The physiochemical functions of proteins in food are continuously being appreciated by many. In addition to their dietary benefits, several studies continue to reveal that they have more to offer than just nourishing the body. Many of the physiochemical functions of most of the naturally occurring proteins are through the actions of peptide sequences that have been encrypted inside the parent proteins. Bioactive peptides refer to peptide sequences within a protein with beneficial activities on various body functions and have the ability to impact human health beyond its known nutritional benefits. Bioactive peptides have the ability to regulate vital body functions through a variety of activities, including antimicrobial, immunomodulatory, and antioxidant, antithrombotic, and mineral binding functions.
The nature of activities that can be performed by bioactive peptides is always a factor of the parent amino acid of the peptide. This is because the bioactive peptides will have to interact with other proteins present in the body as well as modulate other natural processes within the body. The relationship of the structure, as well as the function of the amino acid, may not be well defined at this moment, but it has been observed that both share certain common properties. For example, most of the bioactive peptides have between two and 20 amino acids and they are also very rich in hydrophobic amino acids.
Over past decades, a lot of scientific studies have focused their attention on finding out the differing bioactive sequences that may help in preventing or reducing the risks associated with chronic diseases as well as for providing immune system protection. As a result, the research world has witnessed an increase not just in the studies of, but also in the use of bioactive peptides in the formulation of nutraceuticals as well as functional foods. This has caused a lot of research to be focused on processing and generating bioactive peptides from various food products.
The bioactivity of the peptides could be studied through in vitro-biochemical assays, in vivo studies through animal and human models, and cell cultures. Though the research on the development of food-derived bioactive peptides based on nutraceuticals is first gaining momentum, it is still necessary to spend more time in figuring out how to use the new findings in coming up with practical approaches for the commercial utilization of the bioactive peptides. There are many reasons why this gap may exist, the major ones being: general lack of understanding of the stability of the gastrointestinal tract, lack of proper clinical trials to offer adequate guidance on potential health claims, lack of the operation mechanism of the peptides, and the absence of scalable and reliable methods for the production of bioactive peptides from different types of food and non-food sources.
Production of Bioactive Peptides
Over the years, it has become possible to extract bioactive peptides from a wide range of plant and animal food proteins. Some of the foods that have been widely used for the production of bioactive peptides include milk, eggs, and meat proteins. The bioactive peptides from plant sources have primarily come from pulses, oats, soy wheat, flaxseeds, and hempseeds. Additionally, bioactive peptides have also been obtained from proteins from marine sources such as salmon, squid, fish, oysters, sea urchins, and snow crab.
During manufacturing, food proteins obtained from a variety of sources are digested by the enzyme before it becomes possible to evaluate their biological activities. This is then followed by a series of activity-guided purification and identification which normally lead to the discovery of the most potent sequence. But the process of activity-guided purification and identification is a time-consuming process and most of the studies have failed to supply enough rationale for the choice of their enzyme selection. However, when it comes to the actual production of bioactive peptides, here is a brief look at some of the main methods which are currently being used-:
With the enzyme hydrolysis method, the protein that is supposed to act as the source of the bioactive peptide is subjected to an enzymatic reaction at a specific temperature and pH. One of the benefits of this method is that it can be scaled up quite easily and it features a shorter reaction time compared to other methods such as microbial fermentation. For example, in a study conducted by Gobetti et al., through the process of milk fermentation, it was possible to generate angiotensin-converting enzyme – ACE-inhibitory peptides. This was achieved through the help of bacterial strains such as cremoris, bulgaricus, and Lactobacillus lactis spp.
This, however, contrasted with a study conducted by El-Fattah et al., where it was possible to produce the same bioactive peptide, but through the hydrolysis of milk using protease. In the same study, it was also observed that it was possible to use more than one protease in generating shorter peptides, though it was necessary to optimize both the temperature and the pH for each protease used. Additionally, the protease used as well as the time of the hydrolysis were vital in determining the type of peptides to be generated.
For instance, when rice proteins are hydrolyzed using bacillolysin, it was observed that the resulting product had stronger anti-inflammatory and anti-tyrosine properties when compared to the products of rice proteins hydrolyzed using subtilisin. Also, it was observed that samples hydrolyzed with papin and leucyl aminopeptidase had the least activities. In a similar manner, bovine muscle and porcine plasma generated different bioactive peptide-rich hydrolysates. Several studies have also utilized in-vitro gastrointestinal digestion techniques as a means of producing bioactive peptides from a variety of food proteins. With these methods, the researchers attempted to identify the activity of the peptides that may be noticed in the body following the consumption of particular foods or food proteins.
In fermentation, microorganisms such as fungi, bacteria, or yeasts are cultured in the protein of interest so as to hydrolyze the protein and obtain shorter peptides with their own enzymes. Before the bacteria are harvested, it is vital that they be in the exponential growth phase. Once harvested, they are then washed before being added to glucose with sterile distilled water which will eventually serve as the starting inoculum for the protein substrate. With this method, the degree of hydrolysis will mainly be a factor of microbial strain, the time for the fermentation to complete, and the protein source.
For example, in a study by Ahn et al., it was observed that the strong ACE-inhibitory activity of whey bioactive peptides derived through fermentation with Lactobacillus brevis was stronger than those fermented in L. acidophilus and L. plantarum. Also, in a study conducted by Sanjukta et al., it was observed that when soybean proteins are fermented in Bacillus subtilis, there was a higher degree of hydrolysis for MTCC5480 compared to fermentation with B. subtilis.
Commercialization and quality assurance challenges associated with the production of bioactive peptides
Following the production of the bioactive peptides, the immediate step that follows is to determine the bioactivity of the peptides. Unlike most of the synthetic drug molecules which are usually single entities, the bioactive peptides isolated from foods are never single entities, but rather a mixture of many peptides. To gain a purification of 99% of these peptides, not only will the cost of production increase, but the yields will decline dramatically. Again, such a purification level would also degrade any beneficial additive or synergistic effects when the peptide is considered within the framework of the entire hydrolysate. Additionally, most of the bioactive peptides are hydrophobic in nature, making them less soluble at very high concentrations of purities.
The hydrolysis of food proteins usually features enzymes such as pepsin, bromelain, trypsin, papain, or ficain. Though enzymatic hydrolysis comes with a lot of benefits, including the lack of residual toxic chemicals, and the presence of organic solvents in the final product, the use of enzymatic hydrolysis on an industrial level would significantly increase the cost of production. One solution to curb this problem would be to use cheap sources of enzymes such as certain byproducts of meat processing industries.
Secondly, enzymatic hydrolysis usually leads to the generation of a mixture of peptides. This would not be welcomed on a large scale since it makes the purification process too tedious and time-consuming – in certain cases, you may find that purifying a single peptide may require an exceedingly complex purification protocol.
Contrarily, naturally occurring peptides have a plethora of advantages compared to peptides obtained through enzymatic hydrolysis. This is because naturally occurring peptides are believed to be generally safe. But, since there is no proper technology that can be used for large-scale production, and since such an endeavor would also be very expensive, it has not been easy to commercialize the extraction of naturally occurring peptides from the various food sources where they can be found. Therefore, if the challenges associated with the mass production of bioactive peptides could be overcome, it would be highly beneficial to have them produced on an industrial level.
Oral applications of Bioactive Peptides – considerations and challenges
All the peptides derived from food sources are considered to be natural and as such, they come with a higher perception of acceptance. However, their application as orally ingested products also carries significant challenges and consequences. Here is a brief look at some of them:
The success of ingesting food and medicinal products often depends on flavor. This is usually the first response that the body elicits to any orally ingested substance. When it comes to the taste test, most of the individual peptides and protein hydrolysates usually fail miserably. This is because most of these products are bitter and this greatly hampers their acceptability for oral ingestion. The propensity to the bitterness of these peptides and protein hydrolysates are being linked to increased molecular weights, the degree of electrical charge and the presence of hydrophobic amino acids present on the C-terminal. However, the research world is yet to understand the molecular mechanism of bitterness as well as how it could be regulated.
Various digestive enzymes are responsible for digesting orally ingested substances, with the process starting in the oral cavity, right through to the stomach before final absorption in the small intestines. The human body is host to a myriad of proteolytic enzymes, and some of their actions have the ability to irreversibly change the profile of a peptide. It should be noted that the production of most of the bioactive peptides was produced with the digestive system in mind.
Since some of the bioactive peptides are generated through simulated digestion, most of them are naturally resistant to a bulk of the digestive enzymes. This is considered as a great advantage when it comes to the delivery of bioactive peptides through the oral route since the lack of digestion along the gastrointestinal tract will improve the bioavailability of the cargo, in addition to increasing the chances of creating a significant physiological effect on the body. Contrarily, peptides such as LKPNM which have been derived through the enzymatic digestion of bonito fish protein can still be metabolized into their active components within the GI tract. As such, it is seen as analogous to a pro-therapeutic which first undergoes metabolism before delivering the payload of all its active ingredients.
Absorption along the gastrointestinal tract is necessary before bioactive peptides can cause any systematic biological reaction in the body. Normally, the perception is that all proteins and peptides are digested down to their constituent amino acids since it is believed that it is only the amino acids that have the capability of going through the epithelial walls of the small intestines for absorption. However, with the current knowledge, and insights gained through several studies, it is clear that many peptides have the ability to go through the epithelial walls of the small intestines under normal conditions, and subsequently access circulation where they can then exert systematic effects.
- Fields K., Falla T.J., Rodan K., Bush L. Bioactive peptides: Signaling the future. J. Cosmet. Dermatol. 2009;8:8–13. doi: 10.1111/j.1473-2165.2009.00416.x.
- Moller N.P., Scholz-Ahrens K.E., Roos N., Schrezenmeir J. Bioactive peptides and proteins from foods: Indication for health effects. Eur. J. Nutr. 2008;47:171–182.
- Moldes A.B., Vecino X., Cruz J.M. Nutraceuticals and Food Additives. In: Pandey A., Sanromán M.A., Du G., Soccol C.R., Dussap C.G., editors. Current Developments in Biotechnology and Bioengineering: Food and Beverages Industry. 1st ed. Elsevier; Amsterdam, The Netherlands:
- Haque E., Chand R., Kapila S. Biofunctional properties of bioactive peptides of milk origin. Food Rev. Int. 2009;25:28–4
- Fu Y., Therkildsen M., Aluko R.E., Lametsch R. Exploration of collagen recovered from animal by-products as a precursor of bioactive peptides: Successes and challenges. Crit. Rev. Food Sci. Nutr. 2018:1–17. doi: 10.1080/10408398.2018.1436038.
- Przybylski R., Firdaous L., Châtaigné G., Dhulster P., Nedjar N. Production of an antimicrobial peptide derived from slaughterhouse by-product and its potential application on meat
- Gu Y., Majumder K., Wu J. QSAR-aided in silico approach in evaluation of food proteins as precursors of ACE inhibitory peptides. Food Res. Int. 2011;44:2465–2474.
- Perez Espitia P.J., de Fátima Ferreira Soares N., dos Reis Coimbra J.S., de Andrade N.J., Souza Cruz R., Alves Medeiros E.A. Bioactive Peptides: Synthesis, Properties, and Applications in the Packaging and Preservation of Food. Compr. Rev. Food Sci. Food Saf. 2012;11:187–204. doi: 10.1111/j.1541-4337.2011.00179.
- Gobbetti M., Ferranti P., Smacchi E., Goffredi F., Addeo F. Production of Angiotensin-I-Converting-Enzyme-Inhibitory Peptides in Fermented Milks Started by Lactobacillus delbrueckii subsp. bulgaricus SS1 and Lactococcus lactis subsp. cremoris FT4. Appl. Environ. Microbiol. 2000;66:3898–3904.
- El-Fattah A.M.A., Sakr S.S., El-Dieb S.M., Elkashef H.A.S. Bioactive peptides with ACE-I and antioxidant activity produced from milk proteolysis. Int. J. Food Prop. 2017;20:3033–3042. doi: 10.1080/10942912.2016.1270963
- Ferri M., Graen-Heedfeld J., Bretz K., Guillon F., Michelini E., Calabretta M.M., Lamborghini M., Gruarin N., Roda A., Kraft A., et al. Peptide fractions obtained from rice by-products by means of an environment-friendly process show in vitro health-related bioactivities. PLoS ONE. 2017;12:e0170954.
- Fu Y., Liu J., Hansen E.T., Bredie W.L.P., Lametsch R. Structural characteristics of low bitter and high umami protein hydrolysates prepared from bovine muscle and porcine plasma. Food Chem. 2018;257:163–171. doi: 10.1016/j.foodchem.2018.02.159.
- Aspri M., Leni G., Galaverna G., Papademas P. Bioactive properties of fermented donkey milk, before and after in vitro simulated gastrointestinal digestion. Food Chem. 2018;268:476–484. doi: 10.1016/j.foodchem.2018.06.119.
- Vieira E.F., das Neves J., Vitorino R., Dias da Silva D., Carmo H., Ferreira I.M. Impact of in vitro Gastrointestinal Digestion and Transepithelial Transport on Antioxidant and ACE-Inhibitory Activities of Brewer’s Spent Yeast Autolysate. J. Agric. Food Chem. 2016;64:7335–7341.
- Picariello G., Miralles B., Mamone G., Sánchez-Rivera L., Recio I., Addeo F., Ferranti P. Role of intestinal brush border peptidases in the simulated digestion of milk proteins. Mol. Nutr. Food Res. 2015;59:948–956.
- Rizzello C.G., Lorusso A., Russo V., Pinto D., Marzani B., Gobbetti M. Improving the antioxidant properties of quinoa flour through fermentation with selected autochthonous lactic acid bacteria. Int. J. Food Microbiol. 2017;241:252–261. doi: 10.1016/j.ijfoodmicro.2016.10.035.
- Aguilar-Toalá J.E., Santiago-López L., Peres C.M., Peres C., Garcia H.S., Vallejo-Cordoba B., González-Córdova A.F., Hernández-Mendoza A. Assessment of multifunctional activity of bioactive peptides derived from fermented milk by specific Lactobacillus plantarum strains. J. Dairy Sci. 2017;100:65–75.
- Ahn J.E., Park S.Y., Atwal A., Gibbs B.F., Lee B.H. Angiotensin I-Converting Enzyme (Ace) Inhibitory Peptides From Whey Fermented By Lactobacillus Species. J. Food Biochem. 2009;33:587–602. doi: 10 1111/j.1745-4514.2009.00239.x.
- Sanjukta S., Rai A.K., Muhammed A., Jeyaram K., Talukdar N.C. Enhancement of antioxidant properties of two soybean varieties of Sikkim Himalayan region by proteolytic Bacillus subtilis fermentation. J. Funct. Foods. 2015;14:650–658. doi: 10.1016/j.jff.2015.02.033.
- Chaudhury A., Duvoor C., Reddy Dendi V.S., Kraleti S., Chada A., Ravilla R., Marco A., Shekhawat N.S., Montales M.T., Kuriakose K., et al. Clinical review of antidiabetic drugs: Implications for type 2 diabetes mellitus management. Front. Endocrinol. 2017;8:6. doi: 10.3389/fendo.2017.00006.
- Li-Chan E.C.Y. Bioactive peptides and protein hydrolysates: Research trends and challenges for application as nutraceuticals and functional food ingredients. Curr. Opin. Food Sci. 2015;1:28–37.
- Hanke A.T., Ottens M. Purifying biopharmaceuticals: Knowledge-based chromatographic process development. Trends Biotechnol. 2014;32:210–220. doi: 10.1016/j.tibtech.2014.02.001.
- Cheung I.W.Y., Li-Chan E.C.Y. Angiotensin-I-converting enzyme inhibitory activity and bitterness of enzymatically-produced hydrolysates of shrimp (Pandalopsis dispar) processing byproducts investigated by Taguchi design. Food Chem. 2010;122:1003–1012.
- Kopf-Bolanz K.A., Schwander F., Gijs M., Vergères G., Portmann R., Egger L. Impact of milk processing on the generation of peptides during digestion. Int. Dairy J. 2014;35:130–138.
- Kamdem J.P., Tsopmo A. Reactivity of peptides within the food matrix. J. Food Biochem. 2017:e12489. doi: 10.1111/jfbc.12489.