PARTIAL PURIFICATION AND CHARACTERIZATION OF SWEET CORN GERM LIPOXYGENASE

Lipoxygenase (LPO) extracted from the germ fraction of sweet corn (Zea mays L. var. Jubilee) was purified by acetone powder preparation; extraction with 0.1 M Tris-HCl, pH 8; 40–60% fractionation with ammonium sulfate; and conventional column chromatography on Sephacryl S-300 HR and Fast Protein Liquid Chromatography (FPLC) on a Mono Q column. A 124-fold purification was achieved with 26.3% recovery. Further purification was achieved by FPLC chromatofocusing on a Mono P column and size exclusion chromatography on Superose-12, with a resultant 436-fold purification and 16.8% recovery. The apparent molecular weight and isoelectric point (pI) determined by FPLC on Superose-12 and Mono P columns were 90,500 and 5.06, respectively. Lipoxygenase-catalyzed formation of conjugated dienes was inhibited by both synthetic (BHA, BHT) and natural phenolic antioxidants (quercetin, chlorogenic acid) at a concentration of 0.2 mM with 57.2, 16.3, 61.4 and 32.3% inhibition, respectively. The activation energy for thermal inactivation of sweet corn germ lipoxygenase from the ammonium sulfate preparation at 55–70C was 56.3 kcal/mol.     

Characterization of Seed Lipoxygenase of Phaseolus vulgaris cv, Haricot

Phaseolus vulgaris cv, haricot seed lipoxygenase was extracted and partially purified. The precipitation of an active lipoxygenase fraction with solid ammonium sulfate (at 20–50% of saturation) increased its activity by a factor of 3. The pH for optimum activity was 7.3. The addition of 40 mM potassium cyanide resulted in a doubling of the enzyme activity. Enzyme activity was completely lost on storage at 4°C for 48 hr. The activity of haricot lipoxygenase extract was considerably greater on linoleic acid than on its esters, and decreased in the order: mono- > di > trilinolein. The haricot lipoxygenase shares many of the characteristics of the corresponding enzyme found in several seed sources.     

Purification and some properties of wheat germ lipoxygenase

Lipoxygenase from the germ of bread wheat was purified to near homogeneity by a classical scheme. After extraction at pH 4.5 from defatted germ, lipoxygenase activity was precipitated by 40% saturation (NH₄)₂SO₄ from a 25% saturation supernatant. After dissolution in a phosphate buffer at pH 7 and extensive dialysis against this buffer, the extract was submitted to gel filtration on Ultrogel AcA 34. The final step of DEAE Sephadex A50 chromatography gave three peaks (L₁, L₂ and L₃) with lipoxygenase activity. The total yield of the purification procedure was close to 30% and the degree of purification varied from 200 to 300 depending on the fraction considered. The three isoenzymes were also detected by disc electrophoresis using a specific staining method and were isolated by isoelectric focusing in a liquid medium. The molecular weight for each isoenzyme was determined to be 90 000–95 000 by gel filtration and 110 000 by electrophoresis in a gradient of acrylamide concentration. The stability, pH optimum and calcium effect have been studied. L₂ and L₃ were less stable than L₁. Optimum pH ranged between 6–6.5 and neither isoenzyme activity was affected by calcium ions. L₁ was twice as active towards β-carotene as L₂ and L₃ for the same level of oxygen uptake, but in comparison to lipoxygenase from horsebean the co-oxidising power of wheat lipoxygenase was very poor.     

Optimizing Assay and Extraction of Lipoxygenase in Wheat Germ

Using three-level seven-factor response surface methodology, wheat germ lipoxygenase (LPO) assay conditions were standardized. The important parameters were concentration of the substrate (linoleic acid), and surfactant (Tween 20), pH and temperature. The standardized LPO assay conditions for the 1 mL reaction volume were : 450 μM linoleic acid, 129 μM Tween 20, 175 mM ethanol, 1 mM EDTA, pH 6.2 (phosphate buffer), ionic strength 100 mM and 40°C. LPO extraction conditions were standardized by sequential variation of parameters. Optimum conditions were a single extraction of defatted wheat germ flour at 2–5°C with magnetic stirring of an extractant acetate buffer pH 4.5, ionic strength 100 mM, at buffer-to-solid ratio 10:l.     

Effect of pH,heat, and light treatments on the antioxidant activity of sweet potato leaf polyphenols

In the present study, the antioxidant activity of sweet potato leaf polyphenols was measured, and the effect of different pH values (3.0, 5.0, 7.0, and 8.0), temperatures (55, 65, 80, and 100°C), and light treatments on sweet potato leaf-polyphenols was investigated. Results showed that the O₂^? scavenging activity of sweet potato leaf polyphenols from cultivar Jishu No. 04150 was 5.84 and 6.20 times of that of tea polyphenols and grape seed polyphenols, respectively, at the concentration of 20 μg/mL. The oxygen radical absorbance capacity was 1.28 and 1.27 times of that of tea polyphenols and grape seed polyphenols, respectively. Sweet potato leaf polyphenols dissolved in pH 5–7 solutions showed higher retention rates of antioxidant activity. Heat treatment at 50 and 65°C and light treatment had little effects on sweet potato leaf polyphenols. In conclusion, sweet potato leaf polyphenols possessed high antioxidant activity and processing stability, having the potential to be a new type of natural antioxidant.     

Partial Purification and Characterization of Asparagus Lipoxygenase

Lipoxygenase (LOX) from fresh asparagus was partially purified by extraction of acetone-washed asparagus powder with pH 4.5 potassium phosphate, ammonium sulfate fractionation and carboxymethyl-cellulose (CMC) chromatography. Asparagus LOX purified by ammonium sulfate fractionation had a pH activity optimum of 5.5–6.0 and was stable at pH 4.5–8.0 when stored at 2°C. Asparagus LOX was active on monolinolein as well as linoleic acid, but activity was very low on di- or tri-linolein. The CMC fractions with greatest LOX activity were nearly free of peroxidase activity while the protein fractions which did not bind with CMC at pH 5 were peroxidase active. The LOX activity in the purified asparagus extract was 90% inhibited by 1 mM cyanide when preincubated for 30 min at 25°C.     

Use of a spouted bed to improve the storage stability of wheat germ followed in paper and polyethlyene packages

Stabilization of wheat germ by heating in a spouted bed for 180–540 s with air at 140–200 °C was studied. The lipase activity decreased by 6–65%. Wheat germ processed at 200 °C for 360 s was ranked highest in sensory evaluation, described as having ‘a golden color’ and ‘nutty flavor’, and its lipoxygenase activity had decreased by 91.2%. This product and raw wheat germ were stored in paper, polyethylene and vacuum‐packed polyethylene pouches at 5 °C, room temperature (18–26 °C) and 40 °C, and the moisture contents, water activities, free fatty acid contents and peroxide values were followed for 20 weeks. The increases were faster in paper pouches than in the polyethylene ones; vacuum packaging in polyethylene did not bring about significant improvement. The peroxide values of raw samples exceeded 10 meq O₂ kg^?1 oil after 3–23 days while those of the processed samples stored at room temperature or 5 °C were still less than 10 after 20 weeks. The free fatty acid content and peroxide value changes were expressed by zero order kinetics, resulting in similar activation energies for the raw and processed samples. Copyright © 2005 Society of Chemical Industry     

Identification and characterization of lipoxygenase in fresh culinary herbs

The aim of this project was to study the biochemical characterization of lipoxygenase extracted from basil (Ocimum basilicum L.), rosemary (Rosmarinus officinalis L.), and sage (Salvia officinalis L.) leaves. The lipoxygenase extracted from these culinary herbs was frozen with liquid N₂ and ground into powder before adding ice-cold phosphate buffer (pH 7). The crude extracts from the three aromatic plants showed various specific activities and the highest specific activity and the lowest protein content were observed for the basil extracts. The optimum pH values for lipoxygenase extracted from basil and sage ranged between 5 and 6, while pH 6 was the optimum value for lipoxygenase activity for rosemary. The optimum temperature was 40°C for all of the crude extracts analyzed. Considering the biochemical characteristics evaluated for lipoxygenase from each culinary herb, suitable strategies for the inactivation of the enzyme can be proposed in order to reduce its detrimental effects on fresh aromatic herbs.     

Inactivation of Lipoxygenase in Soybeans with Retention of Protein Solubility

The enzyme lipoxygenase, in soybeans, catalyzes the oxidation of polyunsaturated fatty acids containing a cis, cis 1:4 pcntadiene unit, resulting in rancid off-flavors and poor storage stability. The effects of various processing conditions such as soaking, heating. pH and shearing action on the inactivation of lipoxygenase were investigated. The results showed that lipoxygenase activity was decreased by homogenization and by modcrate heating. Of the three isozymes of lipoxygenase. resistance to inactivation by homogenization and heating was in the order LI > L2 >L3. Total inactivation of soybean lipoxygenase was achicvcd by soaking the seeds at pH 8.5 at about 50°C for a minimum of 3–4 hr. followed by homogenization with water at about 60°C. for 15–30 min at 20,000 rpm.     

Comparison of fumonisin B1 biosynthesis in maize germ and degermed kernels by Fusarium verticillioides

Fusarium verticillioides produces a group of mycotoxins known as fumonisins in maize kernels. Fumonisins are associated with a variety of mycotoxicoses in humans and animals; thus, their presence in food is a considerable safety issue. This study addressed fumonisin B1 (FB1) production in two components of the maize kernel, namely the germ tissues and the degermed kernel. Growth of F. verticillioides was similar in colonized germ tissue and degermed kernels, but FB1 production was at least five times higher in degermed maize kernels than in germ tissue. Expression of the fumonisin polyketide synthase gene, FUM1, as measured by beta-glucuronidase (GUS) and Northern blot analysis, followed the same pattern as FB1 production. Also correlated to FB1 was a concomitant drop in pH of the colonized degermed kernels. A time course experiment showed that degermed kernels inoculated with F. verticillioides became acidified over time (from pH 6.4 to 4.7 after 10 days of incubation), whereas colonized germ tissue became alkaline over the same period (from pH 6.5 to 8.5). Because conditions of acidic pH are conducive to FB1 production and alkaline pH is repressive, the observed correlation between the acidification of degermed kernels and the increase in FB1 provides one explanation for the observed differences in FB1 levels.     

EFFECTS OF ESCULETIN AS A LIPOXYGENASE INHIBITOR IN SOYBEAN EXTRACTS

This study was designed to determine the ability of esculetin to inhibit lipoxygenase activity in soybean. Lipoxygenase was extracted from ground soybean using 0.1 M Tris-HCl buffer (pH 8.6) and centrifuged at 3000 g for 15 min at 4C. The extract was mixed with different volumes of 0.05M esculetin solutions to provide 1.8 × 10⁻⁴M, 3.3 × 10⁻⁴M, 4.6 × 10⁻⁴M and 5.7 × 10⁻⁴M to final assay systems, respectively. Lipoxygenase activity decreased as the concentration of esculetin solution mixed in soybean extract increased. Mixing of 1.8 × 10⁻⁴M esculetin solution to soybean extract showed the least inhibition effect and was significantly different from that of 3.3 × 10⁻⁴M, 4.6 × 10⁻⁴M and 5.7 × 10⁻⁴M which inhibited LOX (lipoxygenase)'s activity of soybean extracts. In the study comparing the inhibition abilities for lipoxygenase activity among selected antioxidants, esculetin was significantly better than BHA (butylated hydroxyanisole) and α-tocopherol.     

On lipoxygenase and enzymes which decompose linoleic acid hydroperoxides in rye (author's transl)]

An enzyme fraction from rye containing lipoxygenase activity was investigated. The molecular weight of lipoxygenase was found to be about 102000. Two bands groups with isoelectric points between 5.1-5.5 and 5.8-6.4 were obtained by isoelectric focusing. Three isoenzymes could be separated by ion exchange chromatography. Lipoxygenase has optimum activity at pH 7.3-7.5 and predominantly forms 13-hydroperoxy-9-cis, 11-trans-octadecadienoic acid (13-LHPO). In rye the 13-LHPO is converted to alpha-ketols by a high molecular protein fraction. This isomerase converts the LHPO formed by rye lipoxygenase predominantly to 12,13-ketohydroxy acids. The Michaelis Constant of isomerase is 3-5 X 10(-5), using LHPO as substrate. At low protein concentrations the reaction velocity of LHPO-conversion increases linearly with protein concentration.     

Injury,Inhibition and Inactivation ofEscherichia coli O157:H7 by Potassium Sorbate and Sodium Nitrite as Affected by pH and Temperature

The effect of potassium sorbate (0–2 g litre⁻¹) and sodium nitrite (0–1 g litre⁻¹) on the growth of four strains of Escherichia coli O157: H7 in tryptic soya broth at various pH levels (pH 4·0–7·0 for sorbate, pH 5·0–8·0 for nitrite) were determined at 37°C and 4°C. Among the pH levels tested, sorbate and nitrite exhibited the highest antimicrobial activity at pH 4·0 and 5·0, respectively. At pH 5·0 and 37°C, the presence of 500 mg litre⁻¹ sorbate or 200 mg litre⁻¹ nitrite completely inhibited the growth of E coli O157: H7. While at higher pH levels, 2 g litre⁻¹ sorbate or 1 g litre⁻¹, nitrite, the highest concentration tested, did not show significant antimicrobial action against the test organisms. At 4°C and pH 5·0, the inoculated test organisms did not showed any significant growth in preservative-free control media. Different degree of inactivation and injury was observed when E coli O157: H7 strain 933 was stored in TSB (pH 5·0) containing 1 g litre⁻¹ sorbate or nitrite at 37°C. At 4°C, inactivation and injury of E coli O157: H7 cells was not observed in the medium containing sorbate or nitrite throughout the 24 h experimental period.     

Biologically Active Components Inactivation and Protein Insolubilization during Heat Processing of Soybeans

Inactivation of lipoxygenase, trypsin inhibitor, urease and retention of protein solubility during water blanching of dehulled soybeans at 90°, 95° and 100°C were investigated. Lipoxygenase was the most heat labile, followed by urease and then trypsin inhibitor. Processing time based on “acceptable inactivation time” (AI) was proposed. AI value was longest for trypsin inhibitor, followed by urease and then lipoxygenase. The combined effect of heat on protein solubility and biologically active components inactivation was expressed as “PDI at acceptable inactivation time” (PDIAI). PDIAI value for the processing “limiting factor,” trypsin inhibitor inactivation, at the three temperatures were 36.7%, 42.5%, and 34.8%, respectively.     

Viscoelastic properties of caseinmacropeptide isolated from cow,ewe and goat cheese whey

Caseinmacropeptide (CMP) is a C‐terminal glycopeptide released from κ‐casein by the action of chymosin during cheese‐making. It is recognised as a bioactive peptide and is thought to be an ingredient with a potential use in functional foods. CMP occurs in sweet cheese whey and whey protein concentrate (WPC). Its composition is variable and depends on the particular whey source and the fractionation technology employed in the isolation. There were no significant (P < 0.05) differences in the relative apparent viscosities between species of CMPs (cow, ewe and goat). Analyses at different pH (2, 4, 7, 10), ionic strength (0, 0.2, 0.4 and 0.7 as NaCl molarity) and protein concentration (50, 100 and 200 g kg^?1) at temperatures from 10 to 90 °C carried out found pH 7 and high protein concentration (200 g kg^?1) conditions to be the best for CMP solutions to keep low and constant relative viscosity values with increasing temperature up to 75 °C. The viscoelastic properties–storage modulus, loss modulus and phase angle–of the different CMPs and WPC solutions were determined. Heat‐induced rheological changes in CMP solutions occurred at moderate temperatures (40–50 °C) with no appreciable differences in viscosity. Gelation took place significantly (P < 0.05) earlier in goat CMP (41 °C), followed by cow CMP (44 °C), ewe CMP (47 °C) and WPC (56 °C). Heating at 90 °C showed that WPC required significantly (P < 0.05) longer times to form gels (>5 min) than the CMPs (<5 min). WPC gels had higher (>20°) phase angle than CMP (<20°), which could be associated with untidy structures, limiting elastic properties of the gel. Copyright © 2006 Society of Chemical Industry     

PARTIAL PURIFICATION AND CHARACTERIZATION OF PEACH PECTINESTERASE

Pectinesterase (EC 3.1.1.11) was extracted from peaches (Prunus persica) and partially purified by preparative free solution isoelectric focusing. On SDS-PAGE gels, protein bands at 36.3 and 33.9 kilodaltons represented the major bands; minor bands were observed at 108.4, 40.7, and 17.0 kilodaltons. The pH optimum for pectinesterase activity in the partially purified extract was 8.0. The enzyme was stable at 30°C for 30 min between pH values of 5 and 8. Peach pectinesterase is stable when heated at 55°C for 5 min in 0.1 M NaCl, 50 mM sodium phosphate, pH 7, buffer. However, residual activity decreased to 23% of 65°C for 5 min and was inactivated at 70°C for 5 min. The energy of activation of peach pectinesterase was determined to be 34, 600 J/mol °K. The Q₁₀ between 30°C and 60°C was estimated to be 1.5–1.6.     

Blanch Time and Cultivar Effects on Quality of Frozen and Stored Corn and Broccoli

Three corn and 2 broccoli cultivars were steam blanched for various times and evaluated for residual enzyme activity. Lipoxygenase was inactivated in 4 min in supersweet corn, while sweet corn required a 6-min blanch; peroxidase was inactivated in 8 min. Inactivation of broccoli lipoxygenase, peroxidase, and cystine lyase was achieved in 90 s. Blanched samples were stored 9 mo at –18 °C, then analyzed for color, texture, hexanal, free fatty acids, and sugars. Firmness increased significantly with blanching in both commodities, then declined. Short blanch treatments targeting lipoxygenase inactivation positively affected color and texture of both corn and broccoli. Changes in current industry practices are recommended.     

Purification of a cyanide-sensitive superoxide dismutase from soya beans: A food-compatible enzyme preparation

A method of purifying superoxide dismutase (SOD; EC 1.15.1.1), free from lipoxygenase (EC 1.13.11.12) and catalase (EC 1.11.1.6), from ethanol-treated soya beans is described, Preparations with the highest activity were obtained from new season's beans. Although not homogeneous by electrophoretic analysis, the preparation was 300-fold purified. The enzyme has a molecular weight of 36 300 and a subunit molecular weight of 18 000. Its sensitivity to cyanide suggests that it is a typical cupro-zinc SOD. Isoelectric focusing showed the presence of three separate charged species with isoelectric points of pH 4·73 (the major species), 4·55 (the minor species), and 4·37. The enzyme withstands 45 min at 65°C without loss of activity, and loses only half its activity in 30 min at 75°C at pH values between 5 and 8. Severe losses in activity at pH 4 and below are observed at 75°C. The enzyme can be stabilised for storage at 4°C in 60% (v/v) glycerol.     

Pectin lyase and polygalacturonase of Aspergillus niger pathogenic for yam tubers (Diascorea rotundata)

Polygalacturonase and pectin lyase of Aspergillus niger partially purified by ethanol, ammonium sulphate precipitation, DEAE-cellulose and Sephadex G-150 column chromatography were characterized. Polygalacturonase gave optimum activity at pH 4–5, and at 35°C. It was stable at pH 3–7 and at 20–50°C. The molecular weight was 38020. For pectin lyase optimum activity occurred at pH 5 and 45°C. The enzyme was stable at pH 3–4 and at 40–50°C. The molecular weight was 30 900. Yam tissue was optimally macerated at pH 4–5 by the enzymes. At pH 4.5, potassium sorbate (0.6 mg/ml), benzoic acid (0.8 mg/ml) and sodium benzoate (1.0 mg/ml) caused complete inhibition of polygalacturonase activity. With pectin lyase, this effect was achieved with potassium sorbate and benzoic acid each at 0.9 mg/ml, but not with sodium benzoate.     

The role of acyl moiety in the formation and reactions of steryl ester hydroperoxides

‘Golden’ is the most cultivated apple group in Europe. In recent years, new mutants have emerged which improve both aspects of production and sensory attributes. In this work, emission of volatile compounds and the activity of lipoxygenase system–related enzymes in ‘Golden Reinders^®’ apples were analysed after 19 and 30 weeks under ultra low oxygen atmosphere (ULO) or under ULO plus different periods in cold air atmosphere and after remaining 1 and 7 days at 20 °C. This study examined modifications in the capacity for volatile ester production, specifically focusing on modification in lipoxygenase and hydroperoxide lyase activity. Volatile compound emission reached a maximum after 30 weeks of cold storage plus 7 days at 20 °C. Straight-chain esters were closely related to lipoxygenase activity in the flesh tissue, leading, suggesting that lipoxygenase enzyme plays an important role in the emission of straight-chain esters: the most characteristic volatile type associated with this apple variety.