Mitochondrial enzyme pathways and their possible role during curing

European Food Research and Technology - The velocity of oxidation of exogeneous ferrocytochrome c by nitrite under anaerobic conditions in the presence of skeletal muscle mitochondria is dependent...     

Mitochondrial enzyme pathways and their possible role during curing.

The velocity of oxidation of exogenous ferrocytochrome c by nitrite under anaerobic conditions in the presence of skeletal muscle mitochondria is dependent upon pH over at least the range 5.6-6.7, increasing markedly as the pH is lowered. A product of the reaction is the complex formed between nitric oxide and ferricytochrome c. At levels up to 20 mM, nitrite inhibits aerobic cytochrome oxidase action; at higher concentrations, however, a partial resuscitation of the oxidation of ferrocytochrome c occurs, the enhancement of reaction velocity being considerably greater at pH 6.0 than at 6.5. Mitochondrial respiration is also inhibited by nitrite but no similar resurgence was, however, observed and thus the oxidation of ferrocytochrome c by high levels of nitrite is considered to be a direct non-enzymic action. Under anaerobic conditions, the rate of increase of the velocity constant of the oxidation of ferrocytochrome c with nitrite concentration in the presence of muscle mitochondria similarly decreased with rise of pH over the same range. The permeability of the muscle mitochondrion to nitrire has been demonstrated by swelling studies and by the rapid conversion of endogenous ferrocytochrome a3 into its nitrosyl-derivative. Over longer periods of anaerobic incubations of mitochondria with nitrite, oxidation of endogenous cytochromes occurs with the formation of nitrosylferricytochrome c. Above a nitrite concentration of 0.3 mM, the mitochondrial enzyme system probably involved is increasingly inhibited but by a concentration of 30 mM a direct non-enzymic oxidation has intervened. Commercial vacuum packed bacons were examined by electron microscopy. Mitochondria were clearly recognisable although they contained fewer cristae than those observed in fresh meat.     

Fatty acid composition of four potential aquatic weeds and their possible use as fish-feed neutraceuticals

A study was conducted to explore the fatty acid composition of four aquatic weeds, namely Salvinia cuculata, Trapa natans, Lemna minor and Ipomoea reptans collected from water bodies of Assam, India. It has been observed that C24:1 (Cis-15 Tetracosenoic acid) followed by C16:1 n-3 (9-Hexadecenoic acid) are the predominant fatty acids in these aquatic weeds and among them, Ipomoea reptans and Trapa natans contain higher proportions of C18:2 and C18:3 PUFA. The ratio of saturated fatty acid to unsaturated fatty acid is extremely low in the case of all four plants and they contain a predominant proportion of highly unsaturated fatty acids (HUFA), essential for the growth and proper performance of fish.     

Possible use of texture characteristics of winegrapes as markers for zoning and their relationship with anthocyanin extractability index

Ribeira Sacra Denomination of Origin is a steep slope viticulture area located in Galicia (northwest of Spain), and it is divided into five different subzones. Taking into account that grape composition depends not only on the grape cultivar used but also on the environmental factors and cultural practices, texture analysis has been proposed to assess the grape mechanical properties of those subzones. With the aid of principal component analysis, berry skin break energy measured on bottom side and berry cohesiveness have been suggested as the best mechanical properties for the subzones differentiation. Moreover, significant correlations among mechanical parameters and anthocyanin extractability index were found. A regression hyperplane allowed estimating anthocyanin extractability from berry skin thickness and Young′s modulus measured on the lateral side of grapes. The speed of mechanical analysis facilitates the planning and management of pressing and maceration processes as a consequence of the drastic reduction in sample treatment time.     

Origins and levels of post pasteurization contamination of milk in the dairy and their effects on keeping quality

Bacterial post pasteurization contamination with psychrotrophic Gram-negative rods (GNR) was measured in commercial milks before and after transfer to retail containers. The tanks of pasteurized milk feeding the filling units contaminated milk less often (39% of samples) but usually at a higher level than the filling units (92% of samples). The number of GNR present had a considerable influence on the shelf life of milk, and the range found in commercially pasteurized milk was reflected in a wide range of shelf lives.     

Norwegian consumers' acceptability of boar tainted meat with different levels of androstenone or skatole as related to their androstenone sensitivity

The aim of work was to study Norwegian consumers' acceptance of pork meat with different levels of skatole and androstenone. One group of androstenone sensitive consumers (N = 46) and one group of non sensitive consumers (N = 55) participated in a home test and evaluated 11 samples with different skatole (range 0–0.35 ppm) and androstenone (range 0–9.0 ppm) levels. Liking of odour during frying and odour and flavour of the fried meat were evaluated. Results showed that the non sensitive consumers accepted all levels of androstenone in the samples. Sensitive consumers gave a significantly lower liking score for androstenone samples containing 3 ppm (and more) than the reference sample when evaluating these samples above the frying pan, but no significant difference were found between 3 ppm samples and reference samples when liking of fried meat was evaluated. This indicated that the sensitive consumers accepted 3 ppm in fried meat, but not if 3 ppm was present in the sample during the frying process. The same consumer's differentiated skatole samples with regard to flavour at 0.15 ppm. The Norwegian established practise with a threshold value of 0.21 ppm skatole is higher than the value accepted by the consumers.     

Vitamin C and sugar levels as simple markers for discriminating Spanish honey sources

In this work, 7 Spanish honeys with different botanical origins were studied. The honey origins were rosemary, chestnut, lavender, echium, thyme, multifloral, and honeydew. The chemical compounds determined were ascorbic acid (vitamin C), hydroxymethylfurfural, and major sugar contents (glucose and fructose). The physicochemical parameters, pH, conductivity, moisture, free acidity, and color, were also measured. Vitamin C is an important antioxidant in food, and the possibility to use it as discriminate parameter among different honeys was studied. The determination of vitamin C in honey samples was carried out by 2 different methods, volumetric and chromatographic comparing the results by both statistically. Vitamin C content was higher in thyme honeys than in the other types; however a wide dispersion in the values was found. Through a linear discriminant analysis (LDA), conductivity, glucose, fructose, and vitamin C content were the most important discriminant parameters. PRACTICAL APPLICATION: Vitamin C content in different honey sources has been determined by a simple and rapid chromatographic method (less than 3 min) in honeys from 6 botanical origins. The results together with glucose and fructose content and some physicochemical parameters have been studied in order to discriminate the botanical origin of honeys and in the future certified their quality. A statistical LDA was applied to the data, and differentiation of honey sources was possible with very good agreement. The vitamin C content found in thymus honeys was significantly higher than in other types. This fact makes vitamin C a special marker for thymus honeys that have a higher antioxidant effect than the others giving it special properties. The identification of honey sources is essential for beekeepers in order to certify honeys for consumers.     

Ultrasonication as a novel processing alternative to pasteurization for camel milk: Effects on microbial load,protein profile,and bioactive properties

Ultrasonic technology presents a promising novel tool in the food industry for the processing of milk and dairy products. In this study, we investigated the effects of ultrasonication (US) as an alternative to thermal pasteurization for stabilization of the bioactive properties of camel milk. Camel and bovine milk samples were subjected to US at 6 different power levels (US1–US6), and 1 set of each type of milk was concurrently subjected to flash heat pasteurization (FHP) for comparative analysis (100 mL; n = 4). The microbiological and bioactive parameters of the samples were analyzed during 7 d of storage at 4°C. In both milk types subjected to US ≥ 140 W (US3), the bacterial load was reduced by almost 4 log cycles and complete reduction of microbial load was achieved with US = 170 W and US = 210 W (US5 and US6 treatments, respectively). No significant changes in protein patterns were observed with either FHP or US treatment. In addition, bioactive properties (cholesteryl esterase and pancreatic lipase inhibition) were either enhanced or retained at US3 or higher. 2,2′-Azino-bis-3-ethylbenzthiazoline-6-sulfonic acid and ferric reducing antioxidant power activities in camel milk were decreased after FHP treatment but increased or retained upon US, particularly at US3 and US4 (160 W). Overall, under our experimental conditions, US4 was effective in completely reducing the microbial count, while concomitantly retaining different bioactive properties of both camel and bovine milk. These outcomes highlight the potential of US at 160 W as an efficient nonthermal alternative processing method for milk.     

Acceptability of entire male pork with various levels of androstenone and skatole by consumers according to their sensitivity to androstenone

Consumer acceptability of entire male pork at eating was assessed in three experiments. The 140 consumers involved in each experiment were classified as insensitive (INSENS) to the odor of pure androstenone or sensitive perceiving it as pleasant (SENS-PLEA) or unpleasant (SENS-UNPL). Entire male pork with very low skatole and androstenone levels (LS-LA) was as well accepted as gilt pork, whatever the consumer category. Entire male pork with elevated levels in both skatole and androstenone (HS-HA) was clearly differentiated from LS-LA pork by SENS-UNPL, but not by SENS-PLEA or INSENS consumers. Whatever the consumer category, entire male pork with elevated levels of androstenone and very low levels of skatole (LS-HA and LS-HHA) were not significantly differentiated from LS-LA pork. The results suggest that, in the conditions of the present experiment, androstenone and skatole totally explain boar taint at eating and that the acceptability threshold for androstenone, in the absence of skatole, is in the range of 2-3 μg/g liquid fat.     

Synthesis,characterisation and use of 2-tetradecylcyclobutanone together with other cyclobutanones as markers for irradiated liquid whole egg

The synthesis and characterisation of 2-tetradecylcyclobutanone (TCB) is described. Both 2-dodecykyclobutanone and TCB were shown to be present in liquid whole egg irradiated at doses of 2.5 and 10.0 kGy. These compounds were absent from the unirradiated pasteurised samples. Using gas chromatography and infrared spectroscopy, there was also evidence for the presence in irradiated egg of 2-tetradeccnyl- and 2-tetradecadienylyclobutanone which are formed from oleic and linoleic acids, respectively. Authentic standards for these unsaturated cyclobutanones were not available commercially but the presence of 2-tetra-decenylcyclobutanone was substantiated by hydrogenating the egg extracts so converting this unsaturated cyclobutanone to TCB. Saturated and unsaturated cyclobutanones appear to be specific products of irradiation and are potential markers for detection of irradiated liquid egg and probably other fat-containing foods.     

Screening of whey protein isolate hydrolysates for their dual functionality: Influence of heat pre-treatment and enzyme specificity

Heat pre-treated and non heat pre-treated whey protein isolate (WPI) were hydrolysed using α-chymotrypsin (chymotrypsin), pepsin and trypsin. The in vitro antioxidant activity, ACE-inhibition activity and surface hydrophobicities of the hydrolysates were measured in order to determine if peptides with dual functionalities were present. Dual functional peptides have both biological (e.g. antioxidant, ACE-inhibition, opioid activities) and technological (e.g. nanoemulsification abilities) functions in food systems. Heat pre-treatment marginally enhanced the hydrolysis of WPI by pepsin and trypsin but had no effect on WPI hydrolysis with chymotrypsin. With the exception of the hydrolysis by trypsin, heat pre-treatment did not affect the peptide profile of the hydrolysates as analysed using size exclusion chromatography, or the antioxidant activity (P > 0.05). Heat pre-treatment significantly affected the ACE-inhibition activities and the surface hydrophobicities of the hydrolysates (P < 0.05), which was a function of the specificity of the hydrolysing enzyme. Extended hydrolysis (up to 24 h) had no significant effect on the DH and the molecular weight profiles (P > 0.05) but in some instances caused a reduction in the antioxidant activity of WPI hydrolysates. The chymotrypsin hydrolysate showed a broad MW size range, and was followed by pepsin and then trypsin. The bioactivities of the hydrolysates generally decreased in the order; chymotrypsin > trypsin > pepsin. This study showed that by manipulating protein conformation with pre-hydrolysis heat treatment, combined with careful enzyme selection, peptides with dual functionalities can be produced from WPI for use as functional ingredients in the manufacture of functional foods.     

Cladonia lichens and their major metabolites as possible natural antioxidant,antimicrobial and anticancer agents

The aim of this study is to investigate in vitro antioxidant, antimicrobial, and anticancer activities of the acetone extracts of the lichens Cladonia furcata, Cladonia pyxidata and Cladonia rangiferina and their atranorin and fumarprotocetraric acid constituents. Antioxidant activity was evaluated by free radical scavenging, superoxide anion radical scavenging, reducing power, and determination of total phenolic and flavonoid compounds. As a result of the study atranorin had largest free radical scavenging activity with IC₅₀ values 131.48 μg/mL. Moreover, the tested samples had effective reducing power and superoxide anion radical scavenging. Total content of phenol and flavonoid in extracts was determined as pyrocatechol equivalent, and as rutin equivalent, respectively. The strong relationships between total phenolic and flavonoid contents and the antioxidant effect of tested extracts were observed. The antimicrobial activity was estimated by determination of the minimal inhibitory concentration by the broth microdilution method. The most active was fumarprotocetraric acid with minimum inhibitory concentration values ranging from 0.031 to 0.125 mg/mL. Anticancer activity was tested against FemX (human melanoma) and LS174 (human colon carcinoma) cell lines using MTT method. All samples were found to be strong anticancer activity toward both cell lines with IC₅₀ values ranging from 10.97 to 41.23 μg/mL.     

Impact of enzyme inactivation conditions during the generation of whey protein hydrolysates on their physicochemical and bioactive properties

The thermal inactivation conditions (75 °C × 35 min, 80 °C × 10 min, 85 °C × 5 min and 90 °C × 5 min) for Protamex? following bovine whey protein concentrate (WPC) hydrolysis was studied with the view to limiting WPC hydrolysate (WPH) aggregation while maintaining bioactivity. A decrease in the amount of large WPH aggregates formed was observed at inactivation temperatures ≤85 °C. However, the WPC appeared to be more hydrolysed on heating at 75 °C × 35 min, as Protamex? was active for longer under these heating conditions. Significantly (P < 0.05), higher WPH antioxidant (oxygen radical absorbance capacity – ORAC) activity was obtained on inactivation at temperatures ≤80 °C. In contrast, the dipeptidyl peptidase IV (DPP‐IV) inhibitory properties of all WPH samples were similar (P > 0.05). A reduction in thermal treatment from 90 °C × 5 min to 85 °C × 5 min was sufficient to decrease the amount of large aggregates formed in the hydrolysate without altering its bioactive properties.     

Simulation of heat transfer for solid–liquid food mixtures in cans and model validation under pasteurization conditions

During processing of canned mixtures of solid–liquid foods, conduction and convection occur simultaneously. The literature lacks in a complete simulation study where a large number of solids are dispersed in the liquid phase, e.g. canned peas. Therefore, the objectives of this study were to determine temperature changes inside a can containing solid–liquid food mixtures. For this purpose, dispersed stationary solids (canned peas in water) in a 2D (axi-symmetrical) configuration were applied. Ansys V11 (Ansys Inc., Canonsburg, PA) was used to solve continuity, energy and momentum equations. For experiments, canned pea samples were prepared in 500 g cans, and heating process was conducted in a retort under pasteurization conditions at ≈98 °C. Temperature changes were measured using needle type thermocouples, and simulation results were validated against experimental data. This study is expected to be a significant contribution to the literature for further optimization studies and to form basis of an industrial project to improve canning process of solid–liquid mixtures.     

Bacterial spore inhibition and inactivation in foods by pressure, chemical preservatives, and mild heat

Sucrose laurates, sucrose palmitate, sucrose stearates, and monolaurin (Lauricidin) were evaluated for inhibitory effects against spores of Bacillus sp., Clostridium sporogenes PA3679, and Alicyclobacillus sp. in a model agar system. The combined treatment of sucrose laurate, high hydrostatic pressure, and mild heat was evaluated on spores of Bacillus and Alicyclobacillus in foods. The minimum inhibitory concentrations of the sucrose esters were higher than that of Lauricidin for all spores tested in the model agar system, but Lauricidin was not the most readily suspended in the test media. The sucrose laurates and sucrose palmitate were more effective and more readily suspended than the sucrose stearates. A combined treatment of sucrose laurate (<1.0%), 392 megaPascals (MPa) at 45 degrees C for 10 to 15 min provided 3- to 5.5-log10 CFU/ml reductions from initial populations of 10(6) CFU/ml for Bacillus subtilis 168 in milk, Bacillus cereus 14579 in beef, Bacillus coagulans 7050 in tomato juice (pH 4.5), Alicyclobacillus sp. N1089 in tomato juice (pH 4.5), and Alicyclobacillus sp. N1098 in apple juice. The most notable change in the appearance of the products was temporary foaming during mixing of the sucrose laurate in the foods. The effect of sucrose laurate appeared to be inhibitory rather than lethal to the spores. The inhibitory effects observed on Bacillus and Alicyclobacillus spores by the combined treatment of pressure, mild heat, and sucrose laurate appear promising for food applications where alternatives to high heat processing are desired.     

Cloning and disruption of the Pichia pastoris ARG1, ARG2, ARG3, HIS1, HIS2, HIS5, HIS6 genes and their use as auxotrophic markers

Screening of a partial genomic database of Pichia pastoris allowed us to identify the ARG1, ARG2, ARG3, HIS1, HIS2, HIS5 and HIS6 genes, based on homology to their Saccharomyces cerevisiae counterparts. Based on the cloned sequences, a set of disruption vectors was constructed, using the previously described PpURA5-blaster as a selectable marker, and the cloned genes were individually disrupted. All disruptants exhibited the expected auxotrophic phenotypes, with only the his2 knockouts displaying a bradytroph phenotype. To allow their use as auxotrophic markers, we amplified the open reading frames and respective promoters and terminator regions of PpARG1, PpARG2, PpARG3, PpHIS1, PpHIS2 and PpHIS5. We then designed a set of integration vectors harbouring cassettes of the ARG pathway as selectable markers, to disrupt the genes of the HIS pathway and vice versa. Employing this strategy, we devised a scheme allowing for the rapid and stable introduction of several heterologous genes into the genome of P. pastoris without the need for recyclable markers or strains with multiple auxotrophies. Furthermore, simple replica-plating, instead of cost-consuming and labour-intensive colony PCR or Southern analysis, can be used to identify positive transformants, making this approach amendable for initial high-throughput applications, which can then be followed up by a more careful analysis of the selected transformants.