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.     

Electrolytic Reduction of Heme Proteins: Attempt to Prepare Stable Natural Colorant for Sausage

Metmyoglobin and methemoglobin were subjected to electrolysis in presence or absence of sodium ascorbate and/or sodium nitrite. Products obtained by electrolysis were dependent on conditions. In absence of sodium ascorbate and sodium nitrite, metmyoglobin and methemoglobin were apparently transformed to red colored hydroxymetderivatives by limited electrolysis (50 mA, 5 min) because of increased pH of electrolytes. They were reduced to oxy-derivatives by prolonged electrolysis (100 mA, ?4 hr) at neutral pH, 7.0–7.5. In presence of sodium ascorbate, met-derivatives were reduced to deoxy-derivatives by limited electrolysis (50 mA, 5 min). In presence of both sodium ascorbate and sodium nitrite, stable nitrosyl derivatives were obtained by limited electrolysis, and by prolonged electrolysis (100 mA, ?4 hr) at neutral pH values especially in presence of 1M sucrose. The electrolytically reduced products appreciably increased redness of sausages.     

Reaction of nitrite with ascorbic acid and its significant role in nitrite-cured food

The decrease in concentration of free nitrite in an aqueous reaction solution made by mixing nitrite and ascorbic acid was studied. This decrease resulted information of a reaction product which transferred NO to ferricytochrome c to form ferrocytochrome c nitroso compound. From the solution containing the reaction product, nitrite could be regenerated spontaneously and suddenly by physical agitation. Oxygen was apparently not necessary for regeneration nitrite from the reaction solution. It does not seem that the regeneration of nitrite was responsible for a bimolecular dismutation of nitrosoascorbic acid in a reverse direction proposed by Fox and Thomson (Biochemistry2, 465; 1963). Heating of the reaction solution at less than 100°C resulted in a loss of nitrite from the solution, which was greater with increasing concentration of ascorbic acid. The reaction product formed between nitrite and ascorbic acid is probably responsible not only for nitrosation reactions, but also for the loss of nitrite observed during curing of meat.     

导数伏安法测定肉类食品中的亚硝酸盐

在盐酸介质中,痕量亚硝酸根对溴酸钾氧化中性红的反应有极强的催化作用,研究了最佳反应条件,发现在氨缓冲溶液中中性红具有良好的导数电流峰,通过悬汞电极跟踪催化反应过程中中性红浓度的变化,建立了测定痕量亚硝酸根的新方法。方法的线性范围为1.2×10~(-7)～5.0×10(~-4)μg·mL~(-1)。检出限为5.4×10~(-8)μg·mL~(-1),应用于肉类食品中亚硝酸根的测定,结果满意。     

Multiple α-galactosidases from <Emphasis Type="Italic">Aspergillus foetidus</Emphasis> ZU-G1: purification,characterization and application in soybean milk hydrolysis

α-Galactosidase had applied in food and feed industries for hydrolyzing raffinose series oligosaccharides (RO) that are the factors primarily responsible for flatulence upon ingestion of soybean-derived products. The objective of the current work was to purify the α-galactosidase of Aspergillus foetidus ZU-G1 and compared the biochemical and hydrolytic properties of three major α-galactosidase forms (α-gal I, α-gal II and α-gal III). The molecular mass of the purified enzyme as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 106.3, 49.7 and 109.9 kDa, respectively. Its optimum reaction temperature was 60 °C and stable below 50 °C. The optimum pH of α-gal I and α-gal III was 5.0 and α-gal II was 4.0. Under 28 °C conditions for 24 h, α-gal I was stable at pH 4.0, α-gal II was stable at pH 6.0, and α-gal III was pH 5.0. α-Galactosidase was completely inhibited by Ag⁺. CuSO₄·5H₂O and SDS were powerful inhibitors of α-gal I and α-gal III but had little effect to α-gal II. EDTA did not strongly affect α-gal I and α-gal III, while strongly affect α-gal II. CaCl₂·2H₂O, MgSO₄·7H₂O and MnSO₄·7H₂O were activation for α-gal I, α-gal II and α-gal III. No significant inhibition of enzymes activity was observed in the presence of raffinose, lactose as well as other sugars tested. Synthetic substrate p-nitrophenyl-α-d-galactopyranoside was not preferentially hydrolyzed than natural substrates, such as melibiose, stachyose and raffinose. Under 40 and 50 °C incubation for 1–5 h, the stachyose of soybean milk was degraded by α-gal I, α-gal II and α-gal III and strongly hydrolyzed by α-gal II, and the raffinose of soybean milk was completely hydrolyzed by α-gal II and weakly hydrolyzed by α-gal I and α-gal III. The distinct hydrolytic and biochemical properties of α-gal I, α-gal II and α-gal III further signify the α-galactosidase of A. foetidus ZU-G1 was propitious to soybean milk and related food industry.     

Growth Inhibition of Listeria monocytogenes by Sodium Polyphosphate as Affected by Polyvalent Metal Ions

Sodium polyphosphate (SPP, average chain length = 13) increased the lag time of L. monocytogenes Scott A (Lm) in brain-heart infusion broth (BHI). Polyvalent metal ions (1–10 mM) reversed inhibition of Lm growth by 0.5% SPP (nominal 3.6 mM). 10 mM Ca²⁺ or Mg²⁺, 5 mM Fe³⁺, 2 mM Mn²⁺ or 1 mM Zn²⁺ added to SPP-containing BHI, pH 6.0, at 19°C resulted in growth comparable to control cultures. Fe²⁺ partially restored growth; Ni²⁺, Co²⁺, Cu²⁺ or Al³⁺ were ineffective. SPP inhibited growth at 28°C in BHI, pH 5.0, and Lm grew upon addition of Ca²⁺, Mg²⁺ or Mn²⁺, but not Zn²⁺ or Fe³⁺. Addition of 0.5% SPP to mineral-rich foods, such as pureed beef, green beans or sweet potatoes, did not delay growth.     

HEAT-INDUCED GELATION AND PROTEIN-PROTEIN INTERACTION OF ACTOMYOSIN1

Natural actomyosin (NAM) and “crude” actomyosin formed gels yielding maximum strengths (from back extrusion force) at pH 5.0 and 5.5, respectively. At pH 6.0, NAM gels had a least protein concentration endpoint (LCE) value of 6 mg/ml. Gel strength increased exponentially with an increase of NAM concentration from 3.75–10 mg/ml. With constant time (30 min)-temperature heating, NAM gel forces increased by 20.5% (NS, P>0.05) in the 30–80°C range. Arrhenius plots of NAM interaction in solution and in gelation at pH 6.0 indicated two different reaction mechanisms within the temperature zones above and below approximately 35°C for solutions and 40°C for gels. Similarity of interaction slopes above the 35–40°C region suggested one reaction mechanism for NAM molecular aggregation in solution and gelation.     

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.     

Characterization of Pullulanase and α-Amylase Activities of a Thermus sp. AMD33

Thermostable Thermus sp. AMD 33 pullulanases (I and II) capable of cleaving α-1,6-links in pullulan as well as α-1,4-glucosidic linkages in amylose were purified to electrophoretically homogeneous states. Relative molecular masses and pI values were determined as 135,000 (I and II) by SDS-PAGE and 4.2 (I) and 4.3 (II) by isoelectric focusing, respectively. The pullulanase and α-amylase activities of the purified enzyme II responded similarly to temperature and pH, with optima at 70°C and pH 5.5–6.0. Both activities were activated by Ca²⁺ and inhibited by Hg²⁺, Fe³⁺, NBS, DBS, SDS and urea to almost the same extent. Both activities were also inhibited competitively by CDs. Enzyme II catalyzed the hydrolysis of α-1,6-glucosidic linkages in maltosyl- and maltotriosyl-α-CD as well as that of α-1,4-bonds in amylose and related linear malto-oligosaccarides larger than maltotriose, but exhibited no action on panose, isopanose or glucosyl α-CD.     

IN VITRO SELF-ASSEMBLY OF SILVER-LINE GRUNT TYPE I COLLAGEN: EFFECTS OF COLLAGEN CONCENTRATIONS, pH AND TEMPERATURES ON COLLAGEN SELF-ASSEMBLY

Collagen self‐assembly of silver‐line grunt type I collagen solution was done at a concentration of 1 mg/mL and at pH 5, 6, 7 and 8 with incubation temperatures of 25, 30 and 35C. The self‐assembly of collagen showed typical sigmoid curves and had a short lag phase at pH 5, 6, 7 and 8. The lowest absorbance was found at pH 5.0, whereas pH 6.0 gave the highest absorbance and the fastest growth phase. The absorbance gradually increased at lower temperature, but rapidly increased at higher temperature. The rate of reconstruction also increased when the temperature increased from 20 to 30C, but the rate decreased when the temperature rose to 35C. The rate increased rapidly with increasing pH from 5.0 to 6.0, but decreased with raising pH from 6.0 to 8.0. The scanning electron micrographs of the collagen fibrils reconstructed at pH 6.0 were thicker, more uniform and more interwoven than fibrils formed at pH 7.0.     

Purification and Some Properties of a Protease from Sorghum Malt Variety KSV8–11

A protease from sorghum malt variety KSV8–11 was purified by a combination of dialysis against 4 M sucrose, ion‐exchange chromatography on Q‐Sepharose (Fast flow), gel filtration chromatography on Sephadex G‐100 and hydrophobic interaction chromatography on Phenyl Sepharose CL‐4B. The enzyme was purified 5‐fold to give a 14.1% yield relative to the total activity in the crude extract and a final specific activity of 1348.9 U mg^?1 protein. SDS‐PAGE revealed a single migrating protein band corresponding to a relative molecular mass of 16 KDa. Using casein as substrate, the purified protease had optimal activity at 50°C and maximal temperature stability between 30°C and 40°C but retained over 64% of its original activity after incubation at 60°C for 30 min. The pH optimum was 5.0 with maximum stability at pH 6.0 but 60% of the activity remained after 24 h between pH 5.0 and 8.0. The protease was inhibited by Ag⁺, Ca²⁺, Co²⁺, Fe²⁺, Mg²⁺, iodoacetic acid (IAA) and p‐chloromercuribenzoate (p‐CMB), stimulated by Cu²⁺, Sr²⁺, phenylmethylsulfonyl‐fluoride (PMSF) and 2‐mercaptoethanol (2‐ME) while Mn²⁺ and ethylenediaminetetraacetic acid (EDTA) had no effect. The purified enzyme had a K_m of 18 mg·mL^?1 and a V_max of 11.1 μmol · mL^?1 · min^?1 with casein as substrate.     

KINETIC ANALYLSIS OF THE FORMATION OF NITROSYLMYOGLOBIN

The formation of nitric oxide myoglobin (nitrosylmyoglobin) was followed in buffered solutions in which the concentrations of ascorbate, nitrite, chloride, myoglobin and hydrogen ion were varied systematically to determine their effect on the rate constants. The rate of formation of nitrosylmyoglobin was zero order with respect to the pigment. The orders for the other reactants were determined by plotting the zero order rate constants as functions of varying orders of each reagent to determine which order gave a linear plot. The results were used to develop a mechanism and a mathematical expression for the reaction. Two reaction sequences involving different nitrosating species were involved; 1, direct action of nitrous acid and 2, the formation of nitrosyl chloride. Both species then nitrosated ascorbate and ascorbic acid, by different mechanisms. The nitric oxide for nitrosylmyoglobin formation came from the nitrosated ascorbate.     

Tomato Peroxidase: Rapid Isolation and Partial Characterization

A rapid method for the isolation of tomato anionic peroxidase (TAPR) was developed using Fast Protein Liquid Chromatography. TAPR was purified 295 times to an RZ value of 0.7. The rate constant for the formation of compound I was 1.53 × 10⁷ M⁻¹ sec⁻¹ and for compound II was 6.93 × 10⁵ M⁻¹ sec⁻¹. TAPR had maximum activity at pH 5.0–5.2 and an isoelectric point at pH 3.5. SDS gel electrophoresis showed three major bands at 16.6, 40.0, and 42.0 kD. TAPR demonstrated a calcium dependancy for activity, exhibiting a maximum at a concentration of 1.0 × 10⁻⁵M.     

Lability and Reactivity of Nonheme Protein-Bound Nitrite

Nitrite treated nonheme protein was separated from the free nitrite of the reaction mixture by Sephadex G-15 column chromatography. Spectral analysis (absorbance at 330 nm) indicated that tryptophyl residues of the protein had been modified with the NO group. The protein-nitrite complex was not stable, decomposing faster at lower pH and higher temperature. As the extent of the decomposition increased with time, an increasing amount of nitrite was detected by the AOAC method, indicating that the Griess reagent reacted with nitrite after it was regenerated from the NO group. When myoglobin was incubated with the nitrosated protein, nitrosyl hemochrome could be extracted from the reaction mixture in the presence of ascorbic acid. The results indicate the potential reversibility of the protein-nitrite reaction.     

PURIFICATION AND CHARACTERIZATION OF THREE POLYPHENOL OXIDASE ISOZYMES FROM LOBSTER (HOMARUS AMERICANUS)

ABSTRACT Three isozymes of polyphenol oxidase (PPO I, PPO II and PPO III) were purified from lobster (Homarus americanus) by ion-exchange chromatography and preparative isoelectric focusing using a Rotofor cell. The purified isozymes migrated as single protein bands in polyacrylamide gels with R_f values corresponding to molecular weights of 32,180, 35,480 and 39,300, respectively. The pI values of the PPO isozymes were 3.89, 4.26 and 4.54, respectively. PPO I was most active at pH 6.5 and most stable from pH 6.0 to 7.0; PPO II was most active within the pH range 6.0 to 7.0, and most stable within the pH range 4.0 to 9.0; while PPO III was most active at pH 7.0 and most stable in the pH range 6.0 to 8.0. The temperature optimum for the PPO-dihydroxyphenylalanine oxidation reaction was 35C with PPO I and 45C with PPO II or III. Lobster PPO I lost about 30% of its initial activity after 30 min incubation at 45C, while PPO II and II retained virtually all their activity after the same heat treatment. The catalytic specificities of the PPO isozymes were relatively higher with dihydroxyphenylalanine as substrate than with chlorogenic acid.     

茶叶浸提液对肉制品中亚硝酸盐的清除效应

研究茶叶浸提液对降低肉制品中亚硝酸盐残留的作用,以提高肉制品食用安全性。根据茶叶浸提温度、浸提时间、茶叶浸提液用量和反应时间、反应液pH对降低肉制品中亚硝酸盐残留的效果,采用正交试验选择茶叶浸提液清除亚硝酸盐的最佳工艺条件。茶叶浸提液[物料比(g/mL)1∶10,90℃水浴浸提60 min]清除亚硝酸盐的最佳条件为12 mL提取液在pH 5.0的反应液中反应20 min,体外清除率最大为56.83%;对肉制品(20 g)中亚硝酸钠的清除效果达到44%以上。此研究为茶叶浸提液降低肉制品中亚硝酸盐残留应用于实际生产提供理论参考。     

Effect of Acid Pretreatment on the Stability of Ascorbic Acid Complexes with Various Iron Sources in a Wheat Flake Cereal

The effect of acid incubation of ascorbic acid with each of five iron sources (ferrous sulfate, ferric chloride, ferric orthophosphate, hydrogen and electrolytically reduced iron) on iron solubilization in a wheat flake cereal was evaluated. Incubation produced more soluble iron at pH 2 but not necessarily at the endogenous pH of the cereal nor at pH 6. At pH 2, Fe⁺² rather than Fe ⁺³ was produced, apparently by a reduction of bound Fe ⁺³ and subsequent release of Fe⁺². At pH 6, the soluble iron was mainly in a complexed form. This indicates that acid incubation with ascorbate might facilitate bioavailability of iron if it were incorporated by fortification techniques.     

Malondialdehyde-Nitrite Interactions in Meat and Model Systems

Nitrite retarded formation of carbonyl compounds in meat when added before cooking, but had no effect after cooking and storage even though the 2-thiobarbituric acid (TBA) number was very low due to reaction of malondialdehyde with nitrous acid. Although tripolyphosphate/ascorbate reacted similary preventing formation of carbonyl compounds, they had no effect on the TBA number when added after cooking. In a model system, nitrite reacted with malondialdehyde at pH 1.3 at room temperature. The percentage of reacting malondialdehyde decreased with higher pH and increased with nitrite concentration. The reaction of malondialdehyde with nitrite yielded high-molecular-weight products. Sulfanilamide prevented the reaction of malondialdehyde with nitrite, but only when added before nitrite.     

N-nitrosamine and mutagenicity formation in Chinese salted fish after digestion.

Salted and dried fish (Nemipterus virgatus), acquired from Hong Kong, was treated with 0.43-110 mM nitrite during in vitro digestion using gastric enzymes and the volatile N-nitrosamine content and mutagenicity on Salmonella typhimurium TA100 assayed without concentration. N-Nitrosodimethylamine (NDMA; the only nitrosamine detected) formation was second order in nitrite concentration. When 10 g of fish was treated with 6.96 mM nitrite, 394 nM NDMA was formed. Thiocyanate was catalytic for NDMA formation at nitrite concentration greater than 0.87 mM and when the ratio of thiocyanate to nitrite was greater than 1. Approximately a 50% inhibition in NDMA formation by ascorbic acid was seen when the ratio of ascorbate to nitrite was approximately 2 or greater and the nitrite concentration was 1.74 mM. Mutagenicity increased with increasing nitrite concentration but the addition of thiocyanate did not increase mutagenicity over nitrite alone. Ascorbate increased mutagenicity even though NDMA formation was inhibited. Even at nitrite concentrations greater than 100-fold higher than expected in vivo, there was insufficient NDMA formed to account for the observed mutagenicity. These data do not exclude the possibility that the observed mutagenicity was due to non-volatile N-nitroso compounds, however, this possibility seems unlikely given the effects of ascorbate and thiocyanate which would be expected to inhibit and enhance non-volatile N-nitroso compound formation.     

姜料中抗坏血酸克吕沃尔菌特性及其产生亚硝酸盐能力研究

目的:研究从姜料分离的抗坏血酸克吕沃尔菌生理生化特性,及其在姜料中产生亚硝酸盐的能力,分析即用姜料产生亚硝酸盐的原因及规律。方法:进行抗坏血酸克吕沃尔菌生化实验,观察该菌在不同冷藏温度、氯化钠浓度和pH的生长情况,用该菌污染姜料后分析姜料的亚硝酸盐变化规律。结果:抗坏血酸克吕沃尔菌硝酸盐还原实验阳性;在1、4℃培养时会逐渐减少直至全部死亡,在8℃会缓慢生长;氯化钠浓度为1%和2%的培养基适合其繁殖;在pH4.0的培养基中会逐步死亡,pH5.0、6.0适合其繁殖;姜料中抗坏血酸克吕沃尔菌总数增长到108CFUg时,多数姜料中亚硝酸盐含量达到峰值。结论:抗坏血酸克吕沃尔菌具有硝酸盐还原能力,在4℃以下不会生长繁殖,其生长的适宜氯化钠浓度为1%～2%,适宜pH为5.0～6.0,抗坏血酸克吕沃尔菌的大量繁殖可以导致姜料中亚硝酸盐大量产生。