Improving the colloidal stability of pectin-phycocyanin complexes by increasing the mixing ratio.

In the food industry, the phycobiliprotein phycocyanin acts as a color pigment or the functional part of the superfood "Spirulina." It is industrially extracted from Arthrospira platensis. Current scientific research is focusing on finding complex partners with the potential to stabilize phycocyanin against its sensitivity toward heating and pH changes. Less attention is paid to the factors that influence complexation. This study focuses on the mixing ratio of phycocyanin with pectin. Phycocyanin concentration was fixed, and the mixing ratios ranged from 0.67 to 2.50 (pectin:phycocyanin). All samples were analyzed for their color, size, microscopic structure, zeta potential, and sedimentation stability before and after heating at 85°C. It was found that increasing the pectin content fostered the initial interactions with the protein and chromophore, resulting in a color shift from blue to turquoise. The size of the complexes decreased from several micrometers to nanometers with increasing pectin concentration. Those smaller complexes that were formed at a mixing ratio of 2.5 showed a higher colloidal stability over a period of ∼2 days. It is suggested that at a low mixing ratio (0.67), phycocyanin cannot be completely entrapped within the complexes and attaches to the complex surface as well. This results in aggregation and precipitation of the complexes upon heating. With increasing aggregation and consequently size as well as density of the complexes, sedimentation was accelerated. PRACTICAL APPLICATION: Under acidic conditions, as found in many foods and beverages (e.g., soft drinks, hard candy), phycocyanin tends to agglomerate and lose its color. Specifically heating, triggers denaturation, causing phycocyanin to aggregate and lose vital protein-chromophore interactions necessary to maintain a blue color. To prevent precipitation of the phycocyanin-pectin complexes, increasing the amount of pectin to a ratio of at least 2.0 is effective. This illustrates how adjusting the mixing ratio improves stability. Conversely, lower mixing ratios  induce color precipitation, valuable in purification processes. Thus, practical use of biopolymer-complexes, requires determination of the optimal mixing ratio for the desired effect.

Ascorbic acid-induced degradation of liposome-encapsulated acylated and non-acylated anthocyanins of black carrot extract.

BACKGROUND: In the presence of ascorbic acid, the degradation of acylated (sinapic, ferulic and p-coumaric acid derivatives of cyanidin-3-xylosylglucosylgalactoside) and non-acylated anthocyanins of black carrot extract (BCE) encapsulated in liposomes was studied. BCEs (0.2% and 0.4% w/w) were encapsulated in liposomes using different lecithin concentrations (1%, 2% and 4% w/w). RESULTS: The liposomes were prepared with particle diameters of less than 50 nm and zeta potentials of about -21.3 mV for extract-containing liposomes and -27.7 mV for control liposomes. The encapsulation efficiency determined using high-performance liquid chromatography (HPLC) showed that increasing lecithin levels increased the efficiency to 59% at the same extract concentration. The concentrations of total anthocyanins and individual anthocyanins were determined for ascorbic acid (0.1% w/w)-degraded extract and liposomes (containing 0.2% w/w extract). Anthocyanin quantification of both liposomal and extract samples was performed by HPLC using cyanidin-3-O-glucoside chloride as standard. Five anthocyanins in the extract and encapsulated liposomes were quantified during 24 h (0-24 h): cyanidin-3-xylosylglucosylgalactoside 1.0-0.51 and 0.82-0.58 mg g-1 , cyanidin-3-xylosylgalactoside 2.5-1.1 and 2.2-1.7 mg g-1 , cyanidin-3-xylosyl(sinapoylglucosyl)galactoside 0.51-0.14 and 0.35-0.28 mg g-1 , cyanidin-3-xylosyl(feruloylglucosyl)galactoside 1.37-0.41 and 1.06-0.98 mg g-1 , and cyanidin-3-xylosyl(coumaroylglucosyl)galactoside 0.28-0.08 mg g-1 for extract and 0.27-0.26 mg g-1 for liposomes, respectively. CONCLUSIONS: This study demonstrates the potential beneficial effect of liposomal encapsulation on individual, particularly acylated, anthocyanins after addition of ascorbic acid during a storage time of 24 h.

Formation and characterization of plant-based emulsified and crosslinked fat crystal networks to mimic animal fat tissue.

Animal fat tissue (that is, pork or beef fat) is composed of liquid and solid fat incorporated in a network of connective tissue. Hence, their rheological and thermal properties may differ significantly from plant-derived fats. Specifically, animal fats have elastic and melting properties that give rise to not only a certain comminution behavior during processing, but also provide meat products such as sausages with certain organoleptic properties. To mimic key properties of animal fat tissue with plant-derived materials, a new structuring approach was used. Canola oil was mixed with <30% (w/w) of fully hydrogenated canola oil at 65 °C, hot-emulsified with a soy protein suspension (8%, w/w) at a lipid content of 70% (w/w) using a high-shear disperser, and cooled to 37 °C. The concentrated, emulsified fat crystal networks were then incubated with transglutaminase for 1 hr to induce protein crosslinking. Microscopy images showed that samples were composed of tightly packed lipid particles with regions of coalesced or unemulsified lipids appearing at higher solid fat concentrations. Texture analysis and rheological measurements showed that crosslinked samples possessed elasticities that decreased with increasing solid fat concentration. Above 30% solid fat, matrices reverted back to exhibiting a mainly plastic behavior. Results were attributed to the formation of either a droplet-filled protein network, a particulate fat crystal network, or a mixture thereof. Taken together, results show that plant-based crosslinked emulsified fat crystal networks are able to mimic mechanical properties of animal fat provided that not too much solid fat (<30% in this study) is used. This makes them useful for the manufacture of meat products or analogues. PRACTICAL APPLICATION: This study introduced a new structuring approach to mimic properties of animal fat tissue with only plant-derived materials. The structured lipids can, for example, be used for the manufacture of processed meat analogues.

Physical and chemical stability of anthocyanin-rich black carrot extract-loaded liposomes during storage.

In this study, the protection effect of black carrot extract on the stability of liposomes during storage was evaluated. The physical and chemical stability of the extract (0.4% w/w) including extract-loaded liposomes with various lecithin content (1%, 2%, and 4% w/w) were demonstrated. Z-average particle diameter and zeta potentials of liposomes were evaluated before and after 21 days of storage. The particle diameter of the samples was found to be lower than 50 nm after storage and no statistical change was determined (p < 0.05). The liposomes with and without extract were visualized by transmission electron microscopy (TEM) after negative staining. The TEM images revealed that unloaded and extract-loaded liposomes are similar in size. The degradation of extract (30%-90%), phenolic content (10%-29%), and antioxidant capacity (4%-33%) of liposomes depending on lecithin content were also determined during storage to evaluate the biochemical stability. A hexanal analysis was performed to investigate the lipid oxidation in liposomes within 60 days. According to the results, the oxidation of polyunsaturated fatty acids may be inhibited with the addition of the extract to liposomes; however, lipid content should be limited according to the extract concentration. The highest protection on oxidation was observed in extract-loaded liposomes containing 1% lecithin. The study provided valuable data on the contribution of an anthocyanin addition to liposomes to overcome oxidation of unsaturated phospholipids.

Impact of liposomal encapsulation on degradation of anthocyanins of black carrot extract by adding ascorbic acid.

Black carrot anthocyanins are known to be relatively stable because they contain acylated anthocyanins. The degradation of vitamin C (l-ascorbic acid) on anthocyanins is a known fact in beverage systems. In this study, the effects of various liposomal systems, including black carrot extract (0.1%, 0.2%, 0.4% w/w) and lecithin (1%, 2%, 4% w/w), on the color and degradation of anthocyanin in different ascorbic acid (0.01%, 0.025%, 0.05%, 0.1% w/w) concentrations were examined via UV/VIS spectroscopy and visual control of the color. The physical characteristics of the liposomal systems resulted in particle diameters of 41-46 nm and zeta-potentials of (-23)-(-20) mV. The encapsulation efficiencies of the liposomal systems increased up to 50% with increasing lecithin concentrations. The encapsulation of black carrot extract in liposomes enhanced the color and stability of the anthocyanins during storage. This study showed that the degradation of anthocyanins due to ascorbic acid can be reduced by liposomes in aqueous solutions.

Initial Droplet Size Impacts pH-Induced Structural Changes in Phase-Separated Polymer Dispersions.

The effect of pH change on the morphology of whey protein isolate (WPI)-pectin dispersions obtained from phase-separated systems after mild shear was studied. The purpose of this study was to examine the impact of mixing speed on the initial particle size of biopolymer complexes and their structure morphology after sequentially changing the pH. Therefore, solutions of WPI and pectin were combined at pH 6.1, allowed to phase separate and were then mildly homogenized at 50, 100, and 150 rpm, respectively, to form a dispersion containing differently sized WPI droplets in a surrounding pectin-rich phase. Each dispersion was then subjected to a pH change, such as 6.1 to 5.2 and 3.2, by slowly adding hydrochloric acid. The systems morphology, size, appearance, rheology, and storage stability was then characterized by optical microscopy, static light scattering, visual inspections, and steady shear rheometry to gain insights into the structural rearrangements. Results indicated substantial changes in the structure of the dispersion when the pH was changed. Formation of core-shell structures from the WPI droplets was observed at an intermediate pH. There, initial droplet size was found to affect structures formed, that is, core-shell type particles would only form if droplets were large (>1.5 µm) prior to pH change. Insights gained may be of importance to food manufacturers intending to create new structures from mixtures of proteins and carbohydrates.

Influence of droplet size on the antioxidant activity of rosemary extract loaded oil-in-water emulsions in mixed systems.

The influence of droplet size on the antioxidant activity of oil-in-water emulsions loaded with rosemary extract in mixed emulsion systems was investigated. Firstly, differently sized hexadecane-in-water model emulsions (10% (w/w) hexadecane, 2% (w/w) Tween 80, pH 5 or 7) containing 4000 ppm rosemary extract in the oil phase or without added antioxidant were prepared using a high shear blender and/or high-pressure homogenizer. Secondly, emulsions were mixed with fish oil-in-water emulsions (10% (w/w) fish oil, 2% (w/w) Tween 80, pH 5 or 7) at a mixing ratio of 1 : 1. Optical microscopy and static light scattering measurements indicated that emulsions were physically stable for 21 days, except for the slight aggregation of emulsions with a mean droplet size d43 of 4500 nm. The droplet size of hexadecane-in-water emulsions containing rosemary extract had no influence on the formation of lipid hydroperoxides at pH 5 and 7. Significantly lower concentrations of propanal were observed for the emulsions loaded with rosemary extract with a mean droplet size d43 of 4500 nm from day 12 to 16 at pH 7. Finally, hexadecane-in-water emulsions containing rosemary extract significantly retarded lipid oxidation of fish oil-in-water emulsions in mixed systems, but no differences in antioxidant efficacy between the differently sized emulsions were observed at pH 5.

Inhibitory effect of liposomal solutions of grape seed extract on the formation of heterocyclic aromatic amines.

The effectiveness of grape seed extract (GSE) encapsulated in liposomes to inhibit the formation of heterocyclic aromatic amines (HAA) during frying of beef patties was assessed. All liposomal systems were prepared by high pressure homogenization at 22 500 psi. A total of six samples (rapeseed oil (control), GSE at 0.1% and 0.2%, and GSE-containing liposomes with 1%, 2%, and 5% soy lecithin) were investigated. MeIQx (2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline), PhIP (2-amino-1-methyl-6-phenylimidazo[4,5b]pyridine), Norharman, and Harman were found after the marinade application and frying. PhIP concentrations decreased upon marination with GSE (0.1%) and GSE-containing liposomes (1% and 5%) (p < 0.05). MeIQx contents decreased in all samples compared to the oil control (p < 0.01) while no effect on ß-carboline formation was observed. Results are in contrast to previous studies that had shown that liposomal encapsulation may enhance effectiveness of polyphenols to inhibit radical reactions. A mechanistic model was proposed to explain the observed differences.

Formation of heterocyclic amines in salami and ham pizza toppings during baking of frozen pizza.

Heterocyclic amines (HAs) are formed as Maillard reaction products in the crust of meat products during heating processes. Two typical pizza toppings--salami and cooked ham--were analyzed for the presence of HAs after baking frozen pizzas at top and bottom temperatures of 250 and 230 °C, respectively. After baking pizza slices for 12 min, MeIQx (2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline; 0.2 ng/g), 4,8-DiMeIQx (2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline; 0.5 ng/g), PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; 0.2 ng/g), norharman (4.5 ng/g), and harman (2.5 ng/g) were found in the ham toppings, whereas only the comutagenic norharman (107.4 ng/g) and harman (11.4 ng/g) were found in the salami toppings. The content of MeIQx and 4,8-DiMeIQx in ham increased from 0.3 to 1.8 ng/g and 0.8 to 1.6 ng/g, respectively, when the recommended baking time was increased from 15 min (manufacturer's specification) to 18 min at 230 °C. MeIQx was formed in salami when the heating time was extended to 18 min. Moreover, higher concentrations of PhIP in salami or ham slices were found when baking temperatures were 250 °C rather than 230 °C (baking time of 12 min). However, sensory tests showed that panelists preferred longer-baked pizzas due to an increased crispiness. Thus, results show that a substantial formation of HAs may occur in pizza toppings such as ham and salami, with ham being particularly susceptible when compared to salami. Formation of HAs increases with increasing baking time and temperature. The occurrence of the cupping of ham or salami slices during baking may also increase the formation of HAs.

Antioxidant capacity and inhibitory effect of grape seed and rosemary extract in marinades on the formation of heterocyclic amines in fried beef patties.

The effect of oil-based marinades containing grape seed extract (Vitis vinifera L.; 0.2, 0.4, 0.6 and 0.8 g/100g) formulated in a water/oil emulsion or rosemary extract (Rosmarinus officinalis; 0.12, 0.2, 0.6, 1.0 and 1.5 g/100g) in oil on the formation of heterocyclic amines (HAs) in fried beef patties was examined. After application of marinades and frying, four HAs MeIQx (2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline), PhIP (2-amino-1-methyl-6-phenylimidazo[4,5b]pyridine), Norharman, and Harman were found at low levels in all fried patties, MeIQx (0.3-1.0 ng/g), and PhIP (0.02-0.3 ng/g). The content of MeIQx and PhIP were significantly reduced by approx. 57% and 90% (p<0.05), respectively, after use of marinades containing the highest extract concentration. The antioxidant capacity of grape seed was about two-times greater than that of rosemary extract. A correlation between inhibition of HAs and Trolox-equivalents (MeIQx, R(2)=0.85, p<0.001; PhIP, R(2)=0.83, p<0.001) was found. Sensory tests showed a high acceptance of flavour and colour for controls and samples.

Influence of interfacial properties on Ostwald ripening in crosslinked multilayered oil-in-water emulsions.

The influence of interfacial crosslinking, layer thickness and layer density on the kinetics of Ostwald ripening in multilayered emulsions at different temperatures was investigated. Growth rates of droplets were measured by monitoring changes in the droplet size distributions of 0.5% (w/w) n-octane, n-decane, and n-dodecane oil-in-water emulsions using static light scattering. Lifshitz-Slyozov-Wagner theory was used to calculate Ostwald ripening rates. A sequential two step process, based on electrostatic deposition of sugar beet pectin onto fish gelatin or whey protein isolate (WPI) interfacial membranes, was used to manipulate the interfacial properties of the oil droplets. Laccase was added to the fish gelatin-beet pectin emulsions to promote crosslinking of adsorbed pectin molecules via ferulic acid groups, whereas heat was induced to promote crosslinking of WPI and helix coil transitions of fish gelatin. Ripening rates of single-layered, double-layered and crosslinked emulsions increased as the chain length of the n-alkanes decreased. Emulsions containing crosslinked fish gelatin-beet pectin coated droplets had lower droplet growth rates (3.1±0.3×10(-26) m(3)/s) than fish gelatin-stabilized droplets (7.3±0.2×10(-26) m(3)/s), which was attributed to the formation of a protective network. Results suggest that physical or enzymatic biopolymer-crosslinking of interfaces may reduce the molecular transport of alkanes between the droplets in the continuous phase.

Influence of droplet size on the efficacy of oil-in-water emulsions loaded with phenolic antimicrobials.

In this study we investigated the effect of droplet size on the antimicrobial activity of emulsions containing two essential oil compounds that are known for their antimicrobial effectiveness: carvacrol and eugenol. Coarse emulsions were prepared by blending a triacylglyceride (Miglyol 812N) containing various concentrations of carvacrol or eugenol (5, 15, 30, 50 wt%) at an oil droplet mass fraction of 10 wt% with an aqueous phase containing 2 wt% Tween 80(®). Premixes were then further dispersed using a high shear blender, a high pressure homogenizer at different pressures or an ultrasonicator to produce droplets with a variety of mean diameters. Microscopy and light scattering storage stability studies over 10 days indicated that manufactured emulsions were stable, i.e. that no aggregation, creaming or other destabilization mechanisms occurred and droplet size distributions remained unchanged. The antimicrobial activity of emulsions was assessed against two model microorganisms, the Gram negative Escherichia coli C 600 and the Gram positive Listeria innocua, by determining growth over time behavior. The analysis yielded the unexpected result that emulsions with larger droplet sizes were more effective at inhibiting growth and inactivating cells than smaller ones. For example, emulsions with a mean oil droplet size of 3000 nm at a concentration of 800 ppm carvacrol completely inhibited L. innocua, while for 80 nm emulsions, only a delay of growth could be observed. Measurements of the concentration of the antimicrobial compounds in the aqueous phase indicated that concentrations of eugenol and carvacrol decreased with decreasing oil droplet sizes. Determination of interfacial tension further showed that eugenol and carvacrol are preferentially located in the oil-water interfaces. Theoretical calculations of Tween 80(®) concentrations needed to saturate interfaces suggested that in small emulsions for the given formulation less Tween 80(®) micelles are present in the aqueous phase. We therefore attribute the fact that antimicrobial nanoemulsions are less active than macroemulsions due to an increased sequestering of antimicrobials in emulsion interfaces and a decreased solubilization in excess Tween 80(®) micelles.

Encapsulation of polyphenolic grape seed extract in polymer-coated liposomes.

Polyphenolic grape seed extract (0.1 w/v%) was encapsulated in liposomes (1% soy lecithin) by high pressure homogenization at 22 500 psi. Liposomes containing grape extract had mean particle diameters of <100 nm compared to control liposomes that contained no extract and that had mean particle diameters of <40 nm. Liposomes with grape seed extract were long-term oxidatively stable, e.g. significantly less hexanal (<15 µmol L(-1)) was formed during storage for 150 days compared to liposomes without extract (>717 µmol L(-1)). Over 80% of the polyphenolic compounds in grape seed extract were incorporated into the liposomal membrane rather than into their interior and compounds remained integrated even if the system pH was changed. Coated liposomes were prepared by alternately adding a cationic polymer (chitosan) and an anionic polymer (citrus pectin). Up to 4 coats could be deposited onto the liposomes without incurring aggregation, i.e. dispersions were physically stable during storage for up to 150 days. Deposition of multiple biopolymer coats onto liposomes decreased the amount of polyphenols that reacted with the Folin-Ciocalteu reagent, suggesting that the polyphenols were no longer exposed to the aqueous phase. Our results hence indicate that polymer-coated liposomes are highly capable carrier systems for polyphenols that may have reduced interactions with ingredients in complex media such as foods.

Inhibitory effect of marinades with hibiscus extract on formation of heterocyclic aromatic amines and sensory quality of fried beef patties.

Heterocyclic aromatic amines (HAA) are carcinogenic compounds found in the crust of fried meat. The objective was to examine the possibility of inhibiting HAA formation in fried beef patties by using marinades with different concentrations of hibiscus extract (Hibiscus sabdariffa) (0.2, 0.4, 0.6, 0.8 g/100g). After frying, patties were analyzed for 15 different HAA by HPLC-analysis. Four HAA MeIQx (0.3-0.6 ng/g), PhIP (0.02-0.06 ng/g), co-mutagenic norharmane (0.4-0.7 ng/g), and harmane (0.8-1.1 ng/g) were found at low levels. The concentration of MeIQx was reduced by about 50% and 40% by applying marinades containing the highest amount of extract compared to sunflower oil and control marinade, respectively. The antioxidant capacity (TEAC-Assay/Folin-Ciocalteu-Assay) was determined as 0.9, 1.7, 2.6 and 3.5 micromol Trolox antioxidant equivalents and total phenolic compounds were 49, 97, 146 and 195 microg/g marinade. In sensory ranking tests, marinated and fried patties were not significantly different (p>0.05) to control samples.

Effect of oil marinades with garlic, onion, and lemon juice on the formation of heterocyclic aromatic amines in fried beef patties.

Effects of the application of oil marinades with garlic, onion, and lemon juice on the formation of heterocyclic aromatic amines (HAAs) in fried beef patties were investigated. Two different statistical design models were used. In the screening experiment, a significant inhibition of formation of MeIQx was determined only by addition of garlic (p < 0.05). When the amount of garlic was changed from 2 to 20 g/100 g of marinade, the estimated MeIQx content in patties was reduced about 70%. MeIQx (0.38-1.22 ng/g), 4,8-DiMeIQx (n.d.-0.45 ng/g), PhIP (n.d.-0.09 ng/g), norharman (0.76-13.5 ng/g), and harman (2.9-21.5 ng/g) were found in fried patties. The results of two-level and three-level fractional factorial design experiments confirmed the first investigation. They showed a stronger reduction in MeIQx in patties with the addition of increasing amounts of garlic (p < 0.01) and onion (p < 0.05) in marinades. A higher content of lemon juice in marinades led to only a marginal reduction in MeIQx (p > 0.05). The optimum amounts of onion, garlic, and lemon juice that achieved a maximum reduction of HAAs were calculated as 31.2%, 28.6%, and 14.6% in marinade.