Winged bean lipoxygenase—Part 2: Physicochemical properties

Winged bean lipoxygenase (linoleate: oxygen oxidoreductase EC 1.13.11.12) isoenzymes FI and FII were isolated and purified according to the method of Truong et al. (1980).FI and FII were both highly specific for linoleic acid. They exhibited optimal activity at pH 6·0 and 5·8, respectively at 30°C. An activation energy of 4·5 kcal mol^?1 was calculated for this lipoxygenase within the temperature range of 30–50°C.At 0·075% Tween 20, FI and FII had K_m values for linoleic acid of 0·44 and 0·37 × 10^?3M, respectively, compared to 0·4 × 10^?3M for the crude enzyme. Maximal activity was obtained at 1·6 × 10^?3M. Higher levels of Tween 20 inhibited the lipoxygenase activity.Both isoenzymes had identical average molecular weight of 80 000 daltons by gel filtration and SDS gel electrophoresis.FI and FII isoenzymes were strongly inhibited by Hg⁺⁺, Mn⁺⁺, Mg⁺⁺ and Fe⁺⁺⁺ and activated by Zn⁺⁺, Co⁺⁺ and Fe⁺⁺. A difference in the degree of inhibition or activation was observed between FI and FII response. Ca⁺⁺ inhibited both FI and FII but the former was more sensitive to Ca⁺⁺. KCN also inhibited the two isoenzymes.Among the antioxidants tested, butylated hydroxytoluene and butylated hydroxyanisole most effectively inhibited both FI and FII at only 10^?6M. Sulphydryl reagents such as iodoacetamide and dithiothreitol have little effect on the lipoxygenase isoenzyme activity.The lipoxygenase isoenzymes were more stable at neutral pH. The enzyme in the crude extract and especially in situ was more stable to heat treatment.     

Oxidation of methionine. Effects of hydrogen peroxide alone and in combination with iodide and selenite

The oxidation of methionine by hydrogen peroxide, and the influence of iodide, pH, amino acids and selenite were studied with free methionine and with casein and fish fillet protein. The concentration levels tested ranged from 0·05 mm to 3·0 mm. Hydrogen peroxide oxidation was not influenced by pH in the range 5·0 to 8·0; at pH 8·5 the rate of oxidation was increased. When iodide was added in amounts equivalent to or less than the amounts of H₂O₂, the reaction was accelerated with free but not protein-bound methionine. At higher levels iodide inhibited the oxidation. An amino acid mixture and proteins inhibited the effect of iodide; this effect seemed to be due to tryptophan. Selenite also accelerated the effect of H₂O₂, both with free and with protein-bound methionine. Cu⁺⁺ catalysed the oxidation by H₂O₂ at low reactant concentration but not at the higher levels. The reaction between methionine and H₂O₂ seemed to be of first order with respect to both reactants.     

The use of sesame oil unsaponifiable matter as a natural antioxidant

Individual components of sesame oil unsaponifiable matter isolated from two different coloured seed varieties (white and brown) were identified and quantified. Unsaponifiables from the brown sesame variety were markedly different in their composition from those of the white variety. The brown variety contained higher amounts of total sterols and tocopherols but lower amounts of sesamin, sesamolin and total hydrocarbons than the white variety. The seeds were roasted at 180 °C for 30 min. Roasting increased some effective antioxidant compounds. These included relatively higher percentages of sesamol, Δ^24,28 ethylidene sterols (Δ⁵ and Δ⁷-avenasterols), squalene, as well as tocopherols and some active browning substances. These antioxidative components are effective via synergistic action. Additionally, unsaponifiable matter from unroasted (USM) and roasted white sesame seeds (RSM) was added individually to sunflower oil at levels of 0.02, 0.05 and 0.1% and their effectiveness was compared with a control (no additives) at 63 °C. Results indicated that both USM and RSM had antioxidant activity which increased with increasing concentration. Compared to USM, the RSM was a better antioxidant in most cases. Moreover, the addition of 0.1% RSM gave a strong antioxidative efficiency and this could be used as an alternative natural antioxidant for food applications.     

Inactivation ofEscherichia coliO157:H7 by combinations of GRAS chemicals and temperature

Reported outbreaks of foodborne illness involvingEscherichia coliO157:H7 have increased in the United States during the last decade, with a variety of food products being implicated as vehicles of infection. Studies were carried out to determine the efficacy of combinations of various GRAS chemicals and moderate temperatures to killE. coliO157:H7. A five-strain mixture ofE. coliO157:H7 of approximately 10⁸cfu ml⁻¹was inoculated into 0·1% peptone solutions containing 1·0 or 1·5% lactic acid plus 0·1% hydrogen peroxide, 0·1% sodium benzoate or 0·005% glycerol monolaurate. The solutions were incubated at 8°C for 0, 15 and 30 min; at 22°C for 0, 10 and 20 min; or at 40°C for 0, 10 and 15 min; populations ofE. coliO157:H7 were determined at each sampling time. At 40°C, the pathogen was inactivated to undetectable levels within 10 min of incubation in the presence of 1·0 or 1·5% lactic acid plus hydrogen peroxide, and within 15 min of incubation in the presence of 1·5% lactic acid plus sodium benzoate or glycerol monolaurate. At 22°C, complete inactivation ofE. coliO157:H7 was observed after 20 min of exposure to 1·5% lactic acid plus 0·1% hydrogen peroxide, whereas a reduction of 5 log₁₀cfu ml⁻¹was observed with a treatment of 1·5% lactic acid plus glycerol monolaurate. None of the treatments resulted in total inactivation of the pathogen at 8°C. The aforementioned treatments could potentially be used to inactivate or reduceE. coliO157:H7 populations on raw produce.     

Chitosan inhibits growth of spoilage micro-organisms in chilled pork products

The aim of this study was to develop novel preservation systems for chilled, comminuted pork products that are sold raw using the natural compound chitosan (polymeric ß -1,4- N -acetylglucosamine). In vitro testing showed that viable numbers of Saccharomycodes ludwigii were reduced by up to 4 log cfu ml⁻¹ on exposure to 0·05% chitosan in 0·9% saline at pH 6·2. Higher concentrations of chitosan (0·25 and 0·5%) were required to achieve similar levels of inactivation withLactobacillus viridescens, Lac. sake and Listeria innocua. Torulaspora delbrueckii and Salmonella enteritidis PT4 were resistant to chitosan at the concentrations tested in this study (up to 0·5%). Trials in real foods showed that dipping of standard and skinless pork sausages in chitosan solutions (1·0%) reduced the native microflora (total viable counts, yeasts and moulds, and lactic acid bacteria) by approximately 1–3 log cfu g⁻¹ for 18 days at 7°C. Chitosan treatment increased the shelf-life of chilled skinless sausages from 7 to 15 days. Addition of 0·3 and 0·6% chitosan to an unseasoned minced pork mixture reduced total viable counts, yeasts and moulds, and lactic acid bacteria by up to 3 log cfu g⁻¹ for 18 days at 4°C compared with the untreated control. The results indicated that chitosan was an effective inhibitor of microbial growth in chilled comminuted pork products.     

High carbon dioxide pressure inactivation kinetics of Escherichia coli in broth

The inactivation kinetics of Escherichia coli by high pressure carbon dioxide was investigated. Inactivation rates increased with increasing pressure (25, 50, 75 and 100 atm), temperature, and exposure time. Microbial inactivation followed first order reaction kinetics, with inactivation rates (k) and decimal reduction times (D) that varied from 0·0848 to 0·4717 min⁻¹and from 4·90 to 27·46 min, respectively, at treatment temperatures (20, 30 and 40°C). The inactivation rates of E. coli were described by the apparent activation volume (ΔV^*) and a ‘pressure z value’, and they were greatly dependent on both temperature and pressure.     

Fractionation of Bolti fish tissue lipids into classes and within the triglyceride class with TLC

By utilising TLC with a developing system of petroleum ether-diethylether-acetic acid (70:30:2 v/v) and allowing the front to travel 18 cm, the best conditions for the fractionation of Bolti tissue lipids were achieved. Four distinct classes—triglycerides, free fatty acids, sterols and phospholipids—were detected. In addition, two other faint classes—hydrocarbons and diglycerides—were identified.Silica gel impregnated with silver nitrate has been employed for the fractionation of the triglycerides into groups. Seven groups were found when 1·5% of ethanol was added to the developing system of chloroform-acetic acid (99·5:0·5 v/v). Raising the ethanol content to 2% resulted in the detection of eight groups.     

Proximate Composition and Seed Lipid Components of Sorghum Cultivars from Argentina

Seeds of 11 sorghum cultivars ( Sorghum bicolor ) from Argentina were analysed for proximate composition, fatty acids and sterols. Oil, protein, carbohydrate and ash contents varied between 41 and 66 g kg⁻¹, 111 and 156 g kg⁻¹, 670 and 730 g kg⁻¹ and 13·8 and 20·6 g kg⁻¹ of dry matter, respectively. Fatty acid profiles revealed that the major acids were palmitic (15·1–24·8%), oleic (29·9–41·8%) and linoleic (35·9–51·3%). Unsaponifiable matter was examined for sterols. Sitosterol was the prominent component in all cultivars (43·8–57·9%), followed by campesterol (18·7–29·1%) and stigmasterol (12·4–20·5%).     

Sensitivity of multidrug-resistant Salmonella typhimurium DT104 to organic acids and thermal inactivation in liquid egg products

A mixture of four Salmonella typhimurium DT104 strains and a mixture of four S. typhimurium non-DT104 strains were examined for their ability to grow in tryptic soy broth (TSB) acidified with acetic, lactic, citric, or malic acids at pH 5·4, 4·4, and 3·7. Significantly (P<0·05) higher numbers of S. typhimurium DT104 cells were detected at pH 4·4 and 4·0 in TSB acidified with acetic acid and at pH 4·4 and 3·7 in TSB acidified with lactic acid compared to non-DT104 cells. Acid-shocked and non-shocked (control) cells were plated on TSA (pH 7·3) acidified with lactic acid at pH 5·4, 4·4, and 4·0 and on TSA (pH 7·0±0·2) containing 0·5, 2·5, and 5% sodium chloride. Populations of acid-shockedS. typhimurium DT104 and non DT104 cells recovered on acidified or salt-supplemented TSA were significantly (P<0·05) lower than those of non-shocked cells. A significantly lower number of acid-shocked non-DT104 cells recovered on TSA at pH 5·4, compared to acid-shocked DT104 cells, suggests that DT104 cells may be more resistant to acid shock and subsequent exposure to acid pH. D values and z values of acid-shocked or non-shocked cells of DT104 and non-DT104 strains in liquid whole egg (WE), egg yolk (EY), egg white (EW), whole egg+10% salt (WES), and egg yolk+10% salt (EYS) were determined. Differences in thermal sensitivity of the two types of cells were few. Rates of thermal inactivation of S. typhimurium DT104 cells indicate that the USDA pasteurization process would eliminate >8 log₁₀cfu ml⁻¹of EW heated at 57°C and >11 log₁₀cfu ml⁻¹of WE, EY, WES, or EYS heated at 61°C. D values of acid-shocked DT104 and non-DT104 cells heated in liquid egg products were significantly (P<0·05) lower than those of respective non-shocked cells.     

Products from the autoxidation of Δ5-avenasterol

Comparison of the products from the oxidation of cholesterol and Δ⁵-avenasterol indicates that similar oxidation products are formed by oxidation in the A and B rings of the sterols during the heating of solutions of these compounds in edible oils. However, the UV spectrum of the oxidation products indicates that at least one compound is formed from Δ⁵-avenasterol which is not analogous to the oxidation products of cholesterol. The formation of 5,24(25),28-stigmastatrien-3β-ol is suggested as a possibility.     

Rapid separation of soft drinks ingredients using high performance liquid chromatography

A method is described which uses high performance liquid chromatography in the reverse phase mode to separate amaranth, quinoline yellow, quinine sulphate, sunset yellow, caffeine, aspartame, saccharin, vanillin, sorbic acid, benzoic acid and green S in 4 min in samples of soft drinks. The final separation was achieved using 17·5% acetonitrile, 12·5% methanol, 70% buffer (0·85% v/v sulphuric acid and 17·5% mm potassium dihydrogen orthophosphate in water with the pH adjusted to 1·8), with a flow rate of 1·35 ml/min through a 100 mm × 4·6 mm column containing 3 μm Spherisorb octylsilane. The octylsilane material is fully capped with monolayer coverage and a carbon loading of 6% w/w, pore diameter of 8 nm and a surface area of 220 m²/g. This material was used in preference to the more widely used octadecylsilane as it provides shorter analysis time and allows for the use of more polar eluting solvents. Detection was at 220 nm, 0·1 AUFSD with an injection of 5 μl samples using a rotary injection valve. The standard solution concentration was 100 mg litre⁻¹ for aspartame; 20 mg litre⁻¹ for quinine sulphate, caffeine, vanillin, sorbic acid and benzoic acid and 5 mg litre⁻¹ for amaranth, quinoline yellow, sunset yellow, green S, tartrazine, indigo carmine and carmoisine.     

Influence of pH and temperature upon calcium accumulation and release by bovine sarcoplasmic reticulum

Sarcoplasmic reticulum (SR) was prepared from fresh beef sternomandibularis muscle and shown to be relatively free from contamination by lysosomes, sarcolemma and mitochondrial membranes. Ca^{2 +} accumulation by SR from fresh and cold-shortened muscle was 51 and 39 nmoles/mg protein, respectively. The Ca^{2 +} accumulating ability of fresh SR vesicles decreased with lowering of pH (7·3, 6·8, 6·2, 5·5 and 5·0) at all temperatures (0, 15 and 38°C). Lowering the temperature from 38 to 0°C at pH 6·6 resulted in the release of 48% of the total accumulated Ca^{2 +}, whereas the corresponding value on lowering the temperature from 38 to 15°C at the same pH was only 12%. Thus, low temperatures accelerate the release of Ca^{2 +} by SR. Although simultaneously lowering pH and temperature also increased Ca^{2 +} release by SR, the amount of Ca^{2 +} released was less than if pH and temperature were altered independently. The findings are discussed in the light of explaining cold shortening.     

The potential application of plant essential oils as natural food preservatives in soft cheese

Investigations were carried out to assess the efficiency of four plant essential oils; bay, clove, cinnamon and thyme as natural food preservatives. The effect of the plant essential oils at concentrations of 0·1, 0·5 and 1% was studied in low-fat and full-fat soft cheese against Listeria monocytogenes and Salmonella enteritidis at 4° and 10°C respectively, over a 14-day period. The composition of the cheese was shown to be an important factor in determining the effectiveness of the plant essential oils. In the low-fat cheese, all four oils at 1% reduced L. monocytogenes to ≤1·0 log₁₀cfu ml⁻¹. In contrast, in the full-fat cheese, oil of clove was the only oil to achieve this reduction. Oil of thyme proved ineffective against S. enteritidis in the full-fat cheese, yet was equally as effective as the other three oils in the low-fat cheese, reducing S. enteritidis to ≤1·0 log₁₀cfu ml⁻¹from day 4 onwards. It is concluded that selected plant essential oils can act as potent inhibitors of L. monocytogenes and S. enteritidis in a food product.     

Comparison of the residence time distributions of water and milk in an experimental UHT sterilizer

The residence time distributions (RTD) of water and milk in an experimental direct-heating UHT plant of the infuser type were measured at the same mean residence time using a dye tracer. Results showed that milk had a broader RTD than water, with minimum residence times of 1·0 s and 1·5 s and σ_t² values of 2·19±0·23 and 1·08±0·18, respectively. The effect of the broader RTD on the sterilization efficiency of the plant is discussed, together with possible reasons for this difference.     

Some studies on the proteins of Carica papaya seeds

Protein isolates and seed meals made from Carica papaya seeds were studied with respect to their composition and functional properties. Studies showed that the seed proteins are most soluble in 5% NaCl (23·77 ± 0·15%). In all concentrations of NaCl tested, the protein has a solubility peak at pH 8·0. Classification of the protein showed that globulins constitute the bulk of the protein (53·9 ± 0·89%). The amino acid pattern of the samples studied is not too different from other plant protein sources. However, the seed appeared deficient in many amino acids.Electrophoretic studies showed that the water extract had only one band with a molecular mass of about 70·7 × 10³ daltons. The 5% NaCl extract gave five bands, with molecular mass ranging from 37·6 to 105·9 × 10³ daltons while the NaOH-soluble fraction gave six bands with a range of 18·2 to 104·0 × 10³ daltons.Compared to soya bean meal and protein concentrate, the papaya products were inferior in terms of functional properties.     

Antibacterial activity of the essential oil of Picea excelsa on Listeria,Staphylococcus aureus and coliform bacteria

Antibacterial activity of essential oil of Picea excelsa was tested with the dilution method against one strain of Listeria ivanovii, six of L. monocytogenes, three of Staphylococcus aureus, three of Escherichia coli, one of Klebsiella oxytoca, one of K. pneumoniae subsp. pneumoniae and one of Enterobacter cloacae. For Gram-positive bacteria in stationary phase, 0·07% of essential oil inhibited about 10⁵colony forming unit per ml; a dose of 0·2–0·3% was bactericidal for 10⁵–10⁷cells contained in 1 ml of liquid culture at 37°C. The coliforms, at a concentration of 10⁵CFU per ml, are resistant whatever the physiological age, since they grow with 8% of essential oil. A simplified technique gives good results for the determination of bactericidal activity against Listeria, only indication against S. aureus.     

The effect of redox potential,and its interaction with sodium chloride concentration,on the probability of growth of Clostridium botulinum type E from spore inocula

A most probable number (mpn) count has been used to assess the effect of redox potential and of redox potential combined with increasing concentrations of sodium chloride on the probability of growth of spores of C. botulinum type E strain Beluga. In the majority of cases the maximum count developed within five days. In medium at pH 6·8 – 7·0, containing 0·1% w/v NaCl, incubated at 20°C, the probability of growth of spores in five days was not significantly lower at an E_h of approx +60 mV, adjusted by the introduction of air, than in strictly anaerobic conditions at E_h ?400 mV. Increasing the E_h above +60 mV resulted in a decrease in the probability of growth of spores, and at a redox potential between approx +122 mV and +164 mV the probability of growth was decreased by a factor of over 10⁵ compared with that at the lowest E_h. The inclusion of 3·25% w/v and 4% w/v NaCl in medium at a redox potential of ?400 mV decreased the probability of growth by factors of 10² and 10⁴ respectively, while the combination of 3·25% w/v NaCl and an E_h between +62 mV and +122 mV decreased the probability of growth by a factor of 10⁶.     

Survival and detection of Escherichia coli O157:H7 and Listeria monocytogenes during the manufacture of dry sausage using two different starter cultures

A sausage batter (35% pork, 35% beef, 30% fat) was inoculated with high (5·46–5·68), medium (3·78–4·54) or low (2·30–2·60 log₁₀cfug⁻¹) levels of Escherichia coli O157:H7 and with high (5·05–5·41) or medium (2·92–3·35 log₁₀cfug⁻¹) levels of Listeria monocytogenes serovar 4b and fermented using starter cultures A (Staphylococcus xylosus DD-34 with bacteriocin-producingPediococcusacidilactici PA-2 and Lactobacillus bavaricus MI-401) and B(S. carnosus MIII withLb. curvatus Lb3). Sausages were manufactured (fermented and dried) in a smoke chamber at 17–23°C for 15 days and further stored at 15–17°C for 34 days. The numbers of E. coli O157:H7 decreased more using starter B than starter A (first experiment P<0·0015, second experiment P<0·0002) but the organism was not eliminated. Small numbers of E. coli O157:H7 were more often detected after enrichment for 18–24 h than for 6 h (P=0·0044) when tested after deep freezing. By contrast, L. monocytogenes decreased more rapidly in the high-inoculum sausages produced with starter A (P<0·0001) but no significant difference was detected between the starters in the medium-inoculum sausages. L. monocytogenes was eliminated from the medium-inoculum sausages after 49 days.     

Selection of starters for a traditional Turkish yayik butter made from yoghurt

Butter is produced from two different materials in Turkey, cream and yoghurt. The butter produced from fresh yoghurt or ‘tulum yoghurt’ (a strained yoghurt produced from cow, goat or sheep milk) is called ‘yayik butter’ and has been traditionally produced in Turkey for centuries. In this research, we attempted to isolate and identify the natural lactic acid bacteria (LAB) of yayik butter and to select the best LAB combination for butter production. Twenty samples of yayik butter were collected from Afyon, Antalya, Isparta and Konya regions in Turkey and determined to have a mean pH of 4·78±0·33, a mean titratable acidity (lactic acid) of 0·23±0·07% and a mean NaCl of 0·55±1·22%. The mean counts of LAB (log₁₀ cfu g⁻¹) were 2·66±0·84 and 1·72±0·82 on MRS agar at 30 and 42°C, 2·44±0·93 and 1·78±0·24 on M17 agar at 30 and 42°C, and 1·64±1·196 on Sodium Azide Leuconostoc agar at 21°C, respectively. Eighty-five different LAB isolates were obtained from 20 yayik butters and identified asStreptococcus salivarius ssp. thermophilus (21·2%), Streptococcus sp. (4·7%), Lactobacillus delbrueckii ssp. bulgaricus (20%), Lactobacillus casei ssp.casei (15·3%), Lactobacillus paracasei ssp. paracasei (2·3%),Enterococcus faecium (18·8%). Leuconostoc pseudomesenteroides (Leucono-stoc mesenteroides ssp. dextranicum) (7·1%), Leuconostoc gelidum (Leuconostoc mesenteroides ssp.mesenteroides ) (4·7%) and Weissella paramesenteroides (Leuconostoc paramesenteroides) (5·9%). Combinations of S. salivarius ssp. thermophilus S51, Lb. delbrueckii ssp.bulgaricus A42, Lb. casei ssp. casei K64, Lb. paracasei ssp. paracasei A27, andLeu. pseudomesenteroides E83 were used as starter bacteria for experimental butter production from cream. Six different groups of butters were produced using different combinations of these bacteria (B, C, D and E samples), commercial culture (F sample), and without culture (A sample). Sensory evaluations showed that the experimentally produced butter sample of group B was more acceptable than the other butters. In addition, the buttermilk of sample B had lowest fact content. LAB counts of experimental butters produced with combined cultures and commercial culture were similar (6·66±1·87–6·83±0·040 and 6·81±0·13 log₁₀ cfu g⁻¹ on MRS agar, respectively).     

Use of surface-smear bacteria for inhibition ofListeria monocytogeneson the rind of smear cheese

Surface-smear micro-organisms isolated from Taleggio cheese were screened for their ability to inhibitListeria monocytogenes. Most of the isolates showing antilisterial activity (19% of the total) consisted of coryneform bacteria, mainly belonging to theMicrobacterium lacticumspecies. The inhibitory activity was observed also after growth at 5°C and pH 6·0. After cross-inhibition tests among inhibitory strains and between these and other pigmented, non-inhibitory strains, two bacterial mixtures were assayed as surface-smear starter of Taleggio cheese. At different ripening stages (1, 20 and 40 days), the cheese surface was contaminated with 2·5×10²cfu cm⁻²L. monocytogenes(Ohio strain) and the growth evaluated over 15 days of incubation at 5°C. Contrary to the laboratory experiments,Listeriacould not be completely inhibited on the cheese surface. With 5·5–6·0 pH range of the cheese rind, (lower than usual values), the growth of surface-smear bacteria was delayed, or even stopped. Nevertheless, a listeriostatic effect was achieved on the surface of cheese samples contaminated at the end of ripening. This seemed to confirm the essential role played by surface-smear bacteria, within the rind microflora, in controlling the development of contaminant micro-organisms, and by competitive bacteria in reducing the overall risks associated with soft cheese. The mechanisms involved in the selection and the growth of competing bacteria on the cheese surface are also discussed.