Ten years post-GAO assessment, FDA remains uninformed of potentially harmful GRAS substances in foods.

It has been approximately 10 years since the Government Accountability Office (GAO) published its report to Congress entitled, FDA Should Strengthen Its Oversight of Food Ingredients Determined to be Generally Recognized as Safe (GRAS), which strongly criticized FDA noting that its "oversight process does not help ensure the safety of all new GRAS determinations." Congress requested GAO to undertake this audit as a result of concerns that GRAS substances added to foods did not require FDA approval. Since 2010, FDA has addressed only a few of the criticisms regarding its process for establishing a food substance as GRAS. However, several of the most important GAO recommendations remain unaddressed, and most critically, FDA has chosen to remain uninformed about food substances self-determined as GRAS by manufacturers. In its 2016 final rule Substances Generally Recognized as Safe, FDA did not take the opportunity to include a provision for creation of a master list of all GRAS chemicals used in food, nor did the FDA request the authority to do so from Congress. FDA cannot fulfill its statutory obligation for ensuring the chemical safety of the U.S. food supply if it does not know which substances, in which quantities, have been added to foods.

Cell-based meat: the need to assess holistically.

Proof-of-principle for large-scale engineering of edible muscle tissue, in vitro, was established with the product's introduction in 2013. Subsequent research and commentary on the potential for cell-based meat to be a viable food option and potential alternative to conventional meat have been significant. While some of this has focused on the biology and engineering required to optimize the manufacturing process, a majority of debate has focused on cultural, environmental, and regulatory considerations. Animal scientists and others with expertise in muscle and cell biology, physiology, and meat science have contributed to the knowledge base that has made cell-based meat possible and will continue to have a role in the future of the new product. Importantly, the successful introduction of cell-based meat that looks and tastes like conventional meat at a comparable price has the potential to displace and/or complement conventional meat in the marketplace.

Biomolecular Interactions Governing Fresh Meat Color in Post-mortem Skeletal Muscle: A Review.

Appearance is an important sensory property that significantly influences consumers' perceptions of fresh meat quality. Failure to meet consumer expectations can lead to rejection of meat products, concomitant loss in value, and potential production of organic waste. Immediately after animal harvest, skeletal muscle metabolism changes from aerobic to anaerobic. However, anoxic post-mortem muscle is biochemically active, and biomolecular interaction between myoglobin, mitochondria, metabolites, and lipid oxidation determines meat color. This review examines how metabolites and mitochondrial activity can influence myoglobin oxygenation and metmyoglobin reducing activity. Further, the review highlights recent research that has examined myoglobin redox dynamics, sarcoplasmic metabolite changes, and/or post-mortem biochemistry.

Effect of 4-hydroxy-2-nonenal on myoglobin-mediated lipid oxidation when varying histidine content and hemin affinity.

The compound 4-hydroxy-2-nonenal (HNE) dissolved in water was examined to remove potential effects of using ethanol to solubilize the aldehyde such as altering protein structure or redox properties of myoglobin (Mb). HNE became covalently bound to sperm whale Mb at up to five sites based on ESI-MS analysis. Adducted Mb promoted lipid oxidation in washed muscle more effectively than non-adducted Mb. Alkylation of P88H/Q152HMb with HNE accelerated metMb formation and subsequent lipid oxidation. P88H/Q152HMb exposed to HNE enhanced lipid oxidation compared to wild-type Mb exposed to HNE. Results using H97A Mb suggested that the combination of HNE and low hemin affinity facilitated rapid decomposition of preformed lipid hydroperoxides to secondary lipid oxidation products. HNE and HHE (4-hydroxy-2-hexenal) facilitated Mb-mediated lipid oxidation similarly. The potential mechanisms by which Mb binding of α,ß-unsaturated aldehydes affect Mb oxidation and the onset of lipid oxidation are discussed.

Quality assessment of commercially processed carbon monoxide-treated tilapia fillets.

Carbon monoxide (CO) has been used to stabilize the color of fish muscle during frozen storage and distribution. This study compared changes in the quality profiles of CO-treated and untreated (UT) tilapia fillets stored at 21 to 22 °C (room temperature), 4 to 5 °C (refrigerated), and 0 °C (iced). Samples (n = 3) were analyzed at different time intervals for chemical, lipid oxidation, microbiological, color, and expert sensory profiles. CO samples contained greater (P < 0.05) apparent ammonia and total volatile base nitrogen (TVB-N) at day 0, with greater (P < 0.05) TVB-N throughout refrigerated and iced storage. At time 0, peroxide values (POV) and thiobarbituric-acid-reactive substances were lower (P < 0.05) for CO samples and continued to have lower trends throughout all storage temperatures. Microbiological analysis at time 0 did not show any differences between UT and CO samples. Redness (a*) color values were greater (P < 0.05) in CO tilapia at time 0; however, treated product showed a more rapid decline in a* throughout all storage temperatures. While expert sensory evaluation showed no statistical differences between UT and CO tilapia at time 0, CO product failed sensory assessment sooner than UT product when stored refrigerated and in ice.

The effects of HNE on ovine oxymyoglobin redox stability in a microsome model.

The effect of 4-hydroxy-2-nonenal (HNE), a secondary lipid oxidation product, on ovine myoglobin (Mb) redox stability was investigated. HNE increased oxymyoglobin (OxyMb) oxidation under all pH/temperature conditions studied. Mono-, di- and tri-HNE adducts were detected by ESI-Q-TOF MS analysis. Sites of adduction, His 120, His 25 and His 65, were determined by ESI-CID-MS/MS analysis. The relationship between ovine Mb (with/without HNE) and lipid oxidation was also studied in a microsome model in the presence of α-tocopherol. Surprisingly, preincubation of Mb with HNE did not affect subsequent Mb redox stability in the microsome model (P<0.05). Microsomes with elevated concentrations of α-tocopherol delayed lipid and Mb oxidations relative to controls. HNE-treated ovine Mb caused greater lipid oxidation compared to control ovine Mb in control microsomes (P<0.05). This study demonstrated an interaction between ovine Mb oxidation and lipid oxidation.

Redox instability and hemin loss of mutant sperm whale myoglobins induced by 4-hydroxynonenal in vitro.

The effects of 4-hydroxy-2-nonenal (HNE) on redox stability of Oxy- and Deoxy- wild-type (WT) and recombinant sperm whale myoglobins (P88H/Q152H, L29F, H97A, and H64F) and hemin loss from Met-myoglobin (Mb) were investigated. HNE induced greater redox instability in WT and mutant Mbs compared to controls (p < 0.05). The extent of HNE-induced OxyMb oxidation was lesser in L29F (p < 0.05) and greater in H97A and P88H/Q152H than in WT (p < 0.05). H64F DeoxyMb was more redox stable than WT DeoxyMb in the presence of HNE (p < 0.05). HNE alkylation occurred exclusively on histidine residues, and histidine 48 was alkylated in all sperm whale myoglobins. HNE alkylation accelerated the protoporphyrin moiety loss only in H97A. Met- forms of WT and L29F but not Deoxy- or Oxy- forms released hemin during storage. Primary structure strongly influenced Mb redox stability in the presence of reactive secondary lipid oxidation products.

Species-specific myoglobin oxidation.

The effect of the lipid oxidation product, 4-hydroxy-2-nonenal (HNE), on oxidation of oxymyoglobin (OxyMb) from seven different meat-producing species was investigated. Relative to controls, HNE increased OxyMb oxidation within all species (p < 0.05) at both 25 and 4 °C, pH 5.6. The relative effect of HNE was greater for myoglobins (Mbs) that contained 12 ± 1 histidine (His) residues than for those that contained 9 His residues (p < 0.05); HNE efficacy in all species except chicken and turkey decreased with time. Mono-HNE adducts were detected in all species except chicken and turkey. In general, HNE alkylation increased the Mbs' ability to accelerate lipid oxidation in a microsome model. However, neither an HNE nor a Mb species dependent effect was observed. Results suggested that microsome model system associated lipid oxidation overshadowed HNE and species effects on OxyMb oxidation observed in lipid-free systems.

Quality assessment of filtered smoked yellowfin tuna (Thunnus albacares) steaks.

UNLABELLED: Filtered smoke (FS) has been used to preserve taste, texture, and/or color in tuna and other fish species. This treatment is particularly important in color preservation during frozen storage. The objective of this study was to compare changes in the quality profiles of FS-treated and untreated (UT) yellowfin tuna (Thunnus albacares) steaks stored in 3 ways: room temperature (21 to 22 °C), refrigerated (4 to 5 °C), and iced (0 °C). FS and UT steaks were processed from the same lot of fish and analyzed for chemical, microbiological, lipid oxidation, color, and sensory profiles. Similar trends were seen for microbial proliferation and accumulation of apparent ammonia and total volatile base nitrogen (TVB-N) during the storage temperatures evaluated. Notable exception in quality profile was found in lipid oxidation which was, as expected, lower for treated samples at all storage temperatures for TBARS (P < 0.05) and lower or significantly (P < 0.05) lower for POV values. FS increased the initial redness value significantly (P < 0.05). Unlike UT product, there was no loss of color value concomitant with quality changes for FS-treated tuna for all storage temperatures evaluated. PRACTICAL APPLICATION: The overall goal of this project was to evaluate filtered smoked tuna steaks as to the impact on the overall quality profile. As a color-stabilizing technology, it could mask deteriorating quality.

Myoglobin and lipid oxidation interactions: mechanistic bases and control.

Lipid oxidation and myoglobin oxidation in meat lead to off-flavor development and discoloration, respectively. These processes often appear to be linked and the oxidation of one of these leads to the formation of chemical species that can exacerbate oxidation of the other. Several investigators have reported preservation of fresh meat color following the inclusion of antioxidant ingredients. An understanding of the complementary oxidation interaction provides a basis for explaining quality deterioration in meat and also for developing strategies to maintain optimal sensory qualities.

Mass spectrometric characterization and redox instability of turkey and chicken myoglobins as induced by unsaturated aldehydes.

Turkey and chicken myoglobins (Mbs) were isolated, purified, and characterized using electrospray ionization mass spectrometry (ESI-MS), and the effect of unsaturated aldehydes (nonenal and hexenal) on their redox stability was investigated in vitro. The deconvoluted spectra from ESI-MS exhibited a molecular mass of 17291 Da for both turkey and chicken Mbs. Significant homogeneity in the fragmentation pattern of both Mbs was indicated by ESI-MS/MS. Both turkey and chicken oxymyoglobins (OxyMbs) were more prone to oxidation at pH 5.8 than at pH 7.4. Metmyoglobin formation was greater in the presence of unsaturated aldehydes than controls (P < 0.05). The results demonstrated that both turkey and chicken Mbs have identical molecular mass and that the effects of alpha,beta-unsaturated aldehydes on their redox stability are consistent with those of mammalian livestock Mbs.

Effect of heating oxymyoglobin and metmyoglobin on the oxidation of muscle microsomes.

Myoglobin (Mb) and its iron have been proposed to be major prooxidants in cooked meats. To understand the mechanisms and differentiate between the prooxidant and antioxidant potential of oxymyoglobin (OxyMb) and metmyoglobin (MetMb), their prooxidant activity, iron content, solubility, free radical scavenging activity, and iron binding capacity were determined as a function of thermal processing. The ability of native and heat denatured OxyMb and MetMb to promote the oxidation of muscle microsomes was different. MetMb promoted lipid oxidation in both its native and denatured states. Conversely, OxyMb became antioxidative when the protein was heated to temperatures >or=75 degrees C. The increased antioxidant activity of heat denatured OxyMb was likely due to a decrease in its prooxidative activity due to its loss of solubility. These data show that the impact on oxidative reactions of Mb is the result of the balance between its antioxidant and prooxidant activities.

Inactivation of Listeria monocytogenes on frankfurters by monocaprylin alone or in combination with acetic acid.

The antilisterial activity of monocaprylin (MC) and its combination with acetic acid (AA) on frankfurters was investigated. Each frankfurter was surface inoculated with a three-strain mixture of Listeria monocytogenes to obtain an inoculation level of 4.0 log CFU per frankfurter, and then dipped for 35 s in sterile deionized water (45 or 50 degrees C) containing 1% ethanol (control), 50 mM MC plus 1% ethanol, 1% AA plus 1% ethanol, or 50 mM MC plus 1% AA plus 1% ethanol. Samples were vacuum packaged, stored at 4 degrees C for 77 days, and analyzed for L. monocytogenes. Sensory odor and color of frankfurters were evaluated using a 9-point hedonic scale. Color was also objectively measured using the Minolta Chroma Meter. From day 0 to day 77, population counts of L. monocytogenes on frankfurters dipped in antimicrobial solutions at 50 degrees C were consistently lower than the control counts. Similar results were observed for samples treated at 45 degrees C. However, L. monocytogenes grew readily on control samples at both temperatures. Dipping of frankfurters in antimicrobial solutions (45 or 50 degrees C) significantly reduced (P < 0.05) the populations of L. monocytogenes. After 70 days of storage, L. monocytogenes was completely killed in samples dipped in MC+AA solution at 50 degrees C. The antimicrobial treatments did not affect the odor or color of the samples (P > 0.05). Overall, results indicated that dipping of frankfurters with MC reduced L. monocytogenes, and inclusion of AA further enhanced MC antilisterial activity, without any negative effect on odor or color.

The effect of freezing and aldehydes on the interaction between fish myoglobin and myofibrillar proteins.

The interaction between fish myoglobin (Mb) and natural actomyosin (NAM) extracted from fresh and frozen fish was studied. The quantity of soluble Mb in Mb-NAM extracted was less in frozen than in fresh fish (P < 0.05). However, no differences were observed in Mb that remained in solution following preparation of Mb-NAM from frozen whole fish vs frozen fillets (P > 0.05). MetMb formation in Mb-NAM was generally greater than that observed in control Mb (P < 0.05); the greatest MetMb content occurred in Mb-NAM extracted from frozen whole fish (P < 0.05). The effect of different aldehyde oxidation products on the interaction between fish Mb and NAM was also studied in vitro. The loss of soluble Mb from NAM:Mb preparations was greater in the presence of hexenal and hexanal (P < 0.05) relative to controls, and the degree of solubility loss varied with aldehyde type. Hexenal caused greater OxyMb oxidation than hexanal (P < 0.05). Whiteness of washed NAM and NAM-Mb mixtures decreased following aldehyde addition (P < 0.05). In the absence of Mb, the Ca2+ -ATPase activity of NAM was lower with added hexenal than with hexanal (P < 0.05). However, no differences in Ca2+ -ATPase activity between hexanal and hexenal-treated samples were observed when Mb was present (P > 0.05). Reducing and nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses suggested that both disulfide and nondisulfide covalent linkages contributed to aldehyde-induced cross-linking between Mb and myofibrillar proteins.

Proteomics of lipid oxidation-induced oxidation of porcine and bovine oxymyoglobins.

Myoglobin (Mb) redox state affects meat color and is destabilized by lipid oxidation products such as 4-hydroxy-2-nonenal (HNE). Our objective was to investigate lipid oxidation-induced oxymyoglobin (OxyMb) oxidation in Mb from two major meat-producing livestock species utilizing MS and proteomics tools. Porcine OxyMb was incubated with HNE and analyzed for metmyoglobin (MetMb) formation. MetMb formation was greater in the presence of HNE than controls at pH 7.4 and 37 degrees C (p <0.05). MALDI-TOF MS was used to identify adduct formation; only mono-adducts of HNE (via Michael addition) with porcine Mb were detected. LC-ESI-MS/MS identified three histidine (HIS) residues in porcine Mb that were readily adducted by HNE (HIS 24, 36 and 119), whereas in bovine Mb seven histidine residues (HIS 24, 36, 81, 88, 93, 119 and 152) were adducted. Quantitation of HNE-adducted peptides using isotope-labeled phenyl isocyanate indicated that, initially, HIS 36 was preferentially adducted in porcine Mb whereas HIS 81, 88 and 93 were the predominant sites of early HNE adduction in bovine Mb. Preferential HNE adduction at the proximal histidine (HIS 93) was observed exclusively in bovine OxyMb and may explain why lipid oxidation-induced OxyMb oxidation appears more extensive in beef, than in pork.

Lipid-oxidation-induced carboxymyoglobin oxidation.

We evaluated the usefulness of the ratio A503/A581 as a browning index (BI) for estimating brown color formation in solutions containing oxymyoglobin (OxyMb) and carboxymyoglobin (COMb). In split-chamber cuvette analyses with different proportions of metmyoglobin (MetMb), COMb and OxyMb, BI was highly correlated (r = 0.93-0.94) with direct estimation of MetMb. Moreover, A503/A581 was not influenced by different COMb-OxyMb proportions. Second, we investigated 4-hydroxy-2-nonenal (HNE)-induced spectral changes in OxyMb and COMb solutions. At pH 7.4 and 37 degrees C, BI was greater in HNE-treated OxyMb and COMb samples than in aldehyde-free controls (P < 0.05). However, at pH 5.6 and 4 degrees C, HNE-induced browning was more pronounced in COMb than in OxyMb. These results indicated that COMb is susceptible to lipid-oxidation-induced browning in a pH- and temperature-dependent manner.

Color stability, reducing activity, and cytochrome c oxidase activity of five bovine muscles.

The objective was to characterize the beef psoas major (PM), longissimus lumborum (LL), superficial semimembranosus (SSM), deep semimembranosus (DSM), and semitendinosus (ST) muscles for differences in instrumental and visual color, metmyoglobin-reducing activity (MRA), total reducing activity (TRA), and cytochrome c oxidase activity. The LL and ST had the most color stability and MRA (p < 0.05), the DSM and PM had the least (p < 0.05), and values for the SSM were intermediate. Visual color (r = -0.66) and a and chroma (r = 0.68) were more correlated with MRA than with TRA (r < 0.14 for all measures). This research supports previous reports that color stability among muscles is variable and that MRA is more useful than TRA for explaining the role of reducing activity in muscle-color stability.

Redox instability induced by 4-hydroxy-2-nonenal in porcine and bovine myoglobins at pH 5.6 and 4 degrees C.

Myoglobin (Mb) redox stability affects meat color and is compromised by lipid oxidation products such as 4-hydroxy-2-nonenal (HNE). Pork lipids are generally more unsaturated and would be expected to oxidize readily and produce more oxidation products than beef. Supranutritional supplementation of vitamin E improves Mb redox stability of beef but not pork. The present study investigated HNE-induced redox instability in porcine and bovine myoglobins at 4 degrees C and pH 5.6. Oxymyoglobin (OxyMb) was incubated with HNE (0.075 mM porcine OxyMb + 0.5 mM HNE; 0.15 mM bovine OxyMb + 1.0 mM HNE). In porcine Mb, only monoadducts formed via Michael addition were detected after 72 h, whereas in bovine Mb both mono- and diadducts were identified. LC-MS-MS identified four histidine residues (His 36, 81, 88, and 152) of bovine Mb that were readily adducted by HNE, whereas in porcine Mb only two histidine residues (His 24 and 36) were adducted. These results suggested that the primary structure of bovine Mb predisposes it to greater nucleophilic attack by HNE and subsequent adduction than is suffered by porcine Mb.

The effects of freeze-thaw and sonication on mitochondrial oxygen consumption, electron transport chain-linked metmyoglobin reduction, lipid oxidation, and oxymyoglobin oxidation.

Mitochondria potentially influence Mb redox stability in meat by (1) decreasing partial oxygen pressure via oxygen consumption, (2) mitochondrial electron transport chain (ETC)-linked reduction of MetMb, and/or (3) oxidation of mitochondrial membrane lipid. The objective of this study was to investigate the effect of freeze-thaw and sonication treatments on mitochondrial oxygen consumption, ETC-dependent MetMb reducing activity, lipid oxidation, and Mb redox stability. Mitochondria were frozen and thawed (-18°C for 2h and 4°C for 0.5h) for 3 cycles, or sonicated for 30s with a sonic dismembrator. State III oxygen consumption rate (OCR) was decreased by both treatments at pH 7.2, and by sonication only at pH 5.6 (P<0.05). There was no effect on state IV OCR (P>0.05). Respiratory control ratio (RCR) was decreased by freeze-thaw and sonication at pH 7.2 and 5.6 (P<0.05). Sonication increased mitochondrial lipid oxidation and MetMb formation (P<0.05); a similar effect was observed in sonicated samples in the presence of ascorbic acid and ferric chloride (P<0.05). Sonication also decreased mitochondrial ETC-dependent MetMb reduction (P<0.05). These results suggested that sonication treatment had the potential to affect Mb stability via mitochondrial lipid oxidation and/or ETC-mediated MetMb reduction, but the effect on myoglobin stability by freeze-thaw treatment was minimal.

Interactions between mitochondrial lipid oxidation and oxymyoglobin oxidation and the effects of vitamin E.

Off-flavor and discoloration of meat products result from lipid oxidation and myoglobin (Mb) oxidation, respectively, and these two processes appear to be interrelated. The objective of this study was to investigate their potential interaction in mitochondria and the effects of mitochondrial alpha-tocopherol concentrations on lipid oxidation and metmyoglobin (MetMb) formation in vitro. The addition of ascorbic acid and ferric chloride (AA-Fe(3+)) increased ovine and bovine mitochondrial lipid oxidation when compared with their controls (p < 0.05); MetMb formation also increased with increased lipid oxidation relative to controls (p < 0.05). Reactions containing Mb and mitochondria with greater alpha-tocopherol concentrations demonstrated less lipid oxidation and MetMb formation than mitochondria with lower alpha-tocopherol concentrations. Greater mitochondrial alpha-tocopherol concentration was also correlated with increased mitochondrial oxygen consumption in vitro and with a more pronounced effect at pH 7.2 than at pH 5.6. Relative to controls, succinate addition to bovine mitochondria resulted in increased concentrations of ubiquinol 10 and alpha-tocopherol and decreased lipid and Mb oxidation (p < 0.05). Mitochondrial lipid oxidation was closely related to MetMb formation; both processes were inhibited by alpha-tocopherol in a concentration-dependent manner.