Lactoferrin (LF) is a glycoprotein secreted by mammary epithelial cells. It is a non-heme iron-binding protein within the transferrin family and is mainly present in the whey protein fraction of milk. The content of lactoferrin in breast milk is approximately 2-3 g/L, while that in cow's milk is approximately 0.8 g/L.
Although lactoferrin has a relatively low content, it has various beneficial effects such as enhancing immunity, having antibacterial properties, antiviral activity, regulating intestinal flora, promoting iron absorption, providing antioxidant protection, exerting anti-inflammatory effects, facilitating growth and development, preventing cardiovascular diseases, and improving oral health. It plays a crucial role in maintaining human health and physiological regulation, and is of great significance for the growth and development of infants and the health of adults.
Lactoferrin is a glycosylated modified peptide chain consisting of 700 amino acids. After folding, it forms two identical globular domains, namely the N-ring and the C-ring. These two domains are connected by a short α-helix, and each domain is further divided into two subdomains (subdomains), namely N1, N2 and C1, C2. Each domain contains an iron-binding site and a glycosylation site. The molecular weight is approximately 80 kDa. Due to its binding to iron ions, the purified lactoferrin appears red. The three-dimensional structure of lactoferrin presents a typical spherical shape with a diameter of about 10 nm. Its surface carries positive charges, which helps it to bind to anionic biological compounds.
Lactoferrin has a variety of important functions and has been used as a food nutrient fortifier in China and the United States for sports and functional foods. Some products have been evaluated as orphan drugs by the European Medicines Agency (EMEA) and the US Food and Drug Administration (FDA).
According to the data, the Dutch DMV company is the world's largest supplier of lactoferrin. The lactoferrin used in most of the European milk powders is provided by this company. The purity of the lactoferrin it produces is as high as 97%, and it is the first lactoferrin raw material in the world to obtain the GRAS certification (a recognized safe substance for use) from the US FDA.
The content of lactoferrin is relatively low. A large number of pretreatment steps are required to prepare the crude product, which is then subjected to further purification.
Reference [1] used bovine colostrum as the starting material. Through centrifugal defatting, gelatinase enzymatic hydrolysis, centrifugal separation of casein precipitation; the centrifugal supernatant was whey protein solution. After ultrafiltration classification, it was segmented into >200KD (casein), >100KD (globulin), >50KD (precursor of lactoferrin), >10KD (casein albumin) sections, to prepare the crude lactoferrin with a molecular weight of 50KD - 100KD. Then, it was purified by cation exchange resin, desalination concentration, and freeze-dried under vacuum at -35℃ to -41℃ with a pressure of 47Pa, to obtain high-purity lactoferrin powder.
Reference [2] used milk as the starting material and employed ammonium sulfate fractional precipitation to remove casein and immunoglobulin. Finally, the centrifuged supernatant was adjusted to a pH of 8.5, and 80% ammonium sulfate precipitation was used to obtain the crude product of lactoferrin. Then, it was prepared using Sephadex G-100 to obtain lactoferrin with a purity of 92%. The influence of the isomer and iron ion binding state was investigated. Lactoferrin exhibited different molecular weight forms. The purified lactoferrin had a maximum absorption peak at 475 nm.
Reference [3] describes the production of lactoferrin N strain from recombinant Bacillus subtilis. Through multiple purification steps including induction culture, cell collection, cell disruption, centrifugation, affinity chromatography, ultrafiltration concentration, and gel chromatography, 23.5 mg of pure protein can be obtained from 1 gram of the cells.
Reference [4] summarizes the comparison of methods for isolating and purifying lactoferrin as follows:
The above methods each have their own advantages and disadvantages.
Ultrafiltration mainly utilizes the difference in molecular weight of proteins for graded separation. The membrane filtration process has a large processing capacity and a high degree of automation, but the purity of the obtained product is low and it is prone to clogging of membrane pores. As long as the problem of membrane filtration blockage is solved, it remains a good method for large-scale preparation of crude lactoferrin.
Although the ion-exchange method is relatively complex to operate, it can be used to prepare highly pure lactoferrin by taking advantage of the different properties of lactoferrin and other proteins.
Affinity chromatography has high selectivity and can directly purify the target protein from crude protein, improving its purity. However, the cost of affinity chromatography materials is relatively high, and it has not yet been applied to the production of large-scale products.
By applying ultrafiltration combined with ion exchange resin method and through pre-treatment of the feed liquid and using the ultrafiltration分级method, the efficiency of the ion exchange resin can be improved.
Wuhan Bona is dedicated to the development of membrane application technologies. In the fields of clarifying and filtering, grading, desalination, and concentration of natural extracted proteins and proteins from fermentation sources, they have encountered and solved numerous material filtration problems, accumulating rich process and engineering experience. The dairy products industry offers a wide range of products and diverse production processes, such as casein concentration, whey protein recovery, whey protein de-sugarization and de-salination, and whey protein grading. A large number of membrane filtration technologies have been applied. With the further division of product types, in aspects such as material pre-treatment, membrane selection, and optimization of membrane filtration parameters, there is still room for continued exploration and improvement.
References:
1] Ma Chuang, Su Zhengbo, Li Ruiguo, et al. Research on the production process of extracting casein, immunoglobulin, milk albumin and lactoferrin from bovine colostrum [J]. Shandong Food Fermentation, 2004(4): 2.
[2] Qu Lianda, et al. "Isolation and Purification of Lactoferrin." Proceedings of the 2nd Meeting of the 6th Council of the Chinese Society of Veterinary Epidemiology and the 6th Congress of the Teaching Professional Committee, 2006.
[3] Jin Liang, Li Lihong, Zhang Rongzhen, et al. Optimization of the N-leaf process for recombinant Bacillus subtilis fermentation production of lactoferrin [J]. Chinese Journal of Biotechnology, 2022, 38(7): 2628-2638.
[4] Wang Yuchui, Sun Cuicui, Du Peng. Research Progress on Isolation, Purification and Functional Characteristics of Lactoferrin [J]. Dairy Products and Human Health, 2023(2): 44-52
