Decomposition of trimethylamine oxide during iced storage of blue whiting (Micromesistius poutassou)

Products of the decomposition of trimethylamine N-oxide (TMAO) during iced storage of blue whiting were monitored over 15?days. Increasing amounts of trimethylamine (TMA) and dimethylamine (DMA) were found in the white muscle with increasing storage time. The production of TMA was interpreted as a consequence of bacterial growth, while DMA production was due to enzymatic activity of trimethylamine N-oxide demethylase (TMAOase). TMAOase activity was monitored with time in different organs. The highest activities always corresponded to kidney and spleen. Also, TMAOase was found to remain active during the 15 days of iced storage. A relationship was found between TMAOase in kidney and DMA concentrations in white muscle.     

Review

Proteins of fish muscle undergo chemical and physical changes during frozen storage which may result in, under certain conditions (i. e. long periods of storage, poor freezing practices, temperature fluctuations, etc), loss of quality, reflected mainly by an unacceptable texture as well as an undesirable flavour, odour and colour. In frozen gadoid fish species, most of these changes are caused by the production of formaldehyde in the muscle. Formaldehyde is produced, along with dimethylamine, by the enzymatic reduction of trimethylamine oxide (TMAO). Many aspects of formaldehyde production by TMAO demethylase (TMAOase) have been studied throughout the last decade. In addition, different approaches have been used to investigate the effect of formaldehyde production on protein denaturation and the associated muscle textural changes. Some insight into the reaction between protein and formaldehyde has clarified the possible mechanism of formaldehyde-mediated denaturation. However, evidence of covalent bonding between proteins and formaldehyde, to form crosslinks, has not explained fully the changes observed in fish proteins during frozen storage. The study of cold-induced denaturation of proteins might give new clues for further investigation of the problem. The implications of formaldehyde in toxicological and nutritional issues is also reviewed, as general concern about the safety of food products is a growing field in food science. Finally, different approaches have been proposed to avoid the detrimental action of formaldehyde during frozen storage of gadoid fish; they are some of the practical applications of the knowledge acquired after years of study of different workers in the field.     

Texture Changes of Frozen Stored Cod and Ocean Perch Minces

During frozen storage, minced cod and ocean perch were examined by expressible moisture (EM), water binding potential (WBP) and computerized texture profile analysis (TPA). The results suggested that a microcomputer interfaced with the Instron efficiently performed TPA. The texture and water loss of all fish minces changed more rapidly at -7°C storage than at -20°C and -40°C. Furthermore, cod, a gadoid with the ability to degrade trimethylamine oxide to dimethylamine and formaldehyde, deteriorated faster than ocean perch, which did not have this ability. EM was useful for examining the water loss of frozen fish at - 7°C only during the first two weeks of storage. The WBP at -7°C storage showed a good correlation with storage time for cod and ocean perch. This suggests that WBP might be a good indicator for examining water holding ability of fish proteins.     

Effect of Mixing and Moisture Modification on Toughening and Dimethylamine Formation in Alaska Pollock Mince during Frozen Storage at −10°C

Alaska pollock minces having reduced, normal and elevated levels of moisture were stored at - 10°C for 2 and 4 wk to determine how toughening was influenced by different amounts of ice in the frozen tissue. Minces containing reduced or elevated amounts of ice exhibited greater toughening than that observed in control minces. Also, some minces received an extra mixing step prior to freezing and this resulted in less toughening during storage, perhaps due to oxygen incorporated and partial inhibition of the enzyme system that converts trimethylamine oxide to dimethylamine and formaldehyde. However, among moisture-modified minces that received the same mixing treatment, development of toughening did not relate in any consistent manner with the development of dimethylamine.     

Decomposition of trimethylamine oxide during iced storage of blue whiting (Micromesistius poutassou)

 Products of the decomposition of trimethylamine N-oxide (TMAO) during iced storage of blue whiting were monitored over 15 days. Increasing amounts of trimethylamine (TMA) and dimethylamine (DMA) were found in the white muscle with increasing storage time. The production of TMA was interpreted as a consequence of bacterial growth, while DMA production was due to enzymatic activity of trimethylamine N-oxide demethylase (TMAOase). TMAOase activity was monitored with time in different organs. The highest activities always corresponded to kidney and spleen. Also, TMAOase was found to remain active during the 15 days of iced storage. A relationship was found between TMAOase in kidney and DMA concentrations in white muscle.     

不同贮藏温度下阿根廷鱿鱼内源性甲醛及相关物质的变化规律

探究不同贮藏温度下阿根廷鱿鱼内源性甲醛及相关物质的变化规律,研究了4 ℃、0 ℃、-20 ℃贮藏条件下鱿鱼体内甲醛(FA)、二甲胺(DMA)、三甲胺(TMA)、氧化三甲胺(TMAO)、氧化三甲胺脱甲基酶活性(TMAOase)、肌原纤维蛋白溶解度、菌落总数的变化。结果表明,随着贮藏时间的延长,鱿鱼体内FA、DMA、TMA和菌落总数呈现上升趋势;TMAO和蛋白溶解度呈现下降趋势;在4 ℃和0 ℃下TMAOase活性呈先上升后平缓的趋势,-20 ℃下TMAOase活性呈先上升后略微降低的趋势;各温度下的甲醛含量和氧化三甲胺脱甲基酶(TMAOase)酶活之间具有显著相关性(r4 ℃=0.791,r0 ℃=0.863,r-20℃=0.825)。本研究为不同贮藏条件下甲醛的控制提供了理论基础。     

Effect of some additives on the inhibition of lizardfish trimethylamine-N-oxide demethylase and frozen storage stability of minced flesh

Effects of some additives on the inhibition of trimethylamine‐N‐oxide demethylase (TMAOase) from lizardfish (Saurida tumbil) muscle were investigated. Sodium citrate and pyrophosphate could inhibit TMAOase activity in a concentration‐dependent manner, most likely because of their chelating property. Sodium alginate was the hydrocolloid possessing the inhibitory activity towards TMAOase (P < 0.05). During the storage of lizardfish mince at ?20 °C for 24 weeks, the addition of 0.5% sodium alginate and 0.3% pyrophosphate in combination with 4% sucrose and 4% sorbitol as the cryoprotectants resulted in the retarded increase in TMAOase activities with the coincidental lowered formation of dimethylamine and formaldehyde (FA), compared with the control (without additives) (P < 0.05). The loss in solubility of muscle proteins was also impeded with the addition of those compounds, suggesting their role in the inhibition of TMAOase as well as the retardation of protein denaturation induced by FA.     

Localization of formaldehyde production during frozen storage of European hake (Merluccius merluccius)

The formation of dimethylamine and formaldehyde from trimethylamine N-oxide by the enzyme trimethylamine N-oxide demethylase in whole hake during frozen storage was studied. The objective was to check if there were parts of the muscle with a higher production of dimethylamine and formaldehyde, and if the presence of kidney during frozen storage had any significant influence on the production. Three variables were examined through one year storage. The first was anatomical location, considering the red muscle and three zones of white muscle, one located right over the kidneys, the dorsal part over the viscera, and the tail. The second variable was the temperature of storage, -11 °C or -18 °C. Finally, the influence of kidneys during storage, comparing fish with and without kidneys, was also evaluated. No differences were found in dimethylamine and formaldehyde production between fish with and without kidneys stored at -18 °C. However at -11 °C the amounts of dimethylamine and formaldehyde detected in fish without kidneys were, in some cases, higher than in those with kidneys. Kidney removal does not have a statistically significant influence on DMA and FA production in frozen storage hake. Differences in dimethylamine and formaldehyde values among different anatomical locations were found, especially in those stored over one year. It was found that, in general, the white muscle located right over the kidneys produced more dimethylamine than other parts of the fish.     

乳酸菌半连续发酵虾头虾壳过程中甲醛的生成规律研究

本文以嗜酸乳杆菌半连续发酵虾头、虾壳回收蛋白质和甲壳素。对半连续发酵过程中氧化三甲胺酶(TMAOase)酶活、甲醛含量以及pH值的变化进行监测,初步探索了发酵过程中甲醛的生成规律。研究结果表明,在发酵过程中,当发酵液pH值为4.5-5.0时,TMAOase活性最高,半连续发酵五个批次的甲醛含量的变化趋势与TMAOase活性变化相一致,说明TMAOase活性是造成甲醛含量增高的重要原因,其中发酵至第五批时甲醛含量最高达30.79 mg/kg。     

Addition of sardine to hake minces and subsequent effect on dimethylamine and formaldehyde formation

Formaldehyde formation and reaction with muscle proteins in lean fish species during frozen storage is considered to be a major factor affecting texture and functionality deterioration. Formaldehyde formation and reaction with muscle compounds was reduced in lean fish minces and model systems when lipids with different degrees of oxidation were added. In order to increase the lipid content and slow down functional and textural changes, hake (Merluccius merluccius) and sardine (Sardina pilchardus) minces mixed in the ratios 3:1; 1:1 and 1:3 (w/w) were stored at ?20 °C and studied for 1 year. Dimethylamine formation and, by deduction, formaldehyde formation increased. However, less free formaldehyde was detected, probably owing to reaction with muscle compounds in the mixed minces. Nevertheless, addition of sardine minces improved the texture, protein solubility and viscosity of the mixed minces compared with the hake minces. In the mixed lots, formation of large protein aggregates was delayed or prevented. This suggests that in the mixed minces formaldehyde reacted with proteins in a different way from that in lean fish or reacted with other muscle components not directly involved in textural changes. © 2002 Society of Chemical Industry     

Review

Proteins of fish muscle undergo chemical and physical changes during frozen storage which may result in, under certain conditions (i. e. long periods of storage, poor freezing practices, temperature fluctuations, etc), loss of quality, reflected mainly by an unacceptable texture as well as an undesirable flavour, odour and colour.In frozen gadoid fish species, most of these changes are caused by the production of formaldehyde in the muscle. Formaldehyde is produced, along with dimethylamine, by the enzymatic reduction of trimethylamine oxide (TMAO). Many aspects of formaldehyde production by TMAO demethylase (TMAOase) have been studied throughout the last decade. In addition, different approaches have been used to investigate the effect of formaldehyde production on protein denaturation and the associated muscle textural changes.Some insight into the reaction between protein and formaldehyde has clarified the possible mechanism of formaldehyde-mediated denaturation. However, evidence of covalent bonding between proteins and formaldehyde, to form crosslinks, has not explained fully the changes observed in fish proteins during frozen storage. The study of cold-induced denaturation of proteins might give new clues for further investigation of the problem.The implications of formaldehyde in toxicological and nutritional issues is also reviewed, as general concern about the safety of food products is a growing field in food science.Finally, different approaches have been proposed to avoid the detrimental action of formaldehyde during frozen storage of gadoid fish; they are some of the practical applications of the knowledge acquired after years of study of different workers in the field.

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Zusammenfassung Die Proteine des Fischmuskels unterliegen während der Gefrierlagerung chemischen und physikalischen Veränderungen, die unter gewissen Bedingungen (lange Lagerzeiten, mangelnde Gefriertechniken, Temperaturschwankungen usw.) zu einem Verlust an Qualität führen können, was sich hauptsächlich in nicht akzeptierbarer Konsistenz und unerwünschten Aromen, Farben und Gerüchen manifestiert. In tiefgefrorenen Gadiden (Kabeljauartigen) werden die meisten dieser Veränderungen durch die Entstehung von Formaldehyd (FA) im Muskel hervorgerufen. FA wird, zusammen mit Dimethylamin (DMA), durch die enzymatische Reduktion von Trimethylaminoxid (TMAO) gebildet. Während der letzten Dekade wurden viele Aspekte der Bildung von FA aus TMAO untersucht. Verschiedene Versuche zur Aufklärung des Effektes der FA-Bildung auf die Proteindenaturierung und die damit verbundenen Veränderungen der Muskelkonsistenz wurden unternommen. Einige Einblicke in die FA-Protein-Reaktionen haben den möglichen Mechanismus der durch FA bewirkten Denaturierung erhellt. Die wahrscheinlich vorliegenden kovalenten Bindungen zwischen Proteinen und FA, die als Quervernetzungsbildner agieren, konnten aber die Veränderungen, die in Fischproteinen während der Gefrierlagerung beobachtet werden, nicht vollständig erklären. Die Untersuchung der Kältedenaturierung von Proteinen könnte neue Anhaltspunkte für eine weiter Untersuchung dieses Systems geben. Die Beteiligung von FA bei toxikologischen und ernährungswissenschaftlichen Fragestellungen wird ebenfalls kritisch betrachtet. Letztlich werden verschiedene Möglichkeiten vorgeschlagen, um die schädliche Wirkung von FA während der Gefrierlagerung von Gadiden zu verhindern.

     

印度洋鸢乌贼氧化三甲胺脱甲基酶的分离纯化及生化特性的研究

对印度洋鸢乌贼肌肉中的氧化三甲胺脱甲基酶(TMAOase)进行了研究。将鸢乌贼肌肉用20 mmol/LTris-醋酸缓冲液萃取,经酸、热处理后通过DE52阴离子交换柱层析和Sephacryl S-300凝胶柱层析得到纯化300倍的TMAOase。纯化得到的TMAOase分子质量为18.2 ku,最适温度和pH分别为55℃和7.0;而且该酶在50℃和pH 7.0～9.0具有很好的稳定性。TMAOase的活性可被茶多酚、植酸和冰醋酸抑制。此外,TMAOase可以将TMAO等量转化成二甲胺(DMA)和甲醛(FA),Km为24.7 mmol/L。     

Textural Changes and Functional Properties of Cod Mince Proteins as Affected by Kidney Tissue and Cryoprotectants

The addition of cryoprotectants, e.g., a mixture of sucrose and sorbitol or polydextrose (PDT) with sodium hexametaphosphate (SHMP), to improve the texture and water retention properties of cod mince with kidney tissue (modified mince) was studied at ?47°C. Kidney tissue provided trimethylamine oxide (TMAO) demethylase to accelerate dimethylamine (DMA) and formaldehyde formation. Expressible moisture (EM), water uptake ability (WUA), protein solubility, cook loss, DMA production, and textural profile analysis were determined to detect changes in modified mince during frozen storage. Modified mince with SHMP and sucrose/sorbitol or PDT at ?14°C had improved EM, WUA, and cook loss and was more tender.     

DISTRIBUTION AND SOME CHARACTERISTICS OF TRIMETHYLAMINE N-OXIDE (TMAO) DEMETHYLASE ACTIVITY OF RED HAKE MUSCLE

It has been suggested by some workers that decomposition of trimethyl N-oxide (TMAO) to dimethylamine (DMA) and formaldehyde in gadoid fishes occurs via enzymic processes while others have suggested a nonenzymic pathway. DMA production in frozen red hake muscle is shown in this study to be enzymic by the necessity of the high molecular weight soluble or insoluble fraction from red hake to convert the low molecular weight components of flounder muscle to DMA. In red hake muscle the TMAO demethylase activity is approximately evenly divided between the high molecular weight soluble and the insoluble fractions: the amount of potential activity in either fraction is 60–100 times that required for the production of DMA that normally occurs during frozen storage of the muscle tissue. The K_m for TMAO of the soluble enzyme was approximately 3 mM; the concentrations of TMAO in red hake muscle range from 60 to 140 mM (calculation based on water content of 80%). Thus, it seems unlikely that TMAO or TMAO demethylase limit the rate of the reaction. On the other hand, the K_m values for flavin mononucleotide and NADH are higher than the concentrations of these components found in the tissue suggesting that the cofactors limit the rate of TMAO breakdown to DMA and formaldehyde in the stored muscle. This supports other studies (Landolt and Hultin 1982; Banda and Hultin 1983) in which the same conclusion is reached based on other considerations.     

The role of oxygen in the production of formaldehyde in frozen minced cod muscle

It is known that formaldehyde is produced by the breakdown of trimethylamine-N-oxide (TMAO) by enzyme action during frozen storage of gadoid muscle. However, little is known of the nature of the enzyme's activity during frozen storage of the fish muscle. Determination of the formaldehyde produced throughout the depth of a block of minced cod muscle during frozen storage has shown that the production of formaldehyde is inhibited near the surface of the mince. Both in-vivo and in-vitro studies using TMAO as substrate have shown that the production of formaldehyde is inhibited by both oxygen and potassium cyanide, and activated by reduced nicotinamide dinucleotide.     

PHYSICOCHEMICAL AND BIOCHEMICAL CHANGES IN WHOLE LIZARDFISH (SAURIDA MICROPECTORALIS) MUSCLES AND FILLETS DURING FROZEN STORAGE

The physicochemical and biochemical changes in whole lizardfish (Saurida micropectoralis) muscles and its fillets kept in air and under vacuum during frozen storage at ?20C for 24 weeks were investigated. The formaldehyde (FA) and dimethylamine contents increased with a concomitant decrease in trimethylamine‐ N‐oxide (TMAO) content as the storage time increased (P < 0.05). The Ca²⁺–adenosine 5′‐triphosphatase activity continuously decreased with a coincidental decrease in salt‐soluble fraction. The disulfide bonds were increasingly formed throughout the storage (P < 0.05). The surface hydrophobicity increased and reached the maximum at week 12 with a subsequent decrease up to the end of storage. In general, the higher changes were observed in samples kept under vacuum than those kept in air. With the same atmosphere used, the whole fish showed slightly higher changes than the fillets. A marked increase in TMAO demethylase (TMAOase) activities was observed up to 12 weeks, followed by the continuous decrease up to 24 weeks of storage. The produced FA might play an important role in inducing protein denaturation and/or aggregation in lizardfish. The TMAOase activity as well as the FA formation could be reduced to some extent with the removal of internal organs and storage in the presence of oxygen. However, a detrimental effect of oxygen, especially on the promotion of lipid oxidation, would be an obstacle.     

氧化三甲胺酶研究进展

氧化三甲胺酶(trimethylamine-N-oxidedemethylase,TMAOase)是许多海洋鱼类体内存在的产生甲醛的重要的酶,与水产品甲醛的本底含量和鱼肉的腐败变质密切相关。本文对TMAOase的分布、活性测定、分离纯化以及酶学特性等方面研究进行了较为全面的综述。     

Effect of trimethylamine-<Emphasis Type="Italic">N</Emphasis>-oxide demethylase from lizardfish kidney on biochemical changes of haddock natural actomyosin stored at 4 and −10 °C

The addition of partially purified trimethylamine-N-oxide demethylase (TMAOase) from lizardfish kidney to haddock natural actomyosin (NAM) in the presence of cofactors (FeCl₂, ascorbate, and cysteine) accelerated formaldehyde (FA) formation throughout the storage either at 4 or −10 °C (p < 0.05). ¹H NMR spectroscopic study revealed that the formation of dimethylamine was enhanced with a concomitant decrease in trimethylamine oxide (TMAO) content when TMAOase was added, particularly at higher concentration. The loss of protein solubility increased as the result of FA formation, which was associated with the increased denaturation/aggregation of proteins. Lipid oxidation determined as hexanal content occurred during extended storage at different degrees. Generally, simulated systems without TMAOase and TMAO contained the highest hexanal content. Differential scanning calorimetry of NAM after storage at 4 and −10 °C for 15 days and for 8 weeks, respectively, showed the lower T _m and enthalpy of endothermic peaks corresponding to myosin and actin, suggesting the conformational changes induced by FA formed. Therefore, TMAOase exhibited the detrimental impact on haddock NAM, mainly caused by FA formation.     

Effect of high pressure processing on the quality of squid (Todarodes pacificus) during refrigerated storage

The influence of high pressure processing (HPP) on the inhibition of trimethylamine-N-oxide demethylase (TMAOase) activity and off-odour production in squid treated at 300 MPa for 20 min was investigated during 12 days of refrigerated storage. TMAOase activity of raw squid (21.5 nkat/g) was significantly decreased to approximately 5 nkat/g after 20 min of HPP. The production of dimethylamine (DMA) in HPP-treated squid for 20 min was significantly decreased to 0.31 μmol/g after 12 days of storage. The decrease in DMA was correlated with the decrease in TMAOase activity. At 300 MPa, the number of total aerobic bacteria in squid was reduced by 1.26 log units after 20 min of HPP. The HPP-treated samples effectively reduced the amount of trimethylamine (TMA). Therefore, the HPP could be used as a promising alternative technology to retard the quality deterioration of squid by inhibiting TMAOase activity and microbial growth.     

Effects of thermal processing and various chemical substances on formaldehyde and dimethylamine formation in squid Dosidicus gigas

BACKGROUND: Trimethylamine oxide (TMAO) in squid is demethylated to dimethylamine (DMA) and formaldehyde (FA) during storage and processing. This study examined the effects of thermal processing and various chemical substances on FA and DMA formation in squid. RESULTS: The thermal conversion of TMAO was assessed by analysing four squid and four gadoid fish species, which revealed that FA, DMA and trimethylamine (TMA) were gradually produced in squid, whereas TMA increased and FA decreased in gadoid fish. A significant increase in both FA and DMA levels was observed in the supernatant of jumbo squid with increased heating temperature and extended heating time at pH 6–7. Ferrous chloride combined with cysteine and/or ascorbate had a significantly positive effect on FA formation in the heated supernatant of jumbo squid. No significant difference was observed in the levels of Cu and Fe in squid and gadoid fish. The capability of Fe²⁺ to promote the formation of FA and DMA was not completely attributable to its reducing power in squid. CONCLUSION: Non‐enzymatic decomposition of TMAO was a key pathway during the thermal processing of jumbo squid, and Fe²⁺ was a crucial activator in the formation of FA and DMA. Copyright © 2012 Society of Chemical Industry