lactoperoxidase (mean 22.8 µg/mL) and lysozyme (mean 0.40 µg/mL)26. Lactoperoxidase is a glycoprotein that possesses antimicrobial activity against Gram-positive and negative bacteria28 by producing reactive oxygen species. Lysozyme functions as an antimicrobial agent by cleaving the peptidoglycan component of bacterial cell walls, thereby leading to celldeath29,30.

Due to their properties, IgG and lactoferrin are processed into different food products to fortify infant formulas, food supplements, high-protein preparations for convalescents and sportsmen as well as in pharmacology and cosmetology31,32,33. Bovine colostrum represents an important source of IgG and lactoferrin. However, the composition of these bioactive proteins in bovine colostrum changes markedly during the lactation period. Therefore, monitoring changes in the concentration of these bioactive proteins in colostrum samples used for research and food processing is critical. This study aims to describe the methods for monitoring the concentration and compositions of the total protein, lactoferrin, and IgG in bovine colostrum during the 6 days after calving.

Protocol

Colostrum samples were collected for 6 days after calving in the noon over the period July-August, from 28 Holstein dairy cows from Uluova Milk Trading Company in Çanakkale, Turkey, and deep-frozen. The samples collected on the same day were pooled according to the day of each sample and analyzed for their total protein, lactoferrin, and IgG concentrations. All samples were assayed in duplicate. 1. Sample preparation Mix 200 µL of bovine colostrum with 40…

Representative Results

Following the protocol, the bovine colostrum samples were analyzed to determine protein, lactoferrin, and IgG concentration. The results of protein, lactoferrin, and IgG analyses of bovine colostrum are shown in Table 4. Table 4: Concentration of protein, lactoferrin, and IgG of bovine colostrum. <st…

Discussion

This study provides information about considerable changes in the protein, lactoferrin, and IgG concentrations in colostrum throughout the transition to mature milk. Detection of changes in the lactoferrin and IgG concentration was carried out by sandwich ELISA, and total protein concentration was analyzed by the BCA assay. Results indicate that early colostrum has the highest protein, lactoferrin, and IgG concentration, that subsequently decreased over the next 3 days. Accurate measurements of these proteins are relevan…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

This study is supported by Uluova Süt Ticaret A.Ş (Uluova Milk Trading Co.). RMD and BMH are employees of Evolve BioSystems, a company focused on restoring the infant microbiome.

Materials

10X Running Buffer (Tris-Glycine-SDS) ClearBand TGS10 SDS-Page analysis
2-mercaptoethanol gibco 31350-010 SDS-Page analysis
Acetic Acid GLACIAL Isolab 901,013,2500 SDS-Page analysis
Bovine IgG ELISA Kit Aviva Systems Biology OKIA00005 Determination of IgG concentration
Bovine LF / LTF / Lactoferrin ELISA Kit LSBio Lifespan Biosciences LS-F4884 Determinaton of lactoferrin concentration
Coomassie Brillant Blue R 250 amresco 0472-25G SDS-Page analysis
Hydrochloric Acid Fuming 37% Isolab 932,103,2501 SDS-Page analysis
Isopropanol Isolab 961,023,2500 SDS-Page analysis
Laemmli Sample Buffer (2X) ClearBand LSB-2x SDS-Page analysis
Methanol Isolab 947,046,2500 SDS-Page analysis
PageRuler Plus Prestained Protein Ladder 10 to 250 Thermo Scientific 26619 SDS-Page analysis
Pierce BCA Protein Assay Kit Thermo Scientific 23225 Determination of protein concentration
Sodium dodecyl sulfate (SDS) BioShop SDS001.500 SDS-Page analysis
SureCast Acrylamide Solution 40% (w/v) Invitrogen HC2040 SDS-Page analysis
SureCast Ammonium persulfate (APS) Thermo Scientific 17874 SDS-Page analysis
SureCast Tetramethylethylenediamine (TEMED) Invitrogen HC2006 SDS-Page analysis
TECAN Infinite M200 Plate Reader Tecan 30035094 Measurement of absorbance
Tris base BioShop TRS001.1 SDS-Page analysis
DOWNLOAD MATERIALS LIST

Riferimenti

  1. Kehoe, S. I., Jayarao, B. M., Heinrichs, A. J. A survey of bovine colostrum composition and colostrum management practices on Pennsylvania dairy farms. Journal of Dairy Science. 90 (9), 4108-4116 (2007).
  2. Levieux, D., Ollier, A. Bovine immunoglobulin G, β-lactoglobulin, α-lactalbumin and serum albumin in colostrum and milk during the early post partum period. Journal of Dairy Research. 66 (3), 421-430 (1999).
  3. Elfstrand, L., Lindmark-Månsson, H., Paulsson, M., Nyberg, L., Åkesson, B. Immunoglobulins, growth factors and growth hormone in bovine colostrum and the effects of processing. International Dairy Journal. 12 (11), 879-887 (2002).
  4. Strekozov, N. I., Motova, E. N., Fedorov, Y. N. Evaluation of the chemical composition and immunological properties of colostrum of cows’ first milk yield. Russian Agricultural Sciences. 34 (4), 259-260 (2008).
  5. Playford, R. J., Weiser, M. J. Bovine colostrum: Its constituents and uses. Nutrients. 13 (1), 265 (2021).
  6. Godhia, M., Patel, N. Colostrum – Its composition, benefits as a nutraceutical: A review. Current Research in Nutrition and Food Science Journal. 1 (1), 37-47 (2013).
  7. Nakamura, T., et al. Concentrations of sialyloligosaccharides in bovine colostrum and milk during the prepartum and early lactation. Journal of Dairy Science. 86 (4), 1315-1320 (2003).
  8. Arain, H. H., Khaskheli, M., Arain, M. A., Soomro, A. H., Nizamani, A. H. Heat stability and quality characteristics of postpartum buffalo milk. Pakistan Journal of Nutrition. 7 (2), 303-307 (2008).
  9. Maunsell, F. P., et al. Effects of mastitis on the volume and composition of colostrum produced by Holstein cows. Journal of Dairy Science. 81 (5), 1291-1299 (1998).
  10. Tittle, D. J. Factors affecting colostrum quality. Cattle Practice. 10 (2), 131-136 (2002).
  11. Zarcula, S., et al. Influence of breed, parity and food intake on chemical composition of first colostrum in cow. Animal Science and Biotechnology. 43 (1), 43 (2010).
  12. McGrath, B. A., Fox, P. F., McSweeney, P. L. H., Kelly, A. L. Composition and properties of bovine colostrum: a review. Dairy Science and Technology. 96 (2), 133-158 (2016).
  13. Bastian, S. E. P., Dunbar, A. J., Priebe, I. K., Owens, P. C., Goddard, C. Measurement of betacellulin levels in bovine serum, colostrum and milk. Journal of Endocrinology. 168 (1), 203-212 (2001).
  14. Zhang, L., et al. Bovine milk proteome in the first 9 days: Protein interactions in maturation of the immune and digestive system of the newborn. PloS One. 10 (2), 0116710 (2015).
  15. Godden, S. Colostrum management for dairy calves. The Veterinary clinics of North America. Food Animal Practice. 24 (1), 19-39 (2008).
  16. Larson, B. L., Heary, H. L., Devery, J. E. Immunoglobulin production and transport by the mammary gland. Journal of Dairy Science. 63 (4), 665-671 (1980).
  17. Ulfman, L. H., Leusen, J. H. W., Savelkoul, H. F. J., Warner, J. O., van Neerven, R. J. J. Effects of bovine immunoglobulins on immune function, allergy, and infection. Frontiers in Nutrition. 5, 52 (2018).
  18. El-Loly, M. M. Bovine milk immunoglobulins in relation to human health. International Journal of Dairy Science. 2 (3), 183-195 (2007).
  19. Korhonen, H., Marnila, P., Gill, H. S. Milk immunoglobulins and complement factors. British Journal of Nutrition. 84 (1), 75-80 (2000).
  20. Arnold, R. R., Brewer, M., Gauthier, J. J. Bactericidal activity of human lactoferrin: Sensitivity of a variety of microorganisms. Infection and Immunity. 28 (3), 893-898 (1980).
  21. Aisen, P., Listowsky, I. Iron transport and storage proteins. Annual Review of Biochemistry. 49 (1), 357-393 (1980).
  22. Zhao, X., et al. The in vitro protective role of bovine lactoferrin on intestinal epithelial barrier. Molecules. 24 (1), 148 (2019).
  23. Chatterton, D. E. W., Smithers, G., Roupas, P., Brodkorb, A. Bioactivity of β-lactoglobulin and α-lactalbumin-Technological implications for processing. International Dairy Journal. 16 (11), 1229-1240 (2006).
  24. Pellegrini, A., Dettling, C., Thomas, U., Hunziker, P. Isolation and characterization of four bactericidal domains in the bovine β-lactoglobulin. Biochimica et Biophysica Acta (BBA) – General Subjects. 1526 (2), 131-140 (2001).
  25. Brück, W. M., et al. rRNA probes used to quantify the effects of glycomacropeptide and α-lactalbumin supplementation on the predominant groups of intestinal bacteria of infant rhesus monkeys challenged with enteropathogenic Escherichia coli. Journal of Pediatric Gastroenterology and Nutrition. 37 (3), 273-280 (2003).
  26. Indyk, H. E., Hart, S., Meerkerk, T., Gill, B. D., Woollard, D. C. The β-lactoglobulin content of bovine milk: Development and application of a biosensor immunoassay. International Dairy Journal. 73, 68-73 (2017).
  27. Swaisgood, H. E. Protein and amino acid composition of bovine milk. Handbook of Milk Composition. , 464-468 (1995).
  28. Seifu, E., Buys, E. M., Donkin, E. F. Significance of the lactoperoxidase system in the dairy industry and its potential applications: A review. Trends in Food Science and Technology. 16 (4), 137-154 (2005).
  29. Wheeler, T. T., Hodgkinson, A. J., Prosser, C. G., Davis, S. R. Immune components of colostrum and milk-A historical perspective. Journal of Mammary Gland Biology and Neoplasia. 12 (4), 237-247 (2007).
  30. Clare, D., Catignani, G., Swaisgood, H. Biodefense properties of milk: The role of antimicrobial proteins and peptides. Current Pharmaceutical Design. 9 (16), 1239-1255 (2003).
  31. Mehra, R., Marnila, P., Korhonen, H. Milk immunoglobulins for health promotion. International Dairy Journal. 16 (11), 1262-1271 (2006).
  32. Gapper, L. W., Copestake, D. E. J., Otter, D. E., Indyk, H. E. Analysis of bovine immunoglobulin G in milk, colostrum and dietary supplements: a review. Analytical and Bioanalytical Chemistry. 389 (1), 93-109 (2007).
  33. Mettler, A. E. Utilization of whey by-products for infant feeding. International Journal of Dairy Technology. 33 (2), 67-72 (1980).
  34. Zenker, H. E., Raupbach, J., Boeren, S., Wichers, H. J., Hettinga, K. A. The effect of low vs. high temperature dry heating on solubility and digestibility of cow’s milk protein. Food Hydrocolloids. 109, 106098 (2020).
  35. Costa, F. F., et al. Microfluidic chip electrophoresis investigation of major milk proteins: Study of buffer effects and quantitative approaching. Analytical Methods. 6 (6), 1666-1673 (2014).
  36. Lönnerdal, B., Du, X., Jiang, R. Biological activities of commercial bovine lactoferrin sources. Biochemistry and Cell Biology. 99 (1), 35-46 (2021).
  37. Belanger, L., Sylvestre, C., Dufour, D. Enzyme-linked immunoassay for alpha-fetoprotein by competitive and sandwich procedures. Clinica Chimica Acta. 48 (1), 15-18 (1973).
  38. Sakamoto, S., et al. Enzyme-linked immunosorbent assay for the quantitative/qualitative analysis of plant secondary metabolites. Journal of Natural Medicines. 72 (1), 32-42 (2018).
  39. Engvall, E. The ELISA, enzyme-linked immunosorbent assay. Clinical Chemistry. 56 (2), 319-320 (2010).
  40. Kohl, T. O., Ascoli, C. A. Immunometric double-antibody sandwich enzyme-linked immunosorbent assay. Cold Spring Harbor Protocols. 2017 (6), (2017).
  41. Shah, K., Maghsoudlou, P. Enzyme-linked immunosorbent assay (ELISA): the basics. British Journal of Hospital Medicine. 77 (7), 98-101 (2016).
  42. Abd El-Fattah, A. M., Abd Rabo, F. H. R., EL-Dieb, S. M., El-Kashef, H. A. Changes in composition of colostrum of Egyptian buffaloes and Holstein cows. BMC Veterinary Research. 8 (1), 19 (2012).
  43. Newby, T. J., Stokes, C. R., Bourne, F. J. Immunological activities of milk. Veterinary Immunology and Immunopathology. 3 (1-2), 67-94 (1982).
  44. Chigerwe, M., et al. Comparison of four methods to assess colostral IgG concentration in dairy cows. Journal of the American Veterinary Medical Association. 233 (5), 761-766 (2008).
  45. Foley, J. A., Otterby, D. E. Availability, storage, treatment, composition, and feeding value of surplus colostrum: A review. Journal of Dairy Science. 61 (8), 1033-1060 (1978).
  46. Mechor, G. D., Gröhn, Y. T., McDowell, L. R., Van Saun, R. J. Specific gravity of bovine colostrum immunoglobulins as affected by temperature and colostrum components. Journal of Dairy Science. 75 (11), 3131-3135 (1992).
  47. Pritchett, L. C., Gay, C. C., Besser, T. E., Hancock, D. D. Management and production factors influencing immunoglobulin G1 concentration in colostrum from Holstein cows. Journal of Dairy Science. 74 (7), 2336-2341 (1991).
  48. Quigley, J. D., Martin, K. R., Dowlen, H. H. Concentrations of trypsin inhibitor and immunoglobulins in colostrum of Jersey cows. Journal of Dairy Science. 78 (7), 1573-1577 (1995).
  49. Bielmann, V., et al. An evaluation of Brix refractometry instruments for measurement of colostrum quality in dairy cattle. Journal of Dairy Science. 93 (8), 3713-3721 (2010).
  50. A Ayar, A., Sıçramaz, H., Çetin, I. The effect of bovine colostrum on the lactic flora of yogurt and kefir. JSM Biotechnology and Biomedical Engineering. 3, 3-8 (2016).
  51. Sobaih, A., Zaki, D. A. Production of novel functional yoghurt fortified with bovine colostrum and date syrup for children. Alexandria Science Exchange Journal. 39, 651-662 (2018).
  52. Saalfeld, M. H., et al. Colostro: a redescoberta de um alimento saudável, nutritivo e com potencial probiótico. Agroecologia e Desenvolvimento Rural Sustentável. 5 (2), 18-24 (2012).
  53. Mouton, E., Aryana, K. J. Influence of colostrum on the characteristics of ice cream. Food and Nutrition Sciences. 06 (05), 480-484 (2015).
  54. Nazir, T., Pal, M. A., Manzoor, A. Effect of admixing varying levels of whole milk to the colostrum on the sensory quality of fermented colostrum product. International Journal of Advanced Research in Science, Engineering and Technology. 7 (4), 156-161 (2018).
  55. Korhonen, H. J. Bioactive milk proteins, peptides and lipids and other functional components derived from milk and bovine colostrum. Functional Foods. , 471-511 (2011).
  56. Cortés-Ríos, J., et al. Protein quantification by bicinchoninic acid (BCA) assay follows complex kinetics and can be performed at short incubation times. Analytical Biochemistry. 608, 113904 (2020).
  57. Johnson, M. Protein quantitation. Materials and Methods. 2, 115 (2012).
  58. Walker, J. M. The Bicinchoninic Acid (BCA) assay for protein quantitation. The Protein Protocols Handbook. , 11-15 (2009).
  59. Wang, R., et al. Sensitive immunoassays based on specific monoclonal IgG for determination of bovine lactoferrin in cow milk samples. Food Chemistry. 338, 127820 (2021).
  60. Kazemi, M. G., Feizy, J. Overview of the important of ELISA technique and application in food industry. Analyzing Microbes. 4 (4), 19-25 (2020).
  61. Verma, J., Saxena, S., Babu, S. G. ELISA-based identification and detection of microbes. Analyzing Microbes. , 169-186 (2013).
  62. Minic, R., Zivkovic, I. Optimization, validation and standardization of ELISA. Norovirus. , (2020).
  63. Drijvers, J. M., Awan, I. M., Perugino, C. A., Rosenberg, I. M., Pillai, S. The enzyme-linked immunosorbent assay. Basic Science Methods for Clinical Researchers. , 119-133 (2017).
  64. Walker, A. Breast milk as the gold standard for protective nutrients. The Journal of Pediatrics. 156 (2), 3-7 (2010).
  65. Patel, K., Rana, R. Pedimune in recurrent respiratory infection and diarrhoea-The Indian experience-The PRIDE study. The Indian Journal of Pediatrics. 73 (7), 585-591 (2006).
  66. Saad, K., et al. Effects of bovine colostrum on recurrent respiratory tract infections and diarrhea in children. Medicina. 95 (37), 4560 (2016).
  67. Buckley, J. D., Brinkworth, G. D., Abbott, M. J. Effect of bovine colostrum on anaerobic exercise performance and plasma insulin-like growth factor I. Journal of Sports Sciences. 21 (7), 577-588 (2003).
  68. Kotsis, Y., et al. A low-dose, 6-week bovine colostrum supplementation maintains performance and attenuates inflammatory indices following a Loughborough Intermittent Shuttle Test in soccer players. European Journal of Nutrition. 57 (3), 1181-1195 (2018).

Tags

Bovine ColostrumProtein ConcentrationBioactive ProteinsLactoferrinImmunoglobulin G (IgG)BCA AssaySDS-PAGEELISA Assay
This article has been published
Video Coming Soon
Keep me updated:

.

PDF
DOI
DOWNLOAD MATERIALS LIST
Citazione di questo articolo
Arslan, A., Duman, H., Kaplan, M., Uzkuç, H., Bayraktar, A., Ertürk, M., Alkan, M., Frese, S. A., Duar, R. M., Henrick, B. M., Karav, S. Determining Total Protein and Bioactive Protein Concentrations in Bovine Colostrum. J. Vis. Exp. (178), e63001, doi:10.3791/63001 (2021).
Scarica citazione
Ristampe e autorizzazioni

View Video

✖

To prove you're not a robot, please enter the text in the image below

CAPTCHA Image
Play CAPTCHA Audio
Refresh Image