Plant and animal protein mixed systems as wall material for microencapsulation of Manuka essential Oil: Characterization and in vitro release kinetics.

Combination of plant and animal protein diet is becoming a valuable source of nutrition in the modern diet due to the synergistic functional properties inherent in these protein complexes. Moreover, the synergy between animal and plant proteins can contribute to the high stability and improved solubility of the encapsulated bioactive ingredients (e.g., essential oils). Therefore, the study was designed to evaluate the plant (pea protein (PP) and lupine protein (LP)) and animal protein (whey protein, WP) mixed systems as a wall material for microencapsulation of manuka essential oil, as an example of bioactive compound. Moreover, physicochemical properties and in vitro release profile of encapsulated manuka essential oil were studied. Manuka essential oil microcapsules exhibited low moisture content (5.3-7.1 %) and low water activity (0.33-0.37) with a solubility of 53.7-68.1 %. Change in wall material ratio significantly affected the color of microcapsules, while microcapsules prepared with 1:1 protein/oil ratio demonstrated a high encapsulation efficiency (90.4 % and 89.4 %) for protein mixed systems (PP + WP and LP + WP), respectively. Microcapsules further showed low values for lipid oxidation with a high oxidative stability and antioxidant activity (62.1-87.0 %). The zero order and Korsmeyer-Peppas models clearly explained the release mechanism of encapsulated oil, which was dependent on the type and concentration of the protein mixed used. The findings demonstrated that the protein mixed systems successfully encapsulated the manuka essential oil with controlled release and high oxidative stability, indicating the suitability of the protein mixed systems as a carrier in encapsulation and application potential in development of encapsulated functional foods.

Anti-inflammatory activity of essential oils from Tunisian aromatic and medicinal plants and their major constituents in THP-1 macrophages.

In this study, the capacity of eight essential oils (EOs), sage (Salvia officinalis), coriander (Coriandrum sativum), rosemary (Rosmarinus officinalis), black cumin (Nigella sativa), prickly juniper (Juniperus oxycedrus), geranium (Pelargonium graveolens), oregano (Origanum vulgare) and wormwood (Artemisia herba-alba), on the inhibition of NF-κB activation was screened at concentrations up to 0.25 µL/mL using THP-1 human macrophages bearing a NF-κB reporter. This screening selected coriander, geranium, and wormwood EOs as the most active, which later evidenced the ability to decrease over 50 % IL-6, IL-1ß, TNF-α and COX-2 mRNA expression in LPS-stimulated THP-1 macrophages. The chemical composition of selected EOs was performed by gas chromatography-mass spectrometry (GC-MS). The two major constituents (>50 % of each EO) were tested at the same concentrations presented in each EO. It was demonstrated that the major compound or the binary mixtures of the two major compounds could explain the anti-inflammatory effects reported for the crude EOs. Additionally, the selected EOs also inhibit>50 % caspase-1 activity. However, this effect could not be attributed to the major components (except for ß-citronellol/geranium oil, 40 %/65 % caspase-1 inhibition), suggesting, in addition to potential synergistic effects, the presence of minor compounds with caspase-1 inhibitory activity. These results demonstrated the potential use of the EOs obtained from Tunisian flora as valuable sources of anti-inflammatory agents providing beneficial health effects by reducing the levels of inflammatory mediators involved in the genesis of several diseases.

Ionic Strength Dependence of the Complex Coacervation between Lactoferrin and ß-Lactoglobulin.

Heteroprotein complex coacervation is an assembly formed by oppositely charged proteins in aqueous solution that leads to liquid-liquid phase separation. The ability of lactoferrin and ß-lactoglobulin to form complex coacervates at pH 5.5 under optimal protein stoichiometry has been studied in a previous work. The goal of the current study is to determine the influence of ionic strength on the complex coacervation between these two proteins using direct mixing and desalting protocols. The initial interaction between lactoferrin and ß-lactoglobulin and subsequent coacervation process were highly sensitive to the ionic strength. No microscopic phase separation was observed beyond a salt concentration of 20 mM. The coacervate yield decreased drastically with increasing added NaCl from 0 to 60 mM. The charge-screening effect induced by increasing the ionic strength is attributed to a decrease of interaction between the two oppositely charged proteins throughout a decrease in Debye length. Interestingly, as shown by isothermal titration calorimetry, a small concentration of NaCl around 2.5 mM promoted the binding energy between the two proteins. These results shed new light on the electrostatically driven mechanism governing the complex coacervation in heteroprotein systems.

Recent trends in design of healthier plant-based alternatives: nutritional profile, gastrointestinal digestion, and consumer perception.

In recent years, various types of plant-based meat, dairy, and seafood alternatives merged in the health-conscious consumer market. However, plant-based alternatives present complexity in terms of nutritional profile and absorption of nutrients after food ingestion. Thus, this review summarizes current strategies of plant-based alternatives and their nutritional analysis along with gastrointestinal digestion and bioavailability. Additionally, regulatory frameworks, labeling claims, and consumer perception of plant-based alternatives are discussed thoroughly with a focus on status and future prospects. Plant-based alternatives become a mainstream of many food-processing industries with increasing alternative plant-based food manufacturing industries around the world. Novel food processing technologies could enable the improving of the taste of plant-based foods. However, it is still a technical challenge in production of plant-based alternatives with authentic meaty flavor. In vitro gastrointestinal digestion studies revealed differences in the digestion and absorption of plant-based alternatives and animal-based foods due to their protein type, structure, composition, anti-nutritional factors, fibers, and polysaccharides. Overall, plant-based alternatives may facilitate the replacement of animal-based foods; however, improvements in nutritional profile and in vitro digestion should be addressed by application of novel processing technologies and food fortification. The specific legislation standards should be necessary to avoid consumer misleading of plant-based alternatives.

Essential Oil Stabilisation by Response Surface Methodology (RSM): Nanoemulsion Formulation, Physicochemical, Microbiological, and Sensory Investigations.

This manuscript aimed to optimise the encapsulation of Thymus capitatus essential oil into nanoemulsion. Response Surface Methodology results were best fitted into polynomial models with regression coefficient values of more than 0.95. The optimal nanoemulsion showed nanometer-sized droplets (380 nm), a polydispersity index less than 0.5, and a suitable Zeta potential (-10.3 mV). Stability results showed that nanoemulsions stored at 4 °C were stable with the lowest d3,2, PolyDispersity Index (PDI), and pH (day 11). Significant ameliorations in the capacity to neutralise DPPH radical after the encapsulation of the antimicrobial efficacy of thyme essential oil were recorded. S. typhimurium growth inhibition generated by nanoencapsulated thyme essential oil was 17 times higher than by bulk essential oil. The sensory analysis highlighted that the encapsulation of thyme essential oil improved enriched milk's sensory appreciation. Indeed, 20% of the total population attributed a score of 4 and 5 on the scale used for milk enriched with nanoemulsion. In comparison, only 11% attributed the same score to milk enriched with bulk essential oil. The novel nanometric delivery system presents significant interest for agroalimentary industries.

Essential Oils in Livestock: From Health to Food Quality.

Using plant essential oils (EOs) contributes to the growing number of natural plants' applications in livestock. Scientific data supporting the efficacy of EOs as anti-inflammatory, antibacterial and antioxidant molecules accumulates over time; however, the cumulative evidence is not always sufficient. EOs antioxidant properties have been investigated mainly from human perspectives. Still, so far, our review is the first to combine the beneficial supporting properties of EOs in a One Health approach and as an animal product quality enhancer, opening new possibilities for their utilization in the livestock and nutrition sectors. We aim to compile the currently available data on the main anti-inflammatory effects of EOs, whether encapsulated or not, with a focus on mammary gland inflammation. We will also review the EOs' antioxidant activities when given in the diet or as a food preservative to counteract oxidative stress. We emphasize EOs' in vitro and in vivo ruminal microbiota and mechanisms of action to promote animal health and performance. Given the concept of DOHaD (Developmental Origin of Health and Diseases), supplementing animals with EOs in early life opens new perspectives in the nutrition sector. However, effective evaluation of the significant safety components is required before extending their use to livestock and veterinary medicine.

Unraveling the molecular mechanisms underlying interactions between caseins and lutein.

Understanding the food protein binding to bioactive compounds is of utmost importance for the development of efficient protein-based delivery systems. The binding of lutein to sodium caseinate (NaCas) or native casein micelle (PPCN) was investigated at pH 7 to evaluate the effect of casein supramolecular structures on the interaction. Fluorescence quenching, UV-vis spectroscopy, and dynamic light scattering were carried out. Under the medium conditions of interaction analysis (DMSO-water and ethanol-water), lutein exists as H-type aggregates. The investigation of lutein/casein interaction showed a predominantly static mechanism of fluorescence quenching and the presence of two fluorophore populations on NaCas and PPCN, but only one accessible to lutein. Moreover, the Scatchard plot indicated that lutein interacted with both caseins in one binding site. The interaction of lutein with caseins occurred with binding constant Kb of 105 M-1, regardless of casein supramolecular structure.

Aroma-retention capacities of functional whey protein aggregates: Study of a strawberry aroma in solutions and in fat-free yogurts.

The aroma-retention capacity of functional whey protein aggregates (WPA) was compared to that of native whey protein isolate (WPI) in aqueous solutions and in fat-free yogurts. The retention of aroma compounds, constituting a model strawberry aroma, was evaluated by calculating gas-matrix partition coefficients using headspace gas chromatography (HS-GC). The retention capacity of WPA differed from the one of WPI for three out of seven aroma compounds detected in HS-GC. Incorporating WPA in fat-free yogurts tended to decrease the release of hydrophobic aroma compounds such as 2-nonanone or methyl-cinnamate. The magnitude of the differences between the partition coefficients of yogurts enriched in WPI or WPA was lower than in aqueous solutions, which is likely to be due to the higher complexity of the food matrix and potential interactions with other ingredients. Overall, the different aroma-retention capacities of native WPI and functional WPA are likely to lead to unbalanced aroma, especially in fat-free dairy products.

Modification of protein structures by altering the whey protein profile and heat treatment affects in vitro static digestion of model infant milk formulas.

Heat treatments induce changes in the protein structure in infant milk formulas (IMFs). The present study aims to investigate whether these structural modifications affect protein digestion. Model IMFs (1.3% proteins), with a bovine or a human whey protein profile, were unheated or heated at 67.5 °C or 80 °C to reach 65% of denaturation, resulting in six protein structures. IMFs were submitted to in vitro static gastrointestinal digestion simulating infant conditions. During digestion, laser light scattering was performed to analyze IMF destabilization and SDS-PAGE, OPA assay and cation exchange chromatography were used to monitor proteolysis. Results showed that, during gastric digestion, α-lactalbumin and ß-lactoglobulin were resistant to hydrolysis in a similar manner for all protein structures within IMFs (p > 0.05), while the heat-induced denaturation of lactoferrin significantly increased its susceptibility to hydrolysis. Casein hydrolysis was enhanced when the native casein micelle structure was modified, i.e. partially disintegrated in the presence of lactoferrin or covered by heat-denatured whey proteins. The IMF destabilization at the end of the gastric digestion varied with protein structures, with larger particle size for IMF containing native casein micelles. During intestinal digestion, the kinetics of protein hydrolysis varied with the IMF protein structures, particularly for IMFs containing denatured lactoferrin, exhibiting higher proteolysis degree (67.5 °C and 80 °C vs. unheated) and essential amino acid bioaccessibility (67.5 °C vs. unheated). Overall, the protein structures, generated by modulating the whey protein profile and the heating conditions, impacted the IMF destabilization during the gastric phase and the proteolysis during the entire simulated infant digestion.

Kinetics of heat-induced denaturation of proteins in model infant milk formulas as a function of whey protein composition.

The process of manufacturing infant milk formulas (IMFs) involves heat treatments that can lead to whey protein denaturation. The objective of the study was to determine how protein composition affects the denaturation kinetics of the whey proteins within IMFs. Three model IMFs (1.3% of cow's milk protein) were produced with a caseins: whey proteins ratio of 40:60, differing only by the whey protein composition. The kinetics of heat-induced denaturation of α-lactalbumin, ß-lactoglobulin and lactoferrin were investigated between 67.5 °C and 80 °C by chromatographic quantification of the residual native proteins. Results showed that the heat-denaturation of α-lactalbumin was reduced when ß-lactoglobulin was absent. The heat-denaturation of lactoferrin was not affected by the composition of the IMFs but its presence enhanced the heat-denaturation of ß-lactoglobulin. This study revealed that, for higher heat treatments (90 °C/15 s, 75 °C/15 min), IMF containing α-lactalbumin and lactoferrin preserved a higher proportion of native whey proteins than IMFs containing ß-lactoglobulin.

Physico-chemical behaviors of human and bovine milk membrane extracts and their influence on gastric lipase adsorption.

Milk fat globule membrane conditions the reactivity and enzymatic susceptibility of milk lipids. The use of bovine membrane extracts to make infant formulas more biomimetic of human milk has been suggested recently. A comparison of the physico-chemical behavior of human and bovine milk membrane extracts and their interaction with gastric lipase is here undertaken using biophysical tools. Milk membrane extracts (70% of polar lipids) were obtained either pooling of mature human milk (n = 5) or bovine buttermilk. Human extract contained more anionic glycerophospholipids, less phosphatidylethanolamine and more unsaturated fatty acids (57% versus 46%) than bovine extract. Human extract presented a higher compressibility, with slower increase of surface pressure, than bovine extract. Micronic liquid condensed (LC) domains were evidenced in both extracts at 10 mN/m, but the evolution differs upon compression. Upon gastric lipase addition, an adsorption preference for liquid expanded phase (LE) was observed for both extracts. However, insertion was more homogeneous in terms of height level in human extract and impacted less its lipid lateral organization than in bovine extract. Both membrane extracts share close physico-chemical properties, however human membrane higher compressibility may favour gastric lipase insertion and higher interfacial reactivity in gastric conditions.

Contrasting Assemblies of Oppositely Charged Proteins.

Oppositely charged proteins can form soluble assemblies that under specific physical chemical conditions lead to liquid-liquid phase separation, also called heteroprotein coacervation. Increasing evidence suggests that surface charge anisotropy plays a key role in heteroprotein complexation, and coacervation. Here, we investigated complexation of an acidic protein, ß-lactoglobulin (BLG), with two basic proteins, rapeseed napin (NAP) and lysozyme (LYS), of similar net charge and size but differing in surface charge distribution. Using turbidity measurements and isothermal titration calorimetry, we confirmed that LYS binds BLG as expected from previous studies. This interaction leads to two types of phase separation phenomena, depending on pH: liquid-solid phase separation in the case of strong electrostatic attraction and liquid-liquid phase separation for weaker attraction. More interestingly, we showed using dynamic light scattering that NAP interacts with BLG, resulting in formation of assemblies in the nanometer size range. The formation of assemblies was also evident when modeling the interactions using Brownian dynamics for both BLG + NAP and BLG + LYS. Similarly, to DLS, BLG and NAP formed smaller assemblies than BLG with LYS. The molecular details rather than the net charge of BLG and NAP may therefore play a role in their assembly. Furthermore, simulated BLG + NAP assemblies were larger than those experimentally detected by DLS. We discuss the discrepancy between experiments and simulations in relation to the limitations of modelling precisely the molecular characteristics of proteins.

Soft-Matter Approaches for Controlling Food Protein Interactions and Assembly.

Animal- and plant-based proteins are present in a wide variety of raw and processed foods. They play an important role in determining the final structure of food matrices. Food proteins are diverse in terms of their biological origin, molecular structure, and supramolecular assembly. This diversity has led to segmented experimental studies that typically focus on one or two proteins but hinder a more general understanding of food protein structuring as a whole. In this review, we propose a unified view of how soft-matter physics can be used to control food protein assembly. We discuss physical models from polymer and colloidal science that best describe and predict the phase behavior of proteins. We explore the occurrence of phase transitions along two axes: increasing protein concentration and increasing molecular attraction. This review provides new perspectives on the link between the interactions, phase transitions, and assembly of proteins that can help in designing new food products and innovative food processing operations.

pH- and ionic strength-dependent interaction between cyanidin-3-O-glucoside and sodium caseinate.

Understanding the mechanism of interaction between food proteins and bioactives constitutes the preliminary step to design food grade nanocarriers. We investigated the interaction between cyanidin-3-O-glucoside (C3G), and 20nm-sized sodium caseinate nanoparticles (NaCas) at pH 7 and pH 2 by fluorescence spectroscopy and dynamic light scattering. The characterization of the C3G-NaCas interaction indicated that the fluorescence quenching mechanism was predominantly static. C3G interacted with two sets of binding sites with association constants Ka of 106 and 105M-1. Electrostatic interactions dominated at pH 7, while hydrophobic effects were the main force at pH 2. Interestingly, the two sets of binding sites were discriminated by ionic strength at pH 7. The binding of C3G slightly modified the average diameter of NaCas nanoparticles without alteration of its surface charge suggesting a complexation of C3G molecules in the internal casein structure. Thus, NaCas constitutes a putative nanocarrier for anthocyanins in new functional foods.

Polar lipid composition of bioactive dairy co-products buttermilk and butterserum: Emphasis on sphingolipid and ceramide isoforms.

Bioactive lipids of the milk fat globule membrane become concentrated in two co-products of the butter industry, buttermilk and butterserum. Their lipid composition is detailed here with special emphasis on sphingolipid composition of nutritional interest, determined using GC, HPLC and tandem mass spectrometry. Butterserum was 2.5 times more concentrated in total fat than buttermilk, with 7.7±1.5vs 19.5±2.9wt% and even more concentrated in polar lipids, with 1.4±0.2vs 8.5±1.1wt%. Both ingredients constitute concentrated sources of sphingomyelin (3.4-21mg/g dry matter) and contained low amounts of bioactive ceramides in a ratio to sphingomyelin of 1:5mol% in buttermilk and 1:10mol% in butterserum. Compared to other natural lecithins, these two co-products are rich in long and saturated fatty acids (C22:0-C24:0), contain cholesterol and could have interesting applications in neonatal nutrition, but also as brain-protective, hepatoprotective and cholesterol lowering ingredients.

How the presence of a small molecule affects the complex coacervation between lactoferrin and ß-lactoglobulin.

Heteroprotein complex coacervation corresponds to the formation of two liquid phases in equilibrium induced by the interaction of two oppositely charged proteins. The more concentrated phase known as coacervate phase, has attracted interest from several fields of science due to its potential applications for example for encapsulation and delivery of bioactives. Prior such application, it is necessary to understand how the presence of small ligands affects the complex coacervation. In this work, we report on the interaction of small ligand with individual proteins ß-lactoglobulin (ß-LG) and lactoferrin (LF) and consequences on their complex coacervation. ANS (8-Anilinonaphthalene-1-sulfonic acid), a fluorescent probe, was used as model ligand. While ANS did not interact with ß-LG, it presented two sets of binding sites with LF inducing its self-aggregation. Depending on its concentration, ANS modulated the shape of ß-LG-LF macromolecular assembly. Coacervates were observed for ANS/LF molar ratio <25 against amorphous aggregates for higher ANS/LF molar ratios. A maximum loading capacity of around 40mg of ANS per gram of LF in the formed heteroprotein coacervates was reached.

Dietary bioactive peptides: Human studies.

Current opinion strongly links nutrition and health. Among nutrients, proteins, and peptides which are encrypted in their sequences and released during digestion could play a key role in improving health. These peptides have been claimed to be active on a wide spectrum of biological functions or diseases, including blood pressure and metabolic risk factors (coagulation, obesity, lipoprotein metabolism, and peroxidation), gut and neurological functions, immunity, cancer, dental health, and mineral metabolism. A majority of studies involved dairy peptides, but the properties of vegetal, animal, and sea products were also assessed. However, these allegations are mainly based on in vitro and experimental studies which are seldom confirmed in humans. This review focused on molecules which were tested in humans, and on the mechanisms explaining discrepancies between experimental and human studies.

Heteroprotein complex coacervation: A generic process.

Proteins exhibit a rich diversity of functional, physico-chemical and biodegradable properties which makes them appealing for various applications in the food and non-food sectors. Such properties are attributed to their ability to interact and assemble into a diversity of supramolecular structures. The present review addresses the updated research progress in the recent field of complex coacervation made from mixtures of oppositely charged proteins (i.e. heteroprotein systems). First, we describe briefly the main proteins used for heteroprotein coacervation. Then, through some selected examples, we illustrate the particularity and specificity of each heteroprotein system and the requirements that drive optimal assembly into coacervates. Finally, possible and promising applications of heteroprotein coacervates are mentioned.

Structure and Dynamics of Heteroprotein Coacervates.

Under specific conditions, mixing two oppositely charged proteins induces liquid-liquid phase separation. The denser phase, or coacervate phase, can be potentially applied as a system to protect or encapsulate different bioactive molecules with a broad range of food and/or medical applications. The optimization of the design and efficiency of such systems requires a precise understanding of the structure and the equilibrium of the nanocomplexes formed within the coacervate. Here, we report on the nanocomplexes and the dynamics of the coacervates formed by two well-known, oppositely charged proteins ß-lactoglobulin (ß-LG, pI ≈ 5.2) and lactoferrin (LF, pI ≈ 8.5). Fluorescence recovery after photobleaching (FRAP) and solid-state nuclear magnetic resonance (NMR) experiments indicate the coexistence of several nanocomplexes as the primary units for the coacervation. To our knowledge, this is the first evidence of the occurrence of an equilibrium between quite unstable nanocomplexes in the coacervate phase. Combined with in silico docking experiments, these data support the fact that coacervation in the present heteroprotein system depends not only on the structural composition of the coacervates but also on the association rates of the proteins forming the nanocomplexes.

Adsorption of gastric lipase onto multicomponent model lipid monolayers with phase separation.

The enzymatic lipolysis of complex natural lipoproteic assemblies such as milk fat globules is central in neonatal digestion. This process first requires the rapid adsorption of a lipolytic enzyme, gastric lipase, onto the membrane enveloping the triglyceride substrate before the onset of catalytic activity. The interactions governing lipase adsorption onto this complex lipid/water interface are not fully elucidated. This study was designed to unravel the interactions of recombinant dog gastric lipase (rDGL) with model monolayers presenting liquid-liquid phase coexistence and mimicking the outer leaflet of the milk fat globule membrane. Combining biophysical tools (ellipsometry, tensiometry and atomic force microscopy), it was evidenced that rDGL partitions toward liquid expanded phase and at phase boundaries. rDGL gets adsorbed at several levels of insertion suggesting molecular cooperation that may favor insertion and strongly impacts on the lipid phase lateral organization. The addition of phosphatidylserine, negatively charged, reinforced adsorption; hence besides hydrophobic interactions and as further investigated through surface potential modeling, rDGL adsorption is favored by electrostatic interactions.