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adoption can occur at the industrial scale. which was 1.9 times higher than that
Economical downstream One of these obstacles is the high cost obtained using the Neutrase® protease
processing of microbial of production, whereby energy-intensive alone [3]. When cellulase and lysozyme
polyunsaturated fatty downstream processing accounts for were used together, the recovery of EPA-
20–60% of the total production cost. rich lipids from Nannochloropsis sp. was
acids Moreover, since PUFAs are easily oxidized higher than that obtained with each en-
1 due to the presence of many double zyme alone [5].
Peng-Wei Huang,
1 bonds, maintaining product quality and
Chun-Xiao Yan,
extending the shelf life is a crucial challenge There is no doubt that organic solvent ex-
Xiao-Man Sun, 1,* and of downstream processing for the com- traction is the most effective method for
He Huang 1,2,* mercial exploitation of microbial PUFAs. extracting microbial PUFAs. Importantly,
Here, we discuss the critical problems in, food-grade solvents should be applied,
and future directions for, the downstream such as hexane, isopropanol, ethanol,
Polyunsaturated fatty acids (PUFAs) processing of microbial PUFA production, butanol, and so on. However, using or-
are important nutrients for humans including extraction, refining, and storage. ganic solvents requires the establishment
and animals. Microorganisms, such of explosion-proof workshops, which in-
as yeast, filamentous fungi, and Developing food-grade lipid creases the cost. Moreover, residual
microalgae, have successfully been extraction strategies solvents can affect the safety of PUFA-
modified to produce PUFAs. Apart As high-value nutrients, PUFA extraction containing products, especially the high
from strain improvement and fer- procedures should be green, safe, and en- toxicity of some polar solvents. Therefore,
vironmentally friendly. A dry or wet route many novel approaches have been devel-
mentation optimization, efficient
can be chosen for PUFA extraction oped to eliminate, or at least reduce, the
and cost-effective downstream pro-
(Figure 1). In the ‘the dry route’, a drying use of organic solvents. Surfactants,
cessing will determine whether
pretreatment is necessary, which is an which have a hydrophobic tail and a hy-
production can advance from the energy-intensive process and, therefore, drophilic head, can interact with cell mem-
laboratory to the factory. this route is not economically feasible in branes and promote cell wall disruption. In
large-scale applications. Therefore, the lipid extraction from wet Nannochloropsis
wet route is usually preferred to assure sp. biomass, it was shown that surfactants
process viability and economy. Wet bio- can completely replace polar organic sol-
Opportunities and challenges for mass requires pretreatment to disrupt the vents [6]. In fact, if the efficiency of cell
microbial PUFA production cell wall, thus facilitating the access of wall disruption and the microbial lipid con-
PUFAs, such as docosahexaenoic acid solvent to lipids. Regardless of the route tent is high enough, lipid recovery can be
(DHA, 22:6), arachidonic acid (ARA, 20:4), chosen, solvent extraction is carried out to achieved using three-phase centrifuges
and eicosapentaenoic acid (EPA, 20:5), collect lipids. In general, the lipid recovery without the use of organic solvents, and
are important nutrients for both humans process can be improved in two aspects: this simple technology has been used in
and animals. They are traditionally extracted enhancing cell disruption efficiency and the industrial extraction of DHA-rich lipids
from animal or plant tissues, but this unsus- reducing the use of solvents. from Schizochytrium sp.
tainable approach cannot meet the rising
market demand. With the development To date, various cell wall disruption Establishing a high-quality
of metabolic engineering, microorganisms methods have been developed, including lipid-refining process
offer excellent alternatives for the produc- bead milling, pulsed electric field, and enzy- Lipid-refining processes generally re-
tion of PUFAs. Notably, microalgae and ole- matic treatment. Among them, enzymatic quire several operation units, including
aginous fungi, such as Schizochytrium sp., treatment is considered to be a mild degumming, deacidification, bleaching,
Yarrowia lipolytica, and Mortierella alpina, method with low energy intensity [4]. and deodorization (Figure 1). In general,
have been successfully developed into Due to differences in cell wall compo- water- and acid-based degumming is tra-
major sources of DHA, EPA, and ARA, re- nents, it is necessary to screen and mix ditionally used to remove the hydratable
spectively [1–3]. Despite the successful specific cell wall-degrading enzymes for and nonhydratable phospholipids (PLs),
commercialization of PUFAs from various each species. For example, a mixture of respectively. For example, most PLs in
microorganisms, there are still many pectinase and papain yielded an ARA-rich Chlorella sp. are nonhydratable; thus, the
challenges to overcome before broader lipid recovery of 96.4% from M. alpina, PL removal rate of acid-based degumming

Trends in Biotechnology, Month 2023, Vol. xx, No. xx 1

 Trends in Biotechnology

Trends in Biotechnology

Figure 1. Downstream process flow diagram of microbial polyunsaturated fatty acid (PUFA)-rich lipid production. The initial process is lipid extraction, the
purpose of which is to obtain crude PUFA-rich lipids. The dry route requires separation of the fermentation medium from the biomass by centrifugation or agglomeration.
The wet biomass then undergoes drying, which is energy intensive. The wet route can directly rupture the cell wall using enzymatic treatment in the cell culture and does not
require drying. The second process is lipid refining, which removes impurities from the crude lipids. This process generally contains several operation units, including
degumming for removing PLs, deacidification for removing FFAs, bleaching for removing pigments, and deodorization for removing undesirable odorous compounds.
The final process is PUFA-rich lipid storage. To lengthen the shelf life of PUFA-rich lipid, various synthetic antioxidants [e.g., tert-butyl hydroquinone (TBHQ), butylated
hydroxyanisole (BHA), α-tocopherol, and butylated hydroxytoluene (BHT)] and natural antioxidants from edible plant materials (e.g., polyphenols and carotenoids) are
added separately or used in combination to improve the oxidative stability of the PUFA-rich lipid. Abbreviation: PL, phospholipid.

reached 83%, while that of water-based
degumming was only 19% [7]. However,
traditional degumming cannot guarantee
residual phosphorus levels of <10 mg/kg,
which inspired the development of more
effective and environmentally friendly enzy-
matic degumming. According to the hydro-
lysis sites in PLs, phospholipase enzymes
can be divided into four major classes: A,
B, C, and D, where A/B are acylhydrolases,
and C/D are phosphodiesterases. Thus,
the selection of the specific enzyme de-
pends on the composition of PLs in lipids.
Jiang et al. investigated the degumming ef-
ficiency of phospholipase A1 and phospho-
lipase C on eight varieties of crude lipids.
The results showed that phospholipase
A1 was suitable for the degumming of lipids
with a high content of nonhydratable PLs
and low content of initial phosphorus,
while the application of phospholipase C
showed no obvious advantage [8].
Chemical deacidification is usually per-
formed to remove free fatty acids (FFAs)
on an industrial scale. In this process,
NaOH is added to neutralize the FFAs,
and the resulting soap is removed by
washing. However, significant lipid loss
could occur due to alkali hydrolysis of
triglycerides. Absorption deacidification
might be an alternative method, because
it is simple, harmless, and reduces lipid
loss. When alkali-pretreated microcrystalline
cellulose was used in the deacidification
of microalgal lipids, the acid value was
reduced from 25 to 2.5 mg KOH/g, and
the lipid recovery rate was >90% [9].
The bleaching process based on solid ad-
sorbents has been applied on a large scale
in industry because no solvents or high
temperatures are involved. Hence, special
attention should be paid to this step to
maintain a high content of PUFAs while
maximizing the removal of impurities.
Lastly, to remove undesirable odorous
compounds, high-temperature steam rang-
ing from 180 to 270°C is used on the indus-
trial scale for the deodorization of lipids, but

2 Trends in Biotechnology, Month 2023, Vol. xx, No. xx

Trends in Biotechnology

PUFAs can be easily oxidized in this pro-
cess. Usually, the maximum limits for the
peroxide value (PV) and p-anisidine value
(p-AV) of PUFA-rich lipids are 5 meq/kg
and 20 meq/kg, respectively. In the de-
odorization of DHA-rich lipids, deoxy-
antioxidant does not mean a stronger anti-
oxidant capacity. According to the Chinese
Standard GB2760-2014, the total amount
of antioxidants that may be added is limited.
For example, for a combination of three
antioxidants, the addition amount of each
it is important to keep in mind the possi-
bility and economic viability of scaling up.
Acknowledgments
This work was supported by the National Natural
Science Foundation of China (No. 22038007).

genated steam can control the PV and
p-AV at 0 meq/kg and 3.5 meq/kg,
much lower than that of nondeoxygenated
steam [10].
Improving oxidative stability during
storage
PUFA oxidation has long been considered
to be the major process affecting the smell
is not allowed to exceed one-third of its
maximum single allowable amount. Thus, a Declaration of interests
The authors declare no financial or commercial conflict
more complex combination of antioxidants
of interest.
requires smaller amounts of each individual
antioxidant but leads to a decrease in anti- 1
School of Food Science and Pharmaceutical Engineering,
oxidant capacity. Nanjing Normal University, Nanjing 210023, People’s Republic
of China
2
College of Biotechnology and Pharmaceutical Engineering,
Concluding remarks Nanjing Tech University, Nanjing 211816, People’s Republic of
An ideal downstream process for micro- China

and nutritional quality of the final product.
Therefore, antioxidants often need to be
added after refining to improve the oxida-
tive stability and prolong the shelf life
(Figure 1). Compared with traditional syn-
thetic antioxidants, natural antioxidants
have the obvious advantage of safety.
Glodde et al. compared the efficiency of
five natural antioxidants and one synthetic
antioxidant in stabilizing DHA and EPA
in dog food; the results showed that four
natural antioxidants could be used as sub-
stitutes for synthetic antioxidants [11].
Different antioxidants have different mech-
anisms, because some act by scavenging
free radicals and quenching singlet oxy-
gen, while others act by removing metal
ions. Therefore, antioxidant cocktails are
usually applied to take advantage of their
synergistic effects. It was reported that
the combination of 80 mg/kg ascorbyl
palmitate, 80 mg/kg vitamin E, 40 mg/kg
phytic acid, and 80 mg/kg tea polyphenols
showed a better antioxidant effect than
adding each antioxidant alone to DHA-
rich algal lipids, and the shelf life was
increased from 7.5 to 28.2 days [12].
However, when using 53.2 mg/kg of
pomegranate and 360 mg/kg of tea
polyphenol palmitate as composite antiox-
idants, the shelf life of DHA-rich algal lipids
was further extended to 34.4 days [13].
bial PUFA production should be highly
*Correspondence:
efficient, low cost, have mild operating xiaomansun@njnu.edu.cn (X.-M. Sun) and
conditions, low solvent residue, and result huangh@njnu.edu.cn (H. Huang).
in a product with a long shelf life. For lipid https://doi.org/10.1016/j.tibtech.2023.01.001
extraction, the maximum lipid extraction © 2023 Elsevier Ltd. All rights reserved.
may not be achieved using only one single
pretreatment method; thus, a combination References
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Therefore, a more complex composite fective and environmentally friendly, and

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