The optimisation of solid–liquid extraction of antioxidants from apple pomace by response surface methodology

Response surface methodology using two food grade solvents, acetone and ethanol, was used to optimise antioxidant extraction from industrially generated apple pomace. Efficiency of extraction was optimised by measuring antioxidant activity, phenol content and three individual polyphenol groups. Conditions for optimal antioxidant activity as measured by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay were 56% ethanol, 80 °C and 31 min. Using these conditions an antioxidant value of 444 mg Trolox/100 g DW was obtained. For acetone extraction the optimal conditions were 65% acetone, 25 °C and 60 min, resulting in an antioxidant value of 436 mg Trolox/100 g DW. Both ethanol and acetone would be suitable to replace methanol for a food grade and more environmental friendly solid–liquid extraction of antioxidants.     

Characterization of protein–polyphenol interactions

Dietary polyphenols have received attention for their biologically significant functions as antioxidants, anticarcinogens or antimutagens, which have led to their recognition as potential nutraceuticals. Polyphenols also characteristically possess a significant binding affinity for proteins, which can lead to the formation of soluble and insoluble protein–polyphenol complexes. Questions remain concerning whether and to what extent the protein–polyphenol interaction influences functionality. For example, is the formation of protein–polyphenol complexes an obstacle to the nutritional bioavailability of either species? This article discusses the development of suitable methodologies to investigate the physicochemical basis of protein–polyphenol interactions and the influence of structure–activity relationships on binding affinities.     

Adsorption of rutin onto a food-grade styrene–divinylbenzene copolymer in a model system

Adsorption of quercetin-3-O-rutinoside (rutin) onto Amberlite^® XAD 16HP was studied applying a modified D-optimal design. Temperature, pH value, rutin concentration and resin amount were studied as independent factors determining rutin adsorption. Using the mathematical model resulting from the analysis of the D-optimal design the optimal conditions for rutin adsorption onto the styrene–divinylbenzene copolymer were determined. Furthermore, the model equation was successfully applied to calculate the Langmuir and Freundlich isotherm parameters of this particular solute–sorbent system. In addition, the data obtained from the D-optimal design allowed the calculation of ΔG, ΔH and ΔS for the adsorption process. The results presented in this study are of utmost importance for a better understanding of adsorption phenomena. The more detailed knowledge of such sorbent systems is required to improve the cost-effectiveness of industrial processes for the recovery of secondary plant metabolites, such as polyphenols, based on resin adsorption technology.     

Effect of temperature and substrate on Pef inactivation of Lactobacillus plantarum in an orange juice–milk beverage

The inactivation kinetics of Lactobacillus plantarum in an orange juice–milk beverage treated by Pulsed Electric Fields (PEF) were studied. Experimental data were fitted to Bigelow and Hülsheger kinetic models and Weibull frequency distribution function. Results indicate that both Hülsheger model and Weibull function fit well the experimental data being Accuracy factor values (Af) closer to 1 and Mean Square Error (MSE) closer to 0. The parameter of the Weibull model can be considered as a kinetic indicator as it expresses the microorganism's resistance to treatment by electric pulses. An increase in temperature favoured the inactivation of L. plantarum by PEF as reflected by a decreased in value. Under the same conditions to those studied by other authors we reached less inactivation of L. plantarum in the beverage used in this study than in substrates with a simpler composition.     

Emulsifying properties of soy whey protein isolate–fenugreek gum conjugates in oil-in-water emulsion model system

Soy whey protein isolate (SWPI)–fenugreek gum conjugates were prepared by Maillard type reactions in a controlled dry state condition (60 °C, 75% relative humidity for 3 days) to improve emulsification properties. SDS-PAGE electropherogram showed that conjugation formed high molecular weight products with the disappearance of 7S fraction, acidic subunits of the 11S fractions and protein band at molecular weight 21 and 24 kDa. However, the amount of protein at molecular weight 30 kDa remained unchanged. The protein solubility of SWPI–fenugreek gum conjugates improved as compared to SWPI and SWPI–fenugreek gum non-conjugated mixture especially at isoelectric point of protein when assessed in the pH range 3–8 at 22 °C. In comparison to SWPI, fenugreek gum and non-conjugated SWPI–fenugreek gum, SWPI–fenugreek gum conjugates had better emulsifying properties near the isoelectric pH of protein. Emulsification at near the isoelectric pH of protein was chosen as at this pH the proteins are prone to aggregate, which could destabilize the emulsion. Heating solutions of the conjugates prior to emulsification further improved their emulsification properties. The conjugates also showed a better emulsifying property at high salt concentration as compared to SWPI alone.     

Effect of β-lactoglobulin A and B whey protein variants on cheese yield potential of a model milk system

Cheese yield mainly depends on the amount and proportion of milk constituents; however, genetic variants of the proteins present in milk may also have an important effect. The objective of this research was to study the effect of the variants A and B of β-lactoglobulin (LG) on cheese yield using a model system consisting of skim milk powder fortified with different levels of a mixture containing α-lactalbumin and β-LG genetic variants (A, B, or A-B) in a 1:2 ratio. Fortified milk samples were subjected to pasteurization at 65°C for 30 min. Miniature cheeses were made by acidifying (pH = 5.9) fortified milk and incubating with rennet for 1 h at 32°C. The clot formed was cut, centrifuged at 2,600 × g for 30 min at 20°C and drained for determining cheese yield. Cheese-yielding capacity was expressed as actual yield (grams of cheese curd per 100 g of milk) and dry weight yield (grams of dried cheese curd per 100 g of milk). Free-zone capillary electrophoresis was used for determining β-LG A or B recovery in the curd during rennet-induced coagulation. The presence of β-LG variant B resulted in a significantly higher actual and dried weight cheese yield than when A or A-B were present at levels ≤0.675% of whey protein (WP) addition. Results of free-zone capillary electrophoresis allowed us to infer that β-LG B associates with the casein micelles during renneting, as shown by an increase in the recovery of this variant in the curd when β-LG B was added up to a maximum at 0.45% (equivalent to 0.675% WP). In general, actual or dried weight cheese yield increased as WP addition was increased from 0.225 to 0.675%. However, when WP addition ranged from 0.675 to 0.90%, a drastic drop in cheese yield was observed. This behavior may be because an increase in the aggregation of casein micelles with a concomitant inclusion of whey protein in the gel occurs at low levels of WP addition, whereas once the association of WP with the casein micelles reach a saturation point at addition levels higher than 0.675%, rearrangements of the gel network result in larger whey expulsion and syneresis. This knowledge is expected to be useful to maximize cheese yield and optimize processing conditions during cheese and cheese analogs manufacturing.     

Characteristics of a calcium–milk coagulum

The texture, microstructure and composition of a milk coagulum obtained by coagulating milk with calcium chloride and heat were evaluated. A direct relationship was established between the microstructure and texture of the calcium–milk coagulum. The microstructure was comprised of casein micelles that were interlinked by appendages or fused with each other depending on the nature of the pre-heat treatment employed. The casein micellar network also entrapped fat globules. The pre-heat treatment of milk and the pH of milk during coagulation affected the hardness, adhesiveness, protein content and microstructure of the calcium–milk coagulum significantly. This study characterises a calcium–milk coagulum as a novel dairy product which differs from an acid coagulum in texture, microstructure and composition.     

Biological activities of Maillard reaction products (MRPs) in a sugar–amino acid model system

The various biological activities of Maillard reaction products (MRPs) from sugar (fructose and glucose) and 20 amino acid model systems were evaluated. Colour development, in vitro antioxidant, α-glucosidase inhibitory, antihypertensive, and antiproliferative activities of aqueous solutions of MRPs produced by heating at 130 °C for 2 h were measured. The fructose–amino acid mixture showed higher UV-absorbance and browning intensity than the glucose–amino acid mixture. The fructose–amino acid model MRPs showed higher DPPH and ABTS radical scavenging and ACE inhibitory activities than the glucose–amino acid model MRPs. The α-glucosidase inhibitory effect of MRPs derived from fructose– and glucose–tyrosine showed higher α-glucosidase inhibitory activity than that of other MRPs. Sugar–amino acid model MRPs inhibited the growth of HCT116 colon cancer cell in a dose-dependent manner (from 0.5 to 1.5 mg/ml). Glucose MRPs showed slightly higher antiproliferative activity than fructose MRPs. In particular, sugar–tryptophan and –tyrosine MRPs exerted higher biological activities than the other MRPs.     

A chemical study of β-carotene oxidation by ozone in an organic model system and the identification of the resulting products

Carotenoids are present in many foods. Due to their polyenic chains, they undergo oxidation reactions which may give several compounds. Ozone, a powerful antimicrobial agent, is applied in the food industry due to its high reactivity and penetrability. This work presents a chemical study of the degradation of β-carotene in solutions, under the influence of ozone. The experiments were carried out at ozone concentrations ranging from 0.8 to 2.5 ppm and the β-carotene solutions were sampled and analysed from zero to seven hours of reaction. The oxidation products were collected in C18 cartridges coated with dinitrophenylhydrazine and the hydrazones formed were analysed by LC-MS. The oxidation reaction was found to follow a zero order kinetic model and the β-carotene decay ranged between 17.2% and 99.8%. Fourteen oxidation products were tentatively identified, amongst them eight which had not been cited yet in the literature as oxidation products of β-carotene.     

Solid-state fermentation of apple pomace using Phanerocheate chrysosporium – Liberation and extraction of phenolic antioxidants

Apple pomace is a by-product from the apple processing industry and can be used for the production of value-added phenolic compounds. A study was carried out to understand the changes and liberation of phenolic compounds and improvement in antioxidant activity during solid-state fermentation of apple pomace using Phanerocheate chrysosporium. The solid-state fermentation of apple pomace using P. chrysosporium mobilised the polyphenolic compounds and improved the nutraceutical properties. The polyphenol content in acetone extract increased and the results were statistically significant (P < 0.05) from 4.6 to 16.12 mg GAE/g dry weight during solid-state fermentation. The effect of various solvents, temperature, time and detergents were also investigated for the extraction of polyphenolics by ultrasonication and microwave-assisted extraction methods. The polyphenol content of the extracts was found to be in the range of 5.78–16.12 mg GAE/g DW of samples, depending on the solvent, extraction time and temperature. Antioxidant activities of polyphenol extracts were tested using the 2,2-diphenyl-1-picryhydrazyl (DPPH) radical methods, where the IC₅₀ ranged from 12.24 to 23.42 μg DW sample, depending on the extraction conditions and the antioxidant activities correlated well with the polyphenol concentrations.     

Analytical techniques for the study of polyphenol–protein interactions

This mini review focuses on advances in biophysical techniques to study polyphenol interactions with proteins. Polyphenols have many beneficial pharmacological properties, as a result of which they have been the subject of intensive studies. The most conventional techniques described here can be divided into three groups: (i) methods used for screening (in-situ methods); (ii) methods used to gain insight into the mechanisms of polyphenol–protein interactions; and (iii) methods used to study protein aggregation and precipitation. All of these methods used to study polyphenol–protein interactions are based on modifications to the physicochemical properties of the polyphenols or proteins after binding/complex formation in solution. To date, numerous review articles have been published in the field of polyphenols. This review will give a brief insight in computational methods and biosensors and cell-based methods, spectroscopic methods including fluorescence emission, UV-vis adsorption, circular dichroism, Fourier transform infrared and mass spectrometry, nuclear magnetic resonance, X-ray diffraction, and light scattering techniques including small-angle X-ray scattering and small-angle neutron scattering, and calorimetric techniques (isothermal titration calorimetry and differential scanning calorimetry), microscopy, the techniques which have been successfully used for polyphenol–protein interactions. At the end the new methods based on single molecule detection with high potential to study polyphenol–protein interactions will be presented. The advantages and disadvantages of each technique will be discussed as well as the thermodynamic, kinetic or structural parameters, which can be obtained. The other relevant biophysical experimental techniques that have proven to be valuable, such electrochemical methods, hydrodynamic techniques and chromatographic techniques will not be described here.     

Time-dependent flow properties of starch–milk–sugar pastes

The time-dependent flow properties of starch–milk–sugar (SMS) pastes have been studied. The flow properties were assessed from the measurement of the shear stress versus time of shearing at constant shear rate. Corn and wheat starches were used in this study, while the sugars were glucose, sucrose, and fructose. The Weltman model was used to evaluate the flow properties of SMS pastes prepared under different conditions. SMS pastes heated at 95 and 85 °C exhibited a thixotropic behavior, while pastes heated at 75 °C behaved like a rheopectic fluid. It was noted that the thixotropy occurred at high shear stress (above 50 Pa), and the rheopexy occurred at low shear stress (below 45 Pa). The degree of thixotropy, as assessed by the Weltman model parameters, increased significantly with starch concentration, and with less pronounced effect with sugar concentration. The effect of sugar type on the degree of thixotropy of SMS pastes heated at 95 °C decreased in the following order: fructose>sucrose>glucose. The type of starch played a role in the time-dependent flow properties of the SMS paste, with a general conclusion that wheat starch had a greater degree of thixotropy than corn starch.     

Casein–whey protein interactions in heated milk: the influence of pH

Heat-treatment of milk causes denaturation of whey proteins, leading to a complex mixture of whey protein aggregates and whey protein coated casein micelles. In this paper we studied the effect of pH-adjustment of milk (6.9–6.35) prior to heat-treatment on the distribution of denatured whey proteins in aggregates and coating of casein micelles. Proteins were fractionated using an alternative fractionation technique based on renneting. Acid- and rennet-induced gelation of these milks were used to obtain more information on the characteristics of the milk. Acid-induced gelation appeared to be mainly influenced by the presence of whey protein aggregates. Rennet-induced gelation was determined by the whey protein coating of the casein micelles. Both the quantity of whey proteins present on the surface of the casein micelles and the homogeneity of the coating were determining the renneting properties. These results extend the current knowledge on pH dependent casein–whey protein interactions. In order to present a clear picture of the changes occuring during heat treatment of milk at various pH, the results are summarized in a model. In this model we propose that heating at a pH>6.6 lead to a partial coverage of the casein micelles and the formation of separate whey protein aggregates. Heating at a pH<6.6 lead to an attachment of all whey proteins to the casein micelles. At pH 6.55 the coverage is rather homogeneous but lowering the pH further lead to an inhomogeneus coverage of the casein micelles. Surprisingly small changes of the pH at which the milk was heated had considerable effects on the gelation behaviour both in renneting and in acid gelation.     

Addition of gelatin enhanced gelation of corn–milk yogurt

Corn–milk yogurt set by a combination of sodium caseinate plus gelatin at concentrations of 0, 0.2, 0.4 and 0.6% (w/v) were studied. The quality of the gels was determined by measurement of acidity, syneresis, texture profile analysis, viscoelasticity, structure scanning electron microscope and microbiology. Texture profile analysis (TPA) showed that increasing levels of gelatin increased hardness, adhesiveness and springiness as well as the acidity of the products. Viscoelastic behaviour displayed similar trends to the TPA characteristics, the storage modulus was less frequency dependent than the loss modulus giving a loss tangent of 0.2 in the high gelatin systems, which might indicate a true gel system. The microstructure was dense and spongy-like with small air cells, in particular, those having a high concentration of gelatin (0.6%, w/v) gave a very firm structure which might impair palatability. The addition of a commercial gelatin at 0.4% (w/v) gave good acceptability for the product (little syneresis of the gels produced). While the gelatin used for this study had a bloom value of 246 g the authors acknowledge that a different commercial gelatin may well result in a different concentration being required.     

Determination of volatile compounds formed in a glucose–selenomethionine model system by gas chromatography–atomic emission detector and gas chromatography–mass spectrometry

In order to gain a better understanding of the formation of organoselenium compounds in food system, the Maillard reaction of selenomethionine and glucose was studied in a model system. The effects of heating time and pH on the volatile compounds formed in a glucose–selenomethionine reaction were also investigated. Nine organoselenium compounds were identified. Pyrazines and dimethyldiselenide are major volatile compounds generated from the glucose–selenomethionine model system. A high pH level favours the formation of pyrazine and dimethyldiselenide. In unbuffered systems, a pH change of three or more pH units may occur, and this may significantly affect the formation of Maillard reaction products.     

Effect of adzuki bean sprout fermented milk enriched in γ-aminobutyric acid on mild depression in a mouse model

This study focused on the ability of adzuki bean (Vigna angularis) sprout fermented milk, which is rich in γ-aminobutyric acid (GABA), to relieve anxiety and mild depression. A high-yield GABA-producing strain, Lactobacillus brevis J1, from a healthy cow was screened, and its physiological and probiotic properties were evaluated. The effect of adzuki bean sprout fermented milk was investigated in vivo in a chronic depression mouse model. The results showed that Lb. brevis J1 had excellent probiotic properties, grew well at low pH and 3% NaCl, and adhered to the surface of HT-29 cells. The GABA-enriched (241.30 ± 1.62 µg/mL) adzuki bean sprout fermented milk prepared with Streptococcus thermophilus, Lactobacillus bulgaricus, and Lactobacillus plantarum, and Lb. brevis J1 can reduce and possibly prevent mild depression-like symptoms in mice (C57/B6) by increasing social interaction and enhancing the pleasure derived from movement. The research revealed that the GABA_B-cyclic AMP-protein kinase A-cAMP-response element binding protein (GABA_B-cAMP-PKA-CREB) signaling pathway was related to the depression-like symptoms and that levels of 5-hydroxytryptamine, norepinephrine, and dopamine in the hippocampus of mice increased after treatment with the adzuki bean sprout fermented milk. Our results suggest that GABA-enriched dairy products have the potential to prevent or treat mild depression-like symptoms in mice, which suggests a new approach for a dietary therapy to treat chronic social stress.     

Elimination of matrix effects in the determination of bisphenol A in milk by solid-phase microextraction–high-performance liquid chromatography

Solid-phase microextraction coupled to high-performance liquid chromatography (SPME–HPLC) with fluorescence detection was employed to determine bisphenol A (BPA) in milk samples. The potential influence of the milk matrix on the determination of BPA by SPME–HPLC were investigated. Optimal conditions to eliminate any matrix effects were as follows: milk samples were deproteinized with trichloroacetic acid, diluted 20-fold with BPA-free Ultrapure water, dissolved in methanol, the precipitated protein was filtered out, rinsed with methanol and evaporated to remove the methanol. Then, a 40.0-ml solution was used for SPME extraction and HPLC analysis. Satisfactory recoveries (milk: 93.1–101%; soybean milk: 93.9–102%) were achieved. The proposed method was successfully applied to real samples, BPA being detected within the range 1.6–2.6 ng ml^?1 in four brands of commercial milk but not in soybean milk.     

Effect of storage conditions on furosine formation in milk–cereal based baby foods

The effect of storage during 9 months at 25, 30 and 37 °C on furosine formation in three milk–cereal based baby foods was studied to evaluate development of the Maillard reaction. Furosine was measured by HPLC-UV. Immediately after the manufacturing process, furosine contents were 310–340 mg/100 g protein and at the 9th storage month were 426–603 mg/100 g protein. Storage time and temperature have a significant increase (p < 0.05) of furosine content during storage. Furosine contents were higher in sample containing honey than in those without honey. Interactions (p < 0.05) between storage time and temperature or type of sample were found. A predictive model equation of the evolution of furosine during storage explaining 80% of the variability in furosine content was obtained. The blockage of lysine through storage calculated using the furosine and total lysine provided values ranged from 9.5% to 18.1% for analysed baby foods.     

Structure and antioxidant activity of Maillard reaction products from α-lactalbumin and β-lactoglobulin with ribose in an aqueous model system

Maillard reaction products (MRPs) were prepared from aqueous model mixtures containing 3% (w/w) ribose and 3% (w/w) of the dairy proteins α-lactalbumin (α-LA) or β-lactoglobulin (β-LG), heated at 95 °C, for up to 5 h. The pH of MRPs decreased significantly during heat treatment of α-LA-Ribose and β-LG-Ribose mixtures from 8.4 to 5.3. The amino group content in MRPs, derived from the α-LA-Ribose and β-LG-Ribose model system, was decreased noticeably during the first hour and did not change thereafter. The loss of free ribose in MRPs was higher for β-LG-Ribose than for α-LA-Ribose. During the Maillard reaction, the concentration of native and non-native α-LA, or β-LG, decreased and the formation of aggregates was observed. Fluorescence intensity of the β-LG-Ribose MRPs reached maximum within 1 h, compared to 2 h for α-LA-Ribose MRPs. Meanwhile, modification of the UV/vis absorption spectra for α-LA and β-LG was mainly due to a condensation reaction with ribose. Dynamic light scattering showed a significant increase in the particle size of the MRPs. Size exclusion chromatography of MRPs revealed the production of both high and low molecular weight material. Electrophoresis of MRPs indicated polymerization of α-LA and β-LG monomers via inter-molecular disulfide bridge, but also via other covelant bonds. MRPs from α-LA-Ribose and β-LG-Ribose exhibited increased antioxidant activities, therefore theses MRPs may be used as natural antioxidants in food products.     

Allergic responses induced by goat milk αS1-casein in a murine model of gastrointestinal atopy

Up to 3% of young children develop milk allergy and this may influence the development of immune-mediated diseases in later life. One protein that has been associated with allergic reactions to ruminant milk is α(S1)-casein (CN). Studies suggest that goat milk with low levels of α(S1)-CN may reduce allergenicity of milk, but the dose response to α(S1)-CN has not been confirmed. In this study, we examined the immune response to varying levels of goat α(S1)-CN in a mouse model of gastrointestinal allergy. BALB/c mice (aged 5 wk) were given intraperitoneal injections with α(S1)-CN and aluminum as adjuvant at 1 and 3 wk to sensitize mice to the antigen. In wk 5, groups of fasting mice (n=8/group) were challenged 4 times on alternate days by intragastric gavage with saline or 2, 10, or 20mg of α(S1)-CN. Serum levels of specific IgE, IgG(1), and IgG(2a) antibodies and mouse mast cell protease-I were determined. Interleukin-4, IL-10, and IFN-γ responses to 48-h activation with antigen were measured in cultured splenocytes. We determined that mice sensitized with α(S1)-CN had higher titers of specific IgG(1) and IgE antibodies compared with controls; however, groups challenged with differing doses of α(S1)-CN did not differ. The group challenged with the highest dose of α(S1)-CN had a 10-fold increase in mouse mast cell protease-I compared with the group challenged with saline. Both IL-4 and IL-10 were produced in a dose-dependent manner by cultured splenocytes incubated with α(S1)-CN. Overall, α(S1)-CN stimulated the production of cytokines associated with allergic disease in a dose-dependent manner. Thus, milk with lower levels of α(S1)-CN should contribute to a lesser antigenic burden.