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.

Multifunctional peptides derived from an egg yolk protein hydrolysate: isolation and characterization.

An egg yolk protein by-product following ethanol extraction of phospholipids (YP) was hydrolyzed with pepsin to produce and identify novel peptides that revealed antioxidant, ACE inhibitory and antidiabetic (α-glucosidase and DPP-IV inhibitory) activities. The peptic hydrolysate of YP was fractionated by ion-exchange chromatography and reversed-phase high-pressure liquid chromatography. Isolated peptides were identified using mass spectrometry (MALDI-ToF) and the Mascot Search Results database. Four peptides of MW ranging from 1,210.62 to 1,677.88 Da corresponded to the fragments of Apolipoprotein B (YINQMPQKSRE; YINQMPQKSREA), Vitellogenin-2 (VTGRFAGHPAAQ) and Apovitellenin-1 (YIEAVNKVSPRAGQF). These peptides were chemically synthesized and showed antioxidant, ACE inhibitory or/and antidiabetic activities. Peptide YIEAVNKVSPRAGQF exerted the strongest ACE inhibitory activity, with IC50 = 9.4 µg/mL. The peptide YINQMPQKSRE showed the strongest DPPH free radical scavenging and DPP-IV inhibitory activities and its ACE inhibitory activity (IC50) reached 10.1 µg/mL. The peptide VTGRFAGHPAAQ revealed the highest α-glucosidase inhibitory activity (IC50 = 365.4 µg/mL). A novel nutraceutical effect for peptides from an egg yolk hydrolysate was shown.

Hydrolysis with Cucurbita ficifolia serine protease reduces antigenic response to bovine whey protein concentrate and αs-casein.

In the present study the effect of hydrolysis with non-commercial Cucurbita ficifolia serine protease on a reduction of the IgE and IgG binding capacity of whey protein concentrate and αs-casein was investigated. The intensity of the protein degradation was analyzed by the degree of hydrolysis, the free amino groups content and RP-HPLC. The ability to bind the antibodies by native proteins and their hydrolysates was determined using a competitive ELISA test. Deep hydrolysis contributed to a significant reduction of immunoreactive epitopes present in WPC. In the case of IgE and IgG present in the serum pool of children with CMA, the lowest binding capacity was detected in the 24 h WPC hydrolysate, where the inhibition of the reaction with native WPC was ≤23 and ≤60 %, respectively. The analysis of the IgG reactivity in the antiserum of the immunized goat showed that the lowest antibody binding capacity was exhibited also by 24 h WPC hydrolysate at a concentration of 1000 µg/ml where the inhibition of the reaction with nWPC was ≤47 %. One-hour hydrolysis of α-casein was sufficient to significant reduction of the protein antigenicity, while the longer time (5 h) of hydrolysis probably lead to the appearance of new epitopes reactive with polyclonal.

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

The 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.

Egg yolk proteins and peptides with biological activity.

Many proteins of food reveal biological activity. In the sequence of these proteins also numerous biologically active peptides are encrypted. These peptides are released during proteolysis naturally occurring in the gastrointestinal tract, food fermentation or during designed enzymatic hydrolysis in vitro. Biopeptides may exert multiple activities, affecting the cardiovascular, endocrine, nervous and immune systems. An especially rich source of bioactive proteins and biopeptides is egg. Bioactive peptides released from egg white proteins have been well described, whereas egg yolk proteins as precursors of biopeptides are less well characterized. This manuscript describes biologically active proteins and peptides originating from egg yolk and presents their potential therapeutic role.

Exploiting the Potential of Powdered Blends of Recovered Sunflower Seed Cake Phenolics and Whey-Development of Sustainable Food Additives.

The management of side streams from the food industry, especially oil and dairy by-products, has become an important issue linked to the European Commission's recommendations for a circular economy. This study aimed to obtain sustainable food additives in the form of soluble-type powders composed of whey and recovered phenolics originating from sunflower seed cake. In order to valorise these di-blend products, the powders were characterised in terms of their physical, chemical, and sensory attributes. Based on the study findings, the addition of sunflower seed cake washouts (SSCWs) to whey (Wh) decreased the dry matter in the feed that affected the viscosity and drying yield. The addition of SSCWs did not have a significant effect on the physical properties of powders, except for colour. By-product management proposed in the study resulted in the production of nutritious and ready-to-use products in powder form with improved functional properties in terms of phenolic compounds and antioxidant capacity. The powders were sensorially appealing with a tangy sourness entwined with a delicate interplay of sweet and salty flavours, which can be easily incorporated into different types of foodstuffs.

Effects of Iron Supplementation on Metabolism in Calves Receiving Whole Milk.

The objective of this study was to determine the effects of feeding a protein-iron complex (PIC) to calves. Specifically, the aim was to understand how it influences productive performance and indicators of iron metabolism, hematology and biochemical and parameters during feeding with whole milk before weaning. The study was carried out on 20 Polish Holstein Friesian calves. The calves were then divided into a control group (CON), fed with full milk (n = 10), and an experimental group (MFe), who received a PIC additive in milk at 16 g/day (n = 10). In order to determine the production parameters, the calves were weighed at the beginning (i.e., on the 7th day of life) and at the end of the experiment (42nd day of life) using an electronic platform scale. Production parameters such as average weight gain (AWG), feed conversion ratio (FCR), and growth rate (GR) were assessed. Blood was collected from an external jugular vein (vena jugularis externa) on the 7th, 14th, 28th, and 42nd days of life. The mean daily gains in body weight (ADG), growth rate (GR), and the feed conversion ratio were highest in the experimental group, MFe. Therefore, it can be concluded that the addition of a protein-iron complex entailed a significant impact on the iron metabolism indicators in the MFe experimental group.

Effect of Two Combined Functional Additives on Yoghurt Properties.

The aim of the research was the analysis of yoghurts enriched with blue honeysuckle berries dry polyphenolic extract and new preparation of resistant starch. The additives were introduced individually at concentration 0.1% (w/v) and in mixture at final concentration of 0.1 and 0.2% of both components. Yogurt microflora, pH, and its physicochemical and antioxidant properties were examined over 14 days of storage under refrigerated conditions. Studies showed that both substances can be successfully used in yoghurt production. Yoghurt microflora es. S. thermophilus and Lb. delbrueckii subsp. bulgaricus counts appeared to be higher in samples supplemented with these additives comparing to control yoghurt by 3-8%. More stimulating effect on their growth, especially on S. thermophilus, revealed resistant starch. Addition of this polysaccharide improved also the rheological properties of yogurts, which showed higher viscosity than samples produced without it. Addition of honeysuckle berries preparation significantly influenced the yogurts' color, giving them deep purple color, and their antioxidant potential. During storage, contents of anthocyanin and iridoid compounds were decreasing, but antioxidant activity in the products remained stable.

Psychosocial stress and cortisol stress reactivity predict breast milk composition.

We studied a sample of 146 Polish, exclusively breastfeeding mothers and their healthy born on time infants to explore the effect of perinatal psychosocial stress on breast milk composition. Maternal perinatal stress was assessed using Recent Life Changes Questionnaire summarizing stressful events from the previous six months. Stress reactivity was determined by administering the cold pressor test and measuring cortisol in saliva samples taken during the test. Breast milk sample was taken to measure energy, protein, fat, lactose, and fatty acid content. Analyses revealed that stress reactivity was positively associated with milk fat and long-chain unsaturated fatty acids and negatively associated with milk lactose. Perinatal psychosocial stress negatively affected energy density, fat as well as medium-chain and long-chain saturated fatty acids in milk. These results, together with previous studies, advocate monitoring maternal psychological status during the peripartum to promote breastfeeding and healthy infant nutrition.

The Effect of Rosa spinosissima Fruits Extract on Lactic Acid Bacteria Growth and Other Yoghurt Parameters.

The aim of the study was to evaluate the effect of purified extract from Rosa spinosissima fruits on the quality characteristics and antioxidant properties of yoghurt. The extract, added to yoghurt at a concentration of 0.1% and 0.2%, contained high quantities of phenolic compounds and exhibited high antioxidant activity due to the presence of anthocyanins flavan-3-ols, flavonols and ellagitannins. Yoghurt physicochemical properties, microbiology and antioxidant properties were evaluated after 1, 7 and 14 days of storage at a temperature of 4 °C. The data revealed a positive influence of rose preparation on yoghurt's microflora and on its other properties. The highest count of traditional yoghurt microflora was observed in samples with 0.2% of extract. Its addition had a positive effect on the yoghurts' color, giving them a characteristic pink color of an intensity dependent on additive concentration. It also significantly affected the yoghurts' antioxidant properties, which were stable during storage, as well as the content of the introduced phenolic compounds.

Reduced IgE and IgG antigenic response to milk proteins hydrolysates obtained with the use of non-commercial serine protease from Yarrowia lipolytica.

The aim of the study was to investigate the use of serine protease from Yarrowia lipolytica yeast for reduction of milk proteins allergenicity. Whey protein concentrate (WPC-80), αs-casein and their hydrolysates were analyzed for the capacity to bind IgE and IgG antibodies present in sera from patients with cow milk protein allergy using a competitive ELISA. The hydrolysis of αs-casein and whey protein concentrate contributed to a significant reduction of their immunoreactive epitopes. In case of IgE antibodies, the lowest binding capacity was detected in the 24 h hydrolysates of both proteins in which the inhibition of the reaction was ≤20 and ≤68% for αs-casein and whey protein concentrate respectively. One hour hydrolysis of WPC-80 reduced the protein antigenicity, while the longer time (5 h) might lead to the exposure of new IgE - reactive epitopes.

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.

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

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.

Application of Asian pumpkin (Cucurbita ficifolia) serine proteinase for production of biologically active peptides from casein.

The main objective of this study was to determine potential application of a serine proteinase derived from Asian pumpkin for obtaining biologically active peptides from casein. The course of casein hydrolysis by three doses of the enzyme (50, 150, 300 U/mg of protein) was monitored for 24 hours by the determinations of: hydrolysis degree DH (%), free amino group content (µmole Gly/g), RP HPLC peptide profiles and by polyacrylamide gel electrophoresis. In all hydrolyzates analyzed antioxidant activities were determined using three tests: the ability to reduce iron ions in FRAP test, the ability to scavenge free radicals in DPPH test, and Fe(2+) chelating activity. The antimicrobial activity of obtained peptide fractions was determined as the ability to inhibit the growth of Escherichia coli, Bacillus cereus and Pseudomonas fluorescens in a diffusion plate test. The deepest degradation, expressed as the DH [%] and the free amino group content (67% and 7528 µmole Gly/mg, respectively), was noted in samples hydrolyzed with 300 U/ml of enzyme for 24 hours, while in other samples the determined values were about three and two times lower. The results were in agreement with the peptide profiles obtained by RP HPLC. The highest antioxidative activities determined in all tests were seen for the casein hydrolysate obtained with 300 U/mg protein of serine proteinase after 24 h of reaction (2.15 µM Trolox/mg, 96.15 µg Fe(3+)/mg, 814.97 µg Fe(2+)/mg). Antimicrobial activity was presented in three preparations. In other samples no antimicrobial activity was detected.

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 Fe(2+) mg(-1), and 549.87 µg Fe(2+) 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.