2. Different Classes of Plant-Derived AMPs
2.1. Defensins
Defensins are universal in the kingdom of plants and are present in all life forms. Defensins are brief peptides that range in size from less than 45 amino acids in length to a molecular weight of about 5 kDa
[11][12][13]. Plant defensins are very basic, and the formation of disulfide linkages has been linked to 8–10 cysteine residues
[14]. Plant defensins are kept together by four disulfide linkages and have a distinct, three-dimensional structure. According to their 3D structure, plant defensins have a parallel, triple-stranded β-sheet and α-helix
[15][16]. Due to their antibacterial and antifungal capabilities, defensins are produced by plants during their reproductive, storage, and stress responses and serve to protect them
[17][18].
2.2. 2S Albumins
The term 2S albumin comes from the sedimentation coefficient of the protein. These are storage proteins that are necessary for plant development and growth. Notably, 2S albumins are made up of a large 18–20 kDa precursor peptide that is post-translationally modified through proteolytic cleavage to yield small active peptide fragments
[19]. Finally, the processed AMP accumulates in storage vacuoles inside seeds, leaves, and vegetative tissues such as tubercles and has a low molecular weight that ranges from 4 to 9 kDa. Importantly, 2S albumins have been discovered to exhibit antibacterial and antifungal properties
[20].
2.3. Glycine-Rich Proteins (GRPs)
GRPs are storage proteins found in the xylem, hypocotyls, stems, and petioles of plants
[21]. They are frequently characterized by several glycine-containing motifs and an overall 70% of glycine percentage in the protein. Plants use GRPs to defend themselves against abiotic and biotic stress through their antifungal and antibacterial properties
[22].
2.4. Lipid Transfer Proteins (LTPs)
LTPs are lipid carriers that bind monomeric lipid units in a hydrophobic pocket and play essential roles in lipid transfer across donor and acceptor membranes
[23]. They are small proteins with a molecular weight of less than 10 kDa. Like defensins, LTPs are cysteine-rich and have four to five α-helices with four disulfide linkages. Several disulfide connections give LTPs their high degree of stability and resistance to heat denaturation
[24][25]. Due to their antibacterial qualities, LTPs are recognized to be essential for plant survival and play significant roles in plant breeding
[26].
2.5. Snakins
Snakins are low-molecular-weight peptides with 12 cysteine residues that are involved in disulfide bond formation and are highly conserved plant AMPs
[27]. They are known to have potent antibacterial and antifungal properties against a variety of plant diseases, as well as being involved in plant development and growth
[28].
2.6. Thionins
Thionins are small peptides consisting of 45–48 amino acids with 3–4 disulfide bonds and about 5 kDa of molecular weight. Thionins are usually found in higher plants and are reported to have potent antibacterial and antifungal activities
[29].
2.7. Cyclotides
Cyclotides are small cysteine-rich AMPs isolated from plants. They have three characteristic disulfide bonds and usually contain 28–37 amino acids. They are known as cyclotides because of their head-to-tail cyclized peptide bone
[30]. Cyclotides have a variety of biological effects, including antibacterial, antifungal, insecticidal, and anticancer effects
[31].
2.8. Napins
Napins are low-molecular-weight plant AMPs, with a molecular weight of less than 15 kDa, while two polypeptide chains are linked by disulfide bonds. Like defensins and other plant AMPs, napins are also cysteine-rich peptides
[32]. Napins are seed storage proteins with high water solubility
[33]. They are reported to have antibacterial, antifungal, and trypsin-inhibiting properties in addition to their role in plant growth and development
[34].
3. Antagonistic Effects of Plant-Derived AMPs against Foodborne Pathogens
Plant AMPs could be a potential alternative for biopreservation applications in the food industry. They can be used for the development of high-yielding genetically modified crops with enhanced resistance against plant and foodborne pathogens. Plant AMPs have already been suggested as potential drug sources for human infectious diseases caused by bacteria, viruses, and parasites, and for the treatment of different types of cancers
[35][36][37]. Although the use of plant AMPs in a clinical setting requires further in-depth experimental studies, they are suitable potential options for use in the food industry as food preservatives against foodborne pathogens. In the natural environment, plants experience different weather and drought conditions, and therefore, their immune system is developed to efficiently fight against various pathogens, where AMPs play essential roles in their protection
[38].
It is interesting to note that using plants and the products in which they are utilized, such as natural medicines and cosmetics, has long been linked to improving human health. Additionally, a number of the pharmaceuticals today are derived from plants
[39]. Furthermore, the numerous antibacterial qualities of AMPs generated from plants suggest their potential use in agricultural production
[40]. Additionally, the promising antifungal and antibacterial properties of AMPs generated from various plant components have the potential to be developed for biotechnological uses in the food business, such as food preservatives
[41][42].
To date, various plant-derived AMPs have been isolated and characterized with enormous structural and functional diversity. PaDef was discovered and extracted from a cDNA library obtained from Mexican avocado fruit. It is a peptide that resembles a defensin. PaDef can be utilized to treat foodborne infections because it has antibacterial action against Escherichia coli and Staphylococcus aureus
[43]. Another defensin, J1, was discovered in a cDNA library of bell pepper fruit and demonstrated potent antifungal activity against the
Colletotrichum gloeosporioides-induced anthracnose disease of transgenic bell pepper
[44][45]. Similar to this, Rs-AFP1 and Rs-AFP2, two defensins isolated from radish seeds, have potent antifungal action
[46].
Further, these two defensins were chemically synthesized and reported to have anti-yeast activity against food spoilage yeast
Zygosaccharomyces bailii in different beverages
[47]. In another study, four closely related cysteine-rich peptides were isolated and characterized from the seeds of
Impatiens balsamina (Balsam), showing antifungal and antibacterial activities
[48]. Interestingly, these cysteine-rich peptides did not show any cytotoxicity against human cells while exhibiting strong activity against enteric foodborne pathogens including
S. aureus,
E. coli,
Salmonella enterica,
Pseudomonas aeruginosa, and
Bacillus cereus [49]. Plant-derived AMPs can also be used for the protection of stored grains in warehouses, as reported in a study where a defensin, Cp-thionin-II, isolated from cowpea seeds, was confirmed to protect stored wheat grains from fungal spoilage caused by
Fusarium culmorum [50]. MsDef1 and MtDef4 are plant defensins isolated from
Medicago sativa, and
M. truncatula showed inhibitory activity against
F. graminearum, a fungal plant pathogen that caused fusarium head blight of wheat and barley
[51].
Another class of AMPs identified in plants is 2S albumin proteins. Pa-AFP-1 is isolated from passion fruit and has been found to efficiently inhibit the development of filamentous fungi,
C. gloeosporioides,
Trichoderma harzianum,
F. oxysporum, and
Aspergillus fumigatus [20][52]. CW-1 is another 2S albumin protein, isolated and characterized from
Malwa perviflora (Cheeseweed), that is reported to have antifungal activity against
F. graminearum [53].
Pg-AMP1 is a glycine-rich peptide that is isolated from guava seeds and has been shown to have potent antibacterial properties against Klebsiella species and Proteus species
[54]. A class of plant AMPs called lipid transfer proteins is effective against bacteria, yeast, and fungi. The lipid transfer protein Ca-LTP1, which is isolated from chili pepper seeds, has high antifungal action against
C. lindemunthianum and
C. tropicalis and may be employed as a food preservative
[55].
Another lipid transfer protein identified from sunflower seeds is called Ha-AP10. Ha-AP10 demonstrates strong inhibitory action against the germination of spores of pathogenic fungal pathogens
F. solani, indicating its potential use in the food business
[56]. Next, a mung bean nsLTP, isolated from mung bean sprouts, has been reported for its potential antifungal activities against various fungi, including
F. solani,
F. oxysporum,
Pythium aphanidermatum, and
Sclerotium rolfsii, as well as its antibacterial activity against
S. aureus.
Snakin-Z is identified and isolated from Jujube fruits and exhibits potential antibacterial and antifungal activities against
S. aureus and
Phomopsis azadirachtae, respectively. Interestingly, snakin-Z does not exhibit any cytotoxicity against RBCs and is suggested as a potential plant AMP for therapeutic or food preservation applications
[57]. MsSN1 is a snakin-1, isolated and characterized from
M. sativa, that exhibits antibacterial and antifungal activity against multiple foodborne pathogens
[58].
Next, CaThi is a thionin-like peptide characterized and isolated from chili. CaThi is reported to have antimicrobial, anti-yeast, and antifungal properties against various pathogenic bacteria, including
S. cerevisiae,
Candida albicans,
C. tropicalis, and
F. solani [59][60]. Another plant thionin is isolated from the wheat endosperm material that showed antibacterial activities against
Corynebacterium michiganense and
Xanthomonas campestris, which are known plant pathogens of tomatoes and peppers
[61][62]. Another study reported a potent antifungal thionin named thionin 2.4, secreted from
Arabidopsis thaliana, which showed antifungal properties against
F. graminearum, a serious crop fungal pathogen
[63]. TuAMP1 and TuAMP2 are thionin-like peptides that are isolated and characterized from the bulbs of
Tulipa gesneriana (Tulip) and exhibit diverse antifungal activity against
Agrobacterium rhizogenes,
A. radiobacter,
Clavibacter michiganensis,
Curtobacterium flaccumfaciens,
F. oxysporum, and
Geotrichum candidum [64].
Cycloviolacin O2 and Cycloviolacin O8 are cyclotides isolated and characterized from
Viola odorata, which showed potential antibacterial activity against various pathogenic bacteria
[65][66]. Additionally, parigidin-br1, a cyclotide isolated from
Palicourea rigida, is reported to have potent insecticidal activity against neonate larvae of Lepidoptera (
Diatraea saccharalis), a sugarcane insect
[67]. This suggests that plant-derived AMPs can also be developed as biopesticides for direct applications in food crops.
An α-hairpin-like peptide, LuffinP1, isolated and characterized from the seed of
Luffa cylindrica (Sponge gourd), showed potential protein translational inhibitory activity
[68]. Bleogen pB1 is a hevein-like peptide isolated from Cactus fruits and has been reported to have potent inhibitory activity against
C. albicans and
C. tropicalis [69]. Next, two hevein-like peptides, EAFP1 and EAP1, isolated and characterized from the bark of
Eucommia ulmoides, exhibit broad inhibitory activity against eight pathogenic fungi from cotton, wheat, potato, tomato, and tobacco
[70]. Another hevein-like peptide, Ee-CBP, was purified and characterized from the bark of
Euonymus europaeus and showed broad-spectrum antifungal activity against various plant pathogenic fungi
[71]. SmAMP3 is a novel hevein-like peptide that is isolated from leaves of
Stellaria media and has been reported to have potent inhibitory activity against plant pathogenic fungi
[72].
Napins are another class of plant AMPs that show promising results to be used as food preservatives. Em2-F18 is a napin-like peptide isolated and characterized from the seed of Jambu fruits, confirmed to have potential antibacterial activities against foodborne pathogens, such as
S. aureus and
S. enteritidis [73]. Another napin, Tn-AFP1, was identified from the Water chestnut and found to have antifungal activity against
F. oxysporum,
Mycosphaerella arachidicola, and
Physalospora piricola through its inhibition of fungal mycelial growth
[74]. Mandal et al. identified and characterized two other napin-like peptides, Cn-AMP1 and Cn-AMP2, from green coconut, which were found to exhibit potent antifungal activity against
C. tropicalis [75].
Knottin-type peptides are another class of plant-derived AMPs that show strong antifungal properties against foodborne pathogens. PAFP-S is a knottin-like peptide isolated from the seeds of
Phytolacca Americana and exhibits antifungal activities against
F. oxysporum and
Pyricularia oryzae, which are common crop pathogens of legumes, rice, and barley
[76]. Mj-AMP1 and Mj-AMP2 are other knottins isolated from the seeds of
Mirabilis jalapa and exhibit potential antifungal activity against major pathogenic fungal species, including
F. oxysporum, which causes significant loss to crops
[77]. Many unclassified plant AMPs, in addition to the known classes of plant AMPs, have the potential to be used in the food sector. A plant AMP called Cn-AMP3 was discovered in coconut water and demonstrated promising antibacterial properties against various bacterial species
[78]. The aforementioned examples collectively show the potential of plant-derived AMPs against several plant diseases and foodborne pathogens, which can be further developed as food preservatives in the future food industry to prevent food spoiling or loss.
4. Applications of Plant-Derived AMPs in the Food Industry
Plant-derived AMPs have been proven to possess strong antibacterial capabilities against bacteria, yeast, and fungi, pointing to the possibility of using them in the future to create food preservatives for the food industry. However, natural plant AMPs have several disadvantages that prevent their usage in culinary applications. The main drawbacks of natural plant AMPs include poor chemical stability, astringent flavor, short-term effectiveness, and cytotoxicity. Despite all the negative aspects, plant AMPs can be altered or enhanced for use as food preservatives through chemical synthesis and the addition of delivery techniques such as encapsulation, nanoparticles, or edible packaging
[41][79]. With recent developments in technology and scientific advancement, some of the natural plant AMPs have been modified and used in the food industry with improved efficacy. In a recent study, glycinin basic polypeptides (GBPs) were isolated from soybean and found to have strong antifungal properties against
A. niger and
Penicillium sp. by inhibiting fungal mycelial growth, spore germination, and plasma membrane disruption via inhibition of ergosterol synthesis. By improving the sensory qualities of fresh, wet noodles, this GBP intriguingly demonstrated possible food preservation characteristics and has been proposed as a potential tool for extending the shelf life of starchy foods
[80]. Ning et al. demonstrated the effects of soybean GPB’s preservation properties on the
Scomberomorus niphonius surimi (Japanese Spanish Mackerel). In a 24-day experiment, it was proven that GBP enhanced texture and prevented microbiological growth, extending the overall shelf life of surimi when stored
[81].
Another study aiming at the evaluation of food preservative capacities of legumes (pea, lentil, and fava bean flours) reported the potent antifungal activity of this native flour mixture against multiple species of
Aspergillus and
Penicillium. Further, purification confirmed the presence of nine native peptides in the legume flour mixture. A purified blend of these nine peptide mixtures was used to make bread under pilot plant conditions, which showed a longer shelf-life in comparison to the control bread
[82]. In another study, the same group found the antifungal activity of pea hydrolysate against
P. roqueforti and found the mixture of active components as a blend of pea defensins, lipid transfer proteins, and other peptides. They synthesized the identified peptides and found that this mixture is efficiently able to enhance the shelf-life of bread up to the storage period of 21 days
[83]. Low-molecular-weight peptides that have the ability to extend the shelf life of starchy foods and exhibit antifungal action were also discovered to be produced during the fermentation of plant products. Palm kernel cake is fermented via solid-state Lacto-fermentation by using
Lactobacillus casei. The generated fermentation product was identified as a mixture of peptides produced by Palm kernels and bacteria. The peptide mixture was reported to have antifungal activity against
Aspergillus sp.,
Fusarium sp., and
Penicillium sp. while also found to enhance the shelf-life of whole wheat bread slices for a storage period of 10 days
[84].
Another method for producing plant-derived AMPs on a large scale is recombinant expression. Huang et. al. confirmed the recombinant expression and production of Ac-AMP2, a plant AMPs, originally produced by
Amaranthus caudatus (an annual flowering plant), and MiAMP1, a highly basic protein from the nut kernel of
Macadamia integrifolia. The recombinant strains of
Pichia pastoris (GS115/Ac-AMP2 and GS115/MiAMP1) expressing these peptides showed potential post-harvest food preservation properties in pears infected with the fungal pathogen
P. expansum [85].
Another method for preserving food during lengthy storage is to use food preservation films coated with AMPs. By using several encapsulation techniques such as liposomes, emulsions, biopolymer particles, nanofibers, and nanofilms, plant AMPs can be employed in active food packaging
[10]. A recent study created and tested double-layered furcellaran/gelatin hydrolysate films containing the Ala-Tyr peptide for the preservation of frozen fish. No bacterial growth or oxidation was seen throughout the lengthy four-month storage period at −18 °C
[86].