Peptides From Protein Hydrolysates

Bioactive peptides have many functional benefits and some of them are proving to be extremely useful as food and pet food ingredients as well as pharmaceutical agents. In the scientific literature, the antimicrobial protein hydrolysates are also known as antimicrobial peptides (AMPs) in some circles. They are oligopeptides which contain between 5 and 100 amino acids. Their range of activity is broad from inhibiting viruses, bacteria, yeasts, moulds etc. It is interesting to see that different protein hydrolysates from the same source can have a diverse range of activity – from antibacterial to clinical benefits to ameliorating for obesity and all from the same source.

Peptides also have sensory properties and a number of them contribute umami (Zhang et al., 2016). Tuna for example is often used to produce a daishi or rich fish soup. 

Alternative Names

We mentioned AMPs but they have also be called cationic host defense peptides, anionic antimicrobial peptides/proteins, cationic amphipathic peptides, cationic AMPs, host defense peptides and α-helical antimicrobial peptides.

Protein hydrolysates are produced by a variety of methods usually requiring an initial protein extraction followed by hydrolysis using a single or mix of proteases. The processes are generally well defined because the production of protein hydrolysates is a common method. The process is often used for the manufacture of nutritional supplements (dietary, medicinal, sport), feed materials for other fermentations or for the manufacture of particular bioactive peptides. All of these materials have been scaled up using conventional methods from laboratory to industrial-scale systems.

Peptide Generation and Isolation

 Acid and enzymic digestion is the most common approach to generating peptides. A typical enzymolysis study will involve a combination of trypsin and alkaline protease in the ratio of 1:2, for 4 hours hydrolysis under 55°C. The solid-liquid ratio is optimal at 1:9.

The range of industrial enzymes includes:-

protamex, trypsin, bromelain, pepsin, acid protease, Flavourzyme^® and alcalase.

 Flavourzyme^® is a broad specificity fungal protease complex with exopeptidase activity that produces degree of hydrolysis (DH) values as high as 50%. This results in hydrolysates containing mostly dipeptides.

Alcalase is a cheap commercial protease from Bacillus licheniformis, (Novozyme Corp) generally used to obtain peptides from food proteins. It is a broad-specificity serine endoprotease hydrolysing most peptide bonds, preferentially those containing aromatic (Doucet et al., 2003) and hydrophobic amino acids (MEROPS database: http://merops.sanger.ac.uk) .

Ultrafiltration is one of the first steps in enriching low molecular weight bioactive peptides from different hydrolysate sources (Cai et al., 2022). An  additional step may involve centrifugation. Different peptides fractions can be obtained using different UF membranes. If a fraction has a particularly useful value it can be further purified using a range of chromatography methods. Gel filtration is a refining method using size to produce peptide fractions of the same size.

The in silico approach is economically effective and time-saving. 

Reversed-phase liquid chromatography is one the methods of choice of purification. Freeze drying is also used to generate a solid.

Sequence analysis is conducted using MALDI-TOF/TOF-MS and by LC-ESI-QTOF-MS.

PeptideRanker is a neural network-based tool used to predict bioactive peptide activity and rank peptide datasets based on bioactivity probabilities.

The Biopep database is used to forecast the known activity of various amino acids.

The antioxidant activity of peptides in reflected in their radical scavenging activity from in vitro studies but also from cell studies on the regulation of antioxidant pathways in vivo. All these peptides, if acting as antioxidants, will show strong 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical-scavenging and lipid peroxidation-inhibiting activity in vitro. 

Algal Derived Antimicrobial Protein Hydrolysates

The first bioactive protein fractions from algae were extracted from the macroalga Saccharina longicruris. Some of these peptides inhibit the growth of Staphyolococcus aureus. The proteins from this algae were extracted and hydrolysed using the protease trypsin. One fraction of peptides which were greater than 10 kDa size decreased the specific growth rate of S. aureus.

The inhibitory peptides appeared to come from different precursors similar in protein structure to histones, ubiquitin, a ribosomal protein and a leucine rich repeat protein. It is thought these types of peptides are related to an innate immune defence of the alga (Beaulieu et al., 2015).

Chia (Salvia hispanica)

Bioactive peptides extracted from chia seed have antioxidant, antihypertensive, and anti-inflammatory activities which are well known (Aguilar-Toala et al., 2018). The peptides also have significant activity against food poisoning bacteria such as Escherichia coli and Listeria monocytogenes. These peptides were produced using microwave and enzymatic hydrolysis which reduced the molecular weights of the proteins. 

Overall, the peptide fraction of  < 3 kDa showed higher antimicrobial activity than both chia seed hydrolysate and peptide fractions in the size range of 3-10 kDa. It was found that the < 3 kDa increased membrane permeability as measured by crystal violet uptake of the cells. The bioactive peptides were found to have multiple effects on these micro-organisms such as causing “a significant extension in the lag phase, decreases in the maximum growth, and growth rate in the bacteria and promoted multiple indentations (transmembrane tunnels), membrane wrinkling, and pronounced deformations in the integrity of the bacterial cell membranes”. The bioactive peptides of less than 3 kDa shared similar characteristics with cationic and hydrophobic properties. Seven of them shared the same amino acid sequence (GDVIAIR). A related study (Coelho et al., 2018) showed these similar sized fractions could inhibit the growth of Staphylococcus aureus.

Cowpea (Vigna unguiculata) yields peptide fractions following hydrolysis of extracted protein which have been shown to interfere in (1) lipid metabolism and cholesterol metabolism (Marques et al., 2015) (2) inhibition of angiotensin-I converting enzyme (ACE-I), antioxidant activity (Segura Campos et al., 2010) (3) antidiabetic potential (Castañeda-Pérez et al., 2019). Alacalase treatment also produces antibacterial peptides (Osman et al., 2021).

Animal Derived Antibacterial Peptides

Chicken (Gallus gallus domesticus) meat as with many meats will produce peptide hydrolysates which have a range of functions too. These are generated by a mix of acid hydrolysis and enzymic hydrolysis. One study showed that chicken liver can produce a peptide WYR which is a potent antioxidant (Chen et al., 2024). Likewise, chicken liver hydrolysate has the capability of soaking up Maillard reaction products. 

Various byproducts from chicken blood produce a range of antibacterial polypeptides (CHb-1 and CHb-2) and an anti-inflammatory peptide ((SNPSVAGVR) in chicken feathers.

Animal liver has been a solid source of antioxidant peptides:-

• PTTKTYFPHF was found in pig (Sus scrofa domesticus) spleen.
• GEHGDSSVPVWSGVN, HLDYYLGK, HLTPWIGK, DTYIRQPW, WDDMEKIWHH, and MYPGIAD found in duck (Anatinae) liver
• LPLPFP was isolated from goose (Anser albifrons) liver.

Some antibacterial compounds come from hydrolysates of animals such as the sea cucumber (Actinopyga lecanora). Various proteases were used to produce these peptide hyrolysates. These were active against Pseudomonas sp., P. aeruginosa and E. coli.

 AMPs from Milk Proteins

Milk proteins including whey and casein are very good sources of bioactive peptides. They are released when enzymatically hydrolysed in the gut from proteins such as lactoglobulin etc,. Many of these are found in protein hydrolysates and fermented dairy foods where they have a range of other properties. They possess opioid, antithrombotic, growth-stimulating and immunomodulatory benefits as well as being antimicrobial. These bioactive peptides are encrypted within the primary structures of the various proteins.

Milk has long been associated with helping people sleep. What is not often commented on is the production of antihypertensive milk peptides using fermentation. In many food fermentation processes, a proteolytic starter culture will produce various proteases which is also the role of the gastrointestinal tract when it begins digesting protein. Two tripeptides, (a.k.a. tactotripeptides), valine-proline-proline (VPP) and isoleucine-proline-proline (IPP) extracted from casein have been shown to significantly reduce high blood pressure in humans (Fekete et al., 2015) and they have been extensively investigated.

Casein has always been a solid source of AMPs. Bellamy et al. (1992) were the first research group to identify an antimicrobial peptide LFcin, released from the N-terminal domain of bovine LF hydrolyzed by pepsin in vitro (Bellamy et al., 1992). 

Whey protein isolate, when treated with trypsin or chymotrypsin enzymes does not produce antibacterial activity implying AMPs are not produced using these enzymes. However, when pepsin is used which is found in the stomach then short peptides are generated. These peptides are fractionated using reversed-phase high performance liquid chromatography. One fragment called 14-18 based on the amino-acid sequence (KVAGT) is derived from beta-lactoglobulin. It is found to be antibacterial and is found in various peptides of many different sources. Other fragments have been found with similar properties (Theolier et al., 2013).

Subsequent studies have shown that probiotic microorganisms such as Lactobacillus plantarum will generate AMPs when they ferment whey (Théolier et al., 2013) and may explain their probiotic benefit in terms of damaging pathogens or having a clinical benefit (Fekete et al., 2015).

Bovine casein was hydrolysed by a serine metalloprotease from the probiotic species Lactococcus lactis. Similar chromatographic separation methods are used to isolate the fractions. They are active against  Listeria innocua and Micrococcus luteus, and two Gram-negative bacteria: Escherichia coli  and Salmonella enteritidis (Bougherra et al., 2017).

Other antioxidant peptides have been isolated from:-

• Monkfish swim bladders (Lophius litulon).
• Skipjack tuna (Katsuwonus pelamis) skins,
• salmon (Salmo salar) muscle,
• yak (Bos grunniens) bone collagen,
• dry-cured Xuanwei ham,
• lotus (Nelumbo nucifera) seeds,
• soybeans (Glycine max)
• finger millet (Eleusine coracana) protein hydrolysate
• Moringa oleifera Lam. leaves
• Watermelon seed protein
• Sea grass (Posidonia autralis)
• Pea protein hydrolysate
• Chinese chestnut  (Castanea mollissima Blume)
• Tuna roe
• Silkworm pupae (Bombyx mori)

Lee et al., (2024) identified antioxidant and anti-inflammatory peptides from Edible Bird’s Nest.

Collagen

Collagen peptide digests are used routinely in food,  medical and cosmetic applications. They have very goof safety, biocompatability and are economic to produce because of the availability of raw substrate.

 Collagen sources include fish (Lee et al., 2017) as well as animals.

References

Aguilar-Toalá, J.E., Deering, A.J., Liceaga, A.M. (2020) New Insights into the Antimicrobial Properties of Hydrolysates and Peptide Fractions Derived from Chia Seed (Salvia hispanica L.). Probiotics Antimicrob Proteins. Dec;12(4) pp. 1571-1581. doi: 10.1007/s12602-020-09653-8. PMID: 32385579 (Article).

Beaulieu, L., Bondu, S., Doiron, K., Rioux, L. E., & Turgeon, S. L. (2015). Characterization of antibacterial activity from protein hydrolysates of the macroalga Saccharina longicruris and identification of peptides implied in bioactivity. Journal of Functional Foods, 17, pp. 685-697 (Article)

Borrajo, P., Pateiro, M., Gagaoua, M., Franco, D., Zhang, W., & Lorenzo, J. M. (2020). Evaluation of the antioxidant and antimicrobial activities of porcine liver protein hydrolysates obtained using alcalase, bromelain, and papain. Applied Sciences, 10(7), 2290.

Bradshaw, J. (2003) Cationic antimicrobial peptides: Issues for potential clinical use. BioDrugs 17, pp. 233–240

Brown, K.L.; Hancock, R.E. Cationic host defense (antimicrobial) peptides. (2006) Curr. Opin. Immunol. 18, pp. 24–30

Castañeda-Pérez, E., Jiménez-Morales, K., Quintal-Novelo, C., Moo-Puc, R., Chel-Guerrero, L., & Betancur-Ancona, D. (2019). Enzymatic protein hydrolysates and ultrafiltered peptide fractions from Cowpea Vigna unguiculata L bean with in vitro antidiabetic potential. Journal of the Iranian Chemical Society, 16, pp. 1773-1781.

Chen, Q., Nie, X., Huang, W., Wang, C., Lai, R., Lu, Q., … & Yu, X. (2024). Unlocking the potential of chicken liver byproducts: Identification of antioxidant peptides through in silico approaches and anti-aging effects of a selected peptide in Caenorhabditis elegans. International Journal of Biological Macromolecules, 272 June (1), 132833 (Article).

Coelho, M.S., Soares-Freitas, R.A.M., Arêas, J.A.G., Gandra, E.A., Salas-Mellado, M.L.M. (2018) Peptides from Chia Present Antibacterial Activity and Inhibit Cholesterol Synthesis. Plant Foods Hum Nutr.  Jun;73(2):101-107. doi: 10.1007/s11130-018-0668-z. PMID: 29679358 (Article).

Djellouli, M., López-Caballero, M. E., Arancibia, M. Y., Karam, N., & Martínez-Alvarez, O. (2020). Antioxidant and antimicrobial enhancement by reaction of protein hydrolysates derived from shrimp by-products with glucosamine. Waste and Biomass Valorization, 11(6), pp. 2491-2505.

Fekete, Á.A.; Givens, D.I.; Lovegrove, J.A. (2015) Casein-derived lactotripeptides reduce systolic and diastolic blood pressure in a meta-analysis of randomised clinical trials. Nutrients, 7, pp. 659–681 (Article)

Ghanbari, R., Ebrahimpour, A., Abdul-Hamid, A., Ismail, A., Saari, N. (2012) Actinopyga lecanora hydrolysates as natural antibacterial agents. Int. J. Mol. Sci. Dec 7;13(12) pp. 16796-811. doi: 10.3390/ijms131216796. PMID: 23222684; PMCID: PMC3546722. (Article).

Groenink, J.; Walgreen-Weterings, E.; van’t Hof, W.; Veerman, E.C.; Nieuw Amerongen, A.V. (1999) Cationic amphipathic peptides, derived from bovine and human lactoferrins, with antimicrobial activity against oral pathogens. FEMS Microbiol. Lett. 179, pp. 217–222

Harris, F.; Dennison, S.R.; Phoenix, D.A. (2009) Anionic antimicrobial peptides from eukaryotic organisms. Curr. Protein Pept. Sci. 10, pp. 585–606

Lee, C. H., Hamdan, N., Nyakuma, B. B., Wong, S. L., Wong, K. Y., Tan, H., … & Lee, T. H. (2024). Purification, identification and molecular docking studies of antioxidant and anti-inflammatory peptides from Edible Bird’s Nest. Food Chemistry, 454, 139797 (Article).

Marques MR, Soares Freitas RA, Corrêa Carlos AC, Siguemoto ÉS, Fontanari GG, Arêas JA. (2015) Peptides from cowpea present antioxidant activity, inhibit cholesterol synthesis and its solubilisation into micelles. Food Chem. Feb 1;168  pp. 288-93. doi: 10.1016/j.foodchem.2014.07.049. Epub 2014 Jul 15. PMID: 25172712.

Osman, A., Enan, G., Al-Mohammadi, A. R., Abdel-Shafi, S., Abdel-Hameid, S., Sitohy, M. Z., & El-Gazzar, N. (2021). Antibacterial peptides produced by Alcalase from cowpea seed proteins. Antibiotics, 10(7), pp. 870

Riedl, S.; Zweytick, D.; Lohner, K. (2011) Membrane-active host defense peptides—challenges and perspectives for the development of novel anticancer drugs. Chem. Phys. Lipids 164, pp. 766–781

Segura Campos, M. R., Chel Guerrero, L. A., & Betancur Ancona, D. A. (2010). Angiotensin‐I converting enzyme inhibitory and antioxidant activities of peptide fractions extracted by ultrafiltration of cowpea Vigna unguiculata hydrolysates. Journal of the Science of Food and Agriculture, 90(14), pp. 2512-2518.

Tkaczewska, J. (2020). Peptides and protein hydrolysates as food preservatives and bioactive components of edible films and coatings-A review. Trends in Food Science & Technology, 106, pp. 298-311.

Théolier, J., Hammami, R., Labelle, P., Fliss, I., & Jean, J. (2013). Isolation and identification of antimicrobial peptides derived by peptic cleavage of whey protein isolate. Journal of Functional Foods,5(2), pp. 706-714 (Article)