J. Dairy Sci.
106:7329–7335
https://doi.org/10.3168/jds.2023-23328
© 2023, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Perspective: A proposal on solutions of modern
supply chain construction for lactoferrin
Ruoyu Mao,1,2,3* Xuanxuan Ma,1,2* Ya Hao,1,2,3 Guihong Pen,1,2 Xueling Zheng,1,2 Na Yang,1,2,3 Da Teng,1,2,3
and Jianhua Wang1,2,3†
1
Key Laboratory of Feed Biotechnology, Ministry of Agriculture and Rural Affairs, Beijing 100081, China
2
Innovative Team of Antimicrobial Peptides and Alternatives to Antibiotics, Gene Engineering Laboratory, Feed Research Institute,
Chinese Academy of Agricultural Sciences, Beijing 100081, China
3
R&D Center, Beijing Shengtai Clouds Bio-Technology Inc., Beijing 100081, China
ABSTRACT
Lactoferrin is an iron-binding glycoprotein of the
transferrin family that is found in most bodily flu-
ids of mammals and has a variety of biological and
beneficial functions, with great importance in health
enhancement as a supplement for humans and other
animals. More than 300 t of lactoferrin were produced
in 2021, and this number is expected to grow yearly
by 10% to 12%, to over 580 t in 2030. With new and
important functions of lactoferrin being revealed and
studied, focus on its industrial production and appli-
cation is increasing accordingly. However, lactoferrin
is mainly sourced from cheese whey or skim milk by
cation-exchange column chromatography, which is a
costly and low-quality method. A potential solution for
lactoferrin global supply chain construction is proposed
in this article as a complement to traditional routes of
purification from whey or skim milk. The large-scale
production of lactoferrin, mainly by recombinant yeast,
mammal, and grain systems, as well as the market
niche and product design, are discussed.
Key words: lactoferrin, supply chain construction,
niche market, recombinant expression
PERSPECTIVE
Lactoferrin (LF) is a multifunctional iron-binding
glycoprotein in many bodily fluids of mammals and has
a variety of biological and beneficial functions. Lacto-
ferrin occurs in high concentrations in human colos-
trum (~7 g/L) and lower concentrations in mature milk
(2–3 g/L). In cow milk, relatively low concentrations of
LF occur in colostrum (0.3–2.1 g/L) and mature milk
(0.03–0.49 g/L), accounting for 20% of the total protein
Received February 3, 2023.
Accepted June 1, 2023.
*These authors contributed equally to this work.
†Corresponding author: wangjianhua@​caas​.cn or wangjianhua.
peking@​qq​.com
7329
of milk (Levay and Viljoen, 1995). As a result, LF is
an important supplement in infant formula milk pow-
der. Worldwide bovine lactoferrin (bLF) production
increased rapidly from less than 80 t per year in 2006
(Wakabayashi et al., 2006) to more than 300 t in 2021
(Krolitzki et al., 2022). Due to huge demands for infant
nutrition, immune health, sports nutrition, cosmetics,
and livestock dam and neonate nutrition (Hao et al.,
2018, 2021; Dierick et al., 2022; Ma et al., 2023), the
bLF market is expected to grow yearly by 10% to 12%
in the upcoming years from approximately €166 million
in 2020 to over €265 billion in 2027 (Hancocks, 2021).
However, the raw material of bLF mainly comes from
cheese whey or skim milk and their byproducts. The
quantity of cheese whey is limited and mainly concen-
trated and distributed in Europe, North America, and
Oceania. The United States and Germany represented
approximately 29% of total exports in 2021 (Index-
Box, 2023), but actual end consumers were distributed
all over the world. In particular, Asia and China are
heavily populated, with a revenue share of more than
42.0% (Grand View Research, 2020); therefore, the gap
between their rigid demand and supply is large. Fac-
ing the pressure of this demand, unstable sources, and
trade disputes, a 10-fold increase in the price of LF in
recent years ($700 USD/kg) is expected to continue.
The bottleneck limiting the continuous development of
LF results from the lack of a stable supply chain of LF
as a raw material supported by modern biotechnology;
thus, breakthroughs are urgently needed.
CURRENT PROCESSING TECHNOLOGY
OF LF FROM WHEY AND SKIM MILK
Looking back on the early foundation and contribu-
tions to the LF industry, 2 milestones and 2 very impor-
tant people should be remembered. Jean-Paul Perraudin
innovated the classic preparation process of LF from
whey or skim milk at the laboratory level in the 1970s;
then, Mamoru Tomita built the industrial production
line in Morinaga Co. (Germany) with Perraudin in the
 Mao et al.: PERSPECTIVE: LACTOFERRIN SUPPLY CHAIN
mid-1980s. Let us look at the more technical elements
in detail, as cation-exchange purification has been the
most widely used process for the production of bLF by
major manufacturers since the 1980s, including Royal
Friesland Campina, MILEI, and Synlait Milk Ltd. (Kus-
sendrager et al., 1997; Tomita et al., 2009). The process
consists of 3 steps: conditioning, chromatography, and
formulation. (1) First, microfiltration (1–2 μm) is used
to remove the microorganism and particulate matter
and fat. (2) Subsequently, the filtrate is put through a
cation-exchange column with a pH of 6.3 to 6.8. Impu-
rities are washed from the column with a low-salt buffer
(1.5–2% sodium chloride), and the target bLF is eluted
with a high-salt buffer (10–15% sodium chloride). (3)
The purified liquid is concentrated in an ultrafiltra-
tion step, followed by desalting via diafiltration. After
pasteurization and spray or freeze drying, the final
powdered product, with a protein content >90% and
purity >95%, is obtained (FDA, 2013a,b, 2016; Tomita
et al., 2009). Although the process of cation exchange–
desalting–pasteurization–spray or freeze drying is used
by all manufacturers, bLF products are differentiated
by iron saturation, glycosylation, and other aspects due
to the different raw sources of whey and skim milk in
the world. Meanwhile, no bLF manufacturer has been
built in China so far, because its total market size and
volume are too small to reach the break-even point, and
few raw materials are distributed in various regions,
despite the fact that China is the largest consumer and
fastest growth market of bLF in the world (Grand View
Research, 2020). Therefore, new stable and large-scale
bLF production processes are urgently needed.
SOLUTION ON TECHNICAL ROUTE
OF MODERN LF SUPPLY CHAIN
Compared with the classical processing of LF from
whey or skim milk, transgenic expression is a more
effective solution to build a stable supply chain with
DNA molecular operation technology with high feasi-
bility; thus far, LF has been expressed in Escherichia
coli (Wang and Tian, 2010; Wang et al., 2010), Pi-
chia pastoris (Iglesias-Figueroa et al., 2016), Aspergillus
awamori (Ward et al., 1995), grains (Lönnerdal, 2002),
mammals (Yang et al., 2008; Wang et al., 2017a), and
gland cells (Kruzel et al., 2013). These successful cases
encouraged us to advance industrialization with in-
creasing expectations. It could realize industrialization
in top-level design and on-demand production, attract-
ing many concerns for years (Table 1). It is expected
that recombinant LF will be designed, expressed, and
supplied on demand by its niche market and consumers,
and the initial framework has been briefly described
in the 15th International Conference on Lactoferrin
Journal of Dairy Science Vol. 106 No. 11, 2023
7330
Structure, Function and Applications (Wang et al.,
2023). This is expected to be a revolutionary solution
and an upgrade from the first era of LF processing
in the 1980s, based on classical separation techniques
with raw materials in the milk industry. Considering
basic requirements and end consumption distribution
in detail, an expression system with the option to meet
various purposes could be developed according to the
following principles: large scale, standardization, fine
differentiation with various target functions, and low
cost, which would weaken or bypass the restrictions of
the primary industry in livestock breeding. Generally,
from the latest reports and levels on DNA recombina-
tion techniques in practice, 3 typical expression systems
of recombinant yeast and aspergilli, mammal and gland
cells, and grains have exhibited industrial potential.
These expressions systems will be described briefly in
the following sections.
Yeast and Aspergilli Expression System
The Pichia pastoris expression system has been
widely and successfully used in the production of many
recombinant proteins with the merits of a high yield
level, corrective post-translational modification and
low cost; this system has been recognized as generally
recognized as safe (GRAS) by the American Food and
Drug Administration (Chen et al., 2004; Werten et al.,
2019) and the Chinese Feed Evaluation Committee (see
“Variety Catalog of Feed Additives: Announcement No.
231 of the Ministry of Agriculture and Rural Affairs of
the P. R. China,” MARA, 2019). It has been reported
that many food-grade enzymes used in the dairy indus-
try, such as buffalo chymosin (300 U/mL) and lactase
(3,600 U/mL), were expressed in P. pastoris (Zou et al.,
2014; Tyagi et al., 2017). The development and estab-
lishment of a cubic-level expression production system
of LF based on the P. pastoris expression system is the
most promising and economical solution to meet the
huge demands for LF in industrial practice if the basic
technical of recombinant LF yeast can be realized on a
large scale in the future. As reported, this system has
been successfully implemented in the production of LF
at the laboratory level (Chen et al., 2004; Jiang et al.,
2008; Bai et al., 2010; Iglesias-Figueroa et al., 2016).
Bovine LF was expressed in P. pastoris KM71-H under
the AOX1 promoter with a yield of 3.5 g/L (Iglesias-
Figueroa et al., 2016). Porcine and human lactoferrin
were also exported into the culture at levels of 0.76
g/L and 1.2 g/L, respectively (Chen et al., 2004; Jiang
et al., 2008). However, there have been few reports on
the large-scale expression of lactoferrin in yeast sys-
tems, despite the technical routes being achieved at
the laboratory level; this is probably related to or lim-
 Mao et al.: PERSPECTIVE: LACTOFERRIN SUPPLY CHAIN 7331
Table 1. Recombinant expression of lactoferrin1

Expression host Source Sequence Vector Yield Reference

Escherichia coli Mice Full length pET28a 17 mg/L Wang and Tian, 2010
BL21(DE3)
E. coli BL21(DE3) Cow Full length pET32a 15.3 mg/L García-Montoya et al., 2013
Pichia pastoris X-33 Cow Full length, pPICZαA Full length: 88 mg/L Bai et al., 2010
bLFA, bLFB, bLFA: 998 mg/L
bLFN bLFB: 56 mg/L
bLFN: 803 mg/L
P. pastoris KM71 Cow Full length pJ902 3.5 g/L Iglesias-Figueroa et al., 2016
P. pastoris GS115 Swine Full length pPICZαC 760 mg/L (cell cytoplasm) Chen et al., 2004
P. pastoris KM71 Human Full length pPIC9K 1.2 g/L Jiang et al., 2008
P. pastoris GS115 Cow Hybrid LFB-HLy pGAPZαA 15.7 mg/L Sun et al., 2019
Aspergillus awamori Cow Full length pPLF-19 2 g/L Ward et al., 1995
Mice Human Full length pBC1 Max yield: 160 g/L Goldman et al., 2012
Average yield: 15.3 g/L
Goat Human Full length pBC1 16 g/L Semak et al., 2019
Cow Human Full length Gene fragment 3 g/L Krimpenfort et al., 1991
transfer by van Berkel et al., 2002
microinjection
Cow Human Full length HLF bacterial 3.4 g/L Yang et al., 2008
artificial chromosome
Cow Human Full length HLF bacterial 4.5–13.6 g/L Wang et al., 2017a
artificial chromosome
Oryza sativa Human Full length pAPI135 5 g of rHLF/kg dehusked rice Nandi et al., 2002
grain weight
O. sativa Human Full length pAPI137 2–4% of the total soluble Suzuki et al., 2003
protein in transgenic calli of
rice
O. sativa Human Full length pCAM1300 0.45% of the total dry weight Lin et al., 2010
of the dehusked rice seeds
Nicotiana tabacum Human Full length 0.7–2.7% of total soluble Choi et al., 2003
protein
Solanum tuberosum Human Full length pPVC702 0.01–0.1% of total soluble Chong and Langridge, 2000
protein
Panax ginseng Human Full length pCGN1578 3% of total soluble protein Kwon et al., 2003
1
HLF = human lactoferrin; rHLF = recombinant HLF; bLFA = the N-lobe and inter-lobe region of bovine lactoferrin (bLF); bLFB = the frag-
ment of bLF from residues 1–284; bLFN = the N-lobe of bLF; LFB-HLy = the hybrid fragment of lactoferrin and lysozyme.

ited by its complex post-transcriptional modification
in yeast. In fact, this bottleneck has been ignored by
previous works (Chen et al., 2004; Jiang et al., 2008;
Bai et al., 2010; Iglesias-Figueroa et al., 2016), and,
thus, special biosynthesis paths to lactoferrin should
be finely designed and optimized. In addition, one
key factor affecting expression and bioactivities must
be considered: the glycosylation side chain is differ-
ent among humans, bovines, and yeast, which is the
bottleneck in this system to solve. Two points should
be focused on: (1) the different glycosylation sites could
be reconstructed and characterized with special prop-
erties and functions required by end consumers; and
(2) The engineered yeast with bovine and humanized
N/O-linked glycosylation should be constructed by
combinatorial genetic libraries, Glycoswitch technology,
synthetic biology, and metabolic engineering to fulfill
and widen the biofunction of recombinant LF. Further-
more, filamentous fungi are also a tool for recombinant
protein expression, and there are 4 cases of lactoferrin
expression in this system (Aspergillus awamori [twice],
Aspergillus nidulans, Aspergillus oryzae) according to
Journal of Dairy Science Vol. 106 No. 11, 2023
the results of data and reference scanning. The yields
vary greatly at concentrations of 5 mg/L (Ward et al.,
1992b), 12 mg/L (Ward et al., 1992a), 2 g/L (Ward et
al., 1995), and 25 mg/L (Ward et al., 1997). Therefore,
more perfect technological improvement and innovation
in the expression host and vectors should be expected
in future works.
Recombinant Mammal and Cell System
Transgenic animals (cattle, sheep) are used as hosts
for the production of LF, which is also considered an
ideal method for the large-scale production of heter-
ologous proteins due to their large capacity for protein
synthesis and efficient secretion; thus, they can be
used as a source of fresh milk to improve LF produc-
tion as expected. An extremely high yield (160 g/L)
of recombinant human LF (rHLF) was produced in
transgenic mice (average production of HLF was 15.3
g/L; Goldman et al., 2012). Transgenic goats produced
rHLF in milk at levels up to 16 g/L (Semak et al.,
2019). Additionally, the expression of rHLF in the milk
 Mao et al.: PERSPECTIVE: LACTOFERRIN SUPPLY CHAIN
of transgenic cows was at concentrations of up to 3
g/L. Based on the observed expression level and an
annual output of 8,000 L of milk per cow, one cow could
produce 24 kg of crude rHLF per year (van Berkel et
al., 2002). The studies from Li’s team are the most
large-scale and are close to industrialization in China
(Yang et al., 2008). Additionally, it has been shown
that the HLF gene did not have any negative influence
on the nutrient compositions of milk and meat (Zhao
et al., 2013). It is expected that there is a long way
to go for the final validation and industrialization of
this system at the farm scale in terms of technical-
ity, safety, evaluation, bioethics, regulation, and more
(Wang et al., 2017b). Meanwhile, the average yields of
lactoferrin in transgenic mammals and P. pastoris were
10 g/L and 1.5 g/L (Table 1), respectively, which were
significantly higher than those in native milk (0.03–0.49
mg/mL). It could be reasonably expected that the cost
of LF production can be reduced significantly by 10
to 100 times when novel and efficient production sys-
tems are used and scaled up over time. Additionally,
animal mammary gland cell reactor systems also have
biosafety advantages in vivo, due to their high homol-
ogy. We know that a stable Chinese hamster ovary cell
line producing >200 mg/L recombinant LF (rLF) was
developed and established via the pTT5 vector, and
it could be purified by a single-step cation-exchange
chromatography procedure (Kruzel et al., 2013). Pure
LF could also be produced in this way with continuous
technological progress.
Recombinant Grain Expression System
Recombinant proteins have been expressed in various
crops, including fruit, seeds, leaves, and tubers. The
expression of LF in plants differs greatly from that in
animal and yeast systems. It was reported that rice
flour containing recombinant LF held a major share
in the market and was valued at over €105 million in
2020 (Hancocks, 2021). A synthetic HLF gene linked
to a rice glutelin 1 (Gt1) promoter and signal sequence
was transformed into rice cells to produce rHLF. The
expression level was up to 5 g of rHLF/kg of dehusked
rice grain weight. Rice grain with derived rHLF was
as biologically active as commercially available HLF
(Nandi et al., 2002). The expression level of rHLF
represented 2% to 4% of the total soluble protein in
transgenic calli of rice (Suzuki et al., 2003). Compa-
rable data were reported by Lin et al. (2010), where the
concentration of rHLF expressed in seeds under the Gtl
promoter reached a level of 0.45% in total dry weight of
the dehusked rice seeds. Apart from the differences in
glycosylation, other physicochemical properties of rHLF
from rice and native HLF are very similar (Nandi et al.,
Journal of Dairy Science Vol. 106 No. 11, 2023
7332
2002; Conesa et al., 2010). Above all, LF from trans-
genic grain can be supplied to special regions where
the iron contents in soil, raw material, or ingredients in
daily intake are not enough or are below normal levels
(Lönnerdal, 2002; Conesa et al., 2010). This is the only
successful large-scale commercial production case and
is the first milestone in the field of recombinant LF
and its modern industrialization from the outstanding
contribution of Bo’s team (Nandi et al., 2002; Suzuki
et al., 2003).
Although various forms of LF (mice, cows, goats,
swine, and humans) have been expressed in the above
expression systems with relatively high yields, rbLF is
more feasible when molecular design and operation,
yield and cost, consumer psychology and dietary hab-
its, bioethics, and other factors are fully considered.
Through our analysis, we believe that the processing of
rbLF would be the fastest way to obtain legal permis-
sion from independent evaluation and official approval
departments. Additionally, according to the logistic of
“easy first, difficult late,” the rLF from grain and mam-
mals should be raw material for direct eating and feed-
ing in the first stage, and the purified products might
be considered later with time in terms of cost, ratio
of performance to cost, market requirement, and other
determinative factors.
Function Niche and Product Design of LF
A total of 19 biological functions of LF have been
evaluated (Ward et al., 2005; García-Montoya et al.,
2012; Vogel, 2012; Actor et al., 2023). All of these func-
tions are not necessary for all users, and thus, niche and
subdivision in detail by requirement is of high impor-
tance for fine nutritional, functional target, industrial
and commercial purposes and is considered the first
step and basis for the molecular design of new LF. Ac-
cording to global bLF market value share statistics,
infant formula milk powder accounts for approximately
38.0%, which is much higher than other applications
(food and sports and functional foods account for 28%
and 22.7%, respectively) and also has growth potential
for the future (Grand View Research, 2020). Conse-
quently, 3 key factors should be first considered. (1)
Milk formula powder: The LF expression systems of
yeast and aspergilli should be established to produce
pure LF with an appropriate degree of glycosylation
and iron saturation, which will be an effective supple-
ment for the additive of infant formula milk powder. (2)
Functional drinks: The LF-rich liquid milk produced
by transgenic mammals could be used as a nutritional
supplement for people who need immunomodifiers. (3)
Foods and feeds: LF-rich rice with high iron content
produced by transgenic plants in the form of raw mate-
 Mao et al.: PERSPECTIVE: LACTOFERRIN SUPPLY CHAIN
Figure 1. Technical route of construction for lactoferrin supply
chain: large-scale production.
rials could serve as food for people with iron deficiency
or other special health needs, or for consumers in some
undeveloped areas, especially regions with low iron con-
tent in soils (Figure 1).
CONCLUSIONS AND FUTURE DIRECTIONS
In general, obtaining LF at an industrial scale by
utilizing transgenic expression systems is feasible in
terms of technical logistics and would be an effective
supplement to classic LF products derived from whey
or skim milk. An initial technical proposal with a high-
er consensus should be discussed, confirmed, and then
tested among LF researchers; this will pave the way for
supporting and solidifying the construction of a modern
LF supply chain with multiple various purposes. More-
over, the appropriate iron content and glycosylation
level in rLF products should be studied based on a
sufficient experimental survey, local background data,
and feedback from more consumer ends. The types and
forms of new rLF products should be checked by the
market. It should be noted that derivatives with small
molecules from LF, such as lactoferricin (Feng et al.,
2006), LFcinB15-W4,10 (Tian et al., 2007), LH (Tian
et al., 2009), LHP7 (Xi et al., 2014), and LF4/5 (Hao
et al., 2017), are not included in this paper. We believe
that the suggestions herein would be beneficial for up-
dating the modern LF industry.
Journal of Dairy Science Vol. 106 No. 11, 2023
7333
ACKNOWLEDGMENTS
The partial viewpoints on “Perspective” and “Func-
tion niche and product design of lactoferrin” were pre-
sented and discussed by corresponding author professor
Jianhua Wang as chair and keynote reporter in the
XVth International Conference on Lactoferrin Struc-
ture, Function and Applications (Report 7, Session 7,
ILC 15, Beijing, virtual) on December 9, 2021. They
were also mentioned briefly in Editorial of Proceedings
of ILC15 (Wang et al., 2023). This study was supported
by the National Natural Science Foundation of China
(No. 31872393; Beijing, China), the Antimicrobial
Peptide Direction of the National Innovation Program
of Agricultural Science and Technology in the Chinese
Academy of Agricultural Sciences (Grant CAAS-
ASTIP-2013-FRI-02; Beijing, China), and the Key
Projects of Alternatives to Antibiotics for Feed (Grant
CAAS-ZDXT2018008; Beijing, China) and Animal Us-
ages (Grant CAAS-ZDRW202111; Beijing, China). The
authors have not stated any conflicts of interest.
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