Skip to main content
NCBI home page
Current Research in Food Science logoLink to Current Research in Food Science
. 2022 Nov 5;5:2162–2170. doi: 10.1016/j.crfs.2022.10.031

Advances in the stability challenges of bioactive peptides and improvement strategies

Jingyan Pei a, Xinchang Gao b,∗∗, Daodong Pan a,c, Ying Hua d, Jun He a, Zhu Liu e,∗∗∗, Yali Dang a,∗
PMCID: PMC9664347  PMID: 36387592

Abstract

Bioactive peptides are widely used in functional foods due to their remarkable efficacy, selectivity, and low toxicity. However, commercially produced bioactive peptides lack quality stability between batches. Furthermore, the efficacies of bioactive peptides cannot be guaranteed in vivo due to gastrointestinal digestion and rapid plasma, liver, and kidney metabolism. The problem of poor stability has restricted the development of peptides. Bioactive peptide stability assessments use different stability assays, so the results of different studies are not always comparable. This review summarizes the quality stability challenges in the enzymatic hydrolysis production of bioactive peptides and the metabolism stability challenges after oral administration. Future directions on the strategies for improving their stability are provided. It was proposed that we use fingerprinting as a quality control measure using qualitative and quantitative characteristic functional peptide sequences. The chemical modification and encapsulation of bioactive peptides in microcapsules and liposomes are widely used to improve the digestive and metabolic stability of bioactive peptides. Additionally, the establishment of a universal stability test and a unified index would greatly improve uniformity and comparability in research into bioactive peptides. In summary, the reliable evaluation of stability is an essential component of peptide characterization, and these ideas may facilitate further development and utilization of bioactive peptides.

Keywords: Bioactive peptide, Quality stability, Property stability, Digestion stability, Metabolism stability, Improvement strategy

Graphical abstract

Image 1

Open in a new tab

Highlights

  • •

    Stability challenges encountered by bioactive peptides were summarized.

  • •

    Strategies to improve the stability of bioactive peptides were provided.

  • •

    A universal stability test and unified index would improve uniformity and comparability in research into bioactive peptides.

  • •

    It was proposed that we use a method of traditional Chinese medicine fingerprinting as a quality control measure.

1. Introduction

Bioactive peptides are protein fragments encrypted in the primary sequences of proteins, and they have a wide array of significant pharmacological effects after their degradation from proteins (Singh et al., 2021). The field of peptide therapy began in 1922 when insulin extracted from the pancreas of animals was first used for medical treatment (Banting et al., 1962), followed by corticotropin in 1949 (Elkinton and Hunt, 1949). In 1953, oxytocin became the first synthetic peptide drug (Vigneaud et al., 1953). At first, it took months to years to synthesize peptides by solution-phase chemistry until the invention of solid-phase synthesis in 1963 (Merrifield, 1965). The peptide industry has developed significantly, accompanied by the maturity of high-performance liquid chromatography (HPLC) purification technology. Due to their remarkable potency, selectivity, and low toxicity, bioactive peptides saw rapid development in the 1970s and 1980s (Muttenthaler et al., 2021). Bioactive peptides derived from foods that prevent the development of chronic diseases were included in the BIOPEP-UWM database. Today, the number of bioactive peptides registered in the BIOPEP-UWM database is 4,299, as of January 2022 (Minkiewicz et al., 2019). The functions of peptides in the BIOPEP-UWM database include antihypertensive, hypolipidemic, hypoglycemic, antioxidant, antithrombotic, immunomodulatory, antimicrobial, and anticancer.

In the past two decades, scientists have applied bioactive peptides to various fields, such as functional foods, biological reagents, biological fertilizers, and biopharmaceuticals. In food science, the conventional methods of producing bioactive peptides are enzymatic hydrolysis and fermentation of different precursor proteins (Iwaniak et al., 2019; Wu et al., 2019). Proteins derived from natural sources, such as plants, bacteria, fungi, and animals, have been extensively studied as sources of bioactive peptides. In addition, bioinformatics, using in silico analysis tools as a new method to discover bioactive peptides, has also made significant progress (Salim and Gan, 2020). Numerous food companies developed food-derived bioactive peptide products and claimed health effects from consuming these functional foods (Hartmann and Meisel, 2007). For example, Calpis Co. (Tokyo, Japan) developed a commercially available sour milk that contained peptides VPP and IPP from β-and κ-casein and declared it had a hypotensive function. The glycomacropeptide sold by Davisco (Minnesota, USA) had anti-cariogenic, antimicrobial, and antithrombotic functions. In non-food matrices, bioactive peptides also provide specific health-promoting effects. For instance, Recaldent™ and Trident™ added bioactive peptide CPP to chewing gum to help remineralize enamel subsurface lesions (Shen et al., 2001). In addition, bioactive peptides also have great potential as drug components. In state-of-the-art peptide drug discovery, display technologies, such as phage display, yeast display, and ribosome display, and integrated venomics are prominent (Muttenthaler et al., 2021). There are around 80 peptide drugs on the global market (Lau et al., 2018), and the global peptide drug market is predicted to exceed $60 billion by 2026.

However, there are stability challenges with the use of bioactive peptides as functional foods or drugs. Therefore, the evaluation of peptide stability is an essential part of peptide characterization (Cavaco et al., 2021). First, scaling up production often results in irreproducible peptides. Unguaranteed quality stability will hinder the commercial production of bioactive peptides. Second, the most convenient and acceptable delivery method of bioactive peptides is oral administration (Yap and Gan, 2020). However, gastrointestinal digestion and rapid metabolism in plasma, liver, and kidney with short half-lives (t1/2) greatly affects the efficacy of bioactive peptides in vivo. Therefore, the application of bioactive peptides will be greatly hindered if high doses and frequent administration are required to ensure efficacy. As stated above, the evaluation of bioactive peptide stability has become critical to the development of this industry (Lee et al., 2019; Panchin et al., 2020).

This review summarizes the stability challenges associated with the production and administration of bioactive peptides, including i) product quality, ii) physicochemical properties, iii) gastrointestinal digestion, and iv) metabolism. In addition, we provide future directions for the improvement of bioactive peptide stability in commercial food health-enhancing compositions and pharmaceutical ingredients. Furthermore, we promote the establishment of a standard stability evaluation test and a unified stability evaluation index.

2. Quality stability of peptide products generated by enzymatic hydrolysis

An increasing number of peptide products appear on the market annually with the gradual verification of many functions of food-derived bioactive peptides. Manufacturers claim their products are functional peptides or contain functional peptides. Japan, the United States, and Europe have taken the lead in launching bioactive peptide products with various functions, forming an industry with great commercial prospects. At the same time, the evaluation of the quality of bioactive peptide products has become an urgent problem.

The most common peptide production method is enzymatic hydrolysis, followed by fermentation of different precursor proteins. Generally, bioactive peptide products derived from natural products, especially food, are not single peptides with high purity. The separation and purification of bioactive peptides from by-product proteins are currently receiving extensive attention. In particular, the by-products of fish and poultry are inhomogeneous, and the ingredients vary greatly, leading to complex protein hydrolysates with variable composition (Mage et al., 2021). In order to improve the quality stability of peptide products, greater consistency from batch to batch is needed. For example, the sequence and content of the peptides obtained by enzymatic hydrolysis should be approximately the same to ensure stable biological activity. Therefore, ensuring the stable quality of different batches of peptide products is worthy of further research. In addition, our experience and the production practices of cooperative companies have demonstrated the challenges in producing precisely the same peptide products generated by enzymatic hydrolysis, even under the same conditions and raw materials. This has been attempted in products, such as yak bone peptides, broccoli stem and leaf peptides, and sea cucumber peptides. Processing conditions, such as temperature, fermentation, and hydrolysis time, can affect the reproducibility of peptides, especially when protein mixtures exist in the substrate (Singh et al., 2021). Even when these factors are controlled and the process is consistent, minuscule and intrinsic batch-to-batch heterogeneity is still unavoidable. Therefore, it is necessary to explore the quality evaluation indicators, such as the content of bioactive peptides, activity stability, and the mechanism of activity stability. The traditional quality evaluation of bioactive peptide products is mainly carried out from the following aspects: the color and appearance, the molecular weight distribution of the peptide, the type and content of amino acids, and the purity and extraction methods for the peptide. However, studies on the functional stability of bioactive peptides are lacking. The functional stability of peptides is related to the quantity and content of specific functional peptides in bioactive peptide products. Therefore, the current research hotspots and difficulties lie in the repeatability and accurate quantification of the characteristic active peptide sequences.

3. Physicochemical property stability of peptides

Proteins are generally composed of a complex network of stable interactions, including hydrophobic interactions, salt bridges, and hydrogen bonds, that result in a unique 3D structure that is significantly stable, compared to other conformations (D’Addio et al., 2016). The bioactive peptides formed after protein degradation appear more “dynamic” in that the energetic barriers to transition among different conformations are usually small, compared with rigid proteins. The link between peptide structure and stability is unique to each individual peptide sequence. Different amino acids vary in their sensitivity to environmental factors, such as heat, pH, and salt. Heat treatment would change the secondary structure of peptides and make the peptide inactive, which might be due to the aggregation (Enciso et al., 2015). pH conditions could cause changes in the ionization properties of peptides and lead to electron transfer. For instance, threonine, serine, and cysteine are unstable under alkaline conditions (Lopez-Sanchez et al., 2016; Wang et al., 2017). The concentration of salt may affect the structure of the bioactive peptides due to changes in the side chains of amino acid residues under high salt concentrations that may result in a decrease in biological activity (Zhu et al., 2014).

The sequences of peptides greatly influence their physicochemical property stability. Guruprasad et al. (1990) reported the correlation between the stability of a protein and its dipeptide composition, which revealed the occurrence of certain dipeptides was significantly different in the unstable proteins, compared with that in the stable ones. The ProtParam tool of the Expasy database was used in this method to estimate the stability of peptides (Gasteiger, 2005). Peptide VLSTSFPPK was purified and identified from Kluyveromyces marxianus protein hydrolysate. If the proline in the antepenultimate position was replaced with cysteine, tyrosine and histidine, the new peptides VLSTSFCPK, VLSTSFYPK, and VLSTSFHPK would have lower stability against the NaCl treatment in DPPH assay (Mirzaei et al., 2020a, Mirzaei et al., 2020b). As a result of exposure to high concentrations of salt, a conformational change in the side chains of cysteine, tyrosine, and histidine may explain the lower stability of VLSTSFCPK, VLSTSFYPK, and VLSTSFHPK (Mirzaei et al., 2020a, Mirzaei et al., 2020b). Peptide KLTWQELYQLKYKGI (KI-15) had exceptional thermal stability, but the stability decreased when the 10th-position leucine was replaced by phenylalanine and valine (Tm from 78.3°C to around 40°C). Computational and experimental studies showed heat resistance was based on the hydrophobic interaction of leucine-7 and leucine-10; therefore, the substitution of leucine in the 10th-position would lead to a substantial decrease in Tm (De Rosa et al., 2021). By replacing the dehydroalanine with dehydrobutyrine at position 5 of antimicrobial peptide Nisin, the resistance to acid-catalyzed chemical degradation of the peptide was enhanced.

In addition, the amino acid sequences affect the structural characteristics of the bioactive peptides, such as α-helices and β-sheets. Structural characteristics play an essential role in the stability of bioactive peptides. For example, the reduction in the peptide-folded helix group was highly correlated with a loss in thermal stability (Diana et al., 2010). Asaduzzaman and Chun (2015) and Udenigwe and Fogliano (2017) also reported bioactive peptides with a high proportion of β-sheet structures would be more sensitive to heat treatments. Mirzaei et al., 2020a, Mirzaei et al., 2020b believed the higher percentage of β-sheet structures in peptide VLSTSFCPK was the reason for its higher sensitivity to heat treatment, compared with other similar peptides. The stability of the physicochemical properties of the peptide itself is the basis for its development and utilization.

4. Gastrointestinal digestion stability

Currently, clinically mature peptide therapy is most commonly administered via intramuscular injection, sustained-release subcutaneous injection, and intranasal delivery. As a typical delivery, hydrophobic depots could increase the duration of action (Mitragotri et al., 2014). However, oral administration is the most convenient and acceptable delivery method of bioactive peptides, especially for the application of peptides as daily therapy for chronic disease management. Many peptides will be digested into inactive peptide fragments or free amino acids and lose activity in vivo after oral administration; however, these same peptides may have significant biological activity in vitro. For example, Li et al. (2018) identified a novel H1N1 virus neuraminidase inhibitory peptide, PGEKGPSGEAGTAGPPGTPGPQGL, from cod skin hydrolysates, and this peptide was almost inactivated after simulated gastrointestinal digestion. Most bioactive peptide drugs, such as insulin, have to be administered by parenteral routes, which is inconvenient and painful (Di, 2015). Therefore, the ability to resist digestion by gastrointestinal protease is critical to bioactive peptides.

4.1. Gastrointestinal digestion of peptides

Susceptibility to the action of gastrointestinal proteases, such as pepsin, trypsin, and chymotrypsin, is one of the important reasons for the short half-life of bioactive peptides (Wang et al., 2019). The cleavage specificity for pepsin (EC 3.4.23.1) lacks specificity, preferentially before or after Trp, Tyr, Leu, or Phe, with other restrictions in hydrophobic amino acid sites. Trypsin (EC 3.4.21.4) was found in pancreatic secretions, which specifically hydrolyzes amino acid residues Lys and Arg of the C-terminal. The chymotrypsin (EC 3.4.21.1) belongs to the serine protease family, and the hydrolysis side chains are composed of Phe, Tyr, and Trp, as well as other large hydrophobic side chains with specificities, such as Met and Leu. The presence of these gastrointestinal cleavage sites on the peptide is an important cause of its gastrointestinal instability. At present, the methods for evaluating the gastrointestinal resistance of bioactive peptides are mainly in silico simulation and in vitro enzymatic hydrolysis assay.

4.2. In silico simulation of gastrointestinal digestion stability

The “Enzyme(s) Action” application in the BIOPEP-UWM database (Minkiewicz et al., 2019) and “PeptideCutter” application in the ExPASy database (Wilkins et al., 1999) are commonly used to predict the hydrolytic actions of gastrointestinal proteases. The activity of chicken breast muscle hydrolysate-activated alcohol dehydrogenase was significantly reduced after intestinal digestion, and in silico assessments subsequently revealed the degradation of peptides KDLFDPVIQ, YPGIADRM, VAPEEHPTLL, and ADGPLKGIL might be responsible for the reduced activity (Xiao et al., 2020). However, there are differences between simulated digestion in silico and actual gastrointestinal digestion. Simulating gastrointestinal digestion in the database, based on the specificity of an enzyme, assumes that all bonds theoretically susceptible to a given proteinase are hydrolyzed (Minkiewicz et al., 2008, 2019). The proteolysis is often incomplete under actual conditions. In silico hydrolysis might not include the conditions, such as pH, temperature, time, the nature of protease-protein interactions, the complexity of the gastrointestinal environment, and other characteristics of enzyme application (Udenigwe, 2014; Vermeirssen et al., 2004). Moreover, the availability of peptide bonds also influences the effectiveness of enzyme action (Vermeirssen et al., 2004). Therefore, there are cases in which sites that could not be cut are cut, and vice versa.

4.3. In vitro simulation of gastrointestinal digestion stability

In vitro simulation of gastrointestinal digestion provides simple and inexpensive way for researchers to estimate the gastrointestinal stability of bioactive peptides. Peptide stability has been evaluated by revealing the peptide sequence and the role of each amino acid (Mirzaei et al., 2021). The consensus is that high molecular weight peptides are more prone to hydrolysis than smaller ones. Chen and Li (2012) proved casein-derived peptides with molecular weight >3 kDa are more likely to be degraded than peptides <3 kDa during two-stage in vitro gastrointestinal digestion. Compared with gastric digestion, peptides are more prone to hydrolysis in the intestine. In addition, bioactive peptides with more acidic amino acids (Picariello et al., 2010) and higher hydrophobicity (Xie et al., 2015) would be more stable during gastrointestinal digestion. In addition, the stability of peptides is influenced by peptide sequences (Mirzaei et al., 2020a, Mirzaei et al., 2020b). A lack of cleavage sites for gastrointestinal proteases is essential for improving gastrointestinal stability. Proline and glutamic acids are conducive to improving the resistance of bioactive peptides to pepsin and pancreatin (Savoie et al., 2005). However, experiments performed by different teams vary in their methods of simulating digestion.

For gastric digestion, Hao et al. (2020) adjusted the pH to 3 and incubated the sample (1 mg/mL) with pepsin (1000 U/mL) at 37°C for 120 min. Differently, Yuan et al. (2018) adjusted the pH of pepsin (enzyme/substrate of 1:35, w/w) to 2.5 and incubated the mixture at 37°C for 60 min. When simulating the gastrointestinal digestion of casein-derived peptides, peptides were adjusted to pH 2 and hydrolyzed with pepsin (2% w/w) at 37°C for 90 min (Chen and Li, 2012). Marseglia et al. (2019) mixed sodium and potassium salts with pepsin (25,000 U/mL) to prepare simulated gastric juice and incubated the sample with simulated gastric juice at 37°C for 2 h after adjusting to pH 3. The digestion stability of peptides is commonly assessed by dividing the remaining amount of peptide after digestion by the amount of peptide before digestion. Different amounts of enzyme added and different reaction conditions (such as pH, incubation time, and solution constituent) will inevitably lead to different results.

In summarizing the reported methods of intestinal digestion, differences still exist among the digestion methods. Furthermore, the source of the enzymes is crucial, but porcine and bovine trypsin were used in different studies to evaluate the digestion stability of peptides. Baptista et al. (2020) prepared pancreatic juice mixed with pancreatin (from porcine pancreas, 800 U/mL) and bile solution in vitro. After adjusting to pH 7, the samples were digested at 37°C for 2 h. Jin et al. (2016) used trypsin (1:100, w/w) from bovine pancreas to simulate the intestinal digestion of yogurt.

Different experimental reagents and methods will affect the results. Ahmed et al. (2022) summarized 31 articles about the simulated digestion stability of peptides, including 27 stable peptides and 66 unstable peptides. However, due to the differences between evaluation methods, it is difficult to directly compare these peptides. For example, if under the same experimental conditions, a peptide that was reported to be unstably incubated for 2 h may be more resistant to gastrointestinal digestion than a peptide that was reported to be stably incubated for 1 h. In addition, a simple mixture solution is different from actual body fluids, which may lead to differences between digestion in vitro and in vivo (Cavaco et al., 2021). Fortunately, more and more studies have reported digestion simulation methods closer to the real human body conditions in recent years, such as the studies by Brodkorb et al. (2019) and Minekus et al. (2014) Moreover, some investigators have developed methods of in vivo digestion in mice instead of in vitro stimulation (Fan et al., 2019; Tu et al., 2019).

4.4. Stability of peptides on the transepithelial transport

The extensive enzymatic system in the gastrointestinal tract, epithelial cells, and mucosa constitute a significant barrier. Whether bioactive peptides can resist hydrolysis by brush border membrane enzymes and travel intact through intestinal epithelial cells is important. Caco-2 cells, human colon cancer cells, are widely used to simulate the transport and stability of bioactive peptides across intestinal epithelial cells (Ohsawa et al., 2008). The apparent permeability coefficient values of bioactive peptides were mainly between 10−8 and 10−6 across the Caco-2 cells monolayer. Pep T1-mediated transport, paracellular diffusion, and transcytosis are primary transfer methods across intestinal epithelial cells (Fig. 1). Several metabolic enzymes on the Caco-2 cells membrane may reflect drug absorption (Teng et al., 2012). The resistance of different peptides against Caco-2 enzymes varies widely. For example, 96% of the ACE-inhibitory peptide QIGLF keep stable after incubation with a Caco-2 cells membrane for 2 h (Ding et al., 2014), while the peptides AAATP, AAPLAP, KPVAAP (Gallego et al., 2016), and YAEERYPIL (Miguel et al., 2008) were almost entirely degraded after 2 h.

Fig. 1.

Fig. 1

Open in a new tab

Stability challenges encountered by bioactive peptides in vivo, including gastrointestinal digestion and metabolism.

5. Metabolism stability

Bioactive peptides show great potential in function foods and pharmaceutical engineering, due to their high potency and low toxicity. However, most peptides have short t1/2 of 2–30 min because of the broad distribution of peptidases in the body (such as plasma, kidney, and liver). After absorption, bioactive peptides are often rapidly degraded by proteases or rapidly cleared in plasma, kidney, and liver. For example, the t1/2 of bioactive peptides VP, VPY, VPYP, and YPQ were less than 5 min (Zheng et al., 2019). Giapreza® (DRVYIHPF), approved by the Food and Drug Administration (FDA) in 2017, is metabolically unstable in the human body and its estimated t1/2 is 30 s. The short t1/2 means that an increase in dosage or repeated administration is required for efficacy, which significantly limits the development of bioactive peptides.

5.1. Metabolism stability in blood

Digestion in the gastrointestinal tract can still be avoided by changing the route of administration, but metabolism after entering the bloodstream is an unavoidable problem. Peptidase genes account for 3.1% of all encoded human proteins, making peptidases one of the most abundant classes of enzymes and widely distributed in the body (Klein et al., 2018). When the bioactive peptides are absorbed into the bloodstream through the epithelial cells of the small intestine, a large number of proteases and peptidases present in the blood will act on the peptides (Fig. 1). Peptidases commonly found in the blood include carboxypeptidase N (cleavage site specificity: Lys, Arg, Ala, and Gly), aminopeptidase N (cleavage site specificity: Ala, Leu, Arg, Trp, Leu, and Phe), DPP4 (cleavage site specificity: Pro and Ala), plasmin (cleavage site specificity: Arg and Lys), furin (cleavage site specificity: Arg, Ser, and Lys), and neprilysin (cleavage site specificity: Phe, Leu, Try, Ile, and Val) (Lai et al., 2021). Bottger et al. (2017) incubated eight peptides with blood, plasma, and serum. Except for one stable peptide, the degradation products of the remaining seven peptides were detected in blood, plasma, and serum. When assessing the serum/plasma stability of bioactive peptides, the assays vary from different study to study, such as the species source of the plasma or serum, commercial plasma or serum and fresh acquisition, peptide/plasma or serum ratio, pH value, and buffer. These all affect the results. For example, to assess its stability, Yang et al. (2017) incubated an antithrombotic peptide RGDWR (200 ng/mL) with rat plasma. Low et al. (2020) used complete human EDTA blood, obtained from the Red Cross Blood Donor Service, to evaluate the plasma stability of a proopiomelanocortin-derived peptide (2 μM). Wang et al. (2021) mixed physiological saline-containing peptide (1 mg/mL) with fresh human plasma (1:1, v/v) to investigate the effect of structural modification of antimicrobial peptides on their plasma stability. Moreover, commercial human serum is to be used with caution because a lack of homogeneity between batches can lead to variable activity (Cavaco et al., 2021).

5.2. Metabolism stability in kidney and liver

Although there are over 44 kinds of tissues, the enzymatic breakdown of bioactive peptides mostly happens in the kidney and liver after reaching organs throughout the body (Hall, 2014). In addition, because the pore size of the glomerulus is about 8 nm and the molecular weight of bioactive peptides is less than 25 kDa, peptides are easily filtered and rapidly removed from systemic circulation by the kidney (Fig. 1) (Katsila et al., 2012). Subsequently, bioactive peptides enter the bladder and excrete from the body because the renal tubules cannot reabsorb (Chia, 2021). The liver is the organ with the most active metabolism and plays an essential role in peptide metabolism. The metabolic rate of bioactive peptides in the liver depends on the amino acid sequence and the specific intake of hepatocytes (Meibohm and Zhou, 2012). At present, there are still few studies on liver and kidney metabolism of food-derived bioactive peptides, as the majority focus on mature peptide drugs. Cetrotide®, a decapeptide containing five unnatural amino acid residues approved by the FDA in 2000, was hydrolyzed by peptidase between Ser-4 and Tyr-5 and became a tetrapeptide before renal elimination (Chia, 2021). Firmagon®, a decapeptide, was degraded in the liver and excreted mainly as tetrapeptide and pentapeptide fragments from the urine (Sonesson and Rasmussen, 2013). Firazyr®, a decapeptide, was metabolized into two inactive fragments through the action of plasma and liver peptidase and excreted by the kidney (Bork et al., 2007; Leach et al., 2015).

6. Strategies to ensure the quality of peptide products

Reproducibility and stability are vital for the entrance of peptide products into the market. Therefore, it is crucial to establish quality control indicators to evaluate product stability. Lopez-Morales et al. (2019) used size exclusion chromatography (SEC), mass spectrometry (MS), and nuclear magnetic resonance spectrometry (NMR) to evaluate the peptide profile, and determined the safety of collagen hydrolysates as a quality indicator. By determining the average molecular weight and polydispersity index with MS and NMR, Vazquez-Leyva et al. (2019) used structural and mass mobility orthogonal analysis to profile complex peptide mixtures. In addition, Mage et al. (2021) studied the potential of using a large Fourier-transform infrared (FTIR) fingerprinting database to obtain new insights on the quality of peptide products.

Traditional Chinese medicines (TCM) are small molecule mixtures with complex component. The fingerprint of TCM is a quality control method for extracts and preparations, which has become an international consensus (Tao et al., 2017). The FDA accepts TCM-style chromatographic fingerprints in application materials. The World Health Organization (WHO) also stipulated in the 1996 guidelines for the evaluation of herbal medicines that if the active ingredients of any given TCM are not clear, chromatographic fingerprints can be provided to prove the product’s quality. Chromatographic, spectroscopic, mass spectrometry, and capillary electrophoresis are widely used in fingerprinting. The application of fingerprints may solve the problems of the quality assessment of TCMs, which often have complex ingredients, unidentified active ingredients, and differences in quality across product batches. Taking the Ginkgo biloba extract preparation as an example, these fingerprints reflect the 33 chemical components (mainly flavonoids and lactones) contained in the preparation and their respective concentrations. Research on the correlation between chemical composition and drug efficacy has shown that extracts composed of about 24% ginkgo flavonoids and about 6% ginkgolide have the best curative effects. The reproducibility of the fingerprint spectrum is good. Crude peptide products generated by enzymatic hydrolysis of protein usually contain different peptide sequences. Learning from the quality control experience of TCM, qualitative and quantitative characteristic function peptide sequences combined with fingerprints may play an essential role in the quality control of crude peptide products.

7. Strategies to improve the stability of bioactive peptides

7.1. Modification

At present, chemical modification is one of the primary measures to improve the stability of bioactive peptides, including D-amino acid or unnatural amino acid substitution, increased molecular mass, cyclization, and N/C-terminal modification or substitution (Fig. 2 & Table 1) (Yao et al., 2018). D-amino acid substitution is a common strategy to improve the stability of peptides. Mao et al. (2021) reported the introduction of D-amino acid promoted the stability of antimicrobial peptides against plasma and liver S9 metabolism when all amino acids of IRIKIRIK were in D configuration. Jia et al. (2017) reported the same D-amino acid substitution strategy to improve the stability of an antimicrobial peptide polybia-CP. A20FMDV2 is a 20-mer peptide, and D-amino acid substitutions could significantly prolong its serum stability (Cardle et al., 2021). Substituting L-amino acid in cationic AMP peptide KRLFKKLLKYLRKF with D-amino acid and unnatural amino acids, the stabilities of the peptides toward proteases were enhanced (Lu et al., 2020). Chia (2021) identified 25 peptide drugs approved by the FAD, 20 of which contain unnatural amino acids. Unnatural amino acid substitution could significantly extend the systemic half-life of bioactive peptides. Not many peptide drugs are composed of natural amino acid residues, as represented by vasopressin, angiotensin II, plecanatide, linaclotide, and oxytocin.

Fig. 2.

Fig. 2

Open in a new tab

Different strategies to improve the stability of bioactive peptides (A) Various chemical modification, (B) Microcapsules, and (C) Liposome.

Table 1.

Summary of the strategies to improve the stability of bioactive peptides.

Strategies Applications Examples Ref.
Chemical modification D-amino acid substitution IRIKIRIK Mao et al. (2021)
Polybia-CP Jia et al. (2017)
A20FMDV2 Cardle et al. (2021)
KRLFKKLLKYLRKF Lu et al. (2020)
Unnatural amino acid substitution Afamelanotide Chia (2021)
Bivalirudin
Cyclosporine
Pasireotide
Increase molecular mass Endostatin-derived peptide Zhou et al. (2009)
RKDVY Tan et al. (2017).
Cyclization Cyclized opioid peptide Piekielna et al. (2013)
Tachyplesin peptides Vernen et al. (2019)
N/C-terminal modification or substitution Antimicrobial peptide Li et al. (2021)
PMAP-36PW Liu et al. (2020)
RLYE Yun et al. (2019)
A20FMDV2 Hung et al. (2017)
Encapsulation Microcapsules Antimicrobial oyster peptides Zhang et al. (2009)
Phaseolus lunatus L. peptides Cian et al. (2019)
α-helical polypeptide Morikawa et al. (2005)
Insulin Aiedeh et al. (1997)
Liposome Antioxidant peptide Ramezanzade et al. (2021)
RLSFNP Zhang et al. (2019)
Insulin Cui et al. (2015)
Open in a new tab

Based on the size-dependent mechanism of renal clearance, increasing the molecular mass of bioactive peptide is an excellent strategy to increase their blood circulation times and extend their half-lives (Maack et al., 1979). Therefore, linking bioactive peptides to large polymers was widely used to avoid renal filtration because polypeptides with molecular mass of 50 to 70 kDa would be retained longer in circulation. Conjugation to polyethylene glycol (PEG) has widely been employed. The t1/2 of bioactive peptides attached to PEG could be extended 10- to 100-fold. A PEGylated analogue of the gut hormone oxyntomodulin is in phase I trials for treating obesity (Bianchi et al., 2013), which has long-term antihyperglycemic, insulinotropic, and anorexigenic effects. Zhou et al. (2009) used methoxy-PEG-aldehyde to modify the N-terminal of an RGD-modified endostatin–derived synthetic peptide, and the t1/2 was prolonged by 5.86-fold. Conjugation to lipidation, plasma proteins, and albumin-binding molecules was used to increase the size of bioactive peptides (Pollaro and Heinis, 2010). Albumin ligand-conjugated thymopentin (RKDVY) became more stable in plasma and had better immune-modulating activity (Tan et al., 2017).

Cyclization is another common method of modification of peptides to improve their stability and activity. Endogenous opioid peptides have great potential in treating pain. However, natural opioid peptide receptors have poor specificity and unstable metabolism, and cannot reach the brain after systemic administration. The cyclized opioid peptide has a longer half-life and higher metabolic stability while retaining its analgesic activity (Piekielna et al., 2013). Vernen et al. (2019) demonstrated backbone cyclization of tachyplesin peptides improved their stability and properties of antimicrobial and anticancer. Methods of cyclizing bioactive peptides have also been reported more in recent years. For example, Zhang et al. (2019) reported a highly chemoselective and simple method of cyclizing bioactive peptides. The reaction was readily accomplished and was found to be capable of tolerating different functionalities. Yamagami et al. (2021) demonstrated the peptide cyclization strategy by combining a soluble-tag-assisted liquid-phase peptide synthesis with a backbone amide linker. In addition to chemical peptide ligation, naturally occurring and engineered enzymes are also used for peptide cyclization (Nuijens et al., 2019). Toplak et al. (2016) reported a peptiligase that could efficiently catalyze head-to-tail peptide cyclization. Thioesterase SurE could cyclize two distinct non-ribosomal peptides (Matsuda et al., 2019).

N/C-terminal modifications are widely used to protect bioactive peptides against endogenous exopeptidases. An antimicrobial peptide reported by Li et al. (2021) was easily degraded in the plasma, and acetylating its N-terminal improved the stability of peptides against pH, plasma, and trypsin degradation. Liu et al. (2020) demonstrated N-terminal myristoylation enhanced the antimicrobial activity of PMAP-36PW. N-terminal acetylated peptide RLYE showed better antitumor activity, due to a decrease in the susceptibility to serum peptidases (Yun et al., 2019). C-terminal modification could impact bioactive peptide stability, membrane permeability, and activity (Arbour et al., 2020). Through generating C-terminal peptide amides of antibacterial peptides, a head-to-tail cyclized antibacterial peptide was synthesized, which exhibited higher proteolytic stability (So et al., 2021). A modified 20-residue peptide A20FMDV2, with N-terminal D-Asn and C-terminal D-Thr, showed improved activity by reducing susceptibility to plasma degradation (Hung et al., 2017).

In addition, side-chain modifications, backbone modification, and alkylation of amide nitrogen are all chemical modifications widely used in bioactive peptides (Pernot et al., 2011). The biochemical modification has received more and more attention in recent years (Gao et al., 2018). Some unmodified bioactive peptides have astonishing resistance to proteolysis, but the determinants of this unusual phenomenon remain mysterious (Bottger et al., 2017; Werner et al., 2016). Demystifying the mechanism behind it is an important direction of future efforts.

7.2. Encapsulation

Modifications are often used for target bioactive peptides that have the potential to be developed as drugs. However, encapsulation methods are more acceptable and inexpensive for bioactive peptides as functional foods.

Microcapsules and microgels are ideal for bioactive peptides delivery applications. The bioactive peptides embedded in microcapsules have a range of advantages, including resistance to digestion in the gastrointestinal system and prevention of chemical and enzymatic degradation to ensure biological activity (Malmsten, 2006). They can also control the release rate and reduce adverse immune side effects (Frokjaer and Otzen, 2005). Zhang et al. (2009) reported alginate/chitosan/starch microcapsules that could deliver antimicrobial oyster peptides to the intestine, and the microcapsules showed a sustained-release effect. Phaseolus lunatus L. peptides embedded in maltodextrin/gum arabic microcapsules preserved their α-glucosidase, α-amylase, and dipeptidyl peptidase-IV inhibitory activity through gastrointestinal digestion (Cian et al., 2019). Morikawa et al. (2005) prepared α-helical polypeptide microcapsules using emulsion-templated self-assembly of amphiphilic poly(γ-benzyl L-glutamate)s (PBLG), which was stable in a dry environment. Chitosan microcapsules could ensure the stable release of insulin over a long period (Aiedeh et al., 1997). The wall material is the primary determinant of microcapsule performance. Natural polymers (such as sodium alginate, chitosan, and cyclodextrins), fully synthetic polymers (such as polylactic acid/glycolic acid copolymer and polycaprolactone), semi-synthetic polymers (such as ethyl cellulose), and inorganic materials (such as calcium carbonate, silicate, double metal hydroxide, phosphate, and clay) have been used to prepare microcapsules. The release of microcapsules depends on pH values, temperature, ionic strength, particle sizes, and additives. At present, the preparation technology of microcapsules focuses on improving the encapsulation efficiency and prolonging the drug release. Furthermore, safer and non-toxic wall materials derived from food ingredients can provide new directions for microcapsules. The development of strategies to better ensure the biological activity of bioactive peptides in microcapsules, particularly those stored at room temperature for extended periods, is also needed in future efforts.

Liposomal packaging is another strategy used to improve bioactive peptide stability. Liposomes, with a microvesicle structure, consist of an aqueous core and an amphiphilic bilayer. Niu et al. (2014) found liposomes containing bile salts showed longer residence time and stronger transmembrane permeability in vivo. Ramezanzade et al. (2021) prepared liposomes coated with chitosan cross-linked with sodium tripolyphosphate and embedded fish-purified antioxidant peptide. The bioavailability of milk-derived ACE-inhibitory peptide RLSFNP after liposome encapsulation was improved and had better intestinal absorption. Parmentier et al. (2011) investigated tetraether lipid liposomes, which could deliver oral peptides. The plant-derived ergosterol liposomes screened by Cui et al. (2015) could significantly improve the oral bioavailability of insulin. Liposomes should maintain their vesicular morphology and avoid early leakage of encapsulated material by resisting destruction by bile salts, pancreatic enzymes, and acidic conditions to improve the oral bioavailability of encapsulated bioactive peptides in the gastrointestinal tract.

8. Conclusions

Bioactive peptides have been developed and advanced over the past two decades and have applications in novel foods and pharmaceuticals. However, their stability continues to be an important challenge to overcome. For the quality control of peptide products, we suggest the method of fingerprint for qualitative and quantitative analysis of specific peptide segments as a quality control index. In order to ensure the efficacy of bioactive peptides, peptide products must have stable physicochemical properties, resistance to gastrointestinal digestion, and specific metabolic stability, so as to achieve an appropriate retention time in the body. Chemical modification and encapsulation in microcapsules or liposomes are two mainstream methods to improve the stability of bioactive peptides. We believe stability studies are essential and will prove highly beneficial to bioactive peptide development. Moreover, we found that different researchers used various methods for mainstream evaluation tests, such as in vitro gastrointestinal digestion and plasma stability, making it difficult to compare the data between different reports. Therefore, establishing a standard stability evaluation test and a unified index are necessary.

CRediT authorship contribution statement

Jingyan Pei: reviewed the literature and wrote the manuscript. Xinchang Gao: with manuscript revised and discussions. Daodong Pan: contributed with discussions. Ying Hua: contributed with discussions. Jun He: contributed with discussions. Zhu Liu: contributed with manuscript revised and discussions. Yali Dang: contributed with manuscript revised and discussions.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by the Science Technology Department of Zhejiang Province (2022C02028), One health Interdisciplinary Research Project, Ningbo University (HZ202202), Zhejiang Provincial Administration for Market Regulation (CY202234), and the National Natural Science Foundation of China (91856126, 31771945).

Handling Editor: Dr. Quancai Sun

Contributor Information

Xinchang Gao, Email: gaoxc20@mails.tsinghua.edu.cn.

Zhu Liu, Email: zliu82@126.com.

Yali Dang, Email: dangyali@nbu.edu.cn.

Data availability

No data was used for the research described in the article.

References

  1. Ahmed Tausif, Sun Xiaohong, Chibuike C., Udenigwe Role of structural properties of bioactive peptides in their stability during simulated gastrointestinal digestion: a systematic review. Trends Food Sci. Technol. 2022;120:265–273. [Google Scholar]
  2. Aiedeh K., Gianasi E., Orienti I., Zecchi V. Chitosan microcapsules as controlled release systems for insulin. J. Microencapsul. 1997;14(5):567–576. doi: 10.3109/02652049709006810. [DOI] [PubMed] [Google Scholar]
  3. Arbour C.A., Mendoza L.G., Stockdill J.L. Recent advances in the synthesis of C-terminally modified peptides. Org. Biomol. Chem. 2020;18(37):7253–7272. doi: 10.1039/d0ob01417f. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Asaduzzaman A.K.M., Chun B.-S. Recovery of functional materials with thermally stable antioxidative properties in squid muscle hydrolyzates by subcritical water. J. Food Sci. Technol.Mysore. 2015;52(2):793–802. doi: 10.1007/s13197-013-1107-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Banting F.G., Best C.H., Collip J.B., Campbell W.R., Fletcher A.A. Pancreatic extracts in the treatment of diabetes mellitus: preliminary report. Can. Med. Assoc. J. 1962;87(20):1062–1067. <Go to ISI>://MEDLINE:20327305. [PMC free article] [PubMed] [Google Scholar]
  6. Baptista D.P., Salgaco M.K., Sivieri K., Gigante M.L. Use of static and dynamic in vitro models to simulate Prato cheese gastrointestinal digestion: effect of Lactobacillus helveticus LH-B02 addition on peptides bioaccessibility. LWT--Food Sci. Technol. 2020;134:11. doi: 10.1016/j.lwt.2020.110229. [DOI] [Google Scholar]
  7. Bianchi E., Carrington P.E., Ingallinella P., Finotto M., Santoprete A., Petrov A.…Pessi A. A PEGylated analog of the gut hormone oxyntomodulin with long-lasting antihyperglycemic, insulinotropic and anorexigenic activity. Bioorg. Med. Chem. 2013;21(22):7064–7073. doi: 10.1016/j.bmc.2013.09.016. [DOI] [PubMed] [Google Scholar]
  8. Bork K., Frank J., Grundt B., Schlattmann P., Nussberger J., Kreuz W. Treatment of acute edema attacks in hereditary angioedema with a bradykinin receptor-2 antagonist (Icatibant) J. Allergy Clin. Immunol. 2007;119(6):1497–1503. doi: 10.1016/j.jaci.2007.02.012. [DOI] [PubMed] [Google Scholar]
  9. Bottger R., Hoffmann R., Knappe D. Differential stability of therapeutic peptides with different proteolytic cleavage sites in blood, plasma and serum. PLoS One. 2017;12(6):15. doi: 10.1371/journal.pone.0178943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Brodkorb A., Egger L., Alminger M., Alvito P., Assuncao R., Ballance S.…Recio I. INFOGEST static in vitro simulation of gastrointestinal food digestion. Nat. Protoc. 2019;14(4):991–1014. doi: 10.1038/s41596-018-0119-1. [DOI] [PubMed] [Google Scholar]
  11. Cardle I.I., Jensen M.C., Pun S.H., Sellers D.L. Optimized serum stability and specificity of an αvβ6 integrin-binding peptide for tumor targeting. J. Biol. Chem. 2021;296 doi: 10.1016/j.jbc.2021.100657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cavaco M., Andreu D., Castanho M. The challenge of peptide proteolytic stability studies: scarce data, difficult readability, and the need for harmonization. Angew. Chem., Int. Ed. 2021;60(4):1686–1688. doi: 10.1002/anie.202006372. [DOI] [PubMed] [Google Scholar]
  13. Chen M., Li B. The effect of molecular weights on the survivability of casein-derived antioxidant peptides after the simulated gastrointestinal digestion. Innovat. Food Sci. Emerg. Technol. 2012;16:341–348. doi: 10.1016/j.ifset.2012.07.009. [DOI] [Google Scholar]
  14. Chia C.S.B. A review on the metabolism of 25 peptide drugs. Int. J. Pept. Res. Therapeut. 2021;27(2):1397–1418. doi: 10.1007/s10989-021-10177-0. [DOI] [Google Scholar]
  15. Cian R.E., Campos-Soldini A., Chel-Guerrero L., Drago S.R., Betancur-Ancona D. Bioactive Phaseolus lunatus peptides release from maltodextrin/gum Arabic microcapsules obtained by spray drying after simulated gastrointestinal digestion. Int. J. Food Sci. Technol. 2019;54(6):2002–2009. doi: 10.1111/ijfs.14031. [DOI] [Google Scholar]
  16. Cui M., Wu W., Hovgaard L., Lu Y., Chen D., Qi J. Liposomes containing cholesterol analogues of botanical origin as drug delivery systems to enhance the oral absorption of insulin. Int. J. Pharm. 2015;489(1–2):277–284. doi: 10.1016/j.ijpharm.2015.05.006. [DOI] [PubMed] [Google Scholar]
  17. D'Addio S.M., Bothe J.R., Neri C., Walsh P.L., Zhang J.T., Pierson E.…Templeton A.C. New and evolving techniques for the characterization of peptide therapeutics. J. Pharmaceut. Sci. 2016;105(10):2989–3006. doi: 10.1016/j.xphs.2016.06.011. [DOI] [PubMed] [Google Scholar]
  18. De Rosa L., Diana D., Di Stasi R., Romanelli A., Sciacca M.F.M., Milardi D.…D'Andrea L.D. Probing the helical stability in a VEGF-mimetic peptide. Bioorg. Chem. 2021;116:8. doi: 10.1016/j.bioorg.2021.105379. [DOI] [PubMed] [Google Scholar]
  19. Di L. Strategic approaches to optimizing peptide ADME properties. AAPS J. 2015;17(1):134–143. doi: 10.1208/s12248-014-9687-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Diana D., Ziaco B., Scarabelli G., Pedone C., Colombo G., D'Andrea L.D., Fattorusso R. Structural analysis of a helical peptide unfolding pathway. Chem.--Eur. J. 2010;16(18):5400–5407. doi: 10.1002/chem.200903428. [DOI] [PubMed] [Google Scholar]
  21. Ding L., Zhang Y., Jiang Y.Q., Wang L.Y., Liu B.Q., Liu J.B. Transport of egg white ACE-inhibitory peptide, Gln-ile-Gly-Leu-phe, in human intestinal caco-2 cell monolayers with cytoprotective effect. J. Agric. Food Chem. 2014;62(14):3177–3182. doi: 10.1021/jf405639w. [DOI] [PubMed] [Google Scholar]
  22. Elkinton J.R., Hunt A.D., Jr. Effects of pituitary adrenocorticotropic hormone therapy. J. Am. Med. Assoc. 1949;141(18):1273–1279. doi: 10.1001/jama.1949.02910180001001. [DOI] [PubMed] [Google Scholar]
  23. Enciso M., Schuette C., Delle Site L. Influence of pH and sequence in peptide aggregation via molecular simulation. J. Chem. Phys. 2015;143(24):2934. doi: 10.1063/1.4935707. [DOI] [PubMed] [Google Scholar]
  24. Fan F.J., Shi P.J., Chen H., Tu M.L., Wang Z.Y., Lu W.H., Du M. Identification and availability of peptides from lactoferrin in the gastrointestinal tract of mice. Food Funct. 2019;10(2):879–885. doi: 10.1039/c8fo01998c. [DOI] [PubMed] [Google Scholar]
  25. Frokjaer S., Otzen D.E. Protein drug stability: a formulation challenge. Nat. Rev. Drug Discov. 2005;4(4):298–306. doi: 10.1038/nrd1695. [DOI] [PubMed] [Google Scholar]
  26. Gallego M., Grootaert C., Mora L., Aristoy M.C., Van Camp J., Toldra F. Transepithelial transport of dry-cured ham peptides with ACE inhibitory activity through a Caco-2 cell monolayer. J. Funct.Foods. 2016;21:388–395. doi: 10.1016/j.jff.2015.11.046. [DOI] [Google Scholar]
  27. Gao Y., Fang H., Fang L., Liu D., Liu J., Su M.…Jiao H. The modification and design of antimicrobial peptide. Curr. Pharmaceut. Des. 2018;24(8):904–910. doi: 10.2174/1381612824666180213130318. [DOI] [PubMed] [Google Scholar]
  28. Gasteiger E. 2005. Protein Identification and Analysis Tools on the ExPASy Server. The Proteomics Protocols Handbook. [DOI] [PubMed] [Google Scholar]
  29. Guruprasad K., Reddy B.V., Pandit M.W. Correlation between stability of a protein and its dipeptide composition: a novel approach for predicting in vivo stability of a protein from its primary sequence. Protein Eng. 1990;4(2):155–161. doi: 10.1093/protein/4.2.155. [DOI] [PubMed] [Google Scholar]
  30. Hall M.P. Biotransformation and in vivo stability of protein biotherapeutics: impact on candidate selection and pharmacokinetic profiling. Drug Metabol. Dispos.: The Biological Fate of Chemicals. 2014;42(11):1873–1880. doi: 10.1124/dmd.114.058347. [DOI] [PubMed] [Google Scholar]
  31. Hao L., Gao X.C., Zhou T.Y., Cao J.X., Sun Y.Y., Dang Y.L., Pan D.D. Angiotensin I-converting enzyme (ACE) inhibitory and antioxidant activity of umami peptides after in vitro gastrointestinal digestion. J. Agric. Food Chem. 2020;68(31):8232–8241. doi: 10.1021/acs.jafc.0c02797. [DOI] [PubMed] [Google Scholar]
  32. Hartmann R., Meisel H. Food-derived peptides with biological activity: from research to food applications. Curr. Opin. Biotechnol. 2007;18(2):163–169. doi: 10.1016/j.copbio.2007.01.013. [DOI] [PubMed] [Google Scholar]
  33. Hung K.-y., Harris P.W.R., Desai A., Marshall J.F., Brimble M.A. Structure-activity relationship study of the tumour-targeting peptide A20FMDV2 via modification of Lys16, Leu13, and N- and/or C-terminal functionality. Eur. J. Med. Chem. 2017;136:154–164. doi: 10.1016/j.ejmech.2017.05.008. [DOI] [PubMed] [Google Scholar]
  34. Iwaniak A., Darewicz M., Mogut D., Minkiewicz P. Elucidation of the role of in silico methodologies in approaches to studying bioactive peptides derived from foods. J. Funct.Foods. 2019;61:14. doi: 10.1016/j.jff.2019.103486. [DOI] [Google Scholar]
  35. Jia F.J., Wang J.Y., Peng J.X., Zhao P., Kong Z.Q., Wang K.R.…Wang R. D-amino acid substitution enhances the stability of antimicrobial peptide polybia-CP. Acta Biochim. Biophys. Sin. 2017;49(10):916–925. doi: 10.1093/abbs/gmx091. [DOI] [PubMed] [Google Scholar]
  36. Jin Y., Yu Y., Qi Y.X., Wang F.J., Yan J.Z., Zou H.F. Peptide profiling and the bioactivity character of yogurt in the simulated gastrointestinal digestion. J. Proteonomics. 2016;141:24–46. doi: 10.1016/j.jprot.2016.04.010. [DOI] [PubMed] [Google Scholar]
  37. Katsila T., Siskos A.P., Tamvakopoulos C. Peptide and protein drugs: the study of their metabolism and catabolism by mass spectrometry. Mass Spectrom. Rev. 2012;31(1):110–133. doi: 10.1002/mas.20340. [DOI] [PubMed] [Google Scholar]
  38. Klein T., Eckhard U., Dufour A., Solis N., Overall C.M. Proteolytic cleavage-mechanisms, function, and "omic" approaches for a near-ubiquitous posttranslational modification. Chem. Rev. 2018;118(3):261–292. doi: 10.1021/acs.chemrev.7b00120. [DOI] [PubMed] [Google Scholar]
  39. Lai X.Y., Tang J.S., ElSayed M.E.H. Recent advances in proteolytic stability for peptide, protein, and antibody drug discovery. Expet Opin. Drug Discov. 2021;16(12):1467–1482. doi: 10.1080/17460441.2021.1942837. [DOI] [PubMed] [Google Scholar]
  40. Lau J.L., Dunn M.K. Therapeutic peptides: historical perspectives, current development trends, and future directions. Bioorg. Med. Chem. 2018;26(10):2700–2707. doi: 10.1016/j.bmc.2017.06.052. [DOI] [PubMed] [Google Scholar]
  41. Leach J.K., Spencer K., Mascelli M., McCauley T.G. Pharmacokinetics of single and repeat doses of icatibant. Clin. Pharmacol.Drug Dev. 2015;4(2):105–111. doi: 10.1002/cpdd.138. [DOI] [PubMed] [Google Scholar]
  42. Lee C.L., Harris J.L., Khanna K.K., Hong J.H. A comprehensive review on current advances in peptide drug development and design. Int. J. Mol. Sci. 2019;20(10) doi: 10.3390/ijms20102383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Li J., Chen Y., Yuan N., Zeng M., Zhao Y., Yu R.…Dong S. A novel natural influenza A H1N1 virus neuraminidase inhibitory peptide derived from cod skin hydrolysates and its antiviral mechanism. Mar. Drugs. 2018;16(10) doi: 10.3390/md16100377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Li D., Yang Y., Li R., Huang L., Wang Z., Deng Q., Dong S. N-terminal acetylation of antimicrobial peptide L163 improves its stability against protease degradation. J. Pept. Sci. 2021;27(9) doi: 10.1002/psc.3337. [DOI] [PubMed] [Google Scholar]
  45. Liu Y., Li S., Shen T., Chen L., Zhou J., Shi S.…Wang C. N-Terminal myristoylation enhanced the antimicrobial activity of antimicrobial peptide PMAP-36PW. Front. Cell. Infect. Microbiol. 2020;10 doi: 10.3389/fcimb.2020.00450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Lopez-Morales C.A., Vazquez-Leyva S., Vallejo-Castillo L., Carballo-Uicab G., Munoz-Garcia L., Herbert-Pucheta J.E.…Medina-Rivero E. Determination of peptide profile consistency and safety of collagen hydrolysates as quality attributes. J. Food Sci. 2019;84(3):430–439. doi: 10.1111/1750-3841.14466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Lopez-Sanchez J., Ponce-Alquicira E., Pedroza-Islas R., de la Pena-Diaz A., Soriano-Santos J. Effects of heat and pH treatments and in vitro digestion on the biological activity of protein hydrolysates of Amaranthus hypochondriacus L. grain. J. Food Sci. Technol.Mysore. 2016;53(12):4298–4307. doi: 10.1007/s13197-016-2428-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Low K., Roulin A., Kunz S. A proopiomelanocortin-derived peptide sequence enhances plasma stability of peptide drugs. FEBS (Fed. Eur. Biochem. Soc.) Lett. 2020;594(17):2840–2866. doi: 10.1002/1873-3468.13855. [DOI] [PubMed] [Google Scholar]
  49. Lu J., Xu H., Xia J., Ma J., Xu J., Li Y., Feng J. D- and unnatural amino acid substituted antimicrobial peptides with improved proteolytic resistance and their proteolytic degradation characteristics. Front. Microbiol. 2020;11 doi: 10.3389/fmicb.2020.563030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Maack T., Johnson V., Kau S.T., Figueiredo J., Sigulem D. Renal filtration, transport, and metabolism of low-molecular-weight proteins: a review. Kidney Int. 1979;16(3):251–270. doi: 10.1038/ki.1979.128. [DOI] [PubMed] [Google Scholar]
  51. Mage I., Bocker U., Wubshet S.G., Lindberg D., Afseth N.K. Fourier-transform infrared (FTIR) fingerprinting for quality assessment of protein hydrolysates. LWT--Food Sci. Technol. 2021;152 doi: 10.1016/j.lwt.2021.112339. [DOI] [Google Scholar]
  52. Malmsten M. Soft drug delivery systems. Soft Matter. 2006;2(9):760–769. doi: 10.1039/b608348j. [DOI] [PubMed] [Google Scholar]
  53. Mao Y., Zhou S., Xu M., Zeng S., Fan W., Yu L., Lin N. The metabolic stability of antimicrobial peptides Ik8 in plasma and liver S9. Applied Sciences-Basel. 2021;11(24) doi: 10.3390/app112411661. [DOI] [Google Scholar]
  54. Marseglia A., Dellafiora L., Prandi B., Lolli V., Sforza S., Cozzini P.…Caligiani A. Simulated gastrointestinal digestion of cocoa: detection of resistant peptides and in silico/in vitro prediction of their ace inhibitory activity. Nutrients. 2019;11(5):17. doi: 10.3390/nu11050985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Matsuda K., Kobayashi M., Kuranaga T., Takada K., Ikeda H., Matsunaga S., Wakimoto T. SurE is a trans-acting thioesterase cyclizing two distinct non-ribosomal peptides. Org. Biomol. Chem. 2019;17(5):1058–1061. doi: 10.1039/c8ob02867b. [DOI] [PubMed] [Google Scholar]
  56. Meibohm B., Zhou H. Characterizing the impact of renal impairment on the clinical pharmacology of biologics. J. Clin. Pharmacol. 2012;52:54S–62S. doi: 10.1177/0091270011413894. [DOI] [PubMed] [Google Scholar]
  57. Merrifield R.B. Solid-phase peptide synthese. Endeavour. 1965;24:3–7. doi: 10.1016/0160-9327(65)90090-6. [DOI] [PubMed] [Google Scholar]
  58. Miguel M., Davalos A., Manso M.A., de la Pena G., Lasuncion M.A., Lopez-Fandino R. Transepithelial transport across Caco-2 cell monolayers of antihypertensive egg-derived peptides. PepT1-mediated flux of Tyr-Pro-lle. Mol. Nutr. Food Res. 2008;52(12):1507–1513. doi: 10.1002/mnfr.200700503. [DOI] [PubMed] [Google Scholar]
  59. Minekus M., Alminger M., Alvito P., Ballance S., Bohn T., Bourlieu C.…Brodkorb A. A standardised static in vitro digestion method suitable for food - an international consensus. Food Funct. 2014;5(6):1113–1124. doi: 10.1039/c3fo60702j. [DOI] [PubMed] [Google Scholar]
  60. Minkiewicz P., Dziuba J., Iwaniak A., Dziuba M., Darewicz M. BIOPEP database and other programs for processing bioactive peptide sequences. J. AOAC Int. 2008;91(4):965–980. Go to ISI>://WOS:000258270400035. [PubMed] [Google Scholar]
  61. Minkiewicz P., Iwaniak A., Darewicz M. BIOPEP-UWM database of bioactive peptides: current opportunities. Int. J. Mol. Sci. 2019;20(23):23. doi: 10.3390/ijms20235978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Mirzaei M., Mirdamadi S., Safavi M., Soleymanzadeh N. The stability of antioxidant and ACE-inhibitory peptides as influenced by peptide sequences. LWT--Food Sci. Technol. 2020;130:9. doi: 10.1016/j.lwt.2020.109710. [DOI] [Google Scholar]
  63. Mirzaei M., Mirdamadi S., Safavib M., Soleymanzadeh N. The stability of antioxidant and ACE-inhibitory peptides as influenced by peptide sequences. LWT--Food Sci. Technol. 2020;130 [Google Scholar]
  64. Mirzaei M., Shavandi A., Mirdamadi S., Soleymanzadeh N., Motahari P., Mirdamadi N.…Goriely S. Bioactive peptides from yeast: a comparative review on production methods, bioactivity, structure-function relationship, and stability. Trends Food Sci. Technol. 2021;118:297–315. doi: 10.1016/j.tifs.2021.10.008. [DOI] [Google Scholar]
  65. Mitragotri S., Burke P.A., Langer R. Overcoming the challenges in administering biopharmaceuticals: formulation and delivery strategies. Nat. Rev. Drug Discov. 2014;13(9):655–672. doi: 10.1038/nrd4363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Morikawa M., Yoshihara M., Endo T., Kimizuka N. alpha-helical polypeptide microcapsules formed by emulsion-templated self-assembly. Chem.--Eur. J. 2005;11(5):1574–1578. doi: 10.1002/chem.200400751. [DOI] [PubMed] [Google Scholar]
  67. Muttenthaler M., King G.E., Adams D.J., Alewood P.E. Trends in peptide drug discovery. Nat. Rev. Drug Discov. 2021;20(4):309–325. doi: 10.1038/s41573-020-00135-8. [DOI] [PubMed] [Google Scholar]
  68. Niu M.M., Tan Y.N., Guan P.P., Hovgaard L., Lu Y., Qi J.P.…Wu W. Enhanced oral absorption of insulin-loaded liposomes containing bile salts: a mechanistic. Int. J. Pharm. 2014;460(1–2):119–130. doi: 10.1016/j.ijpharm.2013.11.028. [DOI] [PubMed] [Google Scholar]
  69. Nuijens T., Toplak A., Schmidt M., Ricci A., Cabri W. Natural occurring and engineered enzymes for peptide ligation and cyclization. Front. Chem. 2019;7 doi: 10.3389/fchem.2019.00829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Ohsawa K., Satsu H., Ohki K., Enjoh M., Takano T., Shimizu M. Producibility and digestibility of antihypertensive beta-casein tripeptides, Val-Pro-Pro and Ile-Pro-Pro, in the gastrointestinal tract: analyses using an in vitro model of mammalian gastrointestinal digestion. J. Agric. Food Chem. 2008;56(3):854–858. doi: 10.1021/jf072671n. [DOI] [PubMed] [Google Scholar]
  71. Panchin A.Y., Khromov-Borisov N.N., Dueva E.V. Drug discovery today: no molecules required. Bmj Evidence-Based Medicine. 2020;25(4) doi: 10.1136/bmjebm-2018-111121corr1. E4-E4. [DOI] [PubMed] [Google Scholar]
  72. Parmentier J., Becker M., Heintz U., Fricker G. Stability of liposomes containing bio-enhancers and tetraether lipids in simulated gastro-intestinal fluids. Int. J. Pharm. 2011;405(1–2):210–217. doi: 10.1016/j.ijpharm.2010.12.005. [DOI] [PubMed] [Google Scholar]
  73. Pernot M., Vanderesse R., Frochot C., Guillemin F., Barberi-Heyob M. Stability of peptides and therapeutic success in cancer. Expet Opin. Drug Metabol. Toxicol. 2011;7(7):793–802. doi: 10.1517/17425255.2011.574126. [DOI] [PubMed] [Google Scholar]
  74. Picariello G., Ferranti P., Fierro O., Mamone G., Caira S., Di Luccia A.…Addeo F. Peptides surviving the simulated gastrointestinal digestion of milk proteins: biological and toxicological implications. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 2010;878(3):295–308. doi: 10.1016/j.jchromb.2009.11.033. [DOI] [PubMed] [Google Scholar]
  75. Piekielna J., Perlikowska R., Gach K., Janecka A. Cyclization in opioid peptides. Curr. Drug Targets. 2013;14(7):798–816. doi: 10.2174/1389450111314070008. [DOI] [PubMed] [Google Scholar]
  76. Pollaro L., Heinis C. Strategies to prolong the plasma residence time of peptide drugs. Medchemcomm. 2010;1(5):319–324. doi: 10.1039/c0md00111b. [DOI] [Google Scholar]
  77. Ramezanzade L., Hosseini S.F., Akbari-Adergani B., Yaghmur A. Cross-linked chitosan-coated liposomes for encapsulation of fish-derived peptide. LWT--Food Sci. Technol. 2021;150:8. doi: 10.1016/j.lwt.2021.112057. [DOI] [Google Scholar]
  78. Salim M., Gan C.Y. Dual-function peptides derived from egg white ovalbumin: bioinformatics identification with validation using in vitro assay. J. Funct.Foods. 2020;64:12. doi: 10.1016/j.jff.2019.103618. [DOI] [Google Scholar]
  79. Savoie L., Gauthier S.F., Marin J., Pouliot Y. In vitro determination of the release kinetics of peptides and free amino acids during the digestion of food proteins. J. AOAC Int. 2005;88(3):935–948. Go to ISI>://WOS:000229529300041. [PubMed] [Google Scholar]
  80. Shen P., Cai F., Nowicki A., Vincent J., Reynolds E.C. Remineralization of enamel subsurface lesions by sugar-free chewing gum containing casein phosphopepticle-amorphous calcium phosphate. J. Dent. Res. 2001;80(12):2066–2070. doi: 10.1177/00220345010800120801. [DOI] [PubMed] [Google Scholar]
  81. Singh B.P., Aluko R.E., Hati S., Solanki D. Bioactive peptides in the management of lifestyle-related diseases: current trends and future perspectives. Crit. Rev. Food Sci. Nutr. 2021;10:14. doi: 10.1080/10408398.2021.1877109. [DOI] [PubMed] [Google Scholar]
  82. So W.H., Bao Y., Chen X., Xia J. On-resin ugi reaction for C-terminally modified and head-to-tail cyclized antibacterial peptides. Org. Lett. 2021;23(21):8277–8281. doi: 10.1021/acs.orglett.1c03014. [DOI] [PubMed] [Google Scholar]
  83. Sonesson A., Rasmussen B.B. In vitro and in vivo human metabolism of degarelix, a gonadotropin-releasing hormone receptor blocker. Drug Metabol. Dispos. 2013;41(7):1339–1346. doi: 10.1124/dmd.113.051706. [DOI] [PubMed] [Google Scholar]
  84. Tan Y., Wang W., Wu C., Pan Z., Yao G., Fang L., Su W. Myristic acid-modified thymopentin for enhanced plasma stability and immune-modulating activity. Int. Immunopharm. 2017;47:88–94. doi: 10.1016/j.intimp.2017.03.025. [DOI] [PubMed] [Google Scholar]
  85. Tao Y., Gu X.H., Li W.D., Wu H., Cai B.C., Zhang B.L. Techniques for biological fingerprinting of traditional Chinese medicine. Trac. Trends Anal. Chem. 2017;97:272–282. doi: 10.1016/j.trac.2017.09.008. [DOI] [Google Scholar]
  86. Teng Z.H., Yuan C.J., Zhang F., Huan M.L., Cao W.D., Li K.C.…Mei Q.B. Intestinal absorption and first-pass metabolism of polyphenol compounds in rat and their transport dynamics in caco-2 cells. PLoS One. 2012;7(1):9. doi: 10.1371/journal.pone.0029647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  87. Toplak A., Nuijens T., Quaedflieg P.J.L.M., Wu B., Janssen D.B. Peptiligase, an enzyme for efficient chemoenzymatic peptide synthesis and cyclization in water. Adv. Synth. Catal. 2016;358(13):2140–2147. doi: 10.1002/adsc.201600017. [DOI] [Google Scholar]
  88. Tu M.L., Liu H.X., Cheng S.Z., Mao F.J., Chen H., Fan F.J.…Du M. Identification and characterization of a novel casein anticoagulant peptide derived from in vivo digestion. Food Funct. 2019;10(5):2552–2559. doi: 10.1039/c8fo02546k. [DOI] [PubMed] [Google Scholar]
  89. Udenigwe C.C. Bioinformatics approaches, prospects and challenges of food bioactive peptide research. Trends Food Sci. Technol. 2014;36(2):137–143. doi: 10.1016/j.tifs.2014.02.004. [DOI] [Google Scholar]
  90. Udenigwe C.C., Fogliano V. Food matrix interaction and bioavailability of bioactive peptides: two faces of the same coin? J. Funct.Foods. 2017;35:9–12. doi: 10.1016/j.jff.2017.05.029. [DOI] [Google Scholar]
  91. Vazquez-Leyva S., Vallejo-Castillo L., Lopez-Morales C.A., Herbert-Pucheta J.E., Zepeda-Vallejo L.G., Velasco-Velazquez M.…Medina-Rivero E. Identity profiling of complex mixtures of peptide products by structural and mass mobility orthogonal analysis. Anal. Chem. 2019;91(22):14392–14400. doi: 10.1021/acs.analchem.9b02873. [DOI] [PubMed] [Google Scholar]
  92. Vermeirssen V., van der Bent A., Van Camp J., van Amerongen A., Verstraete W. A quantitative in silico analysis calculates the angiotensin I converting enzyme (ACE) inhibitory activity in pea and whey protein digests. Biochimie. 2004;86(3):231–239. doi: 10.1016/j.biochi.2004.01.003. [DOI] [PubMed] [Google Scholar]
  93. Vernen F., Harvey P.J., Dias S.A., Veiga A.S., Huang Y.-H., Craik D.J.…Henriques S.T. Characterization of tachyplesin peptides and their cyclized analogues to improve antimicrobial and anticancer properties. Int. J. Mol. Sci. 2019;20(17) doi: 10.3390/ijms20174184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  94. Vigneaud V.D., Ressler C., Swan C., Roberts C.W., Gordon S. The synthesis of an octapeptide with hormonal activity of oxytocin. J. Am. Chem. Soc. 1953;75(19):328–332. [Google Scholar]
  95. Wang X., Yu H., Xing R., Chen X., Liu S., Li P. Optimization of the extraction and stability of antioxidative peptides from mackerel (pneumatophorus japonicus) protein. BioMed Res. Int. 2017 doi: 10.1155/2017/6837285. 2017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  96. Wang B., Xie N.N., Li B. Influence of peptide characteristics on their stability, intestinal transport, and in vitro bioavailability: a review. J. Food Biochem. 2019;43(1):6. doi: 10.1111/jfbc.12571. [DOI] [PubMed] [Google Scholar]
  97. Wang T.R., Zou C.B., Wen N., Liu X.D., Meng Z., Feng S.L.…Wang C.H. The effect of structural modification of antimicrobial peptides on their antimicrobial activity, hemolytic activity, and plasma stability. J. Pept. Sci. 2021;27(5):13. doi: 10.1002/psc.3306. [DOI] [PubMed] [Google Scholar]
  98. Werner H.M., Cabalteja C.C., Horne W.S. Peptide backbone composition and protease susceptibility: impact of modification type, position, and tandem substitution. Chembiochem. 2016;17(8) doi: 10.1002/cbic.201500312. [DOI] [PMC free article] [PubMed] [Google Scholar]
  99. Wilkins M.R., Gasteiger E., Bairoch A., Sanchez J.C., Williams K.L., Appel R.D., Hochstrasser D.F. Protein identification and analysis tools in the ExPASy server. Methods Mol. Biol. 1999;112:531–552. doi: 10.1385/1-59259-584-7:531. Go to ISI>://MEDLINE:10027275. [DOI] [PubMed] [Google Scholar]
  100. Wu S., Wang X., Qi W., Guo Q. Bioactive protein/peptides of flaxseed: a review. Trends Food Sci. Technol. 2019;92:184–193. [Google Scholar]
  101. Xiao C.Q., Zhou F.B., Zheng L., Cai Y.J., Su G.W., Luo D.H., Zhao M.M. Chicken breast-derived alcohol dehydrogenase-activating peptides in response to physicochemical changes and digestion simulation: the vital role of hydrophobicity. Food Res. Int. 2020;136:9. doi: 10.1016/j.foodres.2020.109592. [DOI] [PubMed] [Google Scholar]
  102. Xie N., Wang B., Jiang L., Liu C., Li B. Hydrophobicity exerts different effects on bioavailability and stability of antioxidant peptide fractions from casein during simulated gastrointestinal digestion and Caco-2 cell absorption. Food Res. Int. 2015;76:518–526. doi: 10.1016/j.foodres.2015.06.025. [DOI] [PubMed] [Google Scholar]
  103. Yamagami S., Okada Y., Kitano Y., Chiba K. Peptide head-to-tail cyclization: a "molecular claw" approach. Eur. J. Org Chem. 2021;2021(22):3133–3138. doi: 10.1002/ejoc.202100185. [DOI] [Google Scholar]
  104. Yang L.J., Zhang L.T., Yan L.H., Zheng H.F., Lu P.F., Chen J.J.…Yang T. Stability assessment of a new antithrombotic small peptide, Arg-Gly-Asp-Trp-Arg (RGDWR), and its derivative. Biotechnol. Lett. 2017;39(8):1183–1190. doi: 10.1007/s10529-017-2346-x. [DOI] [PubMed] [Google Scholar]
  105. Yao Jin Feng, Yang Hong, Zhao Y.Z., Xue M. Metabolism of peptide drugs and strategies to improve their metabolic stability. Curr. Drug Metabol. 2018;19(11):892–901. doi: 10.2174/1389200219666180628171531. [DOI] [PubMed] [Google Scholar]
  106. Yap P.G., Gan C.Y. In vivo challenges of anti-diabetic peptide therapeutics: gastrointestinal stability, toxicity and allergenicity. Trends Food Sci. Technol. 2020;105:161–175. doi: 10.1016/j.tifs.2020.09.005. [DOI] [Google Scholar]
  107. Yuan L., Sun L.P., Zhuang Y.L. Preparation and identification of novel inhibitory angiotensin-I-converting enzyme peptides from tilapia skin gelatin hydrolysates: inhibition kinetics and molecular docking. Food Funct. 2018;9(10):5251–5259. doi: 10.1039/c8fo00569a. [DOI] [PubMed] [Google Scholar]
  108. Yun J.-A., Kim J., Baek Y.-Y., Park W., Park M., Kim S.…Kim Y.-M. N-terminal modification of the tetrapeptide arg-Leu-Tyr-Glu, a vascular endothelial growth factor receptor-2 (VEGFR-2) antagonist, improves antitumor activity by increasing its stability against serum peptidases. Mol. Pharmacol. 2019;96(6):692–701. doi: 10.1124/mol.119.117234. [DOI] [PubMed] [Google Scholar]
  109. Zhang L., Liu Y., Wu Z., Chen H. Preparation and characterization of coacervate microcapsules for the delivery of antimicrobial oyster peptides. Drug Dev. Ind. Pharm. 2009;35(3):369–378. doi: 10.1080/03639040802369255. [DOI] [PubMed] [Google Scholar]
  110. Zhang Y., Zhang Q., Wong C.T.T., Li X. Chemoselective peptide cyclization and bicyclization directly on unprotected peptides. J. Am. Chem. Soc. 2019;141(31):12274–12279. doi: 10.1021/jacs.9b03623. [DOI] [PubMed] [Google Scholar]
  111. Zheng L., Xu Q.Y., Lin L.Z., Zeng X.A., Sun B.G., Zhao M.M. In vitro metabolic stability of a casein-derived dipeptidyl peptidase-IV (DPP-IV) inhibitory peptide VPYPQ and its controlled release from casein by enzymatic hydrolysis. J. Agric. Food Chem. 2019;67(38):10604–10613. doi: 10.1021/acs.jafc.9b03164. [DOI] [PubMed] [Google Scholar]
  112. Zhou K., Zheng X., Xu H.-M., Zhang J., Chen Y., Xi T., Feng T. Studies of poly(ethylene glycol) modification of HM-3 polypeptides. Bioconjugate Chem. 2009;20(5):932–936. doi: 10.1021/bc900070r. [DOI] [PubMed] [Google Scholar]
  113. Zhu C.Z., Zhang W.G., Kang Z.L., Zhou G.H., Xu X.L. Stability of an antioxidant peptide extracted from Jinhua ham. Meat Sci. 2014;96(2):783–789. doi: 10.1016/j.meatsci.2013.09.004. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

No data was used for the research described in the article.

Articles from Current Research in Food Science are provided here courtesy of Elsevier

RESOURCES