Aleksandra Zambrowicz

Manufacturing of peptides exhibiting biological activity

Numerous studies have shown that food proteins may be a source of bioactive peptides. Those peptides are encrypted in the protein sequence. They stay inactive within the parental protein until release by proteolytic enzymes (Mine and Kovacs-Nolan in Worlds Poult Sci J 62(1):87–95, 2006; Hartman and Miesel in Curr Opin Biotechnol 18:163–169, 2007). Once released the bioactive peptides exhibit several biofunctionalities and may serve therapeutic roles in body systems. Opioid peptides, peptides lowering high blood pressure, inhibiting platelet aggregation as well as being carriers of metal ions and peptides with immunostimulatory, antimicrobial and antioxidant activities have been described (Hartman and Miesel in Curr Opin Biotechnol 18:163–169, 2007). The biofunctional abilities of the peptides have therefore aroused a lot of scientific, technological and consumer interest with respect to the role of dietary proteins in controlling and influencing health (Möller et al. in Eur J Nutr 47(4):171–182, 2008). Biopeptides may find wide application in food production, the cosmetics industry as well as in the prevention and treatment of various medical conditions. They are manufactured by chemical and biotechnological methods (Marx in Chem Eng News 83(11):17–24. 2005; Hancock and Sahl in Nat Biotechnol 24(12):1551–1557, 2006). Depending on specific needs (food or pharmaceutical industry) different degrees of peptide purifications are required. This paper discusses the practicability of manufacturing bioactive peptides, especially from food proteins.

Egg-yolk protein by-product as a source of ACE-inhibitory peptides obtained with using unconventional proteinase from Asian pumpkin (Cucurbita ficifolia)

UNLABELLED In the present study angiotensin I-converting enzyme (ACE) inhibitory peptides were isolated from egg-yolk protein preparation (YP). Enzymatic hydrolysis conducted using unconventional enzyme from Cucurbita ficifolia (dose: 1000 U/mg of hydrolyzed YP (E/S (w/w)=1:7.52)) was employed to obtain protein hydrolysates. The 4-h hydrolysate exhibited a significant (IC₅₀=482.5 μg/mL) ACE inhibitory activity. Moreover, hydrolysate showed no cytotoxic activity on human and animal cell lines which makes it a very useful multifunctional method for peptide preparation. The compiled isolation procedure (ultrafiltration, size-exclusion chromatography and RP-HPLC) of bioactive peptides from YP hydrolysate resulted in obtaining peptides with the strong ACE inhibitory activity. One homogeneous and three heterogeneous peptide fractions were identified. The peptides were composed of 9-18 amino-acid residues, including mainly arginine and leucine at the N-terminal positions. To confirm the selected bioactive peptide sequences their analogs were chemically synthesized and tested. Peptide LAPSLPGKPKPD showed the strongest ACE inhibitory activity, with IC₅₀ value of 1.97 μmol/L. BIOLOGICAL SIGNIFICANCE Peptides with specific biological activity can be used in pharmaceutical, cosmetic or food industries. Because of their potential role as physiological modulators, as well as theirhigh safety profile, they can be used as natural pharmacological compounds or functional food ingredients. The development of biotechnological solutions to obtain peptides with desired biological activity is already in progress. Studies in this area are focused on using unconventional highly specific enzymes and more efficient methods developed to conduct food process technologies. Natural peptides have many advantages. They are mainly toxicologically safe, have wide spectra of therapeutic actions, exhibit less side effects compared to synthetic drugs and are more efficiently absorbed in the intestinal tract. The complexity of operation of large scale technologies and high cost of purification techniques are limiting factors to the commercialization of food-derived bioactive peptides. Research on the isolation of bioactive peptides in order to reduce the processing time and costs is continuously developing. Bioactive peptides can also be released from protein by-products of the food industry, which reduce the substrate expense and production cost as well as provide the added advantage of an efficient waste disposal. Moreover, proteins as precursors of food-derived peptides are well-tolerated by the human body and therefore their application in drug development may reduce costs and duration of toxicological studies during research, development and clinical trials.

An attractive way of egg white protein by-product use for producing of novel anti-hypertensive peptides

The aim of this study was to (i) examine how enzymatic hydrolysis with a non-commercially available proteinase of fig-leaf gourd fruit (Cucurbita ficifolia) increased the use value of egg white protein preparations, generated as byproducts in the industrial process of lysozyme and cystatin isolation from egg white, and (ii) evaluate the inhibition of angiotensin I-converting enzyme (ACE) by the obtained hydrolysates. Purification procedures including membrane filtration, gel filtration chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC) led to the production of several peptide fractions. Two novel ovalbumin-derived tetrapeptides: SWVE (f 148-151) and DILN (f 86-89) with ACE inhibitory activity were obtained. Study of their inhibitory kinetics revealed a non-competitive binding mode, with an IC50 value against ACE of 33.88 and 73.44 μg for SWVE and DILN, respectively. Synthetic peptides which were designed on the basis of peptide SWVE were examined. A tripeptide sequence of SWV revealed the strongest ACE-inhibitory activity.

Antioxidant and antidiabetic activities of peptides isolated from a hydrolysate of an egg-yolk protein by-product prepared with a proteinase from Asian pumpkin (Cucurbita ficifolia)

Bioactive peptides derived from food have been increasingly popular due to their therapeutic properties. Of particular importance are peptides with a multidirectional activity that can be used in the treatment and prevention of diet-related diseases. This paper attempts to utilize a by-product of phospholipid extraction from egg yolk as a source of peptides with a broad spectrum of biological activity. In addition, in this research we used a non-commercial enzyme obtained from Asian pumpkin, which has not been sufficiently researched in terms of its ability to release biopeptides from food proteins. In the present study the biological properties of peptides, derived from egg-yolk protein by-products (YP) remaining after phospholipid extraction, and their four synthetic analogs were investigated with regard to their antioxidant (radical scavenging capacity, Fe2+ chelating effect, reducing power (FRAP)) and antidiabetic (α-glucosidase and DPP-IV inhibitory activities) properties. One of them, with the sequence LAPSLPGKPKPD, exhibited the highest antioxidant activity (free radical scavenging activity (6.03 μM Troloxeq per mg protein); FRAP (296.07 μg Fe2+ per mg protein)). This peptide also revealed the strongest DPP-IV (361.5 μmol L−1) and α-glucosidase (1065.6 μmol L−1) inhibitory activities, a novel multifunctional effect for peptides from an egg-yolk hydrolysate.

Biological and functional properties of proteolytic enzyme-modified egg protein by-products

Enzymatic hydrolysis led to improve functional properties and biological activity of protein by-products, which can be further used as protein ingredients for food and feed applications. The effects of proteolytic enzyme modification of egg-yolk protein preparation (YP) and white protein preparation (WP), obtained as the by-products left during the course of lecithin, lysozyme, and cystatin isolation on their biological and functional properties, were evaluated by treating a commercial Neutrase. The antihypertensive and antioxidative properties of YP and WP hydrolysates were evaluated based on their angiotensin-converting enzyme (ACE)-inhibitory activity and radical scavenging (DPPH) capacity, ferric reducing power, and chelating of iron activity. The functionality of obtained hydrolysates was also determined. Neutrase caused a degree of hydrolysis (DH) of YP and WP by-products: 27.6% and 20.9%, respectively. In each of them, mixture of peptides with different molecular masses were also observed. YP hydrolysate showed high levels of antioxidant activity. The scavenging capacity, ferric reducing power, and chelating capacity were observed at the level: 0.44 μmol/L Trolox mg−1, 177.35 μg Fe2+ mg−1, and 549.87 μg Fe2+ mg−1, respectively. YP hydrolysate also exhibited significant ACE-inhibitory activity, in which the level was 59.2 μg. Protein solubility was significantly improved as the DH increased. WP hydrolysate showed high water-holding capacity of 43.2. This study indicated that YP and WP hydrolysates could be used in foods as natural antioxidants and functionality enhancers.

Erratum to: Pro-Cognitive Properties of the Immunomodulatory Polypeptide Complex, Yolkin, from Chicken Egg Yolk and Colostrum-Derived Substances: Analyses Based on Animal Model of Age-Related Cognitive Deficits

The online version of the original article can be found under doi: 10.1007/s00005-016-0392-z .

Evaluation of the ACE-Inhibitory Activity of Egg-White Proteins Degraded with Pepsin

Increasing the potency of antihypertensive food-derived peptides is a critical and important step in the development of natural drugs for cardiovascular diseases prevention. We have proposed the egg-white protein precipitate (EWPP) obtained as a byproduct of cystatin and lysozyme isolation as a potential source of ACE-inhibitory peptides derived by pepsin digestion. The results indicated that hydrolysis of EWPP with pepsin produced the ACE inhibitory activity. During 3-h hydrolysis (DH: 38.3%), the IC50 value of EWPP hydrolysate was significantly increased and finally reached IC50=643.1 μg/mL. This hydrolysate was further fractionated by RP-HPLC. The peptide fraction exhibiting the highest ACE inhibitory activity was rechromatographed. Three peptide subfractions exhibiting ACE-inhibitory activities of 69.0, 25.0, and 37.6 μg/mL were further characterised. In each of them, mixtures of peptides with different molecular masses were observed.

A simple and rapid method of isolation of active polypeptide complex, yolkin, from chicken egg yolk

A large number of bioactive peptides isolated from natural sources are known to play important physiological roles in the human body. It is possible to use these as alternative therapy agents. One example is yolkin which can be useful as a food supplement, a natural therapeutic agent for preventing and treating cognitive disorders of various origins, preferably in patients with unsatisfactory responses to known therapies. A new simple method of isolation of yolkin based on precipitation with ethanol or acetone was developed. The best precipitation efficiency of both ethanol and acetone was achieved when stirred into the starting material to a final concentration of 70%. These methods preserved the ability of yolkin to stimulate human whole blood cells to release anti-inflammatory cytokines and neurotrophins. At first we indicated that yolkin displayed a potential neuroprotective effect by the ability to stimulate cells to produce pro-survival brain derived neurotrophic factor (BDNF).

The use of serine protease from Yarrowia lipolytica yeast in the production of biopeptides from denatured egg white proteins

Deriving non-conventional enzymes from cheaper sources than those used for commercially available enzymes may result in the production of hydrolysates with beneficial features, while drastically reducing the cost of hydrolysis. This is especially significant for enzymatic hydrolysis as a method of protein waste utilization. We have previously described the ability of non-commercial serine protease from Yarrowia lipolytica yeast to produce/release bioactive peptides from egg white protein by-products (EP). The enzymatic hydrolysis of EP was carried out for 24 h using the serine protease at an enzyme: substrate ratio of 1:30 (w/w). The obtained hydrolysate was characterized by protein degradation of 38% and also exhibited an antioxidant and cytokine-inducing activity. The isolation procedure (ultrafiltration and RP-HPLC) of bioactive peptides from the EP hydrolysate provided peptide fractions with significant antioxidant and ACE inhibitory activities. Three homogeneous and three heterogeneous peptide fractions were identified using MALDI-TOF/MS and the Mascot Search Results database. The peptides, mainly derived from ovalbumin, were composed of 2-19 amino-acid residues. We have thus demonstrated a novel ability of serine protease from Y. lipolytica to release biopeptides from an EP by-product.

Isolation and Characterization of NP-POL Nonapeptide for Possible Therapeutic Use in Parkinson’s Disease

Colostrum and milk are the initial mammalian nourishment and rich reservoir of essential nutrients for newborn development. Bioactive peptides isolated from natural sources, such as colostrum, serve as endogenous regulators and can be used as alternative therapeutic agents in the treatment of neurodegenerative diseases. One example is the previously unknown NP-POL nonapeptide isolated from Colostrinin. In the present study, we investigated a method of NP-POL nonapeptide isolation using Bio-Gel P2 molecular sieve chromatography. We showed the protective effect of NP-POL on 6-hydroxydopamine- (6-OHDA-) induced neurotoxicity using rat adrenal pheochromocytoma (PC12 Tet On) cells. Treatment of PC12 cells with NP-POL nonapeptide reduced 6-OHDA-induced apoptosis and caused transient phosphorylation of extracellular signal-regulated kinases (ERK 1/2), which were shown to promote cell survival. NP-POL nonapeptide also protected neuronal cells against oxidative injury induced by 6-OHDA. These results showed a potential use of NP-POL in the therapy of Parkinsons disease.