Technical note: A simplified procedure for myofibril hydrophobicity determination

A simple and reliable method for the determination of surface hydrophobicity of nonsolubilized myofibrils (from pig M. longissimus dorsi) was developed and validated. This method is based on the interaction of the hydrophobic chromophore bromophenol blue (BPB) with myofibrillar proteins and the separation of free and bound BPB by centrifugation. The titration of bound BPB is performed by absorption spectroscopy, and the amount of bound BPB is considered as an index of protein hydrophobicity. Heating, which is known to increase protein hydrophobicity, was performed in order to validate this method. Fixation of BPB to myofibrils increased with heating time and temperature, strongly suggesting that it may be closely related to protein hydrophobicity.     

Temperature induced denaturation of myosin: Evidence of structural alterations of myosin subfragment-1

Denaturation of myofibrillar proteins in porcine longissimus thoracis et lumborum muscle was investigated after pre-rigor temperature incubation at 20, 30 and 40 °C. At 24 h myofibrils were isolated and myosin was further cleaved by chymotrypsin. High temperature pre-rigor induced release of myosin S1 (subfragment-1), less (P < 0.05) Ca²⁺-ATPase activity and structural alterations of the region of the myosin molecule that harbors S1. Surface hydrophobicity of myofibrils from the 40 °C group increased (P < 0.001), suggesting a temperature-induced structural rearrangement exposing hydrophobic groups on the surface of myofibrils which in turn may explain the reduced water-holding of PSE meat.     

Kinetics of protein physicochemical changes induced by heating in meat using mimetic models: (2) Effects of fibre type,peroxides and antioxidants

Heating-induced changes in meat proteins were investigated using models made of aqueous suspensions of myofibrils according to muscle fibre types and cellular compounds (oxidants and antioxidants). These changes were evaluated by measurements of carbonyl groups and protein surface hydrophobicity. Model results were compared to trial results obtained on pork meat (M. Longissimus dorsi) heated under the same conditions (45 and 75 °C, from 5 to 120 min). Myofibrillar proteins from α-white fibres were more sensitive to oxidation and thermal denaturation than those from β-red fibres. At 45 °C, there were negligible differences due to peroxide or antioxidant types. At 75 °C, organic peroxides (ROOH) were less oxidative than hydrogen peroxide (H₂O₂), and antioxidant enzymes were less efficient than vitamin E and carnosine at protecting proteins against oxidation. Protein oxidation observed in meat is lower than in the mimetic models and the increase in hydrophobicity remained limited in meat.     

Some functional properties of lupin proteins modified by lactic fermentation and extrusion

The aim of this work was to determine modifying effects of lactic fermentation and extrusion processes on functionality of lupin proteins. Protein content, surface hydrophobicity, water absorption capacity (WAC), water solubility index (WSI) and emulsifying properties (EAI, ESI) of protein preparations obtained from lupin seeds (Lupinus luteus, Lupinus albus, Lupinus angustifolius), with various contents of hull, were analyzed. Changes of protein properties were affected by lupin cultivar, hull content and applied processing method. An increase of soluble protein content after controlled lactic fermentation of lupin seeds, and changes of surface protein hydrophobicity, WAC and WSI values after each treatment and significant worsening of protein emulsifying properties were observed. Correlations were found between parameters examined in this study.     

The Effect of Heat Treatment on Anilinonaphthalene-8-Sulphonate Binding to Rapeseed Albumin (Napin)

Protein surface hydrophobicity can be measured by the fluorescent probe method. The effect of heat treatment on Brassica napus (rapeseed) albumin (napin) interactions with a fluorescent probe, anilinonaphthalene-8-sulphonic acid (ANS) was investigated by fluorescent titration. Upon heating to 100°C for 30 min the number of napin binding sites for ANS (n) increased from 5(±0·7) to 13(±0·5) moles of ANS bound per mole of protein. The ANS-protein dissociation constant (Kd) was 2·0(±0·4)×10^-6 M for the unheated protein and 10·4(±0·1)×10^-6 M for heat-denatured napin. There was also a blue shift in the fluorescence emission spectrum maximum for denatured napin–ANS complex consistent with an increase in the hydrophobicity of the ANS binding sites in the denatured protein. The characteristic fluorescence increase for heat-denatured albumin–ANS mixtures is therefore due to an increase in the number, binding affinity and hydrophobicity of binding sites. Heat treatment of napin leads to the appearance of additional surface hydrophobic sites in the denatured protein. © 1997 SCI.     

Improving water retention of chicken breast meats by CaCl2 combined with pulsed electric fields

Frozen poultry meat is the most widely consumed animal-based food. However, water loss often leads to quality loss of poultry meat. Therefore, the present study sought to investigate the combined effect of calcium chloride (CaCl₂) and pulsed electric fields (PEF) treatment on chicken breast meats and the mechanisms underlying protein degradation. The results showed that the synergistic effect was superior to the single treatment. Compared with the untreated group, the combination of CaCl₂ and PEF increased water holding capacity of chicken breast meats by 16.61% and decreased cooking loss by 28.93%. Low-field nuclear magnetic resonance (LF-NMR) results indicated that the synergistic treatment promoted water molecules' binding capacity in myofibrils of poultry meat, which exhibited higher immobilised water. Additionally, the combination of CaCl₂ and PEF led to increased degradation of proteins of high-molecular weight and surface hydrophobicity of myofibrillar protein. Furthermore, the extension of the protein molecule and microenvironmental changes promoted interaction between protein and water. In conclusion, the synergistic treatment of CaCl₂ and PEF enhanced water retention and improved physicochemical properties of the myofibrillar protein in chicken breast meats.     

Pressure-induced changes of silver carp (Hypophthalmichthys molitrix) myofibrillar protein structure

Silver carp (Hypophthalmichthys molitrix) is one of the major species of freshwater fish in China. Modification of myofibrillar proteins induced by high pressure processing (HPP) has been investigated at pressures ranging from 200 to 500 MPa for 10 min at 20 °C. Influence of high pressure on conformational changes of myofibrils was studied by means of differential scanning calorimetry (DSC), surface hydrophobicity measurement, ultraviolet absorption spectra and second-derivative spectroscopy, intrinsic spectrofluorimetry, Raman spectroscopic analysis and circular dichroism (CD). DSC analyses revealed that the conformational stability of myofibrils is reduced to different extents when they are exposed to a pressure over 200 MPa. More sulfydryl groups as well as hydrophobic regions and amino acid residues which had ultraviolet absorbance had been found significantly after HPP (≥300 MPa). Changes in the Raman spectra were interpreted as the occurrence of secondary structural changes in pressure-induced myofibrillar proteins. CD analysis results demonstrated that α-helix was destroyed after processed at 400 MPa for 10 min. The results implicated that HPP can be used as a possible means of improving the functional properties for growing demand for healthier fish products.     

Functional properties of milk proteins as affected by Maillard reaction induced oligomerisation

Milk proteins were modified by Maillard reaction with glucose, lactose, pectin and dextran and analysed for changes in molar mass distribution and functional properties. The study revealed that oligomeric (20,000–200,000 g/mol) and polymeric (>200,000 g/mol) Maillard reaction products with heterogeneous functional property profiles were generated. Compared to untreated milk proteins, milk protein/saccharide Maillard products formed highly viscous solutions and performed increased antioxidant capacity. Improved heat stability was detected for milk protein/dextran products, higher surface hydrophobicity for milk protein/glucose and milk protein/lactose products, and increased overrun for milk protein/pectin and milk protein/dextran products. Total milk protein/saccharide Maillard products formed outstandingly stable protein foams, Maillard products of total milk protein and glucose, lactose or pectin stable O/W-emulsions. Milk protein/pectin and milk protein/dextran Maillard products were characterized by increased emulsifying activity, and whey protein/saccharide products by enhanced pancreatic in vitro digestibility.The study characterizes Maillard reaction products of milk proteins and makes it possible to specifically select modified proteins for industrial applications according to the requirements towards food proteins, weighing changes in techno- and tropho-functional protein properties.     

Physicochemical changes of myofibrillar proteins during processing of Cantonese sausage in relation to their aggregation behaviour and in vitro digestibility

The physicochemical changes of myofibrillar proteins, especially oxidation behaviour, were measured to determine their mechanism of action on in vitro protein digestibility during Cantonese sausage processing. The results indicated that the carbonyl level significantly increased (p < 0.05) during the process. The SH group level decreased, while S–S group level increased gradually. Protein aggregation was induced by oxidation and heat treatment. Result from Fourier transform infrared (FTIR) spectroscopy confirmed protein aggregation occurred. The analysis of in vitro digestibility showed a highly significant (p < 0.05) correlation between pepsin activity and carbonyl group formation, S–S group level, protein surface hydrophobicity, D_4,3. A negative and highly significant correlation between trypsin, α-chymotrypsin activity and carbonyl group formation was measured, while no significant correlation with S–S groups, protein surface hydrophobicity, D_4,3 was observed. It indicated that not only protein oxidation and aggregation but also degradation by pepsin would influence proteolysis with trypsin and α-chymotrypsin.     

Structural,functional, and sensorial properties of protein isolate produced from salmon,cod, and herring by-products

Nutritional, structural, functional, and sensorial properties of protein isolate developed from salmon (Salmo salar), cod (Gadus morhua), and herring (Clupea harengus) by-products using the pH-shift method was studied. Function of the proteins in an emulsion system in terms of viscoelastic properties was also evaluated. Regardless of origin, the proteins showed satisfying nutritional value as reflected in their high essential amino acid content. The proteins contained significantly (p <?0.05) higher proportion of active sulfhydryl groups and surface hydrophobicity compared to whey and egg white protein reflecting conformational changes caused by the pH-shift process. Solubility, emulsion, and foaming capacity of the proteins showed a trend similar to soy protein and dependent on their origin. Cod protein had better emulsion and foaming capacity than salmon and herring proteins which was in line with its high surface hydrophobicity and myosin heavy chain content. Emulsions developed from cod and salmon proteins showed substantially better viscoelastic properties, with higher stability and viscosity compared to herring protein emulsions. Cod protein scored low for sensorial attributes related to lipid oxidation while herring protein showed high levels of fishy and rancid flavor and odor. Altogether, results showed that the proteins from fish filleting by-products have potential to be used as food ingredients, but their application would be governed by their origin and sensorial properties.     

Hydrolysates of native and modified soy protein isolates: structural characteristics,solubility and foaming properties

The effect of enzymatic hydrolysis on structure characteristics (surface aromatic hydrophobicity and molecular size) of both native and modified soy protein isolates was studied. Effects on thermal behavior and functional properties (solubility and foam formation and stabilization at pH 4.5) were also analysed. Hydrolysates were obtained by bromelain digestion at pH 8 of native (N) and of thermally treated isolates at pH 7 (T7) and at pH 1.6 (T1.6). The differential effect of bromelain on the three isolates produced partially hydrolyzed structures, which exhibited an enhancement of their protein solubility (S_TCA and S_{pH 4.5}). Bromelain digestion was more effective on isolate T7 resulting hydrolysates with improved capacity to form foams at pH 4.5. The different functional behavior at pH 4.5 of hydrolysates was explained through the changes in thermal behavior, surface aromatic hydrophobicity and molecular mass distribution.     

Sarcoplasmic proteins influence water-holding capacity of pork myofibrils

Water-holding capacity (WHC) is one of the main pork quality characteristics. The objective of this study was to determine the influence of (denaturation of) sarcoplasmic proteins on WHC. Myofibrils extracted from red, firm, non-exudative (‘normal’) and PSE (pale, soft, exudative) pork longissimus muscle were combined with sarcoplasmic extracts (with or without proteins) from PSE and normal pork longissimus samples. Weight increase of myofibrils (mg increase mg⁻¹ myofibrillar protein) was used as a measure of WHC. When combined with protein-containing sarcoplasmic extract from normal pork, WHC of myofibrils from PSE (2.6 mg mg⁻¹) and normal (2.8 mg mg⁻¹) pork was higher (P < 0.05) than when combined with sarcoplasmic extract from PSE meat (1.3 mg mg⁻¹ for PSE and 1.9 mg mg⁻¹ for normal myofibrils). Protein-free sarcoplasmic extracts were prepared by heating the extracts for 30 min at 80 °C. WHC of myofibrils combined with protein-free sarcoplasmic extract from PSE and normal pork was not significantly different. WHC of myofibrils combined with protein-free extract was lower than WHC of myofibrils combined with protein-containing extract. Ionic strength or pH could not explain the observed differences. It was concluded that sarcoplasmic proteins do influence WHC. The mechanism of this influence still needs to be determined. © 1999 Society of Chemical Industry     

Surface hydrophobicity of commercial canola proteins mixed with κ-carrageenan or guar gum

The presence of hydrophobic patches on the protein surface can aid in its ability to adsorb at an oil–water or air–water interface. The surface hydrophobicity (S₀) of canola protein isolate (CPI)-hydrocolloid (κ-carrageenan, guar gum) systems was evaluated by fluorometric testing under varied conditions (NaCl, CPI and hydrocolloid concentrations; pH) using 8-Anilino-1-NaphthaleneSulphonate as a fluorescent probe. The S₀ values of CPI-hydrocolloid mixtures increased as hydrocolloid concentrations increased. High S₀ values reflect increased exposure of nonpolar amino acid residues due to changes in protein structure. This effect was greater at high pH and when guar gum was used.     

Effects of isolation technique and conditions on the extractability,physicochemical and functional properties of pigeonpea (Cajanus cajan) and cowpea (Vigna unguiculata) protein isolates. I. Physicochemical properties

Physicochemical properties of pigeonpea and cowpea protein isolates were determined as a function of extraction technique and pH of the extracting medium. Protein extractability by the isoelectric point precipitation (IP) technique was positively correlated within the pH of the NaOH solution used in the pH range 8.5–12.5. The micellization (MP) technique extracted significantly (P<0.05) less protein than the IP technique when extraction pH of the NaOH was 9.5 or higher, and 10.5 or higher from cowpea and pigeonpea, respectively. The subunit composition and electrical mobility of the isolates were not affected by extraction technique and pH conditions. However, it was observed that the IP isolate extracted at pH 12.5 had the lowest proportion of hydrophilic amino acids, suggesting that the pH of the extracting medium exerted a major influence on the hydrophilicity of the isolates. Pigeonpea MP isolate exhibited significantly (P<0.05) higher exposed hydrophobicity than the IP isolates except for those extracted at pH 9.5 and 10.5. However, the cowpea MP isolate exhibited significantly lower exposed hydrophobicity than the IP isolate extracted at pH 8.5 but this was higher than the rest of the IP isolates. For IP isolates, an inverse relationship was apparent between the extraction pH and hydrophobicity. The MP isolates from both legume seeds were significantly lighter in colour than the corresponding IP isolates and, for the latter, the lightness value (L) was inversely correlated with extraction pH. Differential scanning calorimetry showed that the MP isolates exhibited higher transition enthalpy (ΔH) than the IP. For the IP isolates, ΔH decreased with increasing extraction pH.     

Effects of maltodextrin glycosylation following limited enzymatic hydrolysis on the functional and conformational properties of soybean protein isolate

The soy protein hydrolysate (HSPI) was first prepared using Neutrase and then glycosylated with maltodextrin (Md) at different incubation times (120, 180, 240, 270, and 300 min). The effect of glycosylation following limited enzymatic hydrolysis on the physicochemical properties of HSPI was investigated. The sodium dodecylsulphate polyacrylamide gel electrophoresis was used to confirm the covalent conjugation and determine the changes in the molecular weight of soybean protein isolate (SPI) during the structural modification. Surface hydrophobicity (H ₀) measurements revealed that limited hydrolysis as well as glycosylation at 120 min increased H ₀; however, further glycosylation decreased H ₀ due to the shielding effect of the maltodextrin bound. The increased secondary, tertiary conformation stability was confirmed by the far-UV circular dichroism spectroscopy, the intrinsic fluorescence analysis, and the results of differential scanning calorimetry. Subsequently, the functional properties including solubility, heat stability, emulsifying property, as well as antioxidant activities were evaluated. Results indicated that the emulsifying activity index was improved notably from 86.13 ± 1.31 m²/g for the native SPI to 109.07 ± 4.45 m²/g for HSPI–Md conjugates after 270-min incubation. Additionally, the glycosylation had obviously positive effects on the antioxidant activities of the modified SPI proteins. Therefore, HSPI–Md conjugates might be used as potential emulsifiers or multifunctional wall materials for the microencapsulation of bioactive ingredients.     

Glycation of whey protein with dextrans of different molar mass: Effect on immunoglobulin E–binding capacity with blood sera obtained from patients with cow milk protein allergy

A growing concern around the world is the number of people who are suffering from food protein allergies. One potential approach to decrease protein allergenicity is to block IgE-binding epitopes of the protein allergen by attachment of polysaccharides via the Maillard reaction (i.e., glycation). Protein glycation has been extensively studied to modify various functional properties. We wanted to examine whether glycates could reduce IgE binding in patients with cow milk protein allergy and to explore how the size (molar mass; M_W) of the polysaccharide affects this IgE-binding capacity. Glycation was performed using the initial step of the Maillard reaction performed in aqueous solutions. The specific goal of this study was to reduce the IgE-binding capacity of whey protein isolate (WPI) through glycation with dextran (DX). Blood sera were obtained from 8 patients who had been diagnosed with cow milk protein allergy, and a composite sera sample was used for IgE-binding analysis by the ImmunoCap (Phadia, Uppsala, Sweden) method. The WPI was glycated with DX of M_W ranging from 1 to 2,000 kDa, and the M_W of purified glycates was determined using size-exclusion chromatography coupled with multiangle laser light scattering. The WPI to DX molar ratios in the glycates made from DX that had M_W values of 1, 3.5, 10 (G10), 150, 500, and 2,000 kDa were 1:4, 1:3, 1:2, 1:1.5, 1:1, and 1:1, respectively. With the increase in the M_W of DX, there was an increase in the M_W values of the corresponding glycates but a decrease in the number of bound DX. The WPI-DX glycates had lower whey protein IgE-binding capacity than native WPI, with the lowest IgE-binding capacity obtained in the G10 glycate. The DX binding ratios and morphology results from atomic force microscopy images suggested that glycation of WPI with small-M_W DX resulted in extensive protein surface coverage, probably due to the attachment of up to 4 DX molecules per whey protein. The lower IgE binding of the G10 glycate was likely due to greater steric hindrance (or a physical barrier) at the surface of the protein. In summary, our results demonstrate that glycating WPI with DX via Maillard reaction can potentially be used to decrease the allergenicity of whey protein.     

Salt‐ and pyrophosphate‐induced structural changes in myofibrils from chicken red and white muscles

Myofibrils isolated from post‐rigor chicken Pectoralis major (PM, white) and Gastrocnemius (Gas, red) muscles were irrigated with various concentrations of NaCl (0.1–1.0 M ) with or without 10 mM sodium pyrophosphate at pH 5.5 and 6.0. Structural changes were examined using phase contrast microscopy. PM myofibril samples tended to show more definitive H‐zones but obscure Z‐lines compared to Gas myofibrils. Significant myofibril swelling, accompanied by a pronounced protein extraction, occurred in 0.5 M NaCl solution. The extent of swelling as well as protein extraction increased with the NaCl concentration up to about 0.8 M . Addition of pyrophosphate facilitated myofibril swelling and reduced the minimal NaCl concentration for swelling to 0.4 M . Without pyrophosphate, protein extraction for both PM and Gas myofibrils occurred along the A‐band, sometimes starting from the centre, but when pyrophosphate was added, the extraction began from the ends of the A‐band. At pH 5.5, protein extraction was similar for PM and Gas, but at pH 6.0, PM myofibrils were more extractable and their architecture changed more extensively than Gas myofibrils, especially when pyrophosphate was present. The results may explain the different water‐imbibing abilities of white and red meat when processed with salt and phosphate. © 2000 Society of Chemical Industry     

Urea-disc-electrophoresis of actin,tropomyosin, troponin, α-actinin, β-actinin and the extra protein fraction from striated muscle

Purified actin, prepared from both beef and rabbit myofibrils, on being subjected to electrophoresis on gels containing 7m urea produced a diffuse, heterogeneous and lightly-staining band over a range of R_m = 0·26 to 0·33. After protecting against oxidation by the addition of DTT (dithiothreitol), the actin band concentrated sharply at R_m = 0·26 and exhibited little contamination. Tropomyosin prepared from both rabbit and beef muscle and chromatographed on DEAE-cellulose produced heterogeneous electrophoretic patterns with bands always being present at R_m = 0·24 and 0·43. Treatment with DTT or sulphite prior to electrophoresis always produced a single band of R_m = 0·43, which was concluded to be SH-reduced tropomyosin. Rabbit muscle troponin sulphydryl groups were not protected by DTT or sulphite, but evidence suggested that troponin was localised at R_m = 0·88 upon electrophoresis. Both α-actinin and β-actinin were prepared from rabbit muscle. Each sedimented as a single symmetrical peak on ultracentrifugation. Upon urea-disc-gel-electrophoresis, α-actinin produced two bands at R_m = 0·02 and 0·07 while β-actinin exhibited an inconsistent heterogeneous pattern. Extra protein prepared from rabbit myofibrils by washing 8–10 times and chromatographing on DEAE cellulose was eluted from the column into five fractions (I, I-A, II, III and IV) by increasing salt concentrations. Fractions I and IV were shown to be sarcoplasmic- and nucleo-proteins, respectively. Although fractions II and III were not identified, evidence suggests that they were not of myofibrillar origin. Fraction I-A appeared to be identical to troponin. Thus, results suggest that extra protein is an artifact of preparation.     

Structure and functional properties of modified threadfin bream sarcoplasmic protein

Surimi wash-water contains up to 30% of protein from fish muscle that is currently underutilized. This paper describes the effect of acetylation, succinylation, trypsin hydrolysis or pre-heating at 55 °C on the emulsification and foaming properties of a threadfin bream sarcoplasmic protein (TBSP) model for wash-water protein. Multiple regression analysis showed that emulsification and foaming characteristics were differentially affected by TBSP surface hydrophobicity (S₀), solubility in water (S_W) and free amino group (fNH₂) concentration. Emulsification activity index (EAI) for TBSP was most enhanced by succinylation, whereas the foaming capacity (FC) was more effectively extended by trypsin hydrolysis. Structure–function relationships for emulsification were different from those associated with foaming or for ensuring the stability of these food dispersions. This study suggests that surimi wash-water protein functionality can be improved by protein modification. Further strategies may be needed to stabilize fish protein stabilized emulsions and foams.     

Simultaneous determination of bisphenol A and bisphenol B in beverages and powdered infant formula by dispersive liquid–liquid micro-extraction and heart-cutting multidimensional gas chromatography-mass spectrometry

The purpose of this study was to establish a reliable, cost-effective, fast and simple method to quantify simultaneously both bisphenol A (BPA) and bisphenol B (BPB) in liquid food matrixes such as canned beverages (soft drinks and beers) and powdered infant formula using dispersive liquid–liquid micro-extraction (DLLME) with in-situ derivatisation coupled with heart-cutting gas chromatography-mass spectrometry (GC-MS). For the optimisation of the DLLME procedure different amounts of various extractive and dispersive solvents as well as different amounts of the derivative reagent were compared for their effects on extraction efficiency and yields. The optimised procedure consisted of the injection of a mixture containing tetrachloroethylene (extractant), acetonitrile (dispersant) and acetic anhydride (derivatising reagent) directly into an aliquot of beverage samples or into an aqueous extract of powdered milk samples obtained after a pretreatment of the samples. Given the compatibility of the solvents used, and the low volumes involved, the procedure was easily associated with GC-MS end-point determination, which was accomplished by means of an accurate GC dual column (heart-cutting) technique. Careful optimisation of heart-cutting GC-MS conditions, namely pressure of front and auxiliary inlets, have resulted in a good analytical performance. The linearity of the matrix-matched calibration curves was acceptable, with coefficients of determination (r2) always higher than 0.99. Average recoveries of the BPA and BPB spiked at two concentration levels into beverages and powdered infant formula ranged from 68% to 114% and the relative standard deviation (RSD) was <15%. The limits of detection (LOD) in canned beverages were 5.0 and 2.0?ng?l^–1 for BPA and BPB, respectively, whereas LOD in powdered infant formula were 60.0 and 30.0?ng?l^–1, respectively. The limits of quantification (LOQ) in canned beverages were 10.0 and 7.0?ng?l–1 for BPA and BPB, respectively, whereas LOQ in powdered infant formula were 200.0 and 100.0?ng?l^–1, respectively. BPA was detected in 21 of 30 canned beverages (ranging from 0.03 to 4.70?µg?l^–1) and in two of seven powdered infant formula samples (0.23 and 0.40?µg?l^–1) collected in Portugal. BPB was only detected in canned beverages being positive in 15 of 30 samples analysed (ranging from 0.06 to 0.17?µg?l^–1). This is the first report about the presence of BPA and BPB in canned beverages and powdered infant formula in the Portuguese market.