brief introduction

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Lactoferrin was discovered in human milk by scientist Johanson Bengt in 1960. Subsequently, Baggin and other scientists discovered this protein in the body fluids and various cells of some organisms and began extensive research. The research shows that Lactoferrin LF is a natural protein in animal colostrum and a multifunctional protein, which has broad-spectrum antibacterial and antiviral effects and can regulate the balance of iron in the body; Regulate the generation of Myelocyte and promote cell growth; Regulate the body’s immune function and enhance the body’s disease resistance; Inhibition of human tumor cells; It can work synergistically with multiple antibiotics and antifungal agents to treat diseases more effectively.

Distribution and Content

Lactoferrin is widely distributed in human and mammalian milk and a variety of other tissues and their secretions (including tears, semen, bile, synovial fluid and other internal and external secretions and neutrophils), but the content in milk is high, and the content of Lactoferrin in bovine colostrum is the highest. Lactoferrin in the blood is mainly secreted by multinucleated cells, while bone marrow, salivary glands and endometrium can also secrete a small amount of Lactoferrin.

The concentration of Lactoferrin in human milk is about 1.0-3.0mg/mL, 10 times higher than that in cow milk (the content in cow milk is 0.02-0.35mg/mL), accounting for 20% of the total protein in ordinary human milk. During lactation, the content of Lactoferrin changes with the time of lactation. For example, Lactoferrin in human colostrum can reach 6-14mg/mL, and it drops to 1mg/mL during normal lactation. The content of Lactoferrin in colostrum of cows on the first day of postpartum can reach 1mg/mL. When the content of Lactoferrin in the milk of cows at the end of lactation will rise again.

Molecular structure

Lactoferrin (bovine) is a polypeptide chain with 703 amino acid residues, which folds into two basically symmetrical and highly homologous leaf like structures (N-leaf and C-leaf) [5]. Research suggests that this structure may have been formed during evolution due to gene overlap. The N-leaf structure contains amino acids 1-332, while the C-leaf structure contains amino acids 344-703. The two structures are linked through hinge regions, both of which are approximately 40kDa in size. The N-leaf and C-leaf can be correspondingly divided into N1, N2 regions, and C1, C2 regions. Compared to the C-leaf, the N-leaf carries higher positive charges. N-lobe structure has been proved to be related to improving the antibacterial activity of Lactoferrin. C-lobe structure can play a role in the treatment of gastric diseases, diabetes and corneal injury. Lactoferrin has two metal ion binding sites, each site contains two tyrosine, one Aspartic acid and one Histidine, which can reversibly bind one Fe3+ion and one CO32- ion. Because the complex of Lactoferrin and iron ion is red, it is also called “red protein”.

Physical and chemical properties

The affinity between Lactoferrin and iron ions is very high, 250~300 times that of transferrin. According to the difference of Lactoferrin binding iron ions, it can be divided into three types: iron deficient, iron semi saturated and iron saturated. Different types of Lactoferrin have different resistance to pasteurization and heat denaturation, of which the iron saturated type has the strongest resistance and the iron deficient type has the weakest resistance. Lactoferrin can bind not only Fe3+and Fe2+, but also Cu2+, Mn2+and Zn2+.

With regard to the thermal stability of Lactoferrin, the study found that heat treatment at 72 ℃ for 20s or 135 ℃ for 8s hardly affected the iron binding capacity of Lactoferrin, but if the treatment time at both temperatures was extended, the iron binding capacity would be reduced. The heat treatment intensity of 85 ℃ holding for 10 min will not affect the antibacterial activity of Lactoferrin. In addition, some studies showed that under the condition of pH2.0 or pH3.0, after being treated at 120 ℃ for 5min, Lactoferrin was degraded, but the antibacterial activity of the degradation products was higher than that before treatment. This experimental result led people to discover the existence of Lactoferrin active polypeptide. Lactoferrin has antibacterial effect on both Gram-positive bacteria and Gram-negative bacteria, but it has stronger effect on Gram-negative bacteria. The iron saturation of Lactoferrin was negatively correlated with its bacteriostasis. The change of environmental pH value affects the inhibitory effect of Lactoferrin on Gram-positive bacteria, but has little effect on Gram-negative bacteria. When the pH is between 7.5 and 8.0, the antibacterial effect is the best. The antibacterial activity of Lactoferrin was not affected after pasteurization below 70 ℃; Increasing the concentration of HCO3- is beneficial for enhancing its antibacterial activity.

biological function

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Lactoferrin can respond to various physiological and environmental changes, so it is considered to be a key component of the innate defense system. Lactoferrin shows strong antibacterial activity against a variety of bacteria, fungi, yeast, viruses and parasites. It also has anti-inflammatory and anticancer activities, and has various enzyme functions. Lactoferrin plays a key role in maintaining cellular iron level in vivo [5].

antibacterial activity

The biological function of Lactoferrin, which was first discovered and widely studied, is its antibacterial ability. Lactoferrin has the ability of broad-spectrum anti Gram-positive bacteria, Gram-negative bacteria and fungi, and can effectively inhibit the growth of Escherichia coli, Salmonella typhimurium, Streptococcus, Legionella pneumophila, Staphylococcus aureus. In clinical practice, oral Lactoferrin treatment reduces the infection of gastrointestinal bacteria, but does not affect the growth of lactobacilli and bifidobacteria [3].

It is generally believed that Lactoferrin competitively deprives bacteria of iron needed for growth by chelating iron ions, thereby inhibiting bacterial growth. Some studies have also shown that Lactoferrin can bind to lipid A, prompting lipid A to peel off from the bacterial wall of Gram-negative bacteria, leading to bacterial death. The N-terminal of Lactoferrin has a sequence encoding lactoferrin, which is a Cathelicidin with natural antibacterial activity [3].

Antiviral activity

Lactoferrin can help the body against a variety of virus infections. Intestinal infection caused by rotavirus, Enterovirus and adenovirus can be treated with Lactoferrin. At the initial stage of infection, Lactoferrin can inhibit the replication of adenovirus, prevent the virus from adhering to target cells and prevent infection. When studying the Norovirus that causes infectious diarrhea, it was found that Lactoferrin can reduce the damage of cytotoxic, reduce the number of viral cells, prevent the adhesion of virus to cells, inhibit the replication of virus and increase the expression of antiviral cytokines, thereby killing Norovirus. Lactoferrin can inhibit the activities of HIV-1 Reverse transcriptase and splicing enzyme, and prevent HIV virus from infecting cells. In addition, Lactoferrin can also resist the invasion of HSV-1, HCMV, HCV and other viruses to the body. There are also views that Lactoferrin has no direct antiviral effect, but indirectly achieves the purpose of antiviral through the antiviral response of the immune system [3].

Antifungal activity

Human Lactoferrin, bovine milk Ferritin and Lactoferrin derived peptides have in vitro activities against human pathogenic fungi, especially Candida albicans and several other candida species. Lactoferrin can kill Candida albicans and Clostridium cruzi by changing the permeability of cell surface, just like anti bacteria. Lactoferrin can change the permeability of cytoplasm and mitochondrial membrane of cryptococcus neoformans and Candida albicans. The antifungal mechanism of Lactoferrin is achieved through interaction with cell surface rather than iron deprivation [5].

anticancer

In recent years, the anticancer effect of Lactoferrin has been widely reported. Oral administration of bovine milk Ferritin to rodents can inhibit the formation of tumors induced by chemical methods. Clinical trials have confirmed that Lactoferrin can inhibit the occurrence and development of tumors. Lactoferrin has the ability to regulate the production of cytokines in cancer. Lactoferrin induces apoptosis and inhibits tumor growth in vitro. It can also prevent the transition from G1 to S in the cell cycle of malignant cells. In addition, treatment of mouse tumors with recombinant human Lactoferrin can inhibit tumor growth, increase the level of anti-cancer cytokines (such as IL-18), and activate NK cells and CD8+T lymphocytes. Recently, clinical trials have proved that bovine milk Ferritin can inhibit colorectal cancer, while human milk Ferritin can reduce the risk of colon cancer. For breast cancer, Lactoferrin can inhibit the growth of tumor cells. Adding exogenous Lactoferrin to the medium of breast cancer cell line (MDA-MB-231) will lead to cell cycle stagnation in G1/S transition period. In addition, Lactoferrin induced the growth arrest of Smad-2 in HeLa cells. Although the results obtained by researchers show that Lactoferrin has obvious anti-tumor effects, the mechanism of these effects is not completely clear [5].

Immunomodulatory and anti-inflammatory activities

Since Lactoferrin is widely distributed in the exocrine fluid of the body, it can be used as the first natural immune barrier of the body to participate in immune regulation. Lactoferrin is one of the important inflammatory response regulators and an important component involved in regulating the immune system. It can protect the body against microbial infection, reduce the incidence of E. coli and infectious diarrhea, and improve the growth of weaned piglets. For newborns with underdeveloped immune system, colostrum rich in Lactoferrin can help them activate and regulate the function of immune system, and promote the rapid establishment of autoimmune system. Oral administration of bovine milk Ferritin increased the number of cells in lymph nodes and spleen, increased the activity of peritoneal macrophages and spleen NK cells, and enhanced IL-12 and IFY of Th1 type T cells- γ The production of cytokines. Lactoferrin inhibits Programmed cell death of immune cells by blocking the signal passage of apoptosis; It is also possible to regulate the maturation and differentiation of T lymphocytes, as well as the balance of Th1/Th2 cytokines, through interactions with antigen-presenting cells.

enzymatic activity

Lactoferrin can act as an enzyme in some catalytic reactions. Some motifs of Lactoferrin and Ribonuclease A are significantly similar. Lactoferrin has DNA binding characteristics and can play a role in the transcription activation of specific DNA sequences or act as a signal transduction medium. Among all milk proteins, Lactoferrin has the highest Amylase and ATPase activities, however, they are not the only enzymatic activities. The various activities of Lactoferrin are attributed to the changes in its protein characteristics. Lactoferrin has a variety of isoforms, different glycosylation degrees, different tertiary structures and oligomerization degrees [5].

separation method

traditional method

There are many methods to purify Lactoferrin, and many companies choose cation exchange chromatography system to obtain Lactoferrin in a large range. The separation and purification of Lactoferrin began in the 1980s, and many companies are committed to developing processes to obtain high-purity Lactoferrin. The separation and purification methods of Lactoferrin from the initial cation exchange to affinity chromatography to immunological methods, as well as the combination of various methods, have advantages and disadvantages for the separation and purification of Lactoferrin.

Cation exchange chromatography. Using cation exchange chromatography to separate Lactoferrin from raw materials is a traditional method. Its advantages are simple operation, few steps, continuous injection and easy expansion. However, when traditional chromatography is used to rapidly and massively recover Lactoferrin from Whey protein, it shows many shortcomings, such as pollution of chromatographic materials, long cycle time, high pressure of liquid drops when passing through the chromatographic column, and complex control system. Moreover, many miscellaneous proteins with the same points and similar Isoelectric point as Lactoferrin in raw materials are washed off together when separating and purifying Lactoferrin, Therefore, the obtained Lactoferrin has low purity, high cost and relatively low output.

Membrane adsorption. Due to the poor thermal stability of Lactoferrin, membrane separation is a separation process without heating, phase change and chemical reagents. Therefore, this method can maximize the biological activity of Lactoferrin, making it an advantage to separate and recover Lactoferrin. Kerstin Plate and others studied whether membrane technology can be used to recover Lactoferrin in whey and whether laboratory level equipment can be directly applied to industrialization. The membrane area used in the experiment increased from 15cm2 to 4m2, and the experimental results showed that the optimal conditions were to use the Sartobind S system, expand the membrane area to 2m2, and do not need to be cleaned after 8 cycles. Alm é cija et al. used 300 kDa ceramic microporous membrane to separate Lf from whey. Research shows that the optimal solution pH values for obtaining Lactoferrin are 5 and 10 respectively. The former can obtain Lactoferrin, while the latter can retain Lactoferrin in the original whey. In order to overcome the drawback of membrane adsorption being unable to distinguish approximate molecular weights, Brisson et al. used a combination of charged membranes and electric fields, which played an important role in the movement of proteins. Although some purity issues were solved, the electrolytic reaction occurring on the surface of the solution had a negative impact on the separation of Lf.

Electrical separation. Electrical separation technology is a technology that utilizes the differences in molecular size and the charges it carries to separate protein molecules. Current can accelerate the separation speed. Compared with the traditional pressure driven method, the advantage of the application of electric field is that it improves the separation speed of Lactoferrin, but reduces its purity at the same time, because of the migration of other Whey protein and the interaction between proteins while separating Lf. The combination of electric separation and microfiltration membrane can overcome shortcomings such as low selectivity and solution pollution. N Ndiaye et al. used this method to separate Lactoferrin. The optimal separation condition of Lactoferrin was pH=3.0, and the optimal condition was 1.5 when 2 g/L KCl solution was used as the solution × 10-8m2/(V · s); When deionized water is used as the solution, the optimal condition is 3.0 × 10-8m2/(V · s). After 4 hours of treatment with a 500 kDa ultrafiltration membrane, the migration rate reached 46% and the yield was 15%. The disadvantage of this method is that at pH=3.0, β- Lactoglobulin will also be isolated together.

new method

Due to the wide application of Lactoferrin, people have higher and higher requirements for its purity. However, some proteins in whey, such as lactoperoxidase, have similar molecular weight and Isoelectric point with Lactoferrin. In addition, some scholars also pointed out that Lactoferrin binding proteins were purified at the same time as Lactoferrin was purified, including Ribonuclease -4, angiogenin, neutrophil gel enzyme related callipoproteins and Fibroblast growth factor binding proteins. These micro proteins and Lactoferrin may involve many physiological activities of Lactoferrin with functional diversity in vivo. Conventional hydrophobic interactions, affinity chromatography, molecular exclusion, ultrafiltration, and membrane filtration processes are complex, coupled with low selectivity. Therefore, some highly selective methods have been developed.

Hydroxyapatite method. Paul K Ng et al. used a mixed mode chromatography ceramic Hydroxyapatite chromatography. This method can separate Lf, Lp, and other globulins in whey in one step, and then obtain Lf through gradient elution. The test results show that an 80 L hydroxyapatite column can collect 0.32 kg Lf in one hour, and the obtained Lf is verified to have no Lp activity. The high yield, low cost, and simple steps make this operation possess all the characteristics necessary for effectively large-scale biotechnology industries. The purification process of Hydroxyapatite requires a large amount of water to effectively separate macromolecules. The product recovery rate depends on the amount of buffer solution used. This method has no way to distinguish molecules with similar hydrated particle size, so the purity of the product may be affected. In addition, the interactions between different proteins in whey make this process difficult to predict