Sequence alterations affect the antidiabetic attributes of hazelnut peptide fractions during the industrial manufacture and simulated digestion of hazelnut paste.

Press cakes are by-products of cold press oil manufacture and are characterized by significant protein concentrations. Our group has previously demonstrated potential bioactive attributes of hazelnut protein hydrolysates including their antidiabetic activities. Here, an effort was made to utilize DPP-IV (Dipeptidyl peptidase-IV)-inhibitory hazelnut peptides in industrial food manufacture. Hazelnut protein isolates (approx. 95% protein) were obtained via an alkali extraction-isoelectric precipitation method. Papain, bromelain and pepsin were used in the enzymatic hydrolysis and hydrolysates were fractionated via Fast Protein Liquid Chromatography. As a general observation, although fractionation lead to dilution of the samples, fractions were observed to be more bioactive than the total hydrolysates. In vitro antidiabetic activities of the fractions were tested and 3 antidiabetic fractions were added to hazelnut paste. Afterwards simulated gastrointestinal digestion and antidiabetic activity assays were performed. DPP-IV inhibition was the major antidiabetic mechanism in the fractions and digested paste, while some fractions were characterized by comparable IC50 values as the positive controls. Alpha-glucosidase inhibition was limited by digestion trials, whereas alpha-amylase inhibition was only slight in the digested paste (< %6). In silico analyses predicted partial degradation of the peptides, whereas the interactions between DPP-IV or alpha-glucosidase and hazelnut peptides were predicted to be significant (p < 0.05). Consequently hazelnut press cakes were regarded as a potential source of antidiabetic peptides that can be used in industrial manufacture of functional foods, while food processing conditions or gastrointestinal digestion could largely affect peptide bioactivity. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-022-05601-2.

Influence of Maillard reaction conditions and solvent extraction on the surface activity and foaming characteristics of black cumin protein concentrates.

Cold press manufacture of black cumin (BC) oil leads to the formation of BC press cakes that contain significant amounts of protein. Here, an attempt was made to enhance the functionality of BC protein concentrates obtained from cakes based on Maillard conjugation using 3 different of carbohydrates. Molecular weight distribution of the conjugates was determined via electrophoretic techniques. The extent of carbohydrate binding was measured by RP-HPLC-RID. Surface activity and elasticity was studied using drop shape tensiometry. The extent of glucose binding accounted for up to 85% for a protein:glucose ratio of 1:2. Foaming capabilities were moderately enhanced due to Maillard conjugation in the absence of solvent extraction, while due to solvent induced partial denaturation, further enhancement of foaming performance took place. Furthermore, sugar binding capabilities were enhanced upon solvent treatment, while surface pressure and foaming capacity were not necessarily improved. Adsorption rate at the air-water surface and dilational elasticity was highly dependent on molecular size of reacting sugars. In addition, oil remaining in the samples also had a bearing on the extent of Maillard conjugation. Consequently, tailoring of processing conditions could enhance foaming characteristics of BC proteins and ensure their utilization in food foams and other food dispersions.

Identification of Novel Proteins from Black Cumin Seed Meals Based on 2D Gel Electrophoresis and MALDI-TOF/TOF-MS Analysis.

The amount of cold press oil manufacture is globally rising, which in turn leads to the accumulation of deoiled plant seeds at significant quantities and consequent manufacture of plant protein products. In this study, we made an attempt to analyze the protein profile of black cumin seed protein concentrates prepared by the alkali extraction-acid precipitation technique (AE-IP). The analytical strategy relied on gel-based proteome mapping which included two-dimensional gel electrophoresis followed by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF/TOF). 14 different protein bands were identified, and in gel-trypsinolysis was carried out for the corresponding gel spots. Using the MASCOT database, current findings on 10 proteins were compared with the existing data. The highest similarity was 46 which was obtained between the highest pI black cumin protein observed here and the cyclin dependent kinase inhibitor of Arabidopsis thaliana. The molecular mass of the intact protein was determined by linear MALDI-TOF/TOF-MS as 23,711.2186 Da. The peptide constructs of this protein have been further studied in order to identify potential biological activity. Matching sequences generated bioactive peptides in silico such as IR, AL, and SL dipeptides during sequential enzymatic digestion with pepsin and trypsin. Since the majority of bioactivity investigations on black cumin seeds have been related to black cumin oil and its oil soluble components, the structure and bioactivities of black cumin proteins deserve further research.

Hazelnut peptide fractions preserve their bioactivities beyond industrial manufacture and simulated digestion of hazelnut cocoa cream.

Cold press hazelnut cakes represent a concentrated source of proteins that can be industrially exploited. Previously, bioactive attributes of hazelnut protein hydrolysates including antihypertensive and antidiabetic activities were documented. Here, we made an attempt to utilize bioactive hazelnut protein hydrolysates (1 % w/w) in the manufacture of industrial hazelnut cocoa cream and investigate their stability through processing and simulated gastrointestinal digestion. The inclusion of bioactive peptide fractions was a safe practice in the microbiological sense. Proteolysis lowered the potential allergenicity of hazelnut proteins in the cocoa cream products up to about 20 %. In silico trypsinolysis predicted partial degradation for 51.8 % of the peptide sequences (i.e., 43/83) that were present in the hydrolysates. However, partial degradation and mixing of degraded vs non-degraded peptides preserved and/or further elevated bioactive attributes in the digested cocoa cream products in terms of Angiotensin converting enzyme (ACE)-inhibitory (up to about 92 %) and antidiabetic activities (between 7.5 and 44.4 %). In most cases, however, antioxidative activity was < 10 %. While simulated in vitro digestion potentially influenced the bioactive attributes of protein hydrolysates, the influence of cocoa cream processing and food matrix were relatively limited for hydrolysate fractions and more pronounced for protein isolates. Hazelnut press cakes represent a significant resource for the generation and industrial utilization of bioactive peptides, which could preserve their bioactivity beyond industrial manufacture and digestion and lead to slightly reduced allergenicity.

Tombul hazelnut (Corylus avellana L.) peptides with DPP-IV inhibitory activity: In vitro and in silico studies.

Cold press technology generates high quality value-added oil products along with highly stable oilseed cakes. Hazelnut cakes are characterized by high protein concentrations that can be industrially valorized. Here, using an aqueous extraction scheme along with enzymatic proteolysis and FPLC (fast protein liquid chromatography)-based fractionation, a variety of hazelnut peptide fractions with varying bioactive properties were manufactured and their sequences were determined based on mass spectrometry. DPP-IV inhibitory attributes were determined based on an in vitro DPP-IV assay and in silico techniques were administered for for the analysis of overall bioactive potential and DPP-IV inhibitory characteristics of peptides. Based on these investigations, 256 peptides were identified in 81 different fractions. The majority of fractions were characterized with low to moderate DPP-IV inhibitory activity possibly due to their dilute nature. Some hazelnut peptides were characterized by comparable IC50 values as the positive control (Diprotin-A). The most influential 7 peptides were shown to generate higher docking scores than the control. The main interaction mechanism between hazelnut peptides and DPP-IV possibly depended on hydrophobic interactions. While further concentration could enhance the DPP-IV inhibitory potential of hazelnut peptides, hazelnut cakes represent a sustainable resource of potentially antidiabetic peptides.

Delivery of Curcumin Using Skim Milk or Oil in Water Emulsions: Effect of the Matrices on Cellular Uptake.

To enhance the curcumin delivery in a variety of food grade matrices namely spray dried ethanolic curcumin in fresh skim milk (Spray dried Cu-SM), a fresh mixture of ethanolic curcumin and skim milk (Fresh Cu-SM) a powder mixture of curcumin and skim milk powder (Powder Cu-SMP) and oil in water emulsion (Emulsion) were studied. The cellular uptake of curcumin from the respective matrices was studied on Caco-2 cell monolayers. Spray dried Cu-SM showed higher encapsulation efficiency compared to a corresponding Powder Cu-SMP and an oil-in-water emulsion (40% oil) bearing curcumin. Furthermore, ethanolic administration of curcumin in spray dried form enhanced the cellular uptake of curcumin considerably higher than non-ethanolic samples (approx. 4 times). Overall, milk protein based vectors were found to perform better than emulsion samples. These findings highlighted the fact that curcumin uptake may be tailored by fine tuning of curcumin delivery vehicles which highlights possible application of powders as functional foods.

Structural and functional changes in ultrasonicated bovine serum albumin solutions.

Effects of high-intensity ultrasonication on functional and structural properties of aqueous bovine serum albumin (BSA) solutions were investigated. The functional properties of BSA were altered by ultrasonication. Surface activity of BSA increased. Minimal changes were observed in the global structure of BSA but surface charge increased particularly at basic pH values (e.g. pH>9). While dynamic light scattering measurements indicated that the particle size increased up to 3.4 times after 90 min of sonication, no significant increase in the oligomeric state of BSA using blue native PAGE was observed. The amount of free sulfhydryl groups in BSA after 90 min of sonication decreased. The increased particle size and decreased number of free sylfhydryl groups may be attributed to formation of protein aggregates. Surface hydrophobicity increased and circular dichroism spectroscopy and FTIR analysis indicated changes in the secondary structure of BSA. We hypothesize that mechanical, thermal and chemical effects of ultrasonication resulted in structural changes in BSA that altered the functional properties of the macromolecule which may be attributed to the formation of an ultrasonically induced state that differs from a thermally, mechanically or solvent induced state.

Effect of interfacial composition on uptake of curcumin-piperine mixtures in oil in water emulsions by Caco-2 cells.

Encapsulation in lipid particles is often proposed as a solution to improve curcumin bioavailability. This bioactive molecule has low water solubility and rapidly degrades during digestion. In the present study, the uptake of curcumin from oil in water emulsions, prepared with two different emulsifiers, Tween 20 and Poloxamer 407, was investigated to determine the effect of interfacial composition on absorption. Piperine was added to the curcumin to limit the degradation of curcumin because it is known to inhibit ß-glucuronidase activity. The emulsions were administered to Caco-2 cell cultures, which is used as a model for intestinal uptake, and the recovery of curcumin was measured. The curcumin uptake was significantly affected by the type of interface, and the extent of curcumin uptake improved significantly by piperine addition only in the case of oil-in-water emulsions stabilized by Poloxamer 407. This work provides further evidence of the importance of interfacial composition on the delivery of bioactives.

Effect of processing on physicochemical characteristics and bioefficacy of ß-lactoglobulin-epigallocatechin-3-gallate complexes.

Varying amounts of epigallocatechin-3-gallate (EGCG) were encapsulated in ß-lactoglobulin (ß-Lg) nanoparticles, either native or processed, denoted as heated or desolvated protein. The stability, physical properties, and bioactivity of the ß-Lg-EGCG complexes were tested. Native ß-Lg-EGCG complexes showed comparable stability and binding efficacy (EGCG/ß-Lg molar ratio of 1:1) to heated ß-Lg nanoparticles (1% and 5% protein w/w). The sizes of heated and desolvated ß-Lg nanoparticles were comparable, but the latter showed the highest binding affinity for EGCG. The presence of EGCG complexed with ß-Lg did not affect the interfacial tension of the protein when tested at the soy oil-water interface but caused a decrease in dilational elasticity. All ß-Lg complexes (native, heated, or desolvated) showed a decrease in cellular proliferation similar to that of free ECGC. In summary, protein-EGCG complexes did not alter the bioefficacy of EGCG and contributed to increased stability with storage, demonstrating the potential benefits of nanoencapsulation.

Storage stability and physical characteristics of tea-polyphenol-bearing nanoliposomes prepared with milk fat globule membrane phospholipids.

The objective of this work was to better understand the functional properties of milk phospholipids when used as ingredients to prepare liposomes. Liposomal dispersions (10%) were prepared using high-pressure homogenization, and their physical properties as well as their ability to encapsulate tea polyphenols were investigated. The extent of encapsulation, measured by HPLC, increased with tea polyphenol concentration up to about 4 mg·mL(-1). At polyphenol concentrations ≥ 6 mg·mL(-1), the liposome dispersions were no longer stable. The influence of pH (3-7), storage temperature (room temperature or refrigeration), and addition of sugars (0-15%) were studied for liposomes containing 4 mg·mL(-1) polyphenols. The liposomal dispersions were also stable in the presence of peptides. The storage stability of the systems prepared with milk phospholipids was compared to that of liposomes made with soy phospholipids. Soy liposomes were smaller in size than milk phospholipid liposomes, the encapsulation efficiency was higher, and the extent of release of tea polyphenols during storage was lower for milk phospholipid liposomes compared to soy liposomes. The results suggest that milk phospholipids could be employed to prepare tea-polyphenol-bearing liposomes and that the tea catechins may be incorporated in the milk phospholipid bilayer more efficiently than in the case of a soy phospholipid bilayer.

Interactions of chitin nanocrystals with ß-lactoglobulin at the oil-water interface, studied by drop shape tensiometry.

Particle stabilized emulsions have been gaining increasing attention in the past few years, because of their unique interfacial properties. However, interactions between food grade particles and other surfactants at the interface still need to be understood. In this research, the interfacial properties of chitin nanocrystals (ChN) were studied in the presence of a surface active milk protein, ß-lactoglobulin (ß-lg), often used to stabilize oil-in-water emulsions. ChN were prepared by acid hydrolysis of chitin. At low pH (pH 3), ChN and ß-lg do not interact, as demonstrated by light scattering measurements, and both components carry positive charge. The properties of the interface were tested using drop shape tensiometry. Addition of ChN or ß-lg to the aqueous phase reduced the interfacial tension, and ß-lg adsorption was characterized with an increase in the interfacial elasticity. When ß-lg was added to a solution containing 0.1% ChN, the film elasticity increased first and then decreased with increasing ß-lg concentration. The mixed film elasticity was the highest at a combination of 0.1% ChN+0.01% ß-lg, when both molecules were simultaneously added to the aqueous phase. On the other hand, when ß-lg was added after ChN, the protein did not affect the properties of the interface, indicating that the ChN (0.1%) equilibrated film was stable and that protein-protein interactions, normally resulting in an increase in the film elasticity, did not occur.

Utilization of solid lipid nanoparticles for enhanced delivery of curcumin in cocultures of HT29-MTX and Caco-2 cells.

Solid lipid nanoparticles (SLN) have shown potential for encapsulation, protection and delivery of lipophilic functional components. In this study, we have investigated the capabilities of SLN to deliver a hydrophobic polyphenol compound, curcumin, in a coculture system of absorptive Caco-2 and mucus secreting HT29-MTX cells. The cells were grown on transport filters to mimic the human intestinal epithelium. Because of the hydrophobic nature of curcumin, its delivery to the basolateral compartment is expected to take place via a paracellular route. The changes in curcumin concentration in various compartments (i.e., apical, basolateral, mucus, and cell lysates) were evaluated using fluorescence spectroscopy. Two SLN systems were prepared with different emulsifying agents. The encapsulation of curcumin in SLN caused enhanced delivery compared to unencapsulated curcumin. In addition, SLN showed enhanced delivery compared to emulsion droplets containing liquid soy oil. The SLN were retained on the apical mucosal layer to a greater extent than emulsion droplets. The presence of SLN did not affect the integrity of the cellular junctions, as indicated by the TEER values, and the route of transport of the solid particles was simple diffusion, with permeability rates of about 7 × 10(-6) cm s(-1). Approximately 1% of total curcumin was delivered to the basolateral compartment, suggesting that most of the curcumin was absorbed and metabolized by the cell.

Complexation of high methoxyl pectin with ethanol desolvated whey protein nanoparticles: physico-chemical properties and encapsulation behaviour.

Using a desolvation method, whey protein isolate (WPI) nanoparticles were prepared and mixed with high methoxyl pectin (HMP) solutions (DE 72.8) to form WPI-HMP supramolecular complexes at low pH. Aqueous dispersions containing 5% WPI at pH 9 were desolvated with ethanol, and then diluted in HMP solutions at pH 3. Changes in particle size of the HMP-WPI complexes were studied as a function of HMP concentration. Upon dilution of the WPI nanoparticles in 0.05% HMP at pH 3, the average apparent diameter (d(90)) was around 270 nm, and there were no differences with desolvation level. These nanoparticles would undergo coarsening with storage at room temperature. The complexes showed to withstand homogenization and although heating increased aggregation, the particle size of the heated suspensions improved after homogenization. In addition, the suspensions demonstrated higher interfacial pressures (measured by drop tensiometry) compared to the corresponding unprocessed, desolvated or heated WPI solutions, suggesting their employment as surface active ingredients. The encapsulation efficiency of the desolvated WPI suspensions and desolvated WPI-HMP complex suspensions was studied using a model hydrophilic dye. In all cases, appreciable amounts of dye molecule were encapsulated and retained by the nanoparticles during storage at pH 3.

Zinc incorporation capacity of whey protein nanoparticles prepared with desolvation with ethanol.

Whey protein isolate (WPI) nanoparticles were prepared using ethanol desolvation, and their capacity to incorporate ZnCl(2) was analysed. Desolvation was carried out at pH 9 and the volume of added ethanol was 0-3 times the volume of protein solution. The desolvated solutions were dispersed in acidified water (pH 3) immediately after desolvation. The size of the WPI nanoparticles increased with the volume ratio of ethanol:water used, as well as with the amount of ZnCl(2). The nanoparticles showed high incorporation efficiencies, and remained stable after 30 days of storage at 22 °C. The amount of zinc incorporated in the WPI particle suspensions was within the range of daily zinc requirements for healthy adults.

Probing the colloidal properties of skim milk using acoustic and electroacoustic spectroscopy. Effect of concentration, heating and acidification.

In colloidal systems physical-chemical changes are often a function of volume fraction and sample dilutions are critical. While most methods to characterize colloidal particles either require dilution or some disruption, acoustic spectroscopy can be performed in situ, without dilution. Objective of this work was to determine the effects of concentration, heating and acidification on the acoustic and electroacoustic properties of casein micelles in skim milk. The ultrasonic attenuation of skim milk increased with concentration of milk and frequency, and the average size of the colloidal particles calculated from the frequency dependence of attenuation was about 0.15 µm for both unheated and heated milk. When milk was concentrated by ultrafiltration, at 3× and 4× concentration (based on volume reduction), the calculated size deviated from that derived in undiluted or mildly concentrated milk, most likely because of increased particle-particle interactions. Electroacoustic measurements revealed a constant dynamic mobility of the particles in undiluted and concentrated milk, while lower mobilities were observed for milk diluted in permeate. The ζ-potential measured was significantly higher than the values measured using dynamic light scattering, with a value of -45.8 mV for casein micelles in unheated milk. With acidification, the ζ-potential decreased monotonically. Heating profoundly affected the change in charge with pH of the micelles, and it was concluded that the interaction of casein micelles with the whey proteins increased the surface charge of the casein micelles.