This study is supported by Uluova S&#252;t Ticaret A.&#350; (Uluova Milk Trading Co.). RMD and BMH are employees of Evolve BioSystems, a company focused on restoring the infant microbiome.

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 &#199;anakkale, 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
<ol>
	<li>Mix 200 &#181;L of bovine colostrum with 40...

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...

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, &#945;-lactalbumin (&#945;-LA), &#946;-lactoglobulin (&#946;-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 &#945;-lactalbumin and &#946;-lactoglobulin. These proteins are sources of essential amino acids and also have bactericidal activity23,24,25. The mean &#945;-LA and &#946;-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 &#181;g/mL) and lysozyme (mean 0.40 &#181;g/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.

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

determining total protein and bioactive protein concentrations in bovine colostrum

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.

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.
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			<td colspan="4"><st...

Read this Scientific Article Protocol about Determining Total Protein and Bioactive Protein Concentrations in Bovine Colostrum at JoVE.com

Determining Total Protein and Bioactive Protein Concentrations in Bovine Colostrum

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.

Colostrum is a complex biological fluid produced by mammals immediately after parturition. It meets all the nutritional requirements for neonates as a ...