Role of disulphide linkages between protein-coated lipid droplets and the protein matrix in the rheological properties of porcine myofibrillar protein-peanut oil emulsion composite gels

The objective of the study was to establish disulphide interaction between protein-coated oil droplets and the surrounding protein matrix in myofibrillar protein (MP)-emulsion composite gels. An MP-stabilized peanut oil emulsion was treated with 0, 1, 3, 5 and 10 mM N-ethylmaleimide (NEM, a sulphydryl-blocking agent) and subsequently incorporated into a bulk MP sol to produce 5%-lipid, 2%-protein composites at pH 6.2. About 69% of sulphydryls in the emulsion (1% protein) were blocked by 1 mM NEM, and almost all were bound at ≥3 mM NEM. The loss of free sulphydryls resulted in a significant drop in the storage modulus (G') and rupture force of the composite gels. Microstructural examination revealed pores and oil leakage from emulsion droplets by NEM treatments, corresponding to declining rheological properties of the MP-emulsion composites. The results supported the hypothesis that disulphide cross-linking between MP-coated oil droplets and protein matrix contributed to the stabilization and reinforcement of protein-emulsion composite gels formed in comminuted muscle foods.     

Studies of the effect of hydrostatic pressure pretreatment on thermal gelation of chicken myofibrils and pork meat patty

The structures and characteristics of pressure–heat-induced gels of chicken myofibrils and pork patty were investigated. The M-line and Z-line in the chicken myofibril in 0.2 M NaCl were disrupted, and both of the thin and thick filaments were dissociated by pressure treatment. The microstructure of pressure–heat-induced chicken myofibrillar gel was composed of three-dimensional fine strands. Pressurization, at 200 MPa, prior to heating, increased the apparent elasticities of chicken myofibrillar gel and pork patty; however, pressure treatment above 200 MPa decreased it. The apparent elasticity of the pressure-treated (200 MPa) thermal myofibrillar gel was three times higher, and that of pork patty was twice higher than those of the unpressurized ones. The rheological properties of the low salt (1% NaCl) pork sausage can be improved by pressure treatment at 200 MPa prior to heating.     

Mutation analysis of the interaction of B-type cytochrome c oxidase with its natural substrate cytochrome c-551

Heme-copper oxidases in the respiratory chain are classified into three subfamilies: A-, B- and C-types. Cytochrome bo₃-type cytochrome c oxidase from thermophilic Bacillus is a B-type oxidase that is thought to interact with cytochrome c through hydrophobic interactions. This is in contrast to A-type oxidases, which bind cytochrome c molecules primarily through electrostatic forces between acidic residues in the oxidase subunit II and basic residues within cytochromes. In order to investigate the substrate-binding site in cytochrome bo₃, eight acidic residues in subunit II were mutated to corresponding neutral residues and enzymatic activity was measured using cytochrome c-551 from closely related Bacillus PS3. The mutation of E116, located at the interface to subunit I, decreased the k_cat value most prominently without affecting the K_m value, indicating that the residue is important for electron transfer. The mutation of D99, located close to the Cu_A site, largely affected both values, suggesting that it is important for both electron transfer and substrate binding. The mutation of D49 and E84 did not affect enzyme kinetic parameters, but the mutation of E64, E66 and E68 lowered the affinity of cytochrome bo₃ for cytochrome c-551 without affecting the k_cat value. These three residues are located at the front of the hydrophilic globular domain and distant from the Cu_A site, suggesting that these amino acids compose an acidic patch for a second substrate-binding site. This is the first report on site-directed mutagenesis experiments of a B-type heme-copper oxidase.     

Effect of whey protein concentrate and sodium chloride concentrations on the odour profile of sous vide cooked whole-muscle beef from Argentina

Semitendinosus muscles added with whey protein concentrate (WPC) and sodium chloride (NaCl) were submitted to sous vide cooking. Four enhancement treatments and a control were tested: 0.875% WPC (w/w)+0.625% NaCl, 2.625% WPC+0.625% NaCl, 0.875% WPC+1.875% NaCl, 2.625% WPC+1.875% NaCl, and control (non-injected muscles). Odour analyses were carried out with an electronic nose (EN) system. EN data were evaluated applying Principal Component Analysis, Linear Discriminant Analysis and Partial Least Squares algorithm. EN was able to discriminate the odour profiles of cooked enhanced beef as a function of the amount of WPC added. No significant differences in odour profiles were observed regarding NaCl concentration. These results agreed with those obtained when odour profiles were analysed in WPC dispersions. The reported results support the applicability of EN methodology for analysing the impact of processing parameters on beef odour profiles.     

A rapid microwave-assisted derivatization process for the determination of phenolic acids in brewer’s spent grains

A simple and fast method for the TMS derivatization of phenolic acids in a closed vial using microwave irradiation followed by GC/MS analysis is proposed. A full factorial design was used to investigate the effects of two independent variables, namely, time and power of microwave irradiation, on the yield of silylation reaction. The optimal conditions were tested against the classical heating derivatization procedure. No significant differences were found between the classical heating and microwave-assisted derivatization. Chromatographic separation of the nine phenolic acids examined was achieved in 16 min. The mass spectral fragmentation patterns of the derivatives obtained by the proposed method were identical to those from the classical heating. Four different batches of brewer’s spent grain were extracted and analyzed for the total phenolic acid content. Significant differences between the batches of spent grains were found for all analytes. The total phenolic acid content varied between 2688 and 4884 μg/g.     

Effects of pH, temperature, supplementation with whey protein concentrate, and adjunct cultures on the production of exopolysaccharides by Streptococcus thermophilus 1275

Effects of pH, temperature, supplementation with whey protein concentrate (WPC), and non-EPS culture on the exopolysaccharide (EPS) production by Streptococcus thermophilus 1275 were studied. The organism was grown in 10% reconstituted skim milk (RSM) in a Biostat B fermenter for 24 h at various pH (4.5, 5.5 and 6.5) and temperatures (30, 37, 40, and 42 degrees C), and supplementation with WPC 392, and non-EPS producing S. thermophilus 1303 and the amount of EPS produced were determined. Bacterial counts were enumerated and the concentrations of lactic acid, lactose, glucose, and galactose were also determined. A maximum of 406 mg/L of EPS was produced in RSM at 37 degrees C after 24 h of fermentation at pH 4.08 when the pH was not controlled. A pH of 5.5 and temperature of 40 degrees C were found to be optimal for EPS production by S. thermophilus 1275, yielding 458 mg/L. The EPS production increased when RSM was supplemented with WPC 392. At optimum pH and at 37 degrees C with WPC supplementation, the level of EPS increased to 1029 mg/L. Co-culturing S. thermophilus 1275 with non-EPS S. thermophilus 1303 increased EPS production at 37 degrees C and pH 5.5 to 832 mg/L. High temperature (42 degrees C) reduced the amount of EPS production, and EPS production ceased at pH 4.5 when maintained constantly at this pH. The level of lactose utilization and lactic acid production depended on growth conditions of the organism. No glucose was detected, while galactose was found to accumulate in the medium.     

Effect on lamb meat quality of including thyme (Thymus zygis ssp. gracilis) leaves in ewes’ diet

The aim of this study was to investigate the effect of including thyme leaves (TL) in the diet of pregnant sheep on the sensorial characteristics, bacterial spoilage and oxidative stability of lamb meat stored in modified atmosphere (70% O₂:30% CO₂). For this, thirty-six sheep were randomly assigned to three groups: control (basal diet), T₁ (3.7% thyme leaves), T₂ (7.5% thyme leaves). Meat spoilage (TV, PSY, MY, ENT, and LA), TBARS, CIELAB coordinates, metmyoglobin and the sensory characteristics of fresh lamb meat were analyzed on days 0, 7, 14 and 21. The presence of antioxidant compounds in the diet containing TL delayed (P < 0.05) colour deterioration, lipid oxidation and bacterial counts, while at the same time imparting a better appearance to the fresh lamb meat. In general, this effect was more pronounced at the higher level of TL (7.5%). High Pearson’s correlation coefficients were found between the sensory attributes, CIELAB coordinates and TBARS.     

Influence of putrescine,cadaverine, spermidine or spermine on the formation of N-nitrosamine in heated cured pork meat

The influence of biogenic amines (i.e. putrescine, cadaverine, spermidine and spermine) on the N-nitrosamine formation in heated cured lean meat was studied in the presence or absence of sodium nitrite and at different meat processing temperatures. Experimental evidence was produced using gas chromatography with thermal energy analysis detection (GC–TEA). Concentration of N-nitrosamines was modelled as a function of the temperature and the nitrite concentration for two situations, i.e. presence or absence of added biogenic amines to the meat. The significance of the influence of the changing parameters was evaluated by ANOVA (Analysis of Variance).     

Isolation of genes coding for chitin-degrading enzymes in the novel chitinolytic bacterium, Chitiniphilus shinanonensis, and characterization of a gene coding for a family 19 chitinase

Chitiniphilus shinanonensis type strain SAY3(T) is a strongly chitinolytic bacterium, originally isolated from the moat water in Ueda, Japan. To elucidate the chitinolytic activity of this strain, 15 genes (chiA-chiO) coding for putative chitin-degrading enzymes were isolated from a genomic library. Sequence analysis revealed the genes comprised 12 family 18 chitinases, a family 19 chitinase, a family 20 β-N-acetylglucosaminidase, and a polypeptide with a chitin-binding domain but devoid of a catalytic domain. Two operons were detected among the sequences: chiCDEFG and chiLM. The gene coding for the polypeptide (chiN) showed sequence similarity to family 19 chitinases and was successfully expressed in Escherichia coli. ChiN demonstrated a multi-domain structure, composed of the N-terminal, two chitin-binding domains connected by a Pro- and Thr-rich linker, and a family 19 catalytic domain located at the C-terminus. The recombinant protein rChiN catalyzed an endo-type cleavage of N-acetyl-d-glucosamine oligomers, and also degraded insoluble chitin and soluble chitosan (degree of deacetylation of 80%). rChiN exhibited an inhibitory effect on hyphal growth of the fungus Trichoderma reesei. The chitin-binding domains of ChiN likely play an important role in the degradation of insoluble chitin, and are responsible for a growth inhibitory effect on fungi.     

Kinetics of browning onset in white wines: influence of principal redox-active polyphenols and impact on the reducing capacity

The browning capacity of white wines was studied, employing an accelerated test and samples produced and stored under identical conditions. Browning was approached from a kinetic point of view and efforts were focussed on the investigation of plausible correlations with major redox-active polyphenols, including substances with an o-diphenol feature, such as gallic acid, caftaric acid, 2-S-glutathionylcaftaric acid (GRP), caffeic acid, catechin, and epicatechin. Over a period of ten days, browning development was shown to obey zero-order kinetics from the third day of the treatment, and browning rate constants (k) varied from 15.3 to 74.5 × 10⁻³ day⁻¹. Regression analysis between k values and concentration of individual phenolics provided strong evidence that epicatechin is the principal browning agent (r² = 0.8033, P < 0.01). Furthermore, the monitoring of the reducing power (P_R), throughout treatments, indicated that increases in browning are accompanied by a commensurate decline in the reducing ability, raising concerns about the impact of browning reactions on the in vitro antioxidant properties of white wines.     

Removal of polychlorinated dioxins by semi-aerobic fed-batch composting with biostimulation of “Dehalococcoides”

A semi-aerobic, mesophilic, fed-batch composting (FBC) reactor loaded with household garbage was used to remove polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDD/Fs). The reactor was packed with woodchips as the solid matrix and PCDD/F-contaminated soil or flyash and then operated at a waste-loading rate of 0.5 kg (wet wt) day^- 1. All congeners of PCDD/Fs (initial concentration, 200–830 pmol g^- 1 [dry wt]) were totally reduced during the over period of operation, with a half reduction time of 4 months. Direct cell counting and respiratory quinone profiling showed that the reactors at the fully acclimated stage harbored a high population density of bacteria (10¹¹ g^- 1 [dry wt]) with members of the Actinobacteria predominating. Real-time quantitative PCR showed that the population of “Dehalococcoides” and its phylogenetic relatives of Chloroflexi as the possible dechlorinators varied between at the order of 10⁷ to 10⁸ g^- 1 (dry wt). A “Dehalococcoides”-containing dechlorinating culture from the soil-treating reactor was successfully enriched with a model PCDD/F compound, fthalide. 16S rRNA gene-targeted PCR-denaturated gradient gel electrophoresis and clone library analyses showed that this culture comprised at least three major phylogenetic groups of bacteria, Acidaminobacter, “Dehalococcoides,” and Rhizobium. These results suggest that the semi-aerobic FBC process is applicable for the bioremediation of PCDD/Fs and possibly other haloorganic compounds with the biostimulation of “Dehalococcoides” and its relatives as the potent dechlorinators.     

Short communication: characterization of a new genetic variant in the caprine kappa-casein gene

A new polymorphism has been identified in the goat kappa-casein gene by evaluating genomic DNA from the Montefalcone breed in Italy. The polymorphic site consists of a single nucleotide substitution A to G at position 242 of the exon 4 and produces an amino acid substitution Asp/Gly. A polymerase chain reaction-restriction fragment length polymorphism protocol for rapid genotyping of the variant has been developed, using the HaeIII enzyme. Animals from Italian, Spanish, and French breeds have been analyzed to investigate the occurrence of the allele in other populations. The allele appears to be exclusive to the Montefalcone breed.     

Characterisation of a β-N-acetylhexosaminidase from a commercial papaya latex preparation

A β-N-acetylhexosaminidase (β-NAHA) (EC 3.2.1.52) with molecular mass of 64.1 kDa and isoelectric point of 5.5 was purified from a commercial papaya latex preparation. The optimum pH for p-nitrophenyl-N-acetyl-β-d-glucosaminide (pNP-β-GlcNAc) hydrolysis was five; the optimum temperature was 50 °C; the K_m was 0.18 mM, V_max was 37.6 μmol min⁻¹ mg⁻¹ and activation energy (E_a) was 10.3 kcal/mol. The enzyme was thermally stable after holding at 30–45 °C for 40 min, but its activity decreased significantly when the temperature exceeded 50 °C. Heavy metal ions, Ag⁺ and Hg²⁺, at a concentration of 0.25 mM and Zn²⁺ and Cu²⁺, at a concentration of 0.5 mM, significantly inhibited enzyme activity. The β-NAHA had only one active site for binding both pNP-β-GlcNAc and p-nitrophenyl-N-acetyl-β-d-galactosaminide (pNP-β-GalNAc). A prototropic group with pKa value of about five on the enzyme may be involved in substrate binding and transformation, as examined by Dixon–Webb plots.     

Heterotrophic growth and nutritional aspects of the diatom Cyclotella cryptica (Bacillariophyceae): Effect of some environmental factors

To investigate the nutritional value of the diatom Cyclotella cryptica (Reimann, Lewin, and Guillard) as an alternative feed for use in the aquaculture industry, the heterotrophic growth characteristics and resultant fatty acid profile of the microalga were studied when cultivated under a variety of controlled salinity and temperature conditions. In addition, the effects of pH on the growth characteristics were also studied. The maximum specific growth rate was affected by initial pH and cultivation temperature, but not by salinity. The optimal pH and temperature ranges for growth were 7.2 to 8.1 and 22.5 to 25.0 °C, respectively. Lipid accumulation and the fatty acid composition were also affected by cultivation temperature and salinity. The optimal temperature range and salinity level for lipid accumulation were 18.0 to 25.0 °C and 11.2 psu, respectively. In all cases the fatty acid distribution was similar, with the most abundant fatty acids being palmitic acid (16:0), palmitoleic acid (16:1 n-7), stearidonic acid (18:4 n-3, SDA), eicosapentaenoic acid (20:5 n-3, EPA), and decosahexaenoic acid (22:6 n-3, DHA).     

Evaluation of lactic acid bacteria and yeast diversity in traditional white pickled and fresh soft cheeses from the mountain regions of Serbia and lowland regions of Croatia

The goal of this study was the characterisation of indigenous lactic acid bacteria (LAB) and yeasts isolated from nine white pickled (BG) and nine fresh soft (ZG) artisanal cheeses collected in Serbia and Croatia. While LAB were present in all of the cheeses collected, yeasts were found in all BG cheeses but only in three ZG cheese samples. High LAB and yeast species diversity was determined (average H′_L = 0.4 and H′_Y = 0.8, respectively). The predominant LAB species in white pickled (BG) cheeses were Lactococcus lactis, Lactobacillus plantarum, and Leuconostoc mesenteroides, while in fresh soft (ZG) cheeses the most dominant LAB species were L. lactis, Enterococcus faecalis, and Leuconostoc pseudomesenteroides. Among the 20 yeast species found, Debaryomyces hansenii, Candida zeylanoides, and Torulaspora delbrueckii were found to be predominant in BG cheeses, while Yarrowia lipolytica was predominant in ZG cheeses. The characterisation of metabolic and technological potentials revealed that 53.4% of LAB isolates produced antimicrobial compounds, 44.3% of LAB strains showed proteolytic activity, while most of the yeast species possessed either lipolytic or proteolytic activity. In conclusion, the results obtained in this study showed that the composition of LAB and yeast populations in white pickled and fresh soft cheeses is region specific. The knowledge gained in this study could eventually be used to select region specific LAB and yeast strains for the production of white pickled and fresh soft artisanal cheeses with geographically specific origins under controlled conditions.     

Comparison of the total antioxidant status of Bohemian wines during the wine-making process

The total antioxidant status (TAS) of two white and two blue wine varieties from the ?ernoseky wine region (North Bohemia, Czech Republic) during the wine-making process was assessed by measurement with different radical scavenging assays: ABTS, DPPH and DMPD. Significant differences in the antioxidant activity between white and red wines were confirmed and changes of TAS during the wine production process were demonstrated. Moreover, differences were ascertained between individual varieties of red wine. No statistically significant relationship between the results provided by the ABTS and DPPH assays was found, obviously due to the fact that each phenolic substance in wine gives a different response to each specific radical used in the assay. The results obtained supported the assumption that variety plays a considerable role in TAS; the blue wine varieties showed a much higher TAS than did the white wines. The ABTS assay showed higher EQA (equivalents of ascorbic acid, mg/ml) values than the DPPH assay.     

Oxidation of sarcoplasmic proteins during processing of Cantonese sausage in relation to their aggregation behaviour and in vitro digestibility

The physicochemical changes of sarcoplasmic 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 carbonyl level increased (p<0.05) during the process. The fluorescence loss of tryptophan residues was a direct consequence of the oxidative degradation. All the parameters of protein aggregation were highly (p<0.05) correlated with carbonyl level and protein surface hydrophobicity (H(0)), indicating that protein oxidation and thermal denaturation could induce protein aggregation, leading to secondary structural changes. The analysis of in vitro digestibility showed no correlation between pepsin activity and protein oxidation, due to the biphasic response of sarcoplasmic proteins toward proteolysis. However, a highly significant (p<0.05) correlation was observed with trypsin and α-chymotrypsin activity, indicating that protein oxidation induced the changes in H(0), protein aggregation and secondary structure, which further influenced in vitro digestibility.     

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.     

Near-infrared spectroscopic analysis of macronutrients and energy in homogenized meals

Near-infrared (NIR) reflectance spectroscopy was evaluated as a rapid and environmentally benign technique for the simultaneous determination of macronutrients and energy in commercially available, packaged meals. Reflectance spectra (400–2498 nm) of homogenized meals were obtained with a dispersive NIR spectrometer. Protein and moisture were measured by AOAC reference methods, total fat by a semi-automated acid hydrolysis, solvent extraction, gravimetric method and total carbohydrate calculated. Energy was calculated using Atwater factors. Using multivariate analysis software, PLS models (n = 113–115 products) were developed to relate NIR spectra of homogenized meals to the corresponding reference values. The models predicted components and energy in validation samples (n = 37–38 products), overall, with r² of above 0.96. Ratios of deviation to performance were between 3.6 and 6.6, and indicated adequacy of the models for screening, quality control, or process control. Performance of the models varied substantially when used to predict sub-groups of meals within the validation set.