pharmaceutics

Review
Contributions of Glycolipid Biosurfactants and Glycolipid-
Modified Materials to Antimicrobial Strategy: A Review
Qin Shu, Hanghang Lou, Tianyu Wei, Xiayu Liu and Qihe Chen *

Department of Food Science and Nutrition, Zhejiang University, Hangzhou 310058, China;
21713041@zju.edu.cn (Q.S.); louhanghang@zju.edu.cn (H.L.); 21913067@zju.edu.cn (T.W.);
xiayuliu@zju.edu.cn (X.L.)
* Correspondence: chenqh@zju.edu.cn; Tel.: +86-139-6717-1522

Abstract: Glycolipid biosurfactants are natural amphiphiles and have gained particular interest
recently in their biodegradability, diversity, and bioactivity. Microbial infection has caused severe
morbidity and mortality and threatened public health security worldwide. Glycolipids have played
an important role in combating many diseases as therapeutic agents depending on the self-assembly
property, the anticancer and anti-inflammatory properties, and the antimicrobial properties, including
antibacterial, antifungal, and antiviral effects. Besides, their role has been highlighted as scavengers
in impeding the biofilm formation and rupturing mature biofilm, indicating their utility as suitable
anti-adhesive coating agents for medical insertional materials leading to a reduction in vast hospital
infections. Notably, glycolipids have been widely applied to the synthesis of novel antimicrobial
materials due to their excellent amphipathicity, such as nanoparticles and liposomes. Accordingly,



this review will provide various antimicrobial applications of glycolipids as functional ingredients in


medical therapy.
Citation: Shu, Q.; Lou, H.; Wei, T.;
Liu, X.; Chen, Q. Contributions of Keywords: glycolipids; nanocomposites; liposomes; antibacterial mechanism; anti-biofilm
Glycolipid Biosurfactants and
Glycolipid-Modified Materials to
Antimicrobial Strategy: A Review.
Pharmaceutics 2021, 13, 227. 1. Introduction
https://doi.org/10.3390/
Surfactants are a kind of amphiphilic molecules with hydrophilic moieties and hy-
pharmaceutics13020227
drophobic moieties and perform excellent interfacial activity between gas, liquid, and
Academic Editors: Barbara Luppi and
solid surfaces. Since their broad range of applications has been put into the food industry,
Gert Fricker cosmetics, petroleum industry, and sewage treatment, surfactants have become one of
Received: 24 December 2020 the most widely used chemicals in industrial production. However, most chemically-
Accepted: 2 February 2021 synthesized surfactants derived from petroleum products, and the massive consumption
Published: 6 February 2021 of petroleum have caused resource exhaustion and environmental pollution. Thus, the
exploitation of eco-friendly, biodegradable, and renewable alternative to surfactants has to
Publisher’s Note: MDPI stays neutral be settled urgently. In recent years, the emerging of biosurfactants has gained more and
with regard to jurisdictional claims in more attention, and plenty of expectations have been potentiated due to their versatile func-
published maps and institutional affil- tions, such as reducing surface tension, emulsifying activity, and biological properties [1].
iations. Compared to synthetic surfactants, biosurfactants are more popular owing to their higher
biodegradability, lower toxicity, thermostability, and tolerance in extreme conditions [2].
The production of biosurfactants is dependent on microbial fermentation of bacteria, fungi,
and yeast strains based on low-cost carbon and nitrogen source, even industrial waste and
Copyright: © 2021 by the authors. oily byproducts, and the promising and sustainable strategy realizes low energy consump-
Licensee MDPI, Basel, Switzerland. tion in a large scale of production. According to their molecular weight, biosurfactants can
This article is an open access article be segmented into two groups: low molecular weight (LMW) and high molecular weight
distributed under the terms and (HMW), as seen in Figure 1 [3]. Glycolipids [4] and lipopeptides [5] are representative
conditions of the Creative Commons biosurfactants with low molecular weight, such as rhamnolipids [6] and surfactin [7],
Attribution (CC BY) license (https:// and the high molecular weight group contains phospholipids [8], lipoprotein [9], and
creativecommons.org/licenses/by/ emulsan [8] et al.
4.0/).

Pharmaceutics 2021, 13, 227. https://doi.org/10.3390/pharmaceutics13020227 https://www.mdpi.com/journal/pharmaceutics

Pharmaceutics 2021, 13, 227 2 of 22

Figure 1. Main groups of biosurfactants.

Glycolipids are one of the most popular biosurfactants with low molecular weight
and have been investigated thoroughly and intensively for biotechnological applications.
As the name implies, glycolipids comprise two parts: carbohydrate moiety and fatty acid
chains linked by a glycosidic bond, which acts as a hydrophilic role and hydrophobic
role, respectively. The difference of carbohydrate moiety and fatty acid chains cause the
diversity of glycolipids, and the sub-class normally contains rhamnolipids, sophorolipids,
mannosylerythritol lipids, cellobiose lipids, trehalolipids, xylolipids, and so on [10]. Except
for fundamental characteristics of biosurfactants, most glycolipid-producers are microor-
ganisms isolated from oil-contaminated samples, which implied their potential applications
in bioremediation as well as their production on renewable sources. Multiple biological
properties have endowed glycolipids with wide potentiality in various fields. According to
the literature, glycolipids have been confirmed to show potential anticancer effects and
are expected to be efficient candidates for anticancer drugs [5]. For instance, rhamnolipids
have shown a significant anticancer effect on human breast cancer cells MCF-7 in a dose-
dependent manner [11]. Besides, glycolipids exert anti-inflammatory effects on human
immune-related diseases as promising immunomodulators. Sophorolipids could modulate
the immune response to decrease sepsis-related mortality in animal models, the underly-
ing mechanism involves the reduction of nitric oxide and the regulation of inflammatory
cytokines [12]. Remarkably, massive literature reveals that all kind of glycolipids possesses
excellent antimicrobial activity against bacteria, fungi, and virus, and the physiological
changes induced by glycolipids have been deeply investigated aiming at both planktonic
and biofilm state of microorganism [4,6,13–15].
In the previous review [16], abundant work has been devoted to concluding the
biotechnological production, functional properties, and potential applications of glycolipid
biosurfactants. In this review, a comprehensive intensive study will be carried out directing
the contributions of glycolipids and glycolipid-modified materials to antimicrobial therapy.

2. Glycolipids
2.1. Rhamnolipids
Rhamnolipids (RLs) are one of the glycolipid-type biosurfactants, which are pro-
duced mainly by Pseudomonas aeruginosa and most frequently studied due to their effective
surface activity and high yields of production [17]. It was firstly reported in 1946 that
Bergström et al. discovered an oily glycolipid, named pyolipic acid, was produced by Pseu-
Pharmaceutics 2021, 13, 227 3 of 22

domonas pyocyanea (P. aeruginosa) after growing on glucose [18,19]. Soon after that, Jarvis
and Johnson in 1949 have further confirmed the RL isolated from P. aeruginosa contained
two β-hydroxydecanoic acids and two rhamnose moieties, which were linked through a
glycosidic bond [20]. Then Edwards and Hayashi have verified the linkage between the
two rhamnose moieties is an α-1,2-glycosidic linkage in 1965 [21]. Since then, extensive
researches have been conducted on RL including broad aspects. So far, P. aeruginosa has
been thoroughly investigated as a primary source of RLs with titers over 100 g/L [22],
and Pseudomonas species are regarded as the main RL producers. Nevertheless, it has
been reported that many other non-Pseudomonas isolates can produce RLs as well, lead-
ing to the structural diversity of RLs. For instance, there are some studies focused on
the production of rhamnolipids by Burkholderia species, which have been shown to pro-
duce rhamnolipids that have longer alkyl chains than P. aeruginosa, such as Burkholderia
thailandensis [23], Burkholderia plantarii [24], and Burkholderia pseudomallei [25]. Although
the diversity of RL-producers has caused variations in the chemical structures of RL, the
basic structure was composed of rhamnose moiety and lipid moiety, and the number of
rhamnose and the length and number of carbon chain lead to the diversity of RL struc-
ture. Generally, there are mainly four types of RL structures produced by Pseudomonas
species, including mono-rhamno-mono-lipid (Rha-C10), di-rhamno-mono-lipid (Rha-Rha-
Pharmaceutics 2021, 13, x FOR PEER REVIEW 5 of 22
C10), mono-rhamno-di-lipid (Rha-C10-C10), and di-rhamno-di-lipid (Rha-Rha-C10-C10),
as shown in Figure 2.

Figure 2.
Figure 2. Common structures
structures of
of rhamnolipids
rhamnolipids produced
produced by
by Pseudomonas
Pseudomonasspecies.
species.

Earlier
Table in 1971, the
1. Antimicrobial antimicrobial
effects spectrum
of rhamnolipids of rhamnolipids
reported to date. has been studied, sug-
gesting the broad-spectrum antimicrobial activity functioned against common microbes
Producing Microorganism Target Microorganism
including gram-positive Microbial Type
and gram-negative Inhibitory
bacteria, such asEffects Reference
Streptococcus faecalis, Staphy-
Pseudomonas aeruginosa Listeria monocytogenes Gram-positive
lococcus aureus, Bacillus subtilis, and Proteus vulgaris [26]. Whereafter, a large number
Anti-biofilm effects [30] of
PA1 Pseudomonas
intensive and fluorescens Gram-negative
deep researches has been conducted about the microorganism sterilizing
Pseudomonas aeruginosa Staphylococcus aureus Gram-positive
actions of rhamnolipids involving planktonic and biofilm cells, as seen in Table 1. As
Antibacterial effects [27]
OBP1 the most important pathogens,
Klebsiella pneumoniae Gram-negative Staphylococcus aureus and Staphylococcus epidermidis were
reported to beepider-
Staphylococcus influenced as rhamnolipids have suppressed the growth of planktonic cells
at MIC of 0.06 and 0.12 mg/mL, and dispersed pre-formed biofilms up to 93% [27]. Fer-
midis
reira et al. provided evidence that gram-positive pathogens Listeria monocytogenes, Bacillus
Staphylococcus
cereus, and S. aureus wereGram-positive
more sensitiveAnti-biofilm effects
to rhamnolipids, by disrupting
indicating the sterilizing effects
P. aeruginosa DS10-129 aureus [39]
of rhamnolipids were pH-dependent [28]. theSummarily, the inducement of bacterial death is
initial adhesion
Streptococcus
salivarius
Candida tropicalis Fungi
Pseudomonas sp. PS-17 Bacillus subtilis Gram-positive Antibacterial effects [40,41]
Bacillus cereus Gram-positive
Pharmaceutics 2021, 13, 227 4 of 22

attributed to cell lysis along with consequent leakage of cellular components, which was
already confirmed by visualized proofs such as SEM and TEM pictures. As the target of
rhamnolipids to planktonic bacteria, the cell membrane has been altered and damaged
with an increase in cell permeability and reduction on cell surface hydrophobicity [28],
because the unique amphiphilic character allows the interaction between rhamnolipids
and phospholipids [29].
Interestingly, they found that gram-negative Salmonella enterica and E. coli showed
resistance to rhamnolipids at all tested concentrations and all pH levels. Generally speaking,
glycolipids have stronger antibacterial effects on gram-positive bacteria than gram-negative
bacteria, which may be owing to the difference in cell membrane composition. The cell
envelope of gram-negative bacteria consists of the outer membrane (lipopolysaccharides
and phospholipids), peptidoglycan, and internal plasma membrane, which is more complex
and defensive than gram-positive bacteria. The special structure made it difficult for
glycolipids to enter gram-negative bacteria, while there is a common agreement that the
underlying mechanism of antibacterial activity involves reducing membrane permeability,
loss of intracellular constituents, and cell apoptosis induced by membrane lysis.
Generally speaking, the inducement of bacterial death is attributed to cell lysis along
with consequent leakage of cellular components, which was already confirmed by visual-
ized proofs such as SEM and TEM pictures. As the target of rhamnolipids to planktonic
bacteria, the cell membrane has been altered and damaged with an increase in cell perme-
ability and reduction on cell surface hydrophobicity [28], because the unique amphiphilic
character allows the interaction between rhamnolipids and phospholipids [29].
Biofilm is considered as the sessile community of planktonic microorganism fixed
at the solid surface, and plenty of researches have exhibited the anti-biofilm properties
of rhamnolipids. Biofilms are an important concern among food processing industries,
resulting in contamination of pipelines, corrosion of equipment, and final food spoilage.
Araujo et al. discovered the biofilm of P. fluorescens and L. monocytogenes (biofilm-forming
bacteria related to foodborne disease) was reduced up to 79% and 74% by purified rham-
nolipids, respectively, while microbial adhesion was entirely inhibited in the culture
medium [30]. Besides, oral health and hygiene have always been threatened by relat-
ing oral bacteria, and rhamnolipids were studied to eradicate the bacterial biofilm of oral
disease. As one of the major etiological agents in dental caries, the adhesion of Streptococcus
mutans on polystyrene surfaces was reduced by rhamnolipids, and its pre-formed biofilm
was also disrupted according to Abdollahi et al. [31]. Similarly, Elshikh et al. also found
that rhamnolipids from non-pathogenic Burkholderia thailandensis E264 revealed potent
abilities to destruct mature biofilm of some oral pathogens (Streptococcus oralis, Actinomyces
naeslundii, Neisseria mucosa, and Streptococcus sanguinis), forecasting their prospective oral-
related applications against oral-bacteria biofilms [32]. In conclusion, the biofilm-inhibiting
behavior of rhamnolipids was due to blocking the initial adhesion stage. To figure out the
specific role of rhamnolipids playing in the biofilm formation process, Kim et al. investi-
gated the physicochemical interactions between rhamnolipids and Pseudomonas aeruginosa
biofilm layers [33]. They demonstrated that a decrease in surface free energy on the mem-
brane, interaction with some EPS proteins, and loss of EPS amount were several key factors
in rhamnolipid-mediated biofilm reduction.
Furthermore, rhamnolipids also perform excellent inhibiting effects on fungi in-
cluding not only hyphal growth but also spore germination. For detrimental fungi in
the plant, Kim et al. reported that rhamnolipids showed significant antifungal activity
against Phytophthora capsici mainly due to a lytic effect on zoospores at a concentration of
10 mg/mL, and also suppressed the germination of zoospores and the growth of hypha [34].
Goswami et al. also confirmed the effectiveness of rhamnolipid in controlling Colletotrichum
falcatum in vitro as well as in vivo. Results showed 100 mg/mL RL-DS9 exhibited 86.6%
inhibition against C. falcatum spore germination, and in the same concentration, RL-R95
showed 83.3% inhibition. The antifungal mode came from disruption of the fungal mem-
brane and made it possible for its application as an alternative fungicide to control red
Pharmaceutics 2021, 13, 227 5 of 22

rot disease of sugarcane [35]. On the other hand, rhamnolipids are able to combat those
harmful fungi derived from animals or humans. For instance, Sen et al. elucidated that
purified rhamnolipid could effectively suppress spore germination and hyphal prolifera-
tion of Trichophyton rubrum in mice models at a concentration of 500 µg/mL, which can
be a promising candidate to cure dermatophytic infections, known as the most prevalent
superficial mycoses worldwide [36]. Meanwhile, the biofilm of fungi has always been detri-
mental to eliminate microbial contamination because of its high resistance and low living
demands, yet rhamnolipids have outstanding performance in controlling fungi biofilm.
Singh et al. discovered 90% of pre-formed Candida albicans biofilm on polystyrene surfaces
was reduced by rhamnolipids in a dose-dependent manner [37]. Upon yeast type of fungi,
the pre-formed biofilm of Yarrowia lipolytica was removed effectively by rhamnolipids at
low concentrations, reported by Dusane et al. [38].

Table 1. Antimicrobial effects of rhamnolipids reported to date.

Producing
Target Microorganism Microbial Type Inhibitory Effects Reference
Microorganism
Pseudomonas aeruginosa Listeria monocytogenes Gram-positive
Anti-biofilm effects [30]
PA1 Pseudomonas fluorescens Gram-negative
Pseudomonas aeruginosa Staphylococcus aureus Gram-positive
Antibacterial effects [27]
OBP1 Klebsiella pneumoniae Gram-negative
Staphylococcus epidermidis
Staphylococcus
aureus Anti-biofilm effects by
P. aeruginosa DS10-129 Gram-positive [39]
disrupting the initial
Streptococcus adhesion
salivarius
Candida tropicalis Fungi
Pseudomonas sp. PS-17 Bacillus subtilis Gram-positive Antibacterial effects [40,41]
Bacillus cereus Gram-positive
Antibacterial effect
— [28]
Escherichia coli Gram-negative depending on pH
Salmonella enterica Gram-negative
Pseudomonas aeruginosa
Streptococcus mutans Gram-positive Anti-biofilm effects [31]
MN1
— Helicobacter pylori Gram-negative Anti-biofilm effects [42]
Pseudomonas aeruginosa Cercospora kikuchii Inhibiting spore germination
Fungi [34]
strain B5 and hyphal growth
Phytophthora capsici
— Yarrowia lipolytica Fungi Anti-biofilm effects [38]
Pseudomonas aeruginosa
Candida albicans Fungi Anti-biofilm effects [37]
DSVP20
Pseudomonas aeruginosa Inhibiting spore germination
Colletotrichum falcatum Fungi [35]
DS9 and mycelial growth
Pseudomonas aeruginosa Inhibiting spore germination
Trichophyton rubrum Fungi [36]
SS14 and hyphal proliferation

2.2. Sophorolipids
Sophorolipids (SLs) are one of the representative glycolipid biosurfactants owing
to their homogeneous product in high yield, which has been intensively investigated
and commercialized by some companies [43]. The original discovery of sophorolipids
was dated back to 1961 when Gorin et al. firstly revealed that an extracellular glycolipid
mixture was produced by Torulopsis magnoliae, which was corrected as Candida apicola later
 Pharmaceutics 2021, 13, 227 6 of 22

ceutics 2021, 13, x FOR PEER REVIEW 7 of 22

in 1968 [44]. Simultaneously, Tulloch et al. also found extracellular glycolipids were yielded
by Candida bogoriensis in 1968 [45]. In the next decades, sophorolipids have gained extensive
industries, such as poultry,
interest food been
and have preservation,
proved topharmaceutical
be synthesizedindustry, andspecies
by multiple medicalofap-yeast strains like
paratus and instruments.
Starmerella bombicola, Candida riodocensis, Candida stellate, and Wickerhamiella domercqiae,
Aiming atgrowing
pathogenic on fungi, sophorolipids
carbohydrates also have asubstrates
and lipophilic significantwith
influence
titerson spore
over 400 g/L [46–48].
germination, mycelial growth, and biofilm formation. Firstly, sophorolipids show a broad
Moreover, the diversity of producing species has determined the structure of their metabolic
antifungal spectrum
products.involving
Normally, Colletotrichum gloeosporioides,
the molecular structure Fusariumis composed
of sophorolipids verticilliodes,
of a hydrophobic
Fusarium oxysporum f. sp. pisi, Corynespora cassiicola, and Trichophyton rubrum,
fatty acid tail of 16 or 18 carbon atoms and a hydrophilic carbohydrate reported by head sophorose
Suparna et al. and
[57].can
Haque et al. described
be divided the inhibition
into two main of sophorolipids
forms: acidic on hyphal
form and lactonic form, as shown in
growth and biofilm
Figureformation of Candida
3. Specifically, a longalbicans,
chain ofand thoughtfatty
hydroxyl the downregulation of hy-
acid was β-glycosidically attached to
pha specific genes was the reason
the sophorose moiety,forand
blocking biofilm formation
the carboxylic [58]. Later,
tail of the fatty acid isin further
either free (acidic form)
study, they elucidated
or esterified at the 60 - or 600have
that sophorolipids increased
-position the ROS
(lactonic production
form). and expres-
Furthermore, the variation of the
sion of oxidative stress-related genes significantly in Candida albicans, ultimately leading
structure is also reflected in the carbon number, unsaturation, and hydroxylation of the fatty
to cell death by membrane
acid perforation anddepending
chain in sophorolipids, necrosis [59]. To biological
on different kinds control
of carbonof sources
plant in microbial
disease, Chen et al. exposed sophorolipids have a restraining effect on spore germination
fermentation [49]. Thus, the structural difference has strongly influenced the biological
and hyphal tipandgrowth of various plant
physicochemical pathogens,
activities and theanddegree
the result showed pH is
of lactonization solubility of
the key factor. Generally,
sophorolipids lactonized
had influenced their efficacy
sophorolipid shows [58]. For zoonotic
superior surface dermatophyte, sophoro- effects and
activity and antimicrobial
lipids also exerted an antifungal
exhibits and anti-biofilm
more application potential, effect
whichon theTrichophyton mentagrophytesbetter forming
acidic form demonstrates
and are likely to treat cutaneous
capacity mycoses
and solubility [50].[60].

Figure 3.structures
Figure 3. Common Commonofstructures of sophorolipids:
sophorolipids: lactonic formlactonic form
and acidic and R
form. acidic
1 = OHform. R1 = OH3or
or OCOCH , R2 = OH or OCOCH3.
OCOCH3, R2 = OH or OCOCH3.
Sophorolipids have displayed diverse properties including emulsifier, lubricant, mi-
celle formation, detergency, dispersibility, and wettability foaming, and their prominent
antimicrobial activity has been deeply studied and applied in versatile field, seen in Table 2.
Ankulkar et al. found semi-purified sophorolipids exhibited a different degree of antibacte-
rial activity against pathogenic Escherichia coli, Listeria monocytogenes, and Staphylococcus
aureus at minimum inhibitory concentrations (MIC) of 1000, 500, and 250 µg/mL, respec-
tively [51]. Similarly, Fontoura et al. found gram-positive bacteria (Enterococcus faecium,
Staphylococcus aureus, and Streptococcus mutans) were proved to appear more sensitive to
sophorolipids than gram-negative bacteria (Proteus mirabilis, Escherichia coli, Salmonella
enterica subsp. enterica), with difference in treating dose of 500 and 2000 µg/mL [52]. Never-
theless, some researches depicted the potent killing efficacy on both types of strains and
revealed the otherwise working mechanism. For instance, Gaur et al. discovered that
60 mg/L sophorolipids isolated from Candida glabrata CBS138 killed 65.8% Bacillus subtilis
and 4% Escherichia coli, and the further study confirmed the generation of reactive oxygen
species (ROS) induced by sophorolipids has caused cell death [53].
For further practical application, sophorolipids were verified by Solaiman et al. to
successfully inhibit the growth of five representative species of caries-causing oral bacteria:
Lactobacillus acidophilus, Lactobacillus fermentum, Streptococcus mutans, Streptococcus salivarius,
Pharmaceutics 2021, 13, 227 7 of 22

and Streptococcus sobrinus, suggesting the great potential of sophorolipids in oral health
and hygiene [54]. Besides, sophorolipids have made contributions to reduce microbial
contamination of Clostridium perfringens and Campylobacter jejuni in the poultry industry,
which helps to lower enormous economic losses [55]. On the other hand, biofilm often fixes
on the material surface such as medical devices, causes persistent microbial contamination
and drug resistance, and has become prior trouble to be solved urgently. Recent research
has revealed that sophorolipids have succeeded in scavenging biofilm of clinical strains
(Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans) on medical-grade
silicone discs, resulting from their effective anti-adhesive ability [56]. Hence, there is
immense potential to exploit sophorolipids as antimicrobial agents used in a variety of
industries, such as poultry, food preservation, pharmaceutical industry, and medical
apparatus and instruments.
Aiming at pathogenic fungi, sophorolipids also have a significant influence on spore
germination, mycelial growth, and biofilm formation. Firstly, sophorolipids show a broad
antifungal spectrum involving Colletotrichum gloeosporioides, Fusarium verticilliodes, Fusar-
ium oxysporum f. sp. pisi, Corynespora cassiicola, and Trichophyton rubrum, reported by
Suparna et al. [57]. Haque et al. described the inhibition of sophorolipids on hyphal growth
and biofilm formation of Candida albicans, and thought the downregulation of hypha spe-
cific genes was the reason for blocking biofilm formation [58]. Later, in further study,
they elucidated that sophorolipids have increased the ROS production and expression of
oxidative stress-related genes significantly in Candida albicans, ultimately leading to cell
death by membrane perforation and necrosis [59]. To biological control of plant disease,
Chen et al. exposed sophorolipids have a restraining effect on spore germination and
hyphal tip growth of various plant pathogens, and the result showed pH solubility of
sophorolipids had influenced their efficacy [58]. For zoonotic dermatophyte, sophorolipids
also exerted an antifungal and anti-biofilm effect on Trichophyton mentagrophytes and are
likely to treat cutaneous mycoses [60].

Table 2. Antimicrobial effects of sophorolipids.

Producing Microorganism Target Microorganism Microbial Type Inhibitory Effects Reference

Bacillus subtilis Gram-positive
Candida glabrata CBS138 Antibacterial effects [53]
Escherichia coli Gram-negative
Staphylococcus aureus
Candida Gram-positive Antibacterial effects
Listeria monocytogenes [51]
tropicalis RA1
Escherichia coli Gram-negative
Enterococcus faecium
Staphylococcus aureus Gram-positive
Streptococcus mutans
Candida bombicola Antibacterial effects [52]
Proteus mirabilis,
Escherichia coli Gram-negative
Salmonella enterica subsp.
enterica
Lactobacillus acidophilus
Lactobacillus fermentum
Candida bombicola ATCC Gram-positive
Streptococcus mutans Antibacterial effects [54]
22214
Streptococcus salivarius
Streptococcus sobrinus
Starmerella (Candida) Clostridium perfringens Gram-positive
Antibacterial effects [55]
bombicola Campylobacter jejuni Gram-negative
Pharmaceutics 2021, 13, 227 8 of 22

Table 2. Cont.

Producing Microorganism Target Microorganism Microbial Type Inhibitory Effects Reference

Candida bombicola Escherichia coli Gram-negative Antibacterial effects [61]
Staphylococcus aureus Gram-positive
Candida bombicola ATCC
Pseudomonas aeruginosa Gram-negative Anti-biofilm effects [56]
22214
Candida albicans Fungi
Starmerella (Candida) Antifungal and
Candida albicans Fungi [58,59]
bombicola MTCC1910 anti-biofilm effects
Colletotrichum
gloeosporioides
Fusarium verticilliodes
Rhodotorula babjevae YS3 Fusarium oxysporum f. sp. Fungi Antifungal effects [57]
pisi
Corynespora cassiicola
Trichophyton rubrum
Phytophthora infestans
Fusarium sp.
Fusarium concentricum
Fusarium oxysporum
Inhibiting spore
Wickerhamiella domercqiaeY2A Pythium ultimum Fungi germination and mycelial [60]
Pyricularia oryzae growth

Rhizoctorzia solani
Alternaria kikuchiana
Gaeumannomyces graminis
var. tritici
Phytophthora infestans
Antifungal and
Rhodotorula babjevae YS3 Trichophyton mentagrophytes Fungi [62]
anti-biofilm effects

2.3. Mannosylerythritol Lipids

Mannosylerythritol lipids (MELs) are a type of glycolipid biosurfactants produced by
Pseudozyma species mainly as well as Ustilago species [63]. Mannosylerythritol lipids were
initially found and characterized in Ustilago zeae in the 1950s, but relevant researches were
limited and unfocused in the following thirty years [64]. After the 1980s, a variety of strains
were isolated as MEL-producers based on substrates of glucose and oil, such as Pseudozyma
antarctica, Pseudozyma tsukubaensis, and Pseudozyma hubeiensis [65–67]. The basic molecular
structure was composed of the mannose molecule linked to an erythritol residue at C-10
and two fatty acids at the C-20 and C-30 [68]. Due to the different position and quantity of
acetyl groups, these compounds can be divided into four groups: MEL-A (diacetylated
at C-40 and C-60 ), MEL-B (monoacetylated at C-60 ), MEL-C (monoacetylated at C-40 ), and
MEL-D (deacetylated) [69], shown in Figure 4. According to the literature, the structure of
MEL depends on the carbon source primarily, and structural diversity confers to multiple
interfacial and biological properties including emulsibility, non-toxicity, anti-cancer, and
antioxidant activity, biodegradability, anti and environmental compatibility, showing their
great application prospects in medical, environmental, cosmetic, pharmaceutical and food
industries [70].
ctobacillus casei Staphylococcus aureus Gram-positive Anti-biofilm effects [80]
Blumeria graminis f. sp. trit-
ici
Inhibition of conidial germi-
eudozyma yeast Colletotrichum dematium Fungi [79]
Pharmaceutics 2021, 13, 227 nation. 9 of 22
Glomerella cingulata
Magnaporthe grisea

Figure
Figure 4. Different 4. Different
structures structures of mannosylerythritol
of mannosylerythritol lipids: Mannosylerythritol
lipids: Mannosylerythritol lipid (MEL)-A,
lipid (MEL)-A, MEL-B, MEL-C, and MEL-D,
n = 2~18. MEL-B, MEL-C, and MEL-D, n = 2~18.

The bactericidal effects of MELs are of vital importance in their practical applications,
as listed in Table 3. In 1993, Kitamoto et al. firstly pointed out that MELs showed significant
antimicrobial activity, especially against Gram-positive bacteria [71]. Then, Fukuoka et al.
also stated MEL-A and MEL-B have performed high inhibitory effects on Micrococcus luteus,
and the reason was attributed to their solubilizing effect on bilayer biomembranes [72].
Similarly, the growth of gram-positive bacteria (Bacillus megaterium and Bacillus subtilis)
was restrained by MEL-A and MEL-B reported by Recke et al., and they also found MELs
showed moderate antifungal effects on Candida magnoliae [73]. Besides, Okuhira et al.
compared the antimicrobial effects of MEL against two types of bacteria and summarized
MEL selectively inhibited the proliferation of most gram-positive bacteria below the con-
centration of 50 µg/mL, but not gram-negative bacteria [74]. Furthermore, Nashida et al.
synthesized 20 congeners of MELs through a chemical process and compared their inhibi-
tion on microbes, and the results illustrated not only the length of the alkyl chains but also
the pattern of Ac groups on the mannose moiety were important factors for antibacterial
activity [75].
So far, it has been reported that MELs have exhibited excellent sterilizing impact on
foodborne bacteria and is expected to be a novel safe alternative to food preservative in
food storage. Shu et al. investigated the antibacterial efficacy of MELs against Bacillus cereus
and Staphylococcus aureus and concluded the mode of action involved the disruption of
the cell membrane, leakage of cellular contents, the collapse of the whole cytoskeleton
as well as induced cell apoptosis [76,77]. Liu et al. observed the same phenomenon in
Listeria monocytogenes treated by MEL-A (32 µg/mL), and further transcriptome analysis
demonstrated that the differentially expressed genes were enriched in the ABC transporter
system, which verified the disorder of transmembrane protein played a key role in MEL-
mediated cell death [78].
It is worth mentioning MELs are able to reduce the persistent contamination caused by
fungi growth and microbial biofilm. For example, Yoshida et al. exposed the suppressive
effects of MELs on the early infection behaviors of several phytopathogenic fungal conidia
including Blumeria graminis f. sp. tritici (wheat powdery mildew fungi), Colletotrichum
dematium (mulberry anthracnose fungi), Glomerella cingulata (strawberry anthracnose fungi),
and Magnaporthe grisea (rice blast fungi), presumably owing to their inhibition to conidial
Pharmaceutics 2021, 13, 227 10 of 22

germination, and anticipated the future application of MELs as novel agricultural chemical
pesticides [79]. On the other hand, it is evident that MELs showed potent anti-biofilm
activity against Staphylococcus aureus through attachment inhibition and biofilm dispersal,
which was explained as the involvement of biosurfactants in microbial adhesion and
desorption [77,80].

Table 3. Antimicrobial effects of mannosylerythritol lipids.

Producing Microorganism Target Microorganism Microbial Type Inhibitory Effects Reference

Pseudozyma aphidis T-34 Micrococcus luteus Gram-positive Antibacterial effects [72]
Bacillus megaterium
Pseudozyma aphidis CBS Bacillus subtilis Gram-positive
Antibacterial effects [73]
517.83 Staphylococcus aureus
Candida magnoliae Fungi
Pseudozyma aphidis Bacillus cereus Gram-positive Antibacterial effects [76]
Pseudozyma aphidis DSM Antibacterial and
Staphylococcus aureus Gram-positive [77]
70725 anti-biofilm effects
Pseudozyma aphidis DSM
Listeria monocytogenes Gram-positive Antibacterial effects [78]
70,725
Micrococcus luteus
Staphylococcus aureus
Gram-positive Antibacterial effects [75]
Enterococci faecalis
Enterococci faecium
Streptococcus bovis,
Lactobacillus ruminis

Pseudozyma aphidis NBRC Eubacterium ruminantium

Gram-positive Antibacterial effects [74]
10182 Butyrivibirio fibrisolvens
Ruminococcus albus
Ruminococcus flavefaciens
Lactobacillus casei Staphylococcus aureus Gram-positive Anti-biofilm effects [80]
Blumeria graminis f. sp.
tritici
Inhibition of conidial
Pseudozyma yeast Colletotrichum dematium Fungi [79]
germination.
Glomerella cingulata
Magnaporthe grisea

2.4. Cellobiose Lipids

Cellobiose lipids (CLs) are a kind of glycolipid biosurfactants with less research than
other glycolipids. In 1951, cellobiose lipids were firstly reported as ustilagic acid (UA)
with antibiotic activity, produced by phytopathogenic fungi Ustilago maydis [81], and the
structure was then characterized as three forms: CL A, CL B, and CL C, shown in Fig-
ure 5 [82]. Subsequent researches have revealed a diversity of CL-producers including
Cryptococcus huminola, Pseudozyma fusiformata [83], Pseudozyma graminicola [84], Pseudozyma.
flocculosa [85], and Sporisorium scitamineum [86] cultivated on glucose, vegetable oil, and
alkane. The basic structure of cellobiose lipids consist of a cellobiose moiety as the hy-
drophilic group and a fatty acid chain as the hydrophobic group, and the variants can differ
in the presence or absence of hydroxyl group (R = H or OH) or the length of fatty acid
chain as well as an additional easter group (CL C) [82]. The studies suggest the structure of
Pharmaceutics 2021, 13, 227 11 of 22

cellobiose lipids is determined by their producing microorganisms. Cellobiose lipids have

the advantage of biodegradability, low toxicity, emulsibility, surface activity, a wide range
Pharmaceutics 2021, 13, x FOR PEER REVIEW 12 of 22
of pH tolerance and thermostability over chemical surfactants, and have great potential
employed in the food industry, medical field, and environmental protection.

Figure 5.
Figure 5. Structures
Structures of
of cellobiose
cellobiose lipids
lipids isolated
isolated from
from Ustilago
Ustilagomaydis:
maydis: CL-A,
CL-A, CL-B,
CL-B, and
andCL-C.
CL-C.RR== H
H
or OH,
or OH, m
m= = 22 or
or 4.
4.

3. Glycolipid-Modified
The antifungal activity Materials
of cellobiose lipids has been investigated thoroughly since
Haskins et al. firstly
3.1. Nanocomposites discovered the antibiotic activity of ustilagic acid (namely cellobiose
lipids) in 1951 [81]. The targeting fungi contains yeast and filamentous fungi, such as
Nanotechnology is a new burgeoning technology dealing with nanoscale dimensions
Saccharomyces cerevisiae [87], Cryptococcus, and Candida species [83], and the underlying
of particles that can produce novel multifunctional materials and devices with a variety
antifungal mechanism is always associated with the structural and functional disorder of
of applications. The size of nanoparticles ranges from 1 to 100 nm and the combination of
cell membrane caused by cellobiose lipids. For instance, Puchkov et al. illuminated that
two or more compounds of materials, in which at least one compound has a dimension
the intercalation of cellobiose lipids into the liposomal lipid matrix resulted in the increas-
less than 100 nm in size, can be defined as nanocomposites [92]. Compared to normal
ing permeabilization of cytoplasmic membrane, ATP leakage, and high susceptibility of
materials, nanocomposites have the advantage of physicochemical and combined biolog-
targeted cells, and considered this antifungal mode of action was relevant to detergent-like
ical properties and have attracted interest in various technological and environmental ar-
properties [88]. This membrane-damaging activity of cellobiose lipids was also proven
eas such as medicine, energy, cosmetics, electronics, packaging, coatings, and biotechnol-
ogy. As a carrier, nanocomposites are able to deliver substances such as drugs and achieve
the effect of target therapy and release control, which have great potential in practical
applications of antitumor therapy [93]. The synthesis of nanocomposites is usually based
on chemical and physical methods, while the former is the most widely adopted and
Pharmaceutics 2021, 13, 227 12 of 22

by Kulakovskaya et al. that CL has induced K+ leakage and inhibited polyphosphate

accumulation in Saccharomyces cerevisiae cells [89]. Moreover, further study displayed the
sensitivity of Saccharomyces cerevisiae cells to antifungal cellobiose lipids is up to cultura-
tion components, especially carbon source, and speculated that the long-chained polyP
participated in the viability restoring of ethanol-grown cells after treated by cellobiose
lipids [87]. Besides, flocculosin is known as one of the effective components in biological
control agents of powdery mildew fungi and has been confirmed to show inhibitory effects
on pathogenic fungi, and further work revealed that flocculosin caused direct damage to
the membrane surface of sensitive micro-organisms, such as Staphylococcus species and
Candida albicans, which implied the membrane-mediated antimicrobial mechanism was
suitable for gram-positive bacteria [90,91]. Consequently, CLs are defined as a novel safe
and natural fungicide more exactly and allowed to become membrane-penetrating agents
against yeast and fungal cells.

3. Glycolipid-Modified Materials
3.1. Nanocomposites
Nanotechnology is a new burgeoning technology dealing with nanoscale dimensions
of particles that can produce novel multifunctional materials and devices with a variety of
applications. The size of nanoparticles ranges from 1 to 100 nm and the combination of
two or more compounds of materials, in which at least one compound has a dimension
less than 100 nm in size, can be defined as nanocomposites [92]. Compared to normal
materials, nanocomposites have the advantage of physicochemical and combined biological
properties and have attracted interest in various technological and environmental areas
such as medicine, energy, cosmetics, electronics, packaging, coatings, and biotechnology. As
a carrier, nanocomposites are able to deliver substances such as drugs and achieve the effect
of target therapy and release control, which have great potential in practical applications of
antitumor therapy [93]. The synthesis of nanocomposites is usually based on chemical and
physical methods, while the former is the most widely adopted and highly efficient for large-
scale and high-performance production of nanocomposites. Nevertheless, the nanoparticles
are often synthesized along with hazardous and toxic byproducts during the chemical
process, which may pose a threat to public hygiene and environment. As a multifunctional
agent, biosurfactants have gradually gained more attention in recent years and played a
crucial role in the green synthesis of nanocomposites as seen in Figure 6, owing to their
superior biocompatibility, biodegradability, high efficiency of stabilization, and dispersion.
Firstly, biosurfactants could enhance the stability of nanocomposites by reducing the
interfacial tension and facilitating nanoemulsion formation. Furthermore, the presence
of biosurfactants prevents nanoparticles from aggregation and promotes dispersion in
organic solvents or water as a capping agent. On the other hand, biosurfactants could act
as a reducing agent in the synthesis of nanocomposites, which effectively simplifies the
producing procedures with better control [94]. Except for their physical characteristics,
biosurfactants will improve the biological properties of nanocomposites as a functional
adjuvant involving antioxidant, antimicrobial, and anticancer effects.
In the past decade, the rapid development of nanotechnology has been emphasized
and provided more opportunities in the antimicrobial field. Nanomaterials have been
proved to be effective to inhibit cell growth of various microorganisms even multi-drug-
resistant bacteria, and show less possibility to induce microbial resistance [95]. According
to the literature, the antimicrobial modes could be summarized as increasing cell membrane
permeability, inhibiting efflux pump, and generating reactive oxygen species (ROS) [96,97].
Therefore, glycolipids, as a typical kind of biosurfactants, have been employed in nanoma-
terial production due to their excellent emulsifying and antimicrobial properties, as listed
in Table 4.
Das et al. reported that rhamnolipids could be used to stabilize the synthesis of silver
nanoparticles, and the antibacterial and antifungal activity of both silver nanoparticles
and purified rhamnolipids against four strains of bacteria (Staphylococcus aureus, Bacillus
Pharmaceutics 2021, 13, 227 13 of 22

subtilis, Escherichia coli, and Klebsiella pneumoniae) and two fungi (Aspergillus niger and
Aspergillus flavus) was studied [98]. The comparative result suggested silver nanoparticles
are more effective when inhibiting the microbial growth of all kinds than purified rhamno-
lipids. Joanna et al. also synthesized silver nanoparticles (AgNPs) through chemical and
biological processes, respectively, and discovered the existence of rhamnolipid significantly
increased the stability of biogenic AgNPs and enhanced their antimicrobial activities [99].
The rhamnolipid-involved biogenic AgNPs exhibited more effective inhibition on gram-
positive bacteria and phytopathogenic fungi than gram-negative bacteria, probably owing
to the difference in bacterial membrane structure. Furthermore, the interactions between
biogenic AgNPs and DNA were investigated and the DNA particles were significantly
observed to accumulate around the AgNPs densely, potentially due to the strong affinity
between metal nanoparticles and nitrogen bases with the hydrogen bonds. On the other
hand, rhamnolipids help to stabilize the meatal nano-carrier when encapsulating various
substances, such as carvacrol [100]. The rhamnolipid-stabilized carvacrol-loaded zein
nanoparticles have been verified to suppress the growth of phytopathogens Pseudomonas
syringae and Fusarium oxysporum, which might result from the synergistic antimicrobial
effects of carvacrol, zein, and rhamnolipids.
Interestingly, there is often a win-win situation that glycolipid-modified nanocom-
posites exert potent inactivating function against both planktonic cells and persistent
biofilm. Marangon et al. elucidated that a combination of rhamnolipid and chitosan in
nanoparticles boosts their antimicrobial efficacy on Staphylococcus strains, and the reason
was attributed to the increased local delivery of chitosan and rhamnolipid at the cell surface
and, consequently, to their targets in gram-positive bacteria [101]. Especially, although
chitosan could not penetrate deeply in the biofilm through adsorption on the surface, the
nanoparticles enabled the antibacterial rhamnolipid to release and fill the whole biofilm,
rendering devastation of the biofilm structure and death of dormant cells. Another mech-
anism of biofilm eradication for nanomaterials is based on the anti-adhesive property of
glycolipid biosurfactant, and it has been reported that rhamnolipid-coated silver and Fe3 O4
nanocomposites exert more anti-biofilm efficacy against pathogenic Pseudomonas aeruginosa
and Staphylococcus aureus than individual RL or bare nanoparticles [97].
Sophorolipids could also participate in the fabrication of nanomaterials and be consid-
ered as dual roles including a biostabilizer and a biofunctionalizing agent. For instance,
zinc oxide nanoparticles (ZON) modified by sophorolipids have shown stronger inhibitory
activity against Salmonella enterica and Candida albicans compared with naked ZON [96].
Generally, gram-positive bacteria reveal more sensitivity to sophorolipids than gram-
negative bacteria, while sophorolipid capped gold nanoparticles (AuNPs-SL) exhibited
antibacterial properties against both gram-positive and gram-negative bacteria via binding
to cell membrane, disintegrating cell membrane, leaking intracellular constituents, and in-
terfering the enzyme activity [102]. Aiming at biofilm elimination, the acidic sophorolipid
was used to encapsulate hydrophobic curcumin in order to form nanostructures with
better dispersibility in water [102]. The sophorolipid–curcumin nanocomposites have the
capability to scavenge the biofilm of Pseudomonas aeruginosa as a quorum quencher.
Although the researches of MELs involved in nanomaterial synthesis are not as
many as others, their applications in nanotechnology are emerging and advancing latterly.
Firstly, Wu et al. embedded chitosan nanoparticles into essential oils using MEL-A as an
emulsifier and observed an apparent inhibition zone against Staphylococcus aureus around
the chitosan-based nanoparticles [103]. Subsequently, Bakur et al. successfully fabricated
metallic nanomaterials including silver, zinc oxide, and gold nanoparticles mediated by
MEL-A, and all of them exert inhibitory efficacy against pathogenic gram-positive and
gram-negative bacteria [104]. These findings indicated that MELs play a crucial role in the
rapid biosynthesis of metallic nanoparticles and enhance the antimicrobial property, yet
the underlying mechanism of inhibition still needs further study.
 metallic nanomaterials including silver, zinc oxide, and gold nanoparticles mediated by
MEL-A, and all of them exert inhibitory efficacy against pathogenic gram-positive and
gram-negative bacteria [104]. These findings indicated that MELs play a crucial role in the
rapid biosynthesis of metallic nanoparticles and enhance the antimicrobial property, yet
Pharmaceutics 2021, 13, 227 the underlying mechanism of inhibition still needs further study. 14 of 22

Figure 6. Schematic diagram of the synthetic pathway of nanocomposites based on glycolipids.

Figure 6. Schematic diagram of the synthetic pathway of nanocomposites based on glycolipids.

Table 4. Antimicrobial effects of glycolipid-modified nanocomposites.

Name Glycolipid Type Antimicrobial Effects Reference

Rhamnolipid-stabilized Antibacterial and antifungal activity
carvacrol-loaded zein Rhamnolipid against Pseudomonas syringae and [100]
nanoparticles Fusarium oxysporum.
Antibacterial and antifungal activity
against Staphylococcus aureus, Bacillus
Silver nanoparticles Rhamnolipid subtilis, Escherichia coli and Klebsiella [98]
pneumoniae, Aspergillus niger, and
Aspergillus flavus.
Antibacterial activity against
AgNPs Rhamnolipid gram-positive bacteria and antifungal [99]
activity against phytopathogens.
Antibacterial and anti-adhesive
properties against biofilms formed by
Silver and iron oxide nanoparticles Rhamnolipid [97]
Pseudomonas aeruginosa and
Staphylococcus aureus.
Antibacterial and anti-biofilm activity
Chitosan/rhamnolipid nanoparticles Rhamnolipid [101]
against Staphylococcus strains.
Antibacterial activity against Salmonella
ZnO Nanoparticle Sophorolipid [96]
enterica and Candida albicans.
Curcumin-sophorolipid Antibacterial activity anti-biofilm activity
Sophorolipid [105]
nanostructures against Pseudomonas aeruginosa.
Antibacterial and anti-biofilm effects on
Gold Nanoparticles Sophorolipid [102]
Staphylococcus aureus and Vibrio cholerae
Antibacterial activity against
Chitosan Nanoparticles MEL-A [103]
Staphylococcus aureus
Antibacterial activity against Escherichia
MEL@AgNPs, MEL@ZnONPs and
MEL-A coli, Salmonella enterica, Bacillus cereus and [106]
Ag-ZnO/MEL/GA
Staphylococcus aureus.
Antibacterial activity against
Gold nanoparticles (AuNPs) MEL-A gram-positive and gram-negative [104]
bacteria.

3.2. Liposomes
Liposomes are small spherical vesicles and normally possess a closed lipid bilayer
resembling a cell membrane and an aqueous inner chamber, which could encapsulate
hydrophilic and hydrophobic molecules, respectively. As multifunctional drug-carriers,
liposomes have the advantages of biocompatibility, stability, membrane fusion, gene trans-
fection, controlled release, and entrapment protection, widely applied in pharmaceutical,
cosmetics, and food fields [107]. There is a diversity of liposome structures including single
Pharmaceutics 2021, 13, 227 15 of 22

or multiple lipid bilayers, and one or more compartments. The variations of their properties
are depending on the composition, size, surface charge, and producing method. The size of
liposomes is ranging from nanometers to micrometers, and it will be called nanoliposome
when the size is less than 100 nm. Since the first liposome Doxil® was approved by FDA
for cancer therapy in 1995, there are more and more FDA-approved liposome-based phar-
maceuticals applied into clinical treatments in the following decades, involving anticancer,
antimicrobial, and antiviral therapies [108]. For example, rifampicin-loaded liposomes
were frequently reported [109]. For lung inhalation therapy, a polyelectrolyte complex
based on chitosan and carrageenan was used to coat rifampicin-loaded vesicles and ob-
tain a dry powder by spray-drying [110]. Furthermore, inhalable polymer-glycerosomes
were proved to be safe and effective carriers for rifampicin delivery to the lungs, which
enhanced the local pharmacological activity of rifampicin, reduced possible side effects,
and improved drug efficacy [111].
Thereby, the broad encapsulating properties of liposomes come up with a novel
strategy to inactivate microorganisms with reduced resistance. It is easy for natural or
synthetic antimicrobial agents incorporated in liposomes to reach intracellular targets via
the fusion of lipid bilayer and cell membrane, hence exerting high therapeutic efficacy.
Therefore, glycolipids could be embedded into liposomes together with other bactericides,
thereby realizing multiple antimicrobial effects on gram-positive, gram-negative, and fungi.
Nevertheless, this research direction has been rarely investigated.
Except for acting as bioactive substances, glycolipids are able to self-assemble as a
closed lipid bilayer, and the formed spherical vesicles are orderly arranged through exposed
hydrophilic glycosyls and tail-to-tail hydrophobic fatty acid chains. Interestingly, MELs
specialize in self-assembly properties and form vesicle structure with thermodynamic
stability, and have great potential to participate in liposome fabrication and stabilize
the bilayer structure [112]. For example, Wu et al. demonstrated the chitosan-coated
liposome with loading betulinic acid was modified by MEL-A, and the results implied
MEL-A boosted the antioxidant effect of betulinic acid [113]. Fan et al. successfully
prepared stable vesicles consisting of L-α-phosphatidylcholine (PC) and MEL-A by a thin
lipid film hydration technique and evaluated their structural characterization, stability,
and encapsulation efficiency [114]. The consequence showed the addition of MEL-A
changed the dispersibility of PC in water, rendering the formation of vesicle solution with
smaller size and higher encapsulation efficiency. Moreover, anthocyanins embedded in
vesicles exhibited improved antioxidant activity and higher retention rate in simulated
gastrointestinal fluid environment than bare anthocyanins, mainly owing to the protection
of vesicles. In the antimicrobial field, sophorolipid has been reported to formulate noisome
with amphotericin B, and the complex was proved to inhibit the growth of planktonic
cells and destruct mature biofilm of Candida albicans, presumably through downregulating
expressions of the genes responsible for hyphae formation [115]. These findings anticipate
the prospective development of liposomes based on glycolipids.

4. Conclusions and Perspectives

Glycolipids, as a kind of representative biosurfactants, are famous for versatile bioac-
tivities and have immerse potential to be put into practical applications due to their
environmental friendliness compared to chemical surfactants. Deep insight into the pre-
dominant antimicrobial behaviors of glycolipids reveals several underlying mechanisms
when targeting planktonic or biofilm state of microorganisms, as seen in Figure 7, which
provides a theoretical basis for exploiting novel safe antimicrobial agents applied in food,
cosmetics, and medical fields. The induced death of a single cell could be attributed to
the following reasons. The membrane permeabilization is considered as the prior factor
leading to cell lysis and apoptosis. Specifically, glycolipids are able to insert into the
lipid bilayer relying on their unique similar structure, leading to the deformation and
collapse of membrane skeleton physically as well as a conformational change of related
proteins functionally. As the main member of membrane proteins, ATP-binding cassette
Pharmaceutics 2021, 13, 227 16 of 22

(ABC) transporters are responsible for transmembrane migration of material and energy
transformation via ATP hydrolysis, and it has been proven that glycolipids have rendered
the disorder of movement across the membrane and material supplement by regulating
the different expression of related genes, which might be internal reason accounting for
the final cell apoptosis [78,116]. Except for membrane-mediated modes, correlational re-
searches also discovered the generation of ROS in bacteria and fungi [53,59]. Generally,
it is a common phenomenon that ROS is generated by normal cells during oxygen respi-
ration and metabolism, while ROS production is increased due to redox-cycling agents,
membrane disrupters, and antibiotics [117]. As a result, oxidative stress emerges when
a cell is not able to detoxify the excessively accumulated ROS, leading to cell death by
necrosis or apoptosis. On the other hand, eliminating refractory microbial contamina-
tion caused by persistent and resistant biofilm has become a huge challenge to be solved
Pharmaceutics 2021, 13, x FOR PEER REVIEW 17 of 22
urgently. Glycolipid-involved strategy to tackle microbial biofilm is mainly aiming at
blocking the adhesive stage and promoting the dispersal stage during the biofilm forma-
tion [118]. The anti-adhesive behavior can be attributed to the intrinsic antimicrobial and
in hydrophilic/hydrophobic
physiochemical characteristics
characteristics of treated
of glycolipids, causingsurfaces.
reduction Theofpromotion of biofilm
the cell density and
dispersal
changes in is hydrophilic/hydrophobic
related to the permeation of glycolipids into
characteristics the biofilm
of treated matrix,
surfaces. Theresulting
promotion in
detachment of extracellular
of biofilm dispersal matrix
is related to the(EPS) and disintegration
permeation of the
of glycolipids intowhole biofilm
the biofilm commu-
matrix, re-
nity.
sultingAlthough glycolipids
in detachment play extraordinary
of extracellular matrix (EPS)roles
andindisintegration
antimicrobialof work, they behave
the whole biofilm
less friendly inAlthough
community. the human immune system.
glycolipids The literatureroles
play extraordinary revealed rhamnolipids
in antimicrobial has func-
work, they
tioned
behaveas immune
less friendlymodulators
in the humanandimmune
virulence factors,
system. leading
The to rapid
literature necrotic
revealed killing of
rhamnolipids
has functioned as immune
polymorphonuclear leukocytesmodulators and
[119], early virulenceoffactors,
infiltration primaryleading
humanto rapidepithelia
airway necrotic
killingand
[120], of polymorphonuclear leukocytes
killing the myofibroblasts [119], early infiltration of primary human airway
[118].
epithelia [120], and killing the myofibroblasts [118].

Figure 7. Schematic diagram of the antimicrobial mechanism of glycolipids involved in planktonic cells and biofilms.

Figure 7. Schematic diagram of the antimicrobial mechanism of glycolipids involved in planktonic cells and biofilms.

Nano-scale approaches have been rapidly progressed and attract concentration glob-
ally on controlling microbial contamination in medical and healthcare applications. Nano-
Pharmaceutics 2021, 13, 227 17 of 22

Nano-scale approaches have been rapidly progressed and attract concentration glob-
ally on controlling microbial contamination in medical and healthcare applications. Nano-
carriers (such as nanoparticles and liposomes) are regarded as crucial drug delivery systems
due to their great dispersity, high encapsulation efficiency, and stability. Glycolipids have
been reported to play an important role in the modification of these materials both struc-
turally and functionally, relying on their special amphipathicity and self-assembling activity
as well as various biological activities. Therefore, the antimicrobial effect of glycolipid-
modified material has been augmented evidently because of the combination of effective
compounds. The modification of new materials based on glycolipids prompts the exploita-
tion of novel antimicrobial agents applicable to different requirements.
Furthermore, the synergic work of glycolipids and antibiotics or other antimicro-
bials on microorganisms is on trial and has made initial progress. Sophorolipids was
confirmed to work co-jointly with tetracycline to cause swelling and morphological dam-
age of methicillin-resistant Staphylococcus aureus [119]. The underlying mechanism was
speculated that the self-assembled glycolipids could span through the structurally alike
bacterial cell membrane and thereby facilitate the entry of antibiotics [120]. At present,
antimicrobial resistance has posed a serious threat to public health and hygiene, leading to
an increasing number of ineffective antibiotics. Nevertheless, developing new antibiotics
is time- and money-consuming, and difficult, which has the risk of new drug-resistance
emerging. Hence, the great potential of glycolipids to attenuate microbial resistance will be
a hot spot and presents a new strategy to inactivate drug-resistant microorganisms via the
synergy of antibiotics and glycolipids, which will make a break in the antimicrobial field.

Author Contributions: Investigation, H.L. and Q.S.; resources, T.W. and Q.S.; data curation, X.L. and
Q.S.; writing—original draft preparation, Q.S.; writing—review and editing, Q.C. All authors have
read and agreed to the published version of the manuscript.
Funding: This study was financially supported by Public Projects of Zhejiang Province (LGF18C200003),
National Key Research and Development Program of China (2018YFC1200100) China.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.

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