# Lactoferrin Production: A Systematic Review of the Latest Analytical Methods
## 1 Introduction
### Lactoferrin Production  
Due to its unique properties, Lf can be isolated and purified from cheese whey and skimmed milk on an industrial scale [80]. Global Lf production has increased rapidly over the last 20 years, from less than 80 tonnes per year in 2003 to over 300 tonnes per year in 2021 [81; 82]. It is estimated that the market size will exceed 265 million in 2027 [82; 83]. In Europe, the main producers are Germany (170 tonnes per year) and the Netherlands (70 tonnes) [84; 85; 86].  
Commercially, Lf is currently isolated from bovine milk or its by-products primarily due to its good availability and relatively low costs. However, Lf can be isolated from any mammalian secretion [81], and recombinant human Lf has been produced in transgenic animals, fungi, cell cultures, or plants [87; 88]. However, Lf production from bovine milk is expensive due to the large volume of milk or its products required for the chromatographic and ultrafiltration techniques [89]. To purify Lf to supplement grade, milk or other dairy products must undergo an extraction process. The pulled-out Lf is then eluted with a solution of NaCl, then desalted and condensed by ultrafiltration. The obtained product must then be pasteurised and dried to create a powder (Figure 2).  
New techniques of obtaining Lf from sources other than animals are emerging. One of them uses fungi (_Pichia pastoris_) that allows scaling of the production due to the use of bioreactors. In optimal conditions, the yield can reach 2.8 g/L. This method allows for keeping the fungi densely in a limited space, which improves efficiency. However, the initial costs of production infrastructure and equipment are high [90; 91].  
Another method considers transgenic plants. The yield is lower than that of the fungi systems, but the smaller operational costs make it economically advantageous. A downside of using plant material is the reliance on a longer production cycle and environmental variability [92].  
A vital production aspect is to achieve a bioequivalent product, which can be a challenge in transgenic processes, as well as obtaining regulatory approvals. Only then, new methods can be implemented on a mass scale [93].  
Global standardisation in the quality of Lf (bovine lactoferrin) is progressing through Generally Recognised as Safe (GRAS) and Novel Food. These regulations are relatively high, including more than 95% Lf purity in protein [94]. In addition to lactoferrin content, other quality criteria are also assessed. The most important include low levels of microbiological contamination, minimal content of substances left over from technological processes, and limited levels of heavy metals [95]. Total heavy metal content (cadmium, lead, arsenic, mercury, copper) is max. 1 mg/kg. Microbiological guidelines: standard plate count < 1000 cfu/g; _Enterobacteriaceae_ < 10 cfu/g; yeasts < 10 cfu/g; and coagulase-positive _Staphylococcus_: not detected in 1 g [96].  
Due to the broad spectrum of Lf's health-promoting effects, with particular emphasis on antiviral and antibacterial effects, it is often a component of dietary supplements that strengthen immunity. The great interest in pharmaceutical products that prevent infectious diseases means a growing interest in commercial Lf production; therefore, this paper reviews the current Lf production methods.  
Figure 2: Lactoferrin production process.