Purification and characterization of sea bream (Sparus latus Houttuyn) pepsinogens and pepsins

Four pepsinogens (PG-I, PG-II, PG-III, and PG-IV) were identified in sea bream (Sparus latus Houttuyn) stomach and were purified to homogeneity by 20–60% ammonium sulfate fractionation and several chromatographies. The molecular weights of the four purified PGs were 36, 32, 32, and 34 kDa, respectively. All the pepsinogens converted into pepsins within a few minutes under pH 2.0. The molecular weights of all the four pepsins were approximately 30 kDa as determined by SDS–PAGE. Optimum pHs of the four enzymes were 3.0–3.5, and optimum temperatures were 45–50 °C. Western blot analysis revealed that anti-PG-II antibody cross-reacted with all the four pepsins while scarcely any immunological cross-reaction existed between anti-PG-I antibody and other pepsins. From the Lineweaver–Burk double reciprocal plots, the K_ms of pepsins (P-I, P-II, P-III, and P-IV) for hemoglobin were calculated as 8.7 × 10⁻⁸ M, 1.0 × 10⁻⁷ M, 8.6 × 10⁻⁸ M, and 7.3 × 10⁻⁸ M, respectively.     

Isolation and properties of 5′-nucleotidase isolated from jumbo squid (Dosidicus gigas) mantle muscle from the Gulf of California,Mexico

The enzyme 5′-nucleotidase of jumbo squid (Dosidicus gigas) mantle was purified and its SDS–PAGE showed a single band of 33 kDa, whereas a protein with a molecular mass of 107 kDa was detected by gel filtration suggesting a homotrimeric nature of this enzyme. Subunits of the named enzyme were not linked by covalent bonds. Isoelectric focusing of this enzyme showed a pI of 3.6–3.8 and presented a hyperbolic kinetics with V_max of 1.16 μM/min/mg of protein, K_m of 1.49 mM, K_cat of 3.48 μM of P_ι s⁻¹ and K_cat/K_m relation of 356.52 ((mol/L)⁻¹ s⁻¹). Purified enzyme preferred AMP as substrate (by 6.7-folds) than IMP, showing a K_m of 6.34 mM, V_max of 0.19 μM/min/mg of protein a K_cat of 0.3388 mol of P_ι s⁻¹ and K_cat/K_m relation of 53.44 ((mol/L)⁻¹ s⁻¹). The low K_m in relation to purified AMP deaminase of the same organism suggested a high contribution of 5′-nucleotidase in AMP degradation in jumbo squid mantle.     

Multiple forms of polygalacturonase from mango (Mangifera indica L. cv Alphonso) fruit

Polygalacturonase (PG) from mango pulp revealed three isoforms (I, II, III) upon ion exchange and gel filtration chromatography, each having an abundance of 68%, 6% and 26%, and molecular weights (M_r) 40, 51 and 45 kDa, respectively. The pH optimum for the isoforms was between 3 and 4. PG-I was stable over a wide pH range (4–7.5) unlike PG II and III, which were stable at pH 4 and 5, respectively. The optimum temperature was around 40 °C for all the three isoforms. Their apparent K_m for pectic acid was in the range 0.22–0.25 mg ml⁻¹. The V_max for PG I, II and III was 5.7, 3.6 and 4.4 μmol GalA equivalent h⁻¹, respectively. Cd²⁺, Cu²⁺ and Fe²⁺ and EDTA inhibited whereas GalA, Gal, Fuc, Rha and Ara stimulated PG-I activity, in particular. The major endogenous substrates for mango PG were identified to be two rhamnogalacturonans varying in their sugar ratio. These results are discussed in the light of pectin dissolution in vivo in ripening mango.     

Isolation and properties of AMP deaminase from jumbo squid (Dosidicus gigas) mantle muscle from the Gulf of California,Mexico

Adenosine monophosphate (AMP) deaminase was purified from jumbo squid mantle muscle by chromatography in cellulose phosphate, Q-Fast and 5′-AMP sepharose. Specific activity of 2.5 U/mg protein, 4.5% recovery and 133.68 purification fold were obtained at the end of the experiment. SDS–PAGE showed a single band with 87 kDa molecular mass, native PAGE proved a band of 178 kDa, whereas gel filtration detected a 180 kDa protein, suggesting the homodimeric nature of this enzyme, in which subunits are not linked by covalent forces. Isoelectric focusing of this enzyme showed a pI of 5.76, which agrees with pI values of AMP deaminase from other invertebrate organisms. AMP deaminase presented a kinetic sigmoidal plot with V_max of 1.16 μM/min/mg, K_m of 13 mM, K_cat of 3.48 μM.s⁻¹ and a K_cat/K_m of 267 (mol/L)⁻¹.s⁻¹. The apparent relative low catalytic activity of jumbo squid muscle AMP deaminase in the absence of positive effectors is similar to that reported for homologous enzymes in other invertebrate organisms.     

Shear and extensional viscosities of hard wheat flour dough using a capillary rheometer

The rheological behaviour of hard wheat flour dough was investigated for a high shear rate range (10-10⁴ s⁻¹) using a capillary rheometer. The shear viscosity obtained from capillary measurements demonstrated a shear thinning behaviour of dough and described by a power law model with a power law index n = 0.38, a consistency coefficient K = 1387 Pa sⁿ, and a coefficient of determination R² = 0.997. The extensional viscosity obtained from capillary measurements for a high extensional rate range (0.16-154 s⁻¹) showed a tension thinning behaviour and described by a power law model with a power law index m = 0.38, a consistency coefficient K_E = 353,000 Pa s^m, and a coefficient of determination R² = 0.977. Torsional measurements using a parallel plates rheometer for a shear rate range (10⁻³-20 s⁻¹) demonstrated the time or strain dependence of wheat flour dough, no steady state shear viscosity of dough, and the shear viscosity increased with time or strain to a maximum value and then decreased, suggesting a breakdown or rupture of the dough structure.     

Immobilization of pepsin on chitosan beads

In this study, chitosan beads were prepared by using a cross-linking agent and the resulting beads were employed in immobilization process. Studies on free and immobilized pepsin systems for determination of optimum temperature, optimum pH, thermal stability, pH stability, operational stability, storage stability and kinetic parameters were carried out. The optimum temperature interval for free pepsin and immobilized pepsin were 30–40 and 40–50 °C, respectively. Free and immobilized pepsin showed higher activity at pH 2.0–4.0. Apparent K_m = 12.0 g L⁻¹ haemoglobin (1.56 mM tyrosine) and V_max = 5220 μmol (mg protein min)⁻¹ values were obtained for free pepsin, while apparent K_m = 20.0 g L⁻¹ haemoglobin (2.16 mM tyrosine) and V_max = 2780 μmol (mg protein min)⁻¹ values were obtained for immobilized pepsin. Thermal stability and storage stability of immobilized pepsin were higher than that of free pepsin. Milk clotting activity was used for evaluation of the applicability of pepsin immobilization to industrial process. Optimum milk clotting temperature was found as 40 °C for free pepsin and 50 °C for immobilized pepsin.     

Physiochemical properties and kinetics of glucoamylase produced from deoxy-d-glucose resistant mutant of Aspergillus niger for soluble starch hydrolysis

Glucoamylases (GAs) from a wild and a deoxy-d-glucose-resistant mutant of a locally isolated Aspergillus niger were purified to apparent homogeneity. The subunit molecular mass estimated by SDS–PAGE was 93 kDa for both strains, while the molecular masses determined by MALDI-TOF for wild and mutant GAs were 72.876 and 72.063 kDa, respectively. The monomeric nature of the enzymes was confirmed through activity staining. Significant improvement was observed in the kinetic properties of the mutant GA relative to the wild type enzyme. Kinetic constants of starch hydrolysis for A. niger parent and mutant GAs calculated on the basis of molecular masses determined through MALDI-TOF were as follows: k_cat = 343 and 727 s⁻¹, K_m = 0.25 and 0.16 mg mL⁻¹, k_cat/K_m (specificity constant) = 1374 and 4510 mg mL⁻¹ s⁻¹, respectively. Thermodynamic parameters for soluble starch hydrolysis also suggested that mutant GA was more efficient compared to the parent enzyme.     

Adsorption of dark compounds with bentonites in apple juice

The adsorption equilibrium of dark-coloured compounds from apple juice has been investigated as a function of several variables including activation conditions of bentonite (heat and acid treatments), clay concentrations (2×10⁻³-8×10⁻³ kg clay/dm³ apple juice) and temperature (range of 296-336 K). The adsorption efficiency with acid-activated bentonite was greater than that with heat-activated and native bentonite, depending on surface properties; specific surface areas were 95.31, 71.95 and 71.76 m²/g, respectively.Absorbance data at 420 nm were fitted reasonably well with the Langmuir and Freundlich isotherms. The parameters K, Q₀, K_fr and n were determined for different temperatures. Thermodynamic parameters such as Gibbs free energy (ΔG), enthalpy (ΔH) and entropy (ΔS) change of adsorption were determined as about −3.125, 9.43 and 0.039 kJ mol⁻¹ K⁻¹, respectively, for acid-activated bentonite. These parameters were also determined for native and heat-activated bentonites. It was shown that the adsorption process was endothermic, spontaneous and controlled by physical mechanism. The adsorption and desorption rate constants (k_a and k_d) were obtained separately by applying a geometric approach to the first-order Langmuir model. k_a varied from 5.717×10⁻⁴ to 20.667×10⁻³ s⁻¹ and k_d from 1.425×10⁻⁴ to 7.473×10⁻³ s⁻¹. The results showed that acid-activated bentonite is more suitable for the adsorption of dark compounds from apple juice.     

Chitosan-tethered the silica particle from a layer-by-layer approach for pectinase immobilization

“Spherical polyelectrolyte brush” of core–shell structure were prepared by grafting poly (sodium 4-styrenesulphonate) (PSStNa) from SiO₂ nanoparticle via the surface-initiated atom transfer radical polymerization strategy. The colloidal stability was not impeded by the adsorbed proteins despite the fact that up to 316.8 mg of enzyme was adsorbed per gram of the carrier particles. The immobilized pectinase revealed acceptable pH stability over a broad experimental range of 3.0–4.5. The activity half lives for native and bound states of enzyme were found as 13.5 d and 30 d, respectively. The activity of immobilized pectinase adsorbed onto these particles was analyzed in terms of the Michaelis–Menten parameters. Kinetic parameters were calculated as 8.28 and 9.98 g pectin ml⁻¹ for K_m and 1.165 × 10⁻³ g pectin s⁻¹ g enzyme⁻¹ 1.124 × 10⁻³ g pectin s⁻¹ g particle⁻¹ for V_max in the case of free and immobilized enzymes, respectively. Enzyme activity was found to be approximately 49.7% for immobilized enzyme after storage for 1 month.     

Electrooxidation of flavonoids at platinum electrode studied by cyclic voltammetry

Flavonoids are natural vegetable dyes synthesized from phenylalanine. They are responsible for colour of blooming plant portions. Moreover, they are very important for human health due to their activity as free radical acceptors. Cyclic and differential pulse voltammetry was used in the determination of kinetic parameters of flavonoids electrooxidation. Electrochemical measurements of the oxidation of organic compounds can be helpful in understanding how these compounds are metabolised by living organisms. Flavonoids electrochemical oxidation is an irreversible reaction at a platinum electrode. In the case of morin hydrate, rutin, dihydroxyflavone, trihydroxyflavone, hesperidin, quercetin, the first step of the electrooxidation includes an exchange of two electrons during the oxidation of hydroxyl groups in the ring B. Hydroxyl groups in the rings A and C are probably oxidised in subsequent steps. The heterogeneous rate constants (k_bh) determined for the flavonoids electrooxidation are as follows: morin – 3.59 × 10⁻⁴, rutin – 4.42 × 10⁻⁴, dihydroxyflavone – 4.54 × 10⁻⁴, trihydroxyflavone – 4.19 × 10⁻⁴, hesperidin – 4.50 × 10⁻⁴ and quercetin – 4.63 × 10⁻⁴ cm s⁻¹. Their anodic transition coefficient ranged from 0.63 to 0.48 (n = 2). Xanthone and flavone were oxidised easiest and quickest among other substrates at the platinum electrode with the heterogeneous rate constants (k_bh) of 7.08 × 10⁻⁴ and 6.46 × 10⁻⁴ cm s⁻¹, respectively.     

Quality assessment of wild European eel (Anguilla anguilla) stored in ice

The quality assessment of wild European eel (Anguilla anguilla) stored in ice and in boxes without ice (3 ± 1 °C) was investigated by the sensory analysis, levels of nucleotide breakdown products and biogenic amines for up to 19 days. Sensory analysis was assessed using the Tasmanian Food Research Unit Scheme. K and related values (K_i, G, P, H and F_r) were used as freshness indicators. Linear regressions (r²) obtained from K, K_i, G, P, H and F_r were 0.95, 0.96, 0.83, 0.96, 0.99 and 0.96, respectively, for eel stored in ice whereas, for eel kept in boxes without ice, the values were 0.86, 0.86, 0.96, 0.91, 0.98 and 0.86, respectively. When eel stored in ice and in boxes without ice were considered at the limit of acceptability by assessors at ∼12–14 days and ∼5–7 days, respectively, the average K, K_i and P values were ∼70–85%, H values were ∼60% and F_r values were ∼10% for both storage conditions. The level of histamine exceeded the legal limit (5 mg/100 g fish) in eel stored without ice after 6–7 days and, in ice, after 13–14 days of storage, at which time eels were rejected by the sensory panel. The concentrations of biogenic amines were higher in eel stored in boxes without ice than in eel kept in ice. The levels of histamine in the muscle of eel kept in boxes without ice and in ice increased to the maximum levels of 17.9 mg/100 g on day 12 and 12.6 mg/100 g on day 19, respectively.     

Giant Amazonian fish pirarucu (Arapaima gigas): Its viscera as a source of thermostable trypsin

A trypsin was purified from pyloric caeca of pirarucu (Arapaima gigas). The effect of metal ions and protease inhibitors on its activity and its physicochemical and kinetic properties, as well its N-terminal sequence, were determined. A single band (28.0 kDa) was observed by SDS–PAGE. Optimum pH and temperature were 9.0 and 65 °C, respectively. The enzyme was stable after incubation for 30 min in a wide pH range (6.0–11.5) and at 55 °C. The kinetic parameters K_m, k_cat and k_cat/K_m were 0.47 ± 0.042 mM, 1.33 s⁻¹ and 2.82 s⁻¹ mM⁻¹, respectively, using BApNA as substrate. This activity was shown to be very sensitive to some metal ions, such as Fe²⁺, Hg²⁺, Zn²⁺, Al³⁺, Pb²⁺, and was highly inhibited by trypsin inhibitors. The trypsin N-terminal sequence IVGGYECPRNSVPYQ was found. The features of this alkaline peptidase suggest that it may have potential for industrial applications (e.g. food and detergent industries).     

Purification and characterisation of trypsins from the pyloric caeca of mandarin fish (Siniperca chuatsi)

Two trypsins of anionic form (trypsin A) and cationic form (trypsin B) from the pyloric caeca of mandarin fish (Siniperca chuatsi) were highly purified by a series of chromatographies, including DEAE-Sephacel, Sephacryl S-200 HR, Q-Sepharose or SP-Sepharose. Purified trypsins revealed a single band on native-PAGE. The molecular weights of trypsin A and B were 21 kDa and 21.5 kDa, respectively, as estimated by SDS–PAGE, both under reducing and non-reducing conditions. Zymography analysis showed that both trypsins were active in degrading casein. Trypsin A and B exhibited maximal activity at 35 °C and 40 °C, respectively, and shared the same optimal pH of 8.5, using Boc-Phe-Ser-Arg-MCA as substrate. The two trypsins were stable up to 45 °C and in the pH range from 4.5 to 11.0. Trypsin inhibitors are effective on these two enzymes and their susceptibilities were similar. Both trypsins were activated by metal ions such as Ca²⁺ and Mg²⁺ and inactivated by Fe²⁺, Zn²⁺, Mn²⁺, Cu²⁺, Al³⁺, Ba²⁺ and Co²⁺ to different degrees. Apparent K_m values of trypsin A and B were 2.18 μM and 1.88 μM, and K_cat values were 81.6 S⁻¹ and 111.3 S⁻¹ for Boc-Phe-Ser-Arg-MCA, respectively. Immunoblotting analysis using anti-common carp trypsin A positively cross-reacted with the two enzymes, suggesting their similarity. The N-terminal amino acid sequence of trypsin B was determined as IVGGYECEAH, which is highly homologous with trypsins from other species of fish.     

Purification and characterization of polyphenol oxidase from Ferula sp.

Polyphenol oxidase (PPO) of several Ferula sp. was extracted and purified through (NH₄)₂SO₄ precipitation, dialysis, and gel filtration chromatography. Leaf and stem extracts were used for the determination of enzyme properties. Optimum conditions, for pH, temperature, and ionic strength were determined. The best substrates of PPO were catechol for leaf and (−) epicatechin for stem samples. Optimum pH and temperature were determined. K_M and V_max values were 2.34 × 10⁻³ M and 8541 EU/ml for catechol, and 2.89 × 10⁻³ M and 5308 EU/ml for (−) epicatechin. The most effective inhibitor was sodium diethyl dithiocarbamate for leaf samples and sodium metabisulphite for stem samples. Both inhibitors indicated competitive reactions. PPO showed irreversible denaturation after 40 min at 60 °C.     

Emulsifying properties of chickpea protein isolates: Influence of pH and NaCl

Structure and emulsifying properties of chickpea protein isolates (CPI) as a function of protein concentration, oil volume, pH and ionic strength were studied. The optimum protein concentration 2 g l⁻¹ used to determine the emulsifying properties was obtained. Emulsifying activity index (EAI) increased from 244 to 376 m² g⁻¹ with pH from 3.0 to 11.0 except the protein isoelectric point (pI 5.0), where the EAI was 20 m² g⁻¹ and emulsion droplet size was the largest. At lower ionic strengths (0.0–0.1 M NaCl, pH 7.0), EAI decreased from 253 to 72.4 m² g⁻¹; however, it increased from 72.4 to 231.4 m² g⁻¹ at higher ionic strengths (0.1–1.0 M NaCl). A positive relation between EAI and surface hydrophobicity (S₀) of CPI at various ionic strengths was obtained, while EAI was independent of S₀ under different pH values. α-Helix was the major configuration of CPI at the pI or lower ionic strength.     

Migration of α-tocopherol and resveratrol from poly(L-lactic acid)/starch blends films into ethanol

Poly(L-lactic acid) (PLLA)/starch blends with various concentrations of two natural antioxidants, α-tocopherol (α-TOC) and resveratrol, were fabricated by a melt blending and compression molding processes. The effects of the two antioxidants on the optical (color), thermal and mechanical properties of PLLA/starch blends with antioxidants were assessed. PLLA/starch blend films with α-TOC and resveratrol showed a yellowish color influenced by the combined effect of white starch and the brown color of the antioxidants. The glass transition and melting temperatures were significantly reduced with the addition of antioxidants while enhanced thermal stability was observed, which could be a benefit and important for processing and production. The enhanced mechanical properties could be attributed to not only a compatibilization effect based on the chemical linkage between PLLA and starch chains, but also restriction of the chain mobility by antioxidants. The release of resveratrol from PLLA and PLLA/starch blend films into ethanol followed Fickian behavior. The D values of α-TOC were in the range of 0.47–3.95 × 10⁻¹¹ cm² s⁻¹ for PLLA films and 0.70–6.83 × 10⁻¹¹ cm² s⁻¹ for PLLA/starch blend films at 13 °C, 5.67–13.0 × 10⁻¹¹ cm² s⁻¹ for PLLA films and 4.10–24.2 × 10⁻¹¹ cm² s⁻¹ for PLLA/starch blend films at 23 °C, and 89.0–118.0 × 10⁻¹¹ cm² s⁻¹ for PLLA films and 123–282 × 10⁻¹¹ cm² s⁻¹ for PLLA/starch blend films at 43 °C. The D values of resveratrol were in the range of 0.073–0.54 × 10⁻¹⁰ cm² s⁻¹ for PLLA films and 1.42–6.93 × 10⁻¹⁰ cm² s⁻¹ for PLLA/starch blend films at 13 °C, 0.90–3.44 × 10⁻¹⁰ cm² s⁻¹ for PLLA films and 4.16–22.3 × 10⁻¹⁰ cm² s⁻¹ for PLLA/starch blend films at 23 °C, and 24.8–74.1 × 10⁻¹⁰ cm² s⁻¹ for PLLA films and 40.1–309 × 10⁻¹⁰ cm² s⁻¹ for PLLA/starch blend films at 43 °C.     

Spray-dried encapsulation of chia essential oil (Salvia hispanica L.) in whey protein concentrate-polysaccharide matrices

Eight chia essential oil-in-water fresh emulsions (E) variations were prepared using biopolymers blends whey protein concentrate (WPC) with mesquite gum (MG) or gum Arabic (GA), core to wall material ratios (C_o:W_a) of 1:2 and 1:3, and total solids contents (TSC) of 30 and 40 wt%. All E variations displayed volume-weighted mean size (d_4,3) droplet sizes that fell within 2.32 and 3.35 μm and rates of droplet coalescence (k_C) of 10⁻⁸ s⁻¹. E variations were spray-dried and the resulting microcapsules (M) had d_4,3 falling within the range of 13.17–28.20 μm. The encapsulation efficiency (EE) was higher than 70% for all M, but those obtained from E with lower TSC and higher C_o:W_a displayed higher EE and lower surface oil, independently of M particle size. The reconstituted emulsions (RE) exhibited significantly higher d_4,3 and k_C values of the same magnitude as E variations.     

Application and evaluation of mesquite gum and its fractions as interfacial film formers and emulsifiers of orange peel-oil

Mesquite gum was fractionated using hydrophobic interaction chromatography, yielding three fractions (F₁, F₂, F₃) whose average molecular masses ranged from 1.81 × 10⁴ to 5.23 × 10⁵ Da; F₁ had 90% polysaccharide and 1% protein contents, while F₂ and F₃ contained 16 and 46% of protein, respectively. Fractions' ability to form oil–water interfacial films and to stabilize orange peel–oil emulsions was evaluated. The highest interfacial viscosity (321 m Nm⁻¹) and highest instantaneous elastic modulus (E₀) = 0.113 × 10⁻⁴ m Nm⁻¹ were exhibited by F₂ and these values were significantly higher than those exhibited by the whole mesquite gum. F₁ did not exhibit viscoelastic properties. Emulsions made with F₂, F₃, and the whole mesquite gum had coalescence rates of the order of 10⁻⁸ s⁻¹, indicating that these emulsions were very stable. Nevertheless, emulsions made with F₂ were significantly more stable than those made with F₃ and whole mesquite gum, and emulsions made with F₁ broke after 1 day aging. These results indicate that there is a close correlation between emulsion stability, interfacial rheological properties, and an adequate relatively high protein/high polysaccharide balance in the fractions.     

Characterisation of thermostable trypsin and determination of trypsin isozymes from intestine of Nile tilapia (Oreochromis niloticus L.)

Trypsin from intestinal extracts of Nile tilapia (Oreochromis niloticus L.) was characterised. Three-step purification – by ammonium sulphate precipitation, Sephadex G-100, and Q Sepharose – was applied to isolate trypsin, and resulted in 3.77% recovery with a 5.34-fold increase in specific activity. At least 6 isoforms of trypsin were found in different ages. Only one major trypsin isozyme was isolated with high purity, as assessed by SDS-PAGE and native-PAGE zymogram, appearing as a single band of approximately 22.39 kDa protein. The purified trypsin was stable, with activity over a wide pH range of 6.0–11.0 and an optimal temperature of approximately 55–60 °C. The relative activity of the purified enzyme was dramatically increased in the presence of commercially used detergents, alkylbenzene sulphonate or alcohol ethoxylate, at 1% (v/v). The observed Michaelis–Menten constant (K_m) and catalytic constant (K_cat) of the purified trypsin for BAPNA were 0.16 mM and 23.8 s⁻¹, respectively. The catalytic efficiency (K_cat/K_m) was 238 s⁻¹ mM⁻¹.     

Color changes in fish during grilling – Influences of heat transfer and heating medium on browning color

Samples of red sea bream (Pagrus major) were grilled under radiant (far-infrared radiation, FIR) and convective (superheated steam, SHS) heating. The temperature and color (CIE L^∗, a^∗, and b^∗ values) of the sample surface were monitored over time, using SHS, dry air, and N₂ as heating media. The rate of L^∗ changes was evaluated by treating the browning reaction as first-order. Color changes based on a^∗ and b^∗ values were effectively correlated with the L^∗ value, using empirical equations. A slower reduction in L^∗ for heating with SHS rather than FIR was obtained, probably because of different activation energies (31.5 and 50.7 kJ mol⁻¹) and frequency factors (8.2 and 4759 s⁻¹). The order of reductions in L^∗ was dry air > N₂≒$\fallingdotseq$ SHS. The absence of O₂ in the heating medium could be the reason for the delay in the browning reaction during heating using N₂ and SHS.