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. 2020 Oct 27;58(10):3972–3980. doi: 10.1007/s13197-020-04860-1

The significant influences of pH, temperature and fatty acids on meat myoglobin oxidation: a model study

Van-Ba Hoa 1, Soo-Hyun Cho 1, Pil-Nam Seong 1, Sun-Moon Kang 1, Yun-Seok Kim 1, Sung-Sil Moon 2, Yong-Min Choi 3, Jin-Hyoung Kim 1, Kuk-Hwan Seol 1,✉
PMCID: PMC8357901  PMID: 34471321

Abstract

Colour is one of the important quality traits affecting the meat purchasing decision by consumers, and myoglobin is the principal heme protein responsible for the meat colour. This study aimed to investigate the effects of pH (5.3, 5.8, 6.4 and 7.4) and temperature (4 and 25 °C) on oxymyoglobin (OxyMb) oxidation in model reaction mixtures containing OxyMb, fatty acids (C18:2n-6 and C18:3n-3) and vitamin E. A decrease of the OxyMb concentration with increased acidity was observed for all the reaction mixtures with/without fatty acids and vitamin E. After 48 h of storage at 4 °C, the OxyMb concentration decreased by approximately 60–70%, 61–69%, 53.7–53.9% and 40.93–41.84% in the reaction mixtures containing [OxyMb + C18:2n-6 or C18:3n-3] at pH 5.3, 5.8, 6.4 and 7.4, respectively. While, after 48 h at 25 °C, the OxyMb concentration decreased by 95–98% in all the reaction mixtures containing [OxyMb + C18:2n-6 or C18:3n-3] under all the pH conditions. The presence of vitamin E significantly inhibited the OxyMb oxidation in the reaction mixtures containing fatty acids under acidic conditions, but a higher level of vitamin E may be required for meat(s) containing high n-3 fatty acids content that are stored at high temperature.

Keywords: Myoglobin oxidation, pH, Temperature, Discoloration, Fatty acids, Vitamin E

Introduction

Colour is one of the important quality traits affecting the meat purchasing decisions by consumers (Moeller et al. 2010). Myoglobin (Mb) is the principal heme protein responsible for the meat colour (Livingstons and Brown 1981). The colour of meat is heavily dependent on the Mb redox forms such as; oxymyoglobin (OxyMb) and carboxymyoglobin (CoMb) exhibit attractive and preferred bright cherry-red colour while, deoxymyoglobin (DeoxyMb) and metmyoglobin (MetMb) provide purplish-red and brownish colours, respectively (Suman and Joseph 2013). Among these forms, MetMb (Fe3+) is formed as a result of the oxidation of the central iron atom within the heme group of DeoxyMb (Fe2+) or OxyMb (Fe2+) and CoMb (Fe2+), and is known to be the discoloration process occurring in meats during storage and retail (Fautman et al. 2010; Suman and Joseph 2013).

Many endogenous and exogenous factors have been reported to affect the Mb structures as well as their subsequent contributions to the colour development in meats. Amongst these, temperature and pH conditions as well as lipid oxidation are the most important factors affecting the oxidation of DeoxyMb, OxyMb and CoMb to brown MetMb (Mancini and Hunt 2005; Adzitey and Nural 2011; Chauhan and England 2018). Recently, Zareian et al. (2019) reported that when meat pH decreased from 5.8 to 5.4, the total Mb concentration was decreased whereas, MetMb level was increased. These authors also showed that pH was negatively correlated with MetMb level, which means that lowering the pH may result in increased Mb oxidation to MetMb. This is because numerous oxidative processes (e.g., Mb and haemoglobin oxidation) occur more rapidly in a low pH environment (Yin et al. 2017).

Regarding the temperature effect, Wang et al. (2018) also reported that Mb oxidation rate or formed MetMb proportion in rabbit meat markedly increased after just 4 days’ storage at 4 °C whereas, when stored at  − 2 °C, MetMb proportion increased at a slower rate after 10 days. Some researchers reported an interrelationship among the Mb, MetMb and lipid oxidations for instance; in the oxidation reaction, the reduced Mb is converted to MetMb, resulting in formation of superoxide anion radicals (O2−) that can be readily converted to hydrogen peroxide (H2O2) that favours the lipid oxidation via formation of hypervalent heme protein species (Calsen et al. 2005). Meanwhile, pro-oxidant elements such as primary and secondary products (e.g., aldehydes) of lipids oxidation can enhance meat discoloration by favouring the formation of brown MetMb pigment (Sepe et al. 2005; Fautman et al. 2010).

Previously, some attempts have been made to improve meat colour stability, for instance, by supplementation of livestock feed with antioxidants (e.g., vitamin E) or inclusion of antioxidants into fresh meat, which have led to minimization of lipid and OxyMb oxidation (Mitsumoto et al. 1991; Raghavan and Hultin 2006; Suman and Joseph 2013). Numerous studies have also demonstrated that the presences of antioxidants enhance the fresh meat colour stability by promoting the formation of reduced cherry-red OxyMb or purple-red DeoxyMb pigments (Mancini and Hunt 2005; Suman et al. 2011, 2016; Shah et al. 2014).

Practically, pale soft exudative (PSE) and dark firm dry (DFD) meats are two of the major quality defects that the meat industry is facing. Currently, both the PSE and DFD meat conditions can occur in all animal meat species due to their abnormal pH (Loudon et al. 2018; Chauhan and England 2018). The PSE meat is caused by its abnormally low pH (~ 5.3) that results in denaturation of myofibril proteins (e.g., myoglobin etc.) (Smith and Northcutt 2009; Adzitey and Nural 2011; Chauhan and England 2018). Contrastingly, the DFD meat or dark cutting in beef is characterized by a dark colour due to its abnormally high pH (Loudon et al. 2018). These defects usually result in poor quality and profitability loss of meats (Huff-Lonergan et al. 2003; Chauhan and England 2018). From the findings reported in these studies, it may be said that temperature and especially pH are the most important factors dictating the meat quality especially in terms of colour. Therefore, elucidating the mechanisms of myoglobin oxidation under different pH conditions (similar to the PSE, normal and DFD meat) as well as the interaction between the muscle myoglobin with lipid oxidations under retailing/or storage conditions (e.g., temperature and exposure duration) are important to further understand the discoloration phenomenon in meats as well as to identify solutions for minimizing this problem.

Although the oxidation of Mb in meats has extensively been studied as mentioned above, there has been limited published research focusing on quantification of the Mb residual or oxidized Mb under particular pH, temperature and storage time conditions. This model system was conducted aiming to further elucidating: (1) the roles of pH and temperature conditions in the oxidation status of myoglobin, and (2) the effects of lipid oxidation and vitamin E on the oxidation rate of myoglobin under conditions similar to the storage/retailing conditions. To accomplish this, a series of model reactions were designed using muscle myoglobin in the presence or absence of polyunsaturated fatty acids and vitamin E under different pH and temperature conditions.

Materials and methods

Materials

Myoglobin from equine skeletal muscle, vitamin E (α-tocopherol), linolenic acid, linoleic acid, potassium ferricyanide (III), sodium hydrosulphite etc. were purchased from Sigma Chemical Co. (St. Louis, Mo, USA).

Preparation of OxyMb

The preparation of OxyMb was performed using the method of Masuda et al. (2013). Briefly, 200 mg of Mb in 20 mL of 50 mmol/L phosphate buffer (pH = 7.4) was reduced with 500 µL of Na2S2O4 (0.23 mol/L) in the same buffer at 25 °C. The reaction mixture was gently stirred until a fine red OxyMb was formed. The reduction rate was determined by reading the reaction mixture at 582 and 500 nm for OxyMb and MetMb, respectively, using a spectrophotometer (model: Infinite M200, Tecan Life Science) (Tang et al. 2004). The reducing reaction was completed only when its absorbance at the two wavelengths reached a constant value. The residual sodium hydrosulphite was removed by applying the solution to Sephadex-G25 gel column chromatography (Sigma-Aldrich). The same buffer solution at pH 7.4 was used as an eluent buffer in the chromatography. A fraction containing fine red OxyMb was collected (a 10 mL fraction estimated to contain approximately 200 mg of OxyMb was considered as the stock solution) and then used for the oxidation reaction to MetMb.

Oxidation of OxyMb to MetMb under different pH and temperature conditions with/without fatty acids and vitamin E during storage

In the present study, the oxidation of OxyMb to MetMb was carried out at different pH conditions (7.4, 6.4, 5.8 and 5.3) which are similar to the pH values reported for pork muscle tissues, DFD, normal and PSE meats, respectively (Adzitey and Nural 2011). Additionally, the levels of the used substances including OxyMb, fatty acids and vitamin E are almost similar to those reported for pork muscles (Kim et al. 2010, 2015; Turner et al. 2014).

Prior to use, the OxyMb working solution was prepared by diluting its stock solution with 50 mmol/L phosphate buffer at pH: 7.4, 6.4, 5.8 and 5.3 to a concentration of 2.0 mg/mL (~ 120 µmol). The oxidation of OxyMb to MetMb (mimicking the discoloration of meat) under the effects of different pH and temperature conditions with/without fatty acids and/or vitamin E was conducted as follows:

In each reaction tube (3 reaction tubes were prepared for each pH condition or temperature and type of fatty acid), 10 mL of the diluted OxyMb (~ 120 µmol/L) solution, linoleic acid (4.0 mg/mL) or linolenic acid (0.4 mg/mL) and vitamin E (0.004 mg/L) were added (Table 1). The tubes containing OxyMb and/or fatty acids and vitamin E were stored at 2 different temperature conditions: 25 °C and 4 °C (similar to the common chilling storage temperature of meat) for 3, 24 and 48 h before the oxidizing agent (OA: potassium ferricyanide) was added. Particularly, after storage for 3, 24 and 48 h under the designated temperatures, the oxidation of OxyMb in the reaction tubes was initiated by adding 20 mg of potassium ferricyanide (Miura et al. 2014). Thereafter, the reaction tubes were incubated for 30 min (mimicking the meat blooming process) to allow the oxidation reaction to occur. The reaction tubes were not capped to allow air to enter.

Table 1.

The components and conditions used in the model reaction

No Reaction components pH environment Temperature condition Storage time
1 OxyMb (120 µmol) + OA (20 mg) (control) 7.4, 6.4, 5.8 and 5.3 4 and 25 °C 3, 24 and 48 h
2 OxyMb (120 µmol) + C18:2n-6 (4.0 mg/mL) + OA (20 mg) 7.4, 6.4, 5.8 and 5.3 4 and 25 °C 3, 24 and 48 h
3 OxyMb (120 µmol) + C18:2n-6 (4.0 mg/mL) + vitamin E (0.004 mg/mL + OA (20 mg) 7.4, 6.4, 5.8 and 5.3 4 and 25 °C 3, 24 and 48 h
4 OxyMb (120 µmol) + C18:3n-3 (0.4 mg/mL) + OA (20 mg) 7.4, 6.4, 5.8 and 5.3 4 and 25 °C 3, 24 and 48 h
5 OxyMb (120 µmol) + C18:3n-3 (0.4 mg/mL) + vitamin E (0.004 mg/mL + OA (20 mg) 7.4, 6.4, 5.8 and 5.3 4 and 25 °C 3, 24 and 48 h
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OA: Oxidizing agent (at the end of each storing period, the oxidizing agent was added to the reaction tubes and incubated for 30 min before the absorbance was measured)

Measurement of OxyMb oxidation to MetMb

Oxidation was monitored at 570 nm using a spectrophotometer and was calculated using the following equations of Masuda et al. (2013), and expressed as residual percent of MbO2: The residual percent of OxyMb (%) = [initial absorbance of test solution (120 µmol/L) of OxyMb)—measured absorbance of test solution after incubation—absorbance of MetMb (120 µmol/L) × 100]/[initial absorbance of test solution (120 µmol/L—absorbance of MetMb (120 µmol/L)].

Statistical analysis

The percent of OxyMb present in the oxidation model reactions during each period were statistically analysed using the Statistic Analysis System (SAS) package (SAS Institute, Cary, NC, USA, 2007). Means and standard errors were calculated for the variables. The data were analysed by using the General Linear Model procedure considering the reaction mixture and pH group as the main effects. Means were compared using Duncan’s Multiple Range Test. Significant differences were considered at p < 0.05.

Results and discussion

The oxidation rate of OxyMb as affected by pH, fatty acids and vitamin E under chilling condition (4 °C) during storage

Meat(s) that are retailed in shops and supermarkets are usually exposed directly to the air or are simply wrapped with plastic film (aerobically packaged). Under these conditions, the meat usually exhibits the preferred-cherry-red colour resulting from the formed OxyMb by meat Mb and air-derived oxygen. However, this desirable colour then gradually changes to an undesirable and unattractive colour (so-called discoloration) because of the formation of MetMb (McMillin 2008).

In the present model study, the changes in colour of OxyMb in the reaction mixtures with/without fatty acids and vitamin E are shown in Fig. 1. The percent of OxyMb remaining in the reaction mixtures under chilling temperature (4 °C) as affected by different pH conditions and fatty acids during storage are presented in Tables 2 and 3, respectively. After 3 h, approximately 80–85% OxyMb remained in all of the reaction mixtures at all pH conditions. When prolonging the storage time up to 24 and 48 h, the effects of pH and fatty acids on the OxyMb oxidation were observed more clearly. Particularly, in the reaction mixture containing OxyMb only (control), the residual OxyMb level was found to be significantly lower at pH 5.3 (73.98%) compared to those at pH 5.8 (83.92%), 6.4 (85.70%) or 7.4 (85.23%) after 24 h (p < 0.05) (Table 2). As this reaction mixture (OxyMb only) was stored for up to 48 h, the lowest residual OxyMb level was also found at pH 5.3 (67.60%), compared to those at pH 5.8 (71.24%), pH 6.4 (74.75%) and pH 7.4 (74.03%) (p < 0.05). This finding agrees well with that of Tatiyaborworntham et al. (2012), who reported that OxyMb is more stable in neutral and basic environments than in the acidic condition.

Fig. 1.

Fig. 1

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Representative image showing the changes of oxymyoglobin colour in the different reaction mixtures: (1) oxymyoglobin (120 µmol) + oxidizing agent (control); (2): oxymyoglobin (120 µmol) + C18:2n-6 (4.0 mg/mL) + oxidizing agent; (3) oxymyoglobin (120 µmol) + C18:2n-6 (4.0 mg/mL) + vitamin E (0.004 mg/L) + oxidizing agent; (4) oxymyoglobin (120 µmol) + C18:3n-3 (0.4 mg/mL) + oxidizing agent; (5) oxymyoglobin (120 µmol) + C18:3n-3 (0.4 mg/mL) + vitamin E (0.004 mg/L) + oxidizing agent; (6) only oxymyoglobin (120 µmol) without oxidizing agent, after 48 h storage at 4 °C and pH 5.3

Table 2.

Effects of pH, fatty acids and vitamin E on the oxidation of OxyMb under chilling condition (4 °C) after 3 and 24 h storage

Reaction model pH 5.3 pH 5.8 pH 6.4 pH 7.4
After 3 h storage
OxyMb + OA (control) 85.22 ± 0.14aA* 85.42 ± 0.00aA 85.52 ± 0.05aA 85.10 ± 0.10aA
OxyMb + 18:2n6 + OA 80.07 ± 1.63bB 80.99 ± 0.43bD 81.90 ± 1.42abBC 84.81 ± 0.16aA
OxyMb + vitE + C18:2n6 + OA 83.05 ± 0.06aA 82.75 ± 0.43aC 84.39 ± 0.26aA 84.47 ± 0.17aA
OxyMb + C18:3n3 + OA 83.93 ± 0.21aA 83.91 ± 0.30aB 80.87 ± 0.68aC 84.81 ± 0.12aA
OxyMb + vitE + C18:3n3 + OA 84.11 ± 0.10aA 84.05 ± 0.22aB 84.10 ± 0.23aAB 84.23 ± 0.33aA
After 24 h storage
OxyMb + OA (control) 73.98 ± 0.08bAB 83.92 ± 1.86aA 85.70 ± 0.05aA 85.23 ± 0.12aA
OxyMb + 18:2n6 + OA 45.33 ± 0.99bC 53.86 ± 0.50abC 66.83 ± 14.16aAB 65.69 ± 9.66aB
OxyMb + vitE + C18:2n6 + OA 75.98 ± 0.46bA 76.23 ± 0.20bB 80.74 ± 1.23aA 79.18 ± 0.26aAB
OxyMb + C18:3n3 + OA 44.75 ± 0.15bC 49.24 ± 2.81bC 53.41 ± 0.43aB 63.33 ± 4.35aB
OxyMb + vitE + C18:3n3 + OA 72.30 ± 1.55bB 77.50 ± 2.52aB 77.29 ± 0.25aA 77.04 ± 0.32aAB
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*Mean ± SE (percent of OxyMb remaining in the oxidation reaction)

OA: oxidizing agent (at the end of each storage period, the oxidizing agent was added to the reaction tubes and incubated for 30 min before the absorbance was measured); VitE: vitamin E

Means within a row with different letters (a–c) differ significantly (p < 0.05)

Means within a column with different letters (A–D) differ significantly (p < 0.05)

Table 3.

Effects of pH, fatty acids and vitamin E on the oxidation of OxyMb under chilling condition (4 °C) after 48 h storage

Reaction model pH 5.3 pH 5.8 pH 6.4 pH 7.4
OxyMb + OA (control) 67.60 ± 0.09cA* 71.24 ± 1.85bA 74.75 ± 0.02aA 74.03 ± 0.13aA
OxyMb + 18:2n6 + OA 38.40 ± 5.87cB 47.02 ± 0.83bB 46.10 ± 0.14abB 59.07 ± 2.41aC
OxyMb + vitE + C18:2n6 + OA 68.75 ± 1.25cA 70.40 ± 0.39bcA 72.49 ± 0.86abA 73.73 ± 0.16aA
OxyMb + C18:3n3 + OA 30.60 ± 0.93 dB 41.31 ± 0.82cC 46.23 ± 1.53bB 58.16 ± 0.34aC
OxyMb + vitE + C18:3n3 + OA 68.55 ± 1.24cA 73.52 ± 0.64bcA 72.43 ± 0.07aA 69.57 ± 1.44abB
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*Mean ± SE (percent of OxyMb remaining in the oxidation reaction)

OA: oxidizing agent (at the end of each storing period, the oxidizing agent was added to the reaction tubes and incubated for 30 min before the absorbance was measured); VitE: vitamin E

Means within a row with different letters (a–c) differ significantly (p < 0.05)

Means within a column with different letters (A–D) differ significantly (p < 0.05)

It is well recognized that lipid oxidation and its reactive secondary products (e.g., aldehydes) is one of the most important factors compromising meat colour stability by accelerating myoglobin oxidation (Faustman et al. 1999, 2010). Out of polyunsaturated fatty acids (PUFAs) present in the animal meat species, C18:2n-6 and C18:3n-3 are two the most predominant fatty acids (Dominguez et al., 2019). Furthermore, the extent of lipid oxidation has been found to be considerably influenced by the degree of unsaturation in fatty acids; increasing the number of double bonds significantly increases the lipid oxidation for instance; the oxidation rate of C18:2n-6 and C18:3n-3 is approximately 20 and 30 times faster compared to that of the monounsaturated fatty acids (Li and Liu 2012). In the present study, C18:2n-6 and C18:3n-3 was separately added to the reaction mixture containing OxyMb. We observed that the presence of these two fatty acids accelerated the OxyMb oxidation rate compared to the control (OxyMb only) without the fatty acids, regardless of the pH effect. Noticeably, the presence of C18:3n-3 fatty acid generally resulted in the OxyMb oxidation proceeding at a slightly faster rate compared to the C18:2n-6 after storage for 24–48 h. For instance, after 48 h at pH 5.3, the residual OxyMb level (38.40%) in the reaction mixture containing OxyMb + C18:2n-6 was higher than the level (30.60%) in the reaction mixture containing OxyMb + C18:3n-3. This finding aligns with those of Faustman et al. (1999), who showed a positive link between lipid oxidation and OxyMb oxidation. Previously, the mechanisms by which the lipid oxidation accelerates the myoglobin oxidation have been elucidated and are primarily due to the reactivity of lipids-derived primary/secondary products (Faustman et al. 2010; Suman and Joseph 2013; Wang et al. 2018). Particularly, the lipid oxidation-derived reactive aldehydes attack the myoglobin molecules at various histidine positions and cause the oxidation (Suman et al. 2007). Thus, the results indicating the different effects between the C18:2n-6 and C18:3n-3 on the OxyMb oxidation could be related to their differences in unsaturation degree (n-3 is more unsaturated) or levels of reactive primary/secondary products generated between these two fatty acids (Yin and Faustman 1993).

Regarding the effect of pH on the OxyMb in the reaction mixture containing fatty acids, a similar trend to that in the control was also observed, particularly increasing acidity decreased the residual OxyMb levels after 24 to 48 h. In general, after 48 h storage at pH 5.3, the concentration of OxyMb was decreased by approximately 60–70% when the n-6/or n-3 fatty acid was added. Whereas, at the neutral (pH 6.4) or basic condition (pH 7.4), only 53.7–53.9% or 40–41% of OxyMb was decreased in these reaction mixtures containing n-6 or n-3 fatty acid, respectively. These results again confirm that the acidic environment and PUFAs are the main factors favouring the Mb oxidation process.

Vitamin E has long been recognized as a fat-soluble antioxidant that can be used in meat products (Faustman et al. 1989). Our results showed that when vitamin E was added, the oxidation of OxyMb was significantly prevented, resulting in a significantly higher level of residual OxyMb compared to the same reaction mixtures without vitamin E at all of the tested pH conditions (Tables 2, 3). Interestingly, the OxyMb levels remaining in the reaction mixtures containing OxyMb + fatty acids + vitamin E were comparable to the levels in the control (OxyMb only) at pH 6.4 and 7.4 after 24–48 h. This means that the C18:3n-3 and C18:2n-6 fatty acids were mostly protected from the oxidation by the added vitamin E. The mechanism by which vitamin E enhances the OxyMb and meat colour stability is through a protective capacity of polyunsaturated fatty acids against oxidation and thus minimizes the generation of reactive secondary products (Buttriss and Diplock 1988; Suman and Joseph 2013). Furthermore, among the tested pH conditions, these reaction mixtures (OxyMb + C18:2n-6 or C18:3n-3 + vitamin E + OA) at pH 6.4 and 7.4 had significantly higher residual OxyMb levels after 24 to 48 h compared to the levels of the same reaction mixtures at lower pH conditions (5.3 and 5.8) (Tables 2, 3).

Overall, the oxidation of OxyMb was strongly affected by the pH conditions and the fatty acids. After storage for 48 h at pH 5.3, the remaining OxyMb levels among the reaction mixtures were in the following order: [OxyMb + C18:2n-6 + vitamin E + OA] (68.75%) > [OxyMb + C18: n3-3 + vitamin E + OA] (68.55%) > [OxyMb + OA] (67.60%) > [OxyMb + C18:2n-6 + OA] (38.40%) > [OxyMb + C18:3n-3 + OA] (30.60%). This corresponded to a decrease by 31.25, 31.45, 32.40, 61.60 and 69.40% of OxyMb in these reaction mixtures after 48 h, respectively. However, at the basic pH condition (7.4), the remaining OxyMb levels were in the following order: [OxyMb + OA] (74.03%) > [OxyMb + C18:2n-6 + vitamin E + OA] (73.73%)  > [OxyMb + C18: n3-3 + vitamin E + OA] (69.57%) > [OxyMb + C18:2n-6 + OA] (59.07%) > [OxyMb + C18:3n-3 + OA] (58.16%). This corresponded to a decrease by 25.97, 26.27, 30.25, 40.93 and 41.84% of OxyMb in these reaction mixtures after 48 h, respectively.

The oxidation rate of OxyMb as affected by Ph, fatty acids and vitamin E under room temperature conditions (25 °C) during storage

In addition to the storage at 4 °C, the reaction mixtures were also stored at room temperature (25 °C). The percent of OxyMb remaining in the reaction mixtures during storage are shown in Tables 4, 5, respectively. After 3 h under all the pH conditions, approximately 80% of the OxyMb remained in the reaction mixture containing OxyMb only (control). Miura et al. (2014) reported a similar residual OxyMb level (approximately 80%) in a reaction mixture containing the same level of OxyMb (120 µmol/L) and 15–600 µmol/L 2´-S-cysteinylcaffeic acid after 3 h incubation at 37 °C and pH 7.4. After 24 h at pH 5.3, the residual OxyMb levels in the reaction mixtures: [OxyMb + OA], [OxyMb + C18:2n-6 + OA], [OxyMb + C18:2n-6 + vitamin E + OA], [OxyMb + C18: n3-3 + OA] and [OxyMb + C18: n3-3 + vitamin E + OA] were 68.44, 16.41, 60.44, 11.44 and 56.90%, respectively. Noticeably, after 48 h at pH 5.3 only 46.22, 4.89, 45.35, 2.36 and 46.38% of OxyMb remained in these reaction mixtures, respectively. When compared to level (40%) of OxyMb remaining in the reaction mixture containing the same level of OxyMb (120 µmol/L) and 60 µmol/L quercetin (an oxidizing agent) after 3 h incubation at 37 °C and pH 7.3 reported by Inai et al. (2014), in the present study the OxyMb levels remaining in the reaction mixture containing OxyMb (control) after 48 h at all pH conditions were higher. Whereas, the residual OxyMb levels in all of the reaction mixtures containing OxyMb with fatty acids after 24 and 48 h were much lower than the level reported by these authors. These contrasting results could be due to the differences in the oxidizing agents, reaction substances and storage temperature used among the studies.

Table 4.

Effects of pH, fatty acids and vitamin E on the oxidation of OxyMb at room temperature (25 °C) after 3 and 24 h storage

Reaction model pH 5.3 pH 5.8 pH 6.4 pH 7.4
After 3 h storage
O2Mb + OA (control) 80.12 ± 0.08aA* 80.27 ± 0.10aA 80.22 ± 0.15aA 80.00 ± 0.07aA
OxyMb + 18:2n6 + OA 73.41 ± 0.29bC 75.11 ± 1.24bBC 71.51 ± 0.34bB 79.67 ± 1.70aA
OxyMb + vitE + C18:2n6 + OA 74.75 ± 0.98aBC 76.88 ± 0.62aAB 78.51 ± 3.01aAB 80.26 ± 0.19aA
OxyMb + C18:3n3 + OA 69.72 ± 1.35bD 71.68 ± 2.38bC 76.52 ± 0.81aB 78.55 ± 0.40aA
OxyMb + vitE + C18:3n3 + OA 76.75 ± 0.24bB 74.20 ± 1.43bBC 76.72 ± 0.54bB 79.73 ± 0.26aA
After 24 h storage
OxyMb + OA (control) 68.44 ± 0.10bA 69.68 ± 0.11bA 74.09 ± 0.21aA 73.50 ± 0.11aAB
OxyMb + 18:2n6 + OA 16.41 ± 1.59bC 18.77 ± 4.00bC 35.98 ± 0.26aC 42.41 ± 0.91aC
OxyMb + vitE + C18:2n6 + OA 60.44 ± 0.34bB 63.68 ± 2.61abAB 66.88 ± 2.70aB 67.72 ± 0.60aA
OxyMb + C18:3n3 + OA 11.44 ± 2.36bC 15.46 ± 1.42bC 30.09 ± 3.89aC 37.62 ± 0.37aC
OxyMb + vitE + C18:3n3 + OA 56.90 ± 3.08bB 58.65 ± 1.29abB 62.22 ± 2.80abB 64.90 ± 0.50aB
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*Mean ± SE (percent of OxyMb remaining in the oxidation reaction)

OA: oxidizing agent (at the end of each storing period, the oxidizing agent was added to the reaction tubes and incubated for 30 min before the absorbance was measured); VitE: vitamin E

Means within a row with different letters (a–c) differ significantly (p < 0.05)

Means within a column with different letters (A–D) differ significantly (p < 0.05)

Table 5.

Effects of pH, fatty acids and vitamin E on the oxidation of OxyMb at room temperature (25 °C) after 48 h storage

Reaction model pH 5.3 pH 5.8 pH 6.4 pH 7.4
OxyMb + OA (control) 46.22 ± 0.21bA* 46.26 ± 0.12bA 51.15 ± 0.45aA 52.09 ± 1.66aA
OxyMb + 18:2n6 + OA 4.89 ± 0.43aB 3.37 ± 1.43aB 4.64 ± 0.35aB 3.24 ± 1.56aC
OxyMb + vitE + C18:2n6 + OA 45.35 ± 0.58aA 45.91 ± 0.60aA 47.89 ± 0.47aA 47.31 ± 1.43aB
OxyMb + C18:3n3 + OA 2.36 ± 1.28aC 2.30 ± 1.41aB 2.73 ± 0.86aB 1.66 ± 0.41aC
OxyMb + vitE + C18:3n3 + OA 46.38 ± 1.16aA 45.67 ± 0.52aA 44.18 ± 3.44aA 48.29 ± 0.77aAB
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*Mean ± SE (percent of OxyMb remaining in the oxidation reaction)

OA: oxidizing agent (at the end of each storing period, the oxidizing agent was added to the reaction tubes and incubated for 30 min before the absorbance was measured); VitE: vitamin E

Means within a row with different letters (a–c) differ significantly (p < 0.05)

Means within a column with different letters (A–D) differ significantly (p < 0.05)

We observed that the residual OxyMb levels in all of the reaction mixtures at 25 °C were much lower than the levels in the same reaction mixtures at 4 °C under the same pH conditions and storage periods (Table 2, 3). This finding agrees with that of Brown and Mebine (1969), who reported that higher temperatures favour the OxyMb oxidation. Muira et al. (2014) also showed a lower residual OxyMb level (approximately 80%) in the reaction mixture containing OxyMb and 2´-S-cysteinylcaffeic acid at 37 °C compared to level (approximately 90%) in the same reaction mixture at 25 °C. A recent study by Wang et al. (2018) also reported that MetMb proportion formed in rabbit meat was temperature-dependent; the higher the storage temperature, the faster the Mb oxidation rate, and approximately 62% of MetMb was formed after 4 d of storage at 4 °C.

More noticeably, in the reaction mixtures with C18:2n-6 or C18:3n-3, the OxyMb concentration was decreased by approximately 84–90% and 95–98% after 24 and 48 h, respectively. This could be attributed to the increased extent of fatty acids oxidation caused by the high storage temperature (Lu et al. 2014), which resulted in increased levels of oxidative products and subsequently accelerated the OxyMb oxidation (Faustman et al. 2010; Wang et al. 2018). However, when vitamin E was added, only 34–40% and 54–55% of OxyMb in these reaction mixtures were decreased after 24 and 48 h of storage, respectively. Thus, the presence of vitamin E significantly reduced the OxyMb oxidation in the reaction mixtures. This could be due to the protective effect of fatty acids from oxidation by this vitamin as mentioned above. Although vitamin E significantly reduced the OxyMb oxidation, when compared to the residual OxyMb levels (68.55–68.75%) of the same reaction mixtures stored at 4 °C and pH 5.3 after 48 h (Table 3), those stored at 25 °C had much lower levels (Table 5).

Regarding the pH effect, in a weakly acidic environment (pH 5.8, the pH value of normal pork/beef), slightly higher residual OxyMb levels (15.46–69.68%) were found in most reaction mixtures compared to the corresponding reaction mixtures at pH 5.3 after 24 h, but not significantly different (p > 0.05). At higher pH conditions (e.g., 6.4 and 7.4), significantly higher residual OxyMb levels were found in all the reaction mixtures with/or without fatty acids and vitamin E compared to the corresponding reaction mixtures at pH 5.3 and 5.8 after 24 h (p < 0.05) (Table 4). It was also found that approximately 96–98% of OxyMb was decreased in the reaction mixtures containing C18:2n-6 or C18:3n-3 acids at these high pH conditions (6.4 and 7.4) after 48 h (Table 5). Compared to the residual OxyMb levels (46.10–59.07% depending on the type of fatty acids and pH conditions) in the same reaction mixtures stored at 4 °C for 48 h (Table 3), this level (approximately 1–2%) was much lower. Especially, when vitamin E was added, the residual OxyMb (44.18–48.29%) in the reaction mixtures containing fatty acids was significantly higher than levels (1.66–4.64%) in the corresponding reaction mixtures without vitamin E, but these residual levels were still lower than that of the control (OxyMb + OA only) after 48 h (p < 0.05). This implies that the added vitamin E content was mostly used to neutralize the fatty acids-derived oxidative products and thus, a higher vitamin E level may be required to protect the fatty acids and OxyMb from oxidation when incubating at higher temperatures (e.g., 25 °C).

Conclusion

This study for the first time conducted a series of oxidation reaction mixtures containing OxyMb with or without polyunsaturated fatty acids and vitamin E to investigate the effects of different pH and temperature conditions on the myoglobin stability. The oxidation of OxyMb occurred at a faster rate and greater extent in all the reaction mixtures under higher acidity (e.g., pH 5.3) and higher storage temperature (25 °C). The presence of n-6/or n-3 fatty acid significantly promoted the OxyMb oxidation. The addition of vitamin E significantly inhibited the OxyMb oxidation in all the reaction mixtures containing fatty acids under all the tested pH and temperature conditions. Based on the results obtained from this study, it may be concluded that polyunsaturated fatty acids, especially n-3 fatty acids and an acidic environment (e.g., pH 5.3 or 5.8) are among the main factors accelerating the myoglobin oxidation, and this process is enhanced by higher storage temperatures. Furthermore, it may be suggested that to minimize the meat discoloration (such as the pale soft and exudative meat) or maintain the meat colour stability: (1) the final meat pH should range around 6.0 (2) an adequate level of vitamin E in meat tissue is needed, and (3) storage under chilling conditions is required. Insights on the effects of different levels of the fatty acids and vitamin E on the myoglobin oxidation degree under various pH and temperature conditions in relation to the meat discoloration phenomenon will investigated in our future study.

Acknowledegment

This study was supported by 2019-Postdoctorial Fellowship Program of National Institute of Animal Science (Project No. PJ01212502), Rural Development Administration, Republic of Korea.

Compliance with ethical standards

Conflict of interest

This author declared that there is no conflict of interest.

Footnotes

Publisher's Note

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