# Lactoferrin Production: A Systematic Review of the Latest Analytical Methods
## 3 Results and Discussion
### Conclusion Remarks
#### 3.1.1 Conclusion Remarks  
Most of the reviewed methods used bovine milk as a source material. This is due to the fact that cow lactoferrin can reach the widest spectrum of recipients. Supplements containing lactoferrin are based on bovine Lf and can be used by most people. Other \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Matrix** & **Method** & **Yield/Recovery** & **Study** \\ \hline Liquid milk, & & & \\ fermented milk, & UHPLC-UV & Recovery: 83.6–90.8\% & [PERSON] et al. (2022) [97] \\ infant formula & & & \\ \hline Infant formula, milk powder, powder & Optical Biosensor & & \\ reconstituted ready-to-feed basis & Immunoassay & Recovery: 96.1–109.2\% & [PERSON] et al. (2022) [98] \\ \hline Milk, infant formula, whey protein & HPLC-FL & Recovery: 96.3\% for milk, 95.8\% for infant formula, 93.3\% for WPC & [PERSON] et al. (2020) [99] \\ \hline Raw milk & HPLC-UVD & Recovery: 103.44\% & [PERSON] et al. (2021) [100] \\ \hline Skim milk & IEC, RP-HPLC, & Recovery: up to 100\% & [PERSON] [PERSON] et al. (2023) [101] \\ \hline Milk & Spectrophotometry & Yield: \(\geq\)98\% & [PERSON] et al. (2024) [102] \\ \hline Capsules, sachets, milk powder & Spectrophotometry & Recovery: 96.45–104.92\% & [PERSON] et al. (2024) [103] \\ \hline Infant formula & LC-MS/MS & Recovery: 91.7–96.4\% & [PERSON] et al. (2019) [104] \\ \hline Formula powder, paediatric/adult & UHPLC/UV & Recovery: 92.1–97.7\% & [PERSON] et al. (2024) [105] \\ \hline Whey & RP-HPLC-DAD & Recovery: 96–102\% & [PERSON] et al. (2021) [106] \\ \hline Human milk & HPLC, Infrared & & \\ Spectroscopy (FTIR), & & Recovery: 70\% & [PERSON] et al. (2022) [108] \\  & SDS-PAGE & & \\  & electrophoresis & & \\ \hline Human milk & Latex agglutination & & \\ assay, ELISA & Recovery: 90–120\% & [PERSON] et al. (2024) [109] \\ \hline Camel milk & UHPLC-MS/MS & Recovery: 74.5–103.6\% & [PERSON] et al. (2019) [111] \\ \hline \hline \end{tabular} For comparison, the patented cation-exchange chromatography, that is, used for mass scale production, and the yield is 80–96% (Patent No. U5596082A) [114].  
\end{table} 
Table 3: Efficacy of novel techniques of obtaining Lf. sources of Lf are less frequently studied, due to the limited group of recipients of the final product. Camel lactoferrin, although having very similar characteristics to cow lactoferrin, will not be as widely distributed due to the limited areas where camels are bred. It may therefore be a good source of Lf in narrow areas of camel farms, but cow farms are more widespread. Lactoferrin obtained from human milk is used to supplement newborns and infants. It is the safest source of Lf for such young children, which is why some studies focus on human milk. Nevertheless, human milk is a highly valued product with very limited resources. These features make it a less frequently used source and, furthermore, not used in studies as often as cow's milk. The most popular preparation and purification methods used buffers, inorganic reagents, centrifugation, magnetic separation, and filtration through membranes or filter columns, e.g., HiTrap Heparin or strong cation exchanger (Sepharose Big Beads). The most common solution to dilute source products was sodium phosphate, with the pH of the dilutions adjusted to 7-8 [99; 100; 105; 109]. Centrifugation was an essential step to remove fat from the samples heated, vortexed, incubated, or further diluted with designated buffers depending on the method. Commonly used solutions included ultrapure water, acetonitrile, and ammonium chloride [97; 104; 111]. Typically, the solutions were filtered to remove any impurities using nylon or polyethersulfone 0.22-0.2 \(\upmu\)m thick filters [97; 103; 111]. Magnetic microbeads coated with anti-Lf antibodies were also used to elute pure Lf [99]. This method involves using magnetic beads coated with anti-Lf antibodies. The protein bound to its antibody, beads were attracted with a magnet, and the rest of the solution could be discarded, leaving pure Lf to be eluted with fresh buffer [99].