Marek Szołtysik

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.

Biofortification of milk and cheese with microelements by dietary feed bio-preparations.

AbstractThe present work reports studies on biofortification of milk and cheese with microelements. The diet of goats was supplemented with soya-based preparations with Cu(II), Fe(II), Zn(II) and Mn(II), produced by biosorption, instead of mineral salts. In innovative preparations, soya was the biological carrier of microelements. The utilitarian properties of the new preparations were tested in two groups (8 goats in each): experimental and control. The concentration of supplemented microelements was monitored in milk during the experiment. The collected milk was then used to produce cheese by enzymatic and acidic coagulation method. The effect of milk and cheese biofortification in microelements was confirmed. In milk, the level of the following microelements was higher than in the control: Cu(II) – 8.2 %, Mn(II) – 29.2 %, Zn(II) – 14.6 %. In cheese the content of Zn(II) obtained in enzymatic (19.8 %) and in acidic (120 %) coagulation was higher when compared to the control group. By using bio-preparations with microelements it was possible to produce new generation of functional food biofortified with microelements, by agronomic, and thus sustainable and ethically acceptable way. Biofortified milk and cheese can be used as designer milk to prevent from micronutrient deficiencies. Graphical Abstractᅟ

Application of aluminosilicates for mitigation of ammonia and volatile organic compound emissions from poultry manure

Abstract Odor mitigation techniques are widely investigated due to the problem of odor nuisance generated by intensive livestock production. The goal of this research was to investigate the use of aluminosilicate sorbents as filter packs in the air scrubber ODOR1, which enables cleaning of air inside the livestock building. The following sorbents were examined: raw halloysite, roasted halloysite, activated halloysite, raw bentonite, roasted bentonite and expanded vermiculite. The experiment was conducted in chambers where poultry manure was placed, the time of air treatment was 24 hours. A manual SPME (solid-phase microextraction) holder with DVB/Carboxen/PDMS fiber was used for extraction of odor compounds, and analyses were carried out using gas chromatography-mass spectrometry. Ammonia concentrations were determined according to Polish standards (Nessler method) using a spectrophotometer. It was found that all examined aluminosilicates had the potential for removal of ammonia as well as 24 volatile compounds emitted from poultry manure. The highest efficiency was noted for activated halloysite (81%) and roasted bentonite (84%) in the case of ammonia and odors, respectively. Despite the limitations of the study, the results showed the effectiveness of the air scrubber packed with aluminosilicates for the reduction of volatile odorous compounds in the air of livestock buildings. Graphical Abstract

Freeze-Drying Preservation of Yeast Adjunct Cultures for Cheese Production

Freeze-Drying Preservation of Yeast Adjunct Cultures for Cheese Production Four yeast strains: Yarrowia lipolytica PII6a, Candida famata MI1a, C. kefyr PII1b, C. sphaerica FII7a, adjunct cultures for cheese production were preserved by freeze-drying. The effect of process parameters and cryoprotective agents on cell survival and stability of growth characteristics was evaluated. Among three lyophilisation protocols, the process with three-step drying at temperatures of -38°C, -20°C and -10°C assured the highest cell viability. The survival of yeast strains in the presence of multicomponent cryoprotective agents containing skimmed milk, trehalose and sodium glutamate in three combinations (MT, MG, MTG) was significantly higher than in the presence of those agents used alone. The best agent for Y. lipolytica, C. kefyr and C. sphaerica appeared to be MT, while for C. famata - MG. Cell viability of yeast strains directly after freeze-drying was in the range of 74-80% and was relatively stable during one-year storage except C. famata. Initial yeast growth patterns were very well preserved in most of the preparations during 6 months of storage.

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.

Viability and growth promotion of starter and probiotic bacteria in yogurt supplemented with whey protein hydrolysate during refrigerated storage

The effect of whey protein hydrolysate (WPH) addition on growth of standard yoghurt cultures and Bifidobacterium adolescentis during co-fermentation and its viability during storage at 4ºC in yoghurts has been evaluated. WPH was obtained with the use of serine protease from Y. lipolytica yeast. Stirred probiotic yoghurts were prepared by using whole milk standardized to 16% of dry matter with the addition of either whey protein concentrate, skim milk powder (SMP), WPH-SMP (ratio 1:1), WPH. The hydrolysate increased the yoghurt culture counts at the initial stage of fermentation and significantly inhibited the decrease in population viability throughout the storage at 4ºC in comparison to the control. The post-fermentation acidification was also retarded by the addition of WPH. The hydrolysate did not increase the Bifidobacterium adolescentis counts at the initial stage. However, the WPH significantly improved its viability. After 21 days of storage, in the yogurts supplemented with WPH, the population of these bacteria oscillated around 3.04 log10 CFU/g, while in samples where SMP or whey protein concentrate was used, the bacteria were no longer detected.