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...     

Reaction of Nitrite and Cytochrome c in the Presence or Absence of Ascorbate

The reaction of nitrite with cytochrome c in the presence or absence of sodium ascorbate was studied. The amount of ferrocytochrome c nitrosyl compound I (cytochrome c · Fe²⁺-NO⁺) formed in the absence of ascorbate was dependent on pH both at initial equal concentrations of nitrite or nitrous acid; the amount formed was very small at pH above 5.0. The presence of ascorbate significantly enhanced the extent of the reaction in the pH range 5.0–6.0. The reaction decreased rapidly at pH above 6.0, but application of heat was effective in giving a large formation of the product. Ascorbate reduced compound I to ferrocytochrome c nitrosyl compound II (cytochrome c · Fe²⁺-NO). The relative amounts of the two compounds formed was influenced by pH; at pH below 3.6, a mixture of compound I and II was formed, but only compound II was formed above pH 3.6. Compound II easily gave rise to ferrocytochrome c Cytochrome c probably reacts with nitrite in cured meat if ascorbate or erythorbate is used.     

Nitrite Mode of Action: Inhibition of Yeast Pyruvate Decarboxylase (E.C. 4.1.1.1) and Clostridial Pyruvate:Ferredoxin Oxidoreductase (E.C. 1.2.7.1) by Nitric Oxide

The effects of nitric oxide (NO) on the nonoxidative decarboxylation of pyruvate were studied using yeast pyruvate decarboxylase (PD) as a model. PD was inhibited by NO (50% inhibition 0.5 mM) under anaerobic but not under aerobic conditions. Prior inhibition by NO was not reversed by aerobic conditions. Sodium nitrite (0.5 mM) was not inhibitory. Inhibition of PD by NO was markedly enhanced in the presence of ascorbate. A preparation of pyruvate:ferredoxin oxidoreductase (PFO) from Clostridium perfringens (3624 A) was inhibited by NO (50% inhibition 0.025 mM) but not by sodium nitrite (1.0 mM). These results suggest that the thiamin dependent decarboxylation of pyruvate may be an additional site for the antimicrobial mode of action of nitrite independent of the iron-sulfur center of PFO and supports the concept that the active principle is NO.     

N-nitrosodimethylamine formation by free-chlorine-enhanced nitrosation of dimethylamine

The formation of N-nitrosodimethylamine (NDMA) by the nitrosation of dimethylamine (DMA) is greatly enhanced by the presence of free chlorine (HOCl). The effect of HOCl appears at first to be contrary because HOCl rapidly oxidizes nitrite and hence should reduce NDMA formation from a mechanism involving classical nitrosation. The enhanced nitrosation by the presence of HOCl is, however, consistent with a mechanism that involves the formation of a highly reactive nitrosating intermediate such as dinitrogen tetroxide (N2O4) formed during the oxidation of nitrite to nitrate. This mechanism is quite unlike another recently proposed NDMA formation pathway involving the rate-limiting oxidation of DMA directly by monochloramine. NDMA formation by the proposed HOCl-enhanced nitrosation pathway is inhibited by the presence of ammonia and occurs very quickly, only during the short period during which nitrite oxidation occurs. The general importance of this NDMA formation mechanism in actual drinking water appears to be limited by the amount of DMA and nitrite typically present. The mechanism described here, however, suggests the potential involvement of other nitrogen redox reactions that may produce reactive intermediates leading to the indirect and incidental formation of NDMA in the presence of appropriate organic nitrogen precursor.     

Impact of citric acid on the tenderness,microstructure and oxidative stability of beef muscle

Acidification of meat can improve texture however it also increases susceptibility to lipid oxidation. The effect of injection and marination of citric acid to acidify and sodium carbonate or sodium tri-polyphosphate to increase pH of beef on tenderness, microstructure and oxidative stability was determined. Water-holding capacity and tenderness of beef semitendinosus muscle increased significantly at pH 3.52 upon addition of citric acid and returned to the level of untreated sample after pH was increased (pH ∼5.26) by sodium tri-polyphosphate. The microstructure of the muscle was lost upon acidification but reformed upon increasing muscle pH. Lipid oxidation was inhibited in cooked beef blocks and ground muscle acidified with citric acid. Lipid oxidation was also inhibited in citric acid acidified beef that was readjustment to pH values equal to or greater than the raw beef muscle with sodium tri-polyphosphate or sodium carbonate. In addition, citric acid that was adjusted to the pH of the raw beef so that it did not alter the pH of the beef also inhibited lipid oxidation. These results indicate that citric acid and not sodium tri-polyphosphate or pH adjustment was responsible for inhibiting lipid oxidation in beef. These results suggest that the best acid marination technique for beef would be citric acid since it is effective at both improving texture and inhibiting lipid oxidation.     

Chemical nitrite oxidation in acid solutions as a consequence of microbial ammonium oxidation

In long-term experiments with membrane aerated biofilm reactors we observed complete nitrite oxidation in highly concentrated ammonium nitrite solutions with a contaminant pH decrease to values below 3. The maximum initial concentration for ammonium was 42 mM and for nitrite was 41 mM. We hypothesized that (1) acid-tolerant ammonium oxidizing bacteria were responsible for the pH decrease, and (2) chemical processes caused complete nitrite oxidation at low pH values. To test this hypothesis we set up a mechanistic computer model based on kinetic data from literature and we validated the model with additional experiments. The simulations fitted the measurements very well. Additionally, an experiment with the inhibitor allylthiourea showed that ammonium-oxidizing bacteria were active at pH values far below 5.5. Experiments in a sterile reactor confirmed the chemical nitrite oxidation to nitrate. Nitrogen balances revealed that 8 +/- 4% of the initial nitrogen (ammonium, nitrite, and nitrate) were lost during the cycles. On the basis of measurements and simulations we concluded that volatilization was responsible for the significant nitrogen loss. We estimated that about half of the lost nitrogen volatilized as nitrous acid HNO2. The rest mainly volatilized as dinitrogen N2 and nitrous oxide N2O.     

The reactivity of intermediates in non-enzymic browning reactions towards nitrite ion

The intermediates of non-enzymic browning reactions, 4, 5-dihydroxy-2-oxopentanal and 2-oxo-4,5,6-trihydroxyhexanal, show significant reactivity towards nitrite ion at pH 4.7 and 7.0, respectively.     

New processes in the environmental chemistry of nitrite: nitration of phenol upon nitrite photoinduced oxidation

The role of nitrite as an environmental factor has been widely recognized. Nitrite is a relevant source of *OH in the atmosphere, both in the gas phase via photolysis of gaseous HNO2 and in atmospheric hydrometeors by photolysis of NO2-. In aqueous systems, *OH production through nitrite photolysis can be negligible due to the competition for light absorption by dissolved Fe(III), colloidal iron oxides, and nitrate. These photoexcited oxidants interact with NO2- and HNO2 to form *NO2, either directly or via formation of *OH. As a consequence, nitrite and nitrous acid may act as *NO2 rather than *OH sources. The radical *NO2 is involved in the nitration of many aromatic compounds, of which phenol is a model in this work. Kinetic measurements using 2-propanol as *OH scavenger show that the direct production of *OH by aqueous Fe(III) species decreases as pH increases. At slightly acidic and neutral pH values, oxidation of nitrite occurs by direct electron transfer to photoexcited Fe(III)aq species or colloidal iron oxides, in addition to the *OH-mediated oxidation of NO2-. The reported findings suggest a completely new role of nitrite in aquatic environments.     

Oxygen consumption rate of permeabilized cells and isolated mitochondria from pork M. masseter and liver examined fresh and after freeze-thawing at different pH values

Abstract: The oxygen consumption rate (OCR) of 2 types of permeabilized tissues and their corresponding isolated mitochondria from porcine M. masseter and liver, resulting in 4 systems, was studied at different pH values (5.0 to 7.1) using fresh samples and samples frozen directly in liquid nitrogen (N₂) or air‐frozen at ?20°C. A protocol with the additive sequence rotenone–succinate–ADP (adenosine diphosphate)–cytochrome c–FCCP (carbonyl cyanide p‐trifluoromethoxyphenylhydrazone) was used to study respiration changes. The OCR of liver respiring on succinate (OCR _S ) was higher than that of muscle tissue. pH had a larger effect on OCR_S than freeze‐thawing. Low pH was associated with reduced OCR_S. The OCR_S of isolated muscle mitochondria appeared to be an underestimated relative to the OCR_S of permeabilized muscle cells. Increasing pH, following prior subjection to pH 5.0, showed partial reversibility of the OCR_S. The freeze‐thaw cycle increased the OCR_S when muscle systems were frozen and examined above pH 6.0; this effect was less apparent for liver tissue. A response to cytochrome c addition, indicating a defective outer mitochondrial membrane, was observed for all 4 systems. The response was, however, lowest for permeabilized cells. The ADP/FCCP additive pair indicated partial coupling for isolated liver and muscle mitochondria. These additives gave weak responses for the permeabilized liver cells while the OCR seemed to be inhibited for permeabilized muscle fibers when ADP/FCCP was added. Practical Application: The mitochondrial state is believed to be important for myoglobin reduction, development of flavor, and possibly other meat qualities. By monitoring the oxygen consumption in mitochondria and meat we can better understand and control such processes following freezing and thawing.     

Control of bovine hepatic fatty acid oxidation

Fatty acid oxidation by bovine liver slices and mitochondria was examined to determine potential regulatory sites of fatty acid oxidation. Conversion of 1-[14C]palmitate to 14CO2 and total [14C]acid-soluble metabolites was used to measure fatty acid oxidation. Oxidation of palmitate (1 mM) was linear in both liver slice weight and incubation time. Carnitine stimulated palmitate oxidation; 2 mM dl-carnitine produced maximal stimulation of palmitate oxidation to both CO2 and acid-soluble metabolites. Propionate (10 mM) inhibited palmitate oxidation by bovine liver slices. Clofenapate, an inhibitor of fatty acid esterification, alone increased palmitate oxidation and was able to prevent the propionate-induced inhibition of palmitate oxidation by liver slices. Propionate (.5 to 10 mM) had no effect on palmitate oxidation by mitochondria, but malonyl Coenzyme A, the first committed intermediate of fatty acid synthesis, inhibited mitochondrial palmitate oxidation (inhibition constant = .3 microM). Liver mitochondrial carnitine palmitoyltransferase (EC 2.3.1.21) exhibited Michaelis constants for palmitoyl Coenzyme A and l-carnitine of 11.5 microM and .59 mM, respectively. Long-chain fatty acid oxidation in bovine liver is regulated by mechanisms similar to those in rats but adapted to the unique digestive physiology of the bovine.     

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.     

Curtailing Oxidation‐Induced Loss of Myosin Gelling Potential by Pyrophosphate Through Shielding the S1 Subfragment

In muscle food processing, where oxidation is inevitable, phosphates are usually added to improve water binding. This present study attempted to investigate the interactive roles of protein oxidation and pyrophosphate (PP) during thermal gelation of myosin. Myosin isolated from pork muscle was solubilized in 0.5 M NaCl at pH 6.2 then oxidatively stressed with an iron‐redox cycling system that produces hydroxyl radicals with or without 1 mM PP and 2 mM MgCl₂ at 4 °C for 12 or 24 h then heated to 50 °C at 1.3 °C/min. Protein conformational stability was measured by differential scanning calorimetry, and covalent cross‐linking was examined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis following chymotrypsin digestion. The binding of PP to myosin suppressed disulfide bond formation in myosin subfragments 1 and 2 and partially inhibited oxidation‐initiated cross‐linking of heavy meromyosin during myosin gelation with a lesser effect on light meromyosin. In the presence of PP, myosin exhibited less loss of conformational integrity upon oxidation than myosin without PP. Rheological analysis from 20 to 75 °C indicated up to 32% decreases (P < 0.05) in elastic modulus (G′) of myosin gels due to oxidation. However, the presence of 1 mM PP, which also lowered the gelling capacity of myosin, inhibited the oxidation‐induced G′ by nearly half (P < 0.05). These results suggest that the protection of myosin head from oxidative modification by PP can be a significant factor for the minimization of gelling property losses during cooking of comminuted meats.     

Effects of anka rice, nitrite, and phosphate on warmed-over flavor and palatability characteristics in roast beef

Effects of anka rice, nitrite, and phosphate used in surface curing of roast beef on chemical analysis, lipid oxidation, sensory evaluation, physical measurements, and microbial growth after refrigerated storage were studied. TBA values indicated that nitrite retarded lipid oxidation and inhibited development of warmed-over flavor; however, anka rice had a flavor masking effect on oxidation; and phosphate did not improve physical measurements due to surface curing but slightly inhibited oxidation. There was a synergistic effect among anka rice, nitrite, and phosphate in some measurements.     

INHIBITION OF AEROBIC AND ANAEROBIC GROWTH OF STAPHYLOCOCCUS AUREUS IN A MODEL SAUSAGE SYSTEM

Sodium nitrite, potassium sorbate, and glycerol monolaurate all inhibited anaerobic growth of Staphylococcus aureus more than its aerobic growth in an agar-meat model sausage system, and all were more inhibitory when lactic acid was added. Whereas anaerobic growth of S. aureus was inhibited by concentrations of 100, 2500, and 2500 ppm, respectively, of nitrite, potassium sorbate, and glycerol monolaurate, corresponding concentrations of 150, 5000, and 5000 ppm were required to inhibit aerobic growth ((20 meq lactic acid added).
Sorbic acid, a 3:1 mixture of sorbic acid and glycerol monolaurate by weight, and butylated hydroxyanisole (BHA), however, did not show differential inhibitory effects toward aerobic and anaerobic growth of S. aureus and also were more effective inhibitors with addition of lactic acid. Sorbic acid suppressed staphylococcal growth in the model sausage system at 500 ppm, the mixture of sorbic acid and glycerol monolaurate at 750 ppm, and BHA at 10,000 ppm (20 meq lactic acid added).     

Effect of Phospholipid Hydrolysis on Lipid Oxidation in Flounder Muscle Microsomes

Endogenous phospholipase A activity was observed to be associated with the microsomal fraction of flounder muscle. Preincubation of microsomes with exogenous phospholipase A 2 resulted in an inhibition of both enzymic and nonenzymic lipid oxidation systems contrary to the generally accepted assumption that fatty acids are more susceptible to oxidation when free than when esterified. Removal of free fatty acids from the membrane led to a partial restoration of oxidative activity in both systems. Preincubation of microsomes with phospholipase C lowered enzymic oxidation but did not affect non-enzymic oxidation, whereas, preincubation with 1% sodium desoxycholate led to inhibition of both oxidation systems. This study suggests a direct role of phospholipid hydrolysis in the inhibition of lipid oxidation in an isolated membrane fraction of fish muscle and the possibility that such hydrolysis in situ may be a controlling factor in lipid oxidation and its subsequent effects.     

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.     

亚硝酸钠对自然发酵哈尔滨风干肠微生物生长、脂肪氧化及挥发性化合物的影响

研究不同添加水平的亚硝酸钠（NaNO2）对自然发酵哈尔滨风干肠微生物生长、脂肪氧化和挥发性化合物的影响。结果表明,添加NaNO2对风干肠发酵过程中的pH值、水分活度、总菌落数和乳酸菌菌落数无显著影响（P＞0.05）,但添加0.10?g/kg以上的NaNO2可以显著降低风干肠中大肠杆菌菌落数（P＜0.05）。色差分析表明当NaNO2添加量达到0.10?g/kg时可以起到良好的发色作用,过氧化物值和硫代巴比妥酸反应产物显示NaNO2具有良好的抗氧化作用,NaNO2残留量随发酵时间的延长而逐渐降低,发酵结束后,各实验组间差异不显著（P＞0.05）。与此同时,挥发性化合物的分析结果显示,NaNO2添加量不影响风干肠中由碳水化合物发酵和氨基酸降解所产生的挥发性化合物的相对含量（P＞0.05）,但显著影响由脂肪氧化所产生的醛类和酮类化合物的相对含量（P＜0.05）,NaNO2添加量越大,对脂肪氧化产物的抑制作用越显著。     

Sweetpotato α- and β-Amylases: Characterization and Kinetic Studies with Endogenous Inhibitors

Sweetpotato α- and β-amylases were characterized to assist optimization of direct hydrolysis of starch by endogenous amylases. In kinetic studies purified starch was substrate, and ascorbate, oxalate, phenolics, phytate and sweetpotato extracts were assayed for inhibitory activity. α-Amylase had optimum pH between 5.8 and 6.4 and was stable from pH 5.0 to 9.0. Optimum activity occurred at 71.5°, but it was inactivated by heat in the absence of Ca²⁺ at > 63°. The Km for soluble starch was 2.08 mg/mL. The molecular weight was 45000 daltons. α-Amylase activity was reduced up to 70% by 0.2 mM K-ascorbate and moderately by Na-oxalate and Na-phytate. β-Amylase had optimum pH between 5.3 and 5.8, and was stable from pH 4.0 to 8.0. Its maximum activity was at 53° and it was inactivated at 60°. Km for soluble starch was 3.71 mg/mL. At 0.08 mM, K-ascorbate strongly inhibited β-amylase activity.     

Carbon Monoxide as a Colorant in Cooked or Fermented Sausages

ABSTRACT:  The study aimed at substituting nitrite with carbon monoxide (CO) in cooked or fermented meat batter products by investigating color and color stability in myoglobin solutions, model meat systems, and full-scale hotdog and salami sausages of pork and beef. For cooked model meat systems and hotdogs at 75 to 80 °C core temperatures, direct flushing with a 1% CO gas mixture during the last stage of the batter chopping produced an initial red color equal to nitrite or more intense than with nitrite. For fermented model meat systems and salami sausages with a final pH of 4.7, pretreatment and storage of ground raw meat in a 1% CO mixture, and later use of the pretreated meat in batters, also formed an initial red color in the final products. The color stability during air and light display of cooked and fermented meat products with CO was inadequate compared to products with nitrite, although the red color of CO products was largely maintained by anaerobic packaging and storage. Spectra of carboxy- and nitrosomyoglobin at pH 4.7 demonstrated higher absorbance for carboxymyoglobin.     

Hemoglobin-mediated lipid oxidation of protein isolates obtained from cod and haddock white muscle as affected by citric acid,calcium chloride and pH

Acid or alkali solubilization followed by isoelectric precipitation can be used to isolate proteins with good functional properties from muscle tissue. Both acid (pH 3.0) and alkali (pH 10.5) treatment of the muscle decreased lipid oxidation catalyzed by hemoglobin. Citric acid and calcium chloride improved the oxidative stability of both acid- and alkali-solubilized muscle protein isolates when added to the homogenized muscle before separating the membrane or directly to the isolated membranes in the assay. Citric acid may have functioned in part by lowering the pH and destroying preformed peroxides. Exposing the muscle and the hemoglobin together at pH 3 promoted lipid oxidation, while addition of citric acid/calcium chloride or press juice to washed cod prior to solubilization inhibited lipid oxidation even when the tissue and hemoglobin were acidified together.