A. Polanowski

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.

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.

Immunomodulatory activity accompanying chicken egg yolk immunoglobulin Y

Abstract Immunity transfer from a mother to the newborn does not depend exclusively on immunoglobulins. Peptides, which are characterized by immunoregulatory properties that accompany IgG2, known as proline- rich polypeptide complex (PRP), have been discovered for the first time in ovine colostrum. In this report we present new data showing that some immunoregulatory peptides associated with the main immunoglobulin class, IgY, are also present in the avian immune system. Cytokine-inducing activity of particular fractions obtained from ovine colostrum, IgG+ (IgG2 containing PRP), IgG− (IgG2 free of PRP), and purified PRP, was compared with that of crude egg yolk IgY (IgY+), additionally purified egg yolk IgY (IgY−), and polypeptides accompanying IgY named Yolkin (Y), using an ex vivo model of whole human blood cells. It was shown that both IgG+ fraction and PRP, but not IgG−, stimulated the whole blood cells to release tumor necrosis factor-α and interleukin-1β cytokines. Similar experiments performed with hens egg IgY preparations showed that IgY+ and Y samples showed higher cytokine-inducing activity than samples additionally purified with the use of size exclusion chromatography (IgY−). The IgY+ at a dose of 100 μg was even more active than the positive lipopolysaccharide control. It was also found that Y is able to stimulate macrophage cell line J774.2 to release nitric oxide. The results obtained suggest that IgY, the main chicken immunoglobulin fraction, is accompanied by additional polypeptides and plays a role of a transporter of biologically active substances, which was observed in the case of colostral IgG.

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 .

Peptides accompanying chicken egg yolk IgY--alternative methods of isolation and immunoregulatory activity.

In the hen immune system the egg content plays as significant a role in the development of the chick as colostrum does in newborn mammals. One of the most important proteins in this system seems to be the main yolk immunoglobulin IgY. It has been shown that IgY is accompanied by an immunostimulatory polypeptide complex named yolkin. In this report the biological activities of yolkin separated by means of four different procedures are presented. It was shown that yolkin acts as an inducer rather than a modulator of cytokine and nitric oxide release, and does not participate in the protection of cells against destructive effects of reactive oxygen species. However, using the perchloric acid procedure it is possible to obtain a peptide fraction with higher inducing activity, stronger antioxidant properties and ability to decrease the NO level induced by lipopolysaccharide. The results obtained show that it is feasible to select one of the presented methods of yolkin isolation that yields a product of particular activity. The properties of yolk peptides not only indicate their roles in the development of chicks, but can also be useful for the regulation of some immunological disturbances.

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).

Effects of Immunomodulatory Peptide Y On Cognitive Functions in Animal Models

Despite the great intensity of studies to explain mechanisms of the Alzheimer’s disease, there’s still lack of effective prevention and treatment. As many specimens had been unadmitted because of their adverse side effects, thus recent attempts to follow natural biological protective mechanisms might open a range of possibilities in developing novel drugs to slow down processes of dementia and cognitive impairment. Promising results were achieved in patients treated with herbal agents, such as Gingko biloba extract, as well as animal derived preparations, i.a. PRP/Colostrinine (proline rich polipeptyde) complex isolated from ovine and bovine colostrum with confirmed immunomodulating properties and pro-cognitive action on memory and learning functions. The contribution of egg yolk (vitellus) ingredients to chicks development appeared similar to mammalian colostrum role in growth and immunity of newborns. Recently discovered polipeptyde complex associated with IgY, named Yolkine,has been put to the series of experiments in order to determine the effects of egg yolk preparations on cognitive functions in young and old rats as a model of cognitive decline and process of brain aging. To estimate Yolkine efficiency, different doses and routes of administration were applied, along with wide range of aging-sensitive behavioral tests, i.a. novel object recognition task and Morris water maze. The authors will present the results of the ongoing study and key findings.

Procognitive Properties of Cysteine Protease Inhibitor – Ovocystatin in Alzheimer's Disease Mice Model.

Background The results obtained in the last decade suggest that cystatin C plays an important role in the development of Alzheimer´s disease (AD) by inhibiting the aggregation of β-amyloid and its deposition. Cystatin C also demonstrate protective role via inhibition of cysteine proteases or by induction of autophagy and induction of proliferation (1–3). Aims/Objectives The aim of the study concerns the influence of the ovocystatin derived from the new generation of eggs on cognitive decline in AD mice model. Methods Animals: Mice B6C3-Tg(APPswe, PSEN1dE9)85Dbo/Mmjax–Genotype HEMI and NCAR (The Jackson Laboratories). Specimen and administration: Ovocystatin [40μg/mouse] were administered with drinking water by 22 weeks. The placebo group received drinking water. The locomotor activity were tested by IR Actimeter. The evaluation of learning and memory was determined by Morris Water Maze test (MWM) (4). The study „Innovative technologies of bio-preparations’ production on the base of new generation of eggs” was co-financed by the European Union from the European Regional Development Fund under the Operational Program Innovative Economy, 2007-2013. Results Mice from group with ovocystatin administration with drinking water have traveled statistically longer distance [D%] at Target quadrant than Placebo mice in MWM (p>0,05). Conclusion Ovocystatin given with drinking water has influence on learning and memory in HEMI group. 1. Gauthier S, Kaur G, Mi W, Tizon B, Levy E. Front Biosci (Schol Ed), 2011; 1(3):541-554. 2. Kaur G, Levy E. Front Mol Neurosci, 2012; 6(5):79. 3. Zerovnik E. BioEssays, 2009; (6):597-599. 4. Bromley-Brits, K., Deng, Y., Song, W. J Vis Exp, 2011, (53):e2920.