2025 年 31 巻 6 号 p. 525-536
This study investigated the effects of retort sterilization and oxygen on meat coloration during storage of beef meatballs. Beef meatballs were prepared and retort-sterilized under different oxygen concentrations, and color changes were observed after 12 weeks of storage. In the beef meatballs processed in 21 % oxygen, the center cross-section became remarkably pinked with significantly increased a* value, resembling the undesirable persistent pinking in beef. In addition, the outer cross-section turned brown. The oxidation-reduction potential value of the pink cross-section was significantly lower than that of the brown cross-section, although the pH did not change. In addition, oxygen consumption appeared to occur after retort sterilization of the meatballs, and an assay of thiol group concentration, carboxyl compounds, and iron ion valence indicated the oxidation of salt-soluble proteins in browned meatballs. These findings suggest that the observed pinking phenomenon resulted from alterations in the oxidation-reduction state of the meat during storage.
Meat is an excellent source of various nutrients, particularly proteins, and is widely consumed. Thermal processing is essential for eliminating microorganisms and pathogens responsible for foodborne illnesses, including Salmonella spp., Listeria monocytogenes, pathogenic Escherichia coli O157, and Clostridium botulinum (Borch and Arinder, 2002). To ensure food safety, specific internal temperature conditions for cooking meat have been established to effectively eliminate these pathogens by maintaining at 74 ℃ for 15 s (King and Whyte, 2006) or 71 ℃ ⅰ)–ⅲ) (Suman et al., 2016; Faustman et al., 2023). In the case of retorted products, to ensure commercially sterile conditions, the thermal process is performed under heat sterilization conditions that are sufficient to eliminate Clostridium botulinum Jimenez et al., 2023, Codex Alimentarius Commission [(CAC/RCP 23-1979)]. Meat color is a critical factor that influences consumers’ purchasing decisions. During cooking, myoglobin (Mb) undergoes denaturation, resulting in a dull brown coloration. The internal color of meat changes depending on the degree of cooking, and consumers frequently use the transition from red (raw) to brown (cooked) as an indicator of doneness and safety (Holownia et al., 2003; Mancini and Hunt, 2005; King and Whyte, 2006; Suman et al., 2014; Suman et al., 2016; Faustman et al., 2023). Thus, post-heatingcolor changes in meat are a key parameter in meat processing.
Retort sterilization is an effective processing that allows long-term storage at room temperature while maintaining safety and quality. To ensure microbiologically safe products, this processing is often used to sterilize foodstuffs packed in sealed containers at a high temperature of 115 ℃ (240 °F) to 121.1 ℃ (250 °F) or higher (Jimenez et al., 2023, Codex Alimentarius Commission [CAC/RCP 23-1979] ). Retort sterilization is also used in meat processing, and various canned meat products are commercially available (Cheon et al., 2015; Abzhanova et al., 2022). Although retort sterilization of meat and meat products at high temperatures eliminates microorganisms, especially pathogens, it can also negatively affect consumer quality attributes such as nutritional composition. This occurs because high-temperature processing affects biochemically important components, such as proteins, fats, vitamins, and minerals, which are the most abundant components in meat. In addition, these changes affect the texture, flavor, color, and nutritional value of the product. The previous study has demonstrated the effects of sterilization on the quality of meat products and examined optimal sterilization conditions (Cheon et al., 2015); however, the observed changes in composition, physical properties, and meat color have not been investigated in detail.
Thermal processing influences meat color through multiple factors, including meat pH(Trout, 1989; Ahn and Maurer, 1990; King et al., 2006; Suman et al., 2016; Faustman et al., 2023), fresh meat storage conditions with oxygen (King and Whyte, 2006; Faustman et al., 2023), the use of nitrite/nitrate as a color-forming agent in curing (Binkerd and Kolari, 1975; King and Whyte, 2006; Faustman et al., 2023) and the amount of Mb content (Faustman et al., 2023). However, most studies examining the impact of thermal processing on meat color have focused on heat treatment conditions below 100°C or in an atmospheric environment, and changes in meat color due to long-term storage after retort sterilization remain unexplored. In addition, oxygen has a high affinity for Mb, which is associated with meat color. Typically, meat color is influenced by the balancebetweenthe oxygenated form and the oxidized form metmyoglobin (MetMb). Thus, oxygen is believed to be a crucial factor in determining meat color. To the best of our knowledge, few studies have demonstrated the oxygen factors related to changes in meat color during long-term storage after retort sterilization.
This study aimed to investigate the effects of retort sterilization and oxygen treatment on meat color changes in beef meatballs during storage. We prepared retort-sterilized beef meatballs and examined color changes after 12 weeks of storage. In addition, the underlying mechanisms of these changes were investigated with a particular focus on the redox state.
Beef meatball preparation Fresh round beef (US Angus cattle) was obtained from Marudai Foods Co., Ltd. (Ibaraki, Japan) and was stored at 0 ℃ until use. Ground meat was prepared from the obtained beef using a meat chopper (Bonny Model BK-220, Osaka, Japan; pore size 3.2 mm). One percent NaCl (w/w) was added to the ground meat and mixed in a food processor (Model MK-78; National, Osaka, Japan) for 20 s. Next, 25 g of the mixture was molded into spheres to form the beef meatball samples. The samples were then placed in boiling water at 98°C for 10 min and subsequently cooled in water. The cooled samples were placed in a retort pouch (4-layer standing pouch, including aluminum, 110 × 160 mm; Toyo Seikan Co., Ltd., Shinagawa, Japan) and vacuum-packaged (gauge-pressure -0.1 MPa, Model N46-P60, Yoshikawa Kogyo Co., Ltd., Kita-kyusyu, Japan). A nitrogen-mixed gas (Iwatani Fine Gas Co., Ltd., Amagasaki, Japan), with an oxygen concentration adjusted to 0 %, 21 %, and 100 %, was added in 50 mL before the pouches were heat-sealed again. The retort process was carried out at 120°C for 23 to 26 min (F0 = 6–9 min, z = 10 °C) while monitoring the central temperature. After processing, the samples were stored at 23°C until evaluation. To assess antioxidant effects, a batch of meatballs was prepared with 0.5 % ascorbic acid (w/w) (Happou-Shokusan Co., Ltd., Osaka, Japan). All other preparation steps were identical.
Beef meatball appearance evaluation and color measurement The appearance of the tested beef meatballs was evaluated by photographing the cross-section and exterior surfaces. Photographs were taken under the same conditions in a dark room, with LED lamps positioned at a 45° angle on both sides. A camera (D60, Nikon Co., Ltd., Shinagawa, Japan) was used with an aperture value of F18 and a shutter speed of 1/2.5 s. The color of the beef meatballs was photographed using a color chart (CASMATCH, Bear Medic Co., Ltd., Kuji, Japan) after adjusting the white balance, and the obtained image was subjected to color correction based on a color chart. Color corrections were performed using Photoshop (Adobe Systems, Inc., Shinagawa, Japan). In addition, the lightness (L*), redness (a*), and yellow (b*) values of the beef meatballs were measured using a spectrophotometer (Model CM-600d, Konica Minolta Co., Ltd., Chiyoda, Japan) configured with a D65 light source, reflectance rejection, and a 10° field of view for reflectance measurement through a transparent wrap film.
Oxygen consumption measurement in the beef meatball Headspace gases were collected in a glass bottle with a septum containing a saturated NaCl solution with approximately 0.1 % citric acid to prevent the dissolution of gases, such as oxygen andcarbon dioxide. The collected headspace gas volume was measured using a glass bottle scale, and the oxygen concentration was analyzed using an oxygen analyzer (Model CheckMate 3; Mocon Dansensor, Ringsted, Denmark).
The oxygen volume and percentage of pouch-packed beef meatballs were measured before the retort process as the initial oxygen, as follows:
Initial oxygen volume (mL) = headspace volume before retort process (mL) × oxygen concentration (%) ・・・ Eq. 1
The volume and percentage of stored samples were measured as follows:
After storage oxygen volume (mL) = after storage headspace volume (mL) × oxygen concentration (%) ・・・・・・ Eq. 2
The oxygen consumption level of the beef balls was estimated using the following formula:
Oxygen consumption (mL) = initial oxygen volume (mL) - after stored oxygen volume (mL) ・・・・・・ Eq. 3
pH and oxidation-reduction potential (ORP) of the beef meatball The pH and ORP values of the beef meatballs were directly measured using a pH meter testo 206-pH2 (Testo Co., Ltd., Kanagawa, Japan) and an ORP sensor (OR-101S; Kasahara Chemical Instruments Corp., Saitama, Japan) equipped with a pH/ORP meter KP-10F (Kasahara Chemical Instruments Corp.), respectively.
Determination of the thiol group concentration of the beef meatball To prepare the extracts from the central and outer portions of the beef meatball, 5.0 g of the tested meatball was suspended in 25 mL distilled water or 0.6 mol/L KCl solution and homogenized with a Polytron homogenizer at 8 000 rpm for 30 s, repeated three times, which the distilled water and 0.6 mol/L KCl were used to extract crude sarcoplasmic proteins and crude myofibrillar proteins, respectively. The thiol group concentration was measured using the method described by the previous studies (Ahhmed et al., 2019; Takeda et al., 2024; Kanda et al., 2025) with some modifications. A 0.4 % 5,5'-dithiobis(2-nitrobenzoic acid) (Fujifilm Wako Pure Chemicals Corp., Osaka, Japan) solution was prepared by dissolving the acid in 50 mmol/L tris(hydroxymethyl)aminomethane (Fujifilm Wako Pure Chemicals Corp., Osaka, Japan) buffer (pH 7.0) containing 2 % sodium dodecyl sulfate (Fujifilm Wako Pure Chemicals Corp., Osaka, Japan), 48 % urea (Fujifilm Wako Pure Chemicals Corp., Osaka, Japan), and 0.29 % ethylenediaminetetraacetic acid (Dojindo, Kami-mashiki, Japan). The 0.5 mL of extracts with distilled water or 0.6 mol/L KCl solution was mixed with 2.5 mL of tris(hydroxymethyl)aminomethane buffer (50 mmol/L, pH 7.0) and 20 µL of 0.4 % 5,5'-dithiobis(2-nitrobenzoic acid) (Fujifilm Wako Pure Chemicals Corp., Osaka, Japan) solution. The mixture was maintained in the dark at approximately 25°C for 1 h and subsequently centrifuged at 3 300×g for 7 min. The absorbance of the supernatant was measured at 412 nm using a spectrophotometer (Cary 3 500 UV-Vis, Agilent Technologies Inc., Santa Clara, CA, USA), and the thiol group concentration was calculated using the following formula:
Thiol group (μmol/g) = 73.53 × absorbance at 412 nm/ protein content (g) ・・・・・・ Eq. 4
Determination of total protein carbonyls Total protein carbonyls were determined according to previous reports (Oliver et al., (1987), Rodríguez-Carpena et al., (2011)), 1 g of meatball sample was minced and then homogenized 1:10 (weight/volume) (w/v) in 20 mmol/L sodium phosphate buffer containing 0.6 mol/L NaCl (pH 6.5) using a Polytron homogenizer at 8 000 rpm for 30 s. Two equal aliquots of 0.2 mL were taken from the homogenates and dispensed in 2 mL Eppendorf tubes. Proteins were precipitated by cold 1 mL of 10 % trichloroacetic acid (TCA) and subsequently centrifuged for 5 min at 2 100×g. One pellet was treated with 1 mL of 2 mol/L HCl (for protein concentration measurements) and the other with an equal volume of 0.2 % (w/v) dinitrophenylhydrazine (DNPH) (Fujifilm Wako Pure Chemicals Corp., Osaka, Japan) in 2 mol/L HCl (for carbonyl concentration measurements). Both the samples were incubated for 1 h at room temperature. Subsequently, samples were precipitated with 1 mL of 10 % TCA and washed twice with 1 mL ethanol:ethyl acetate (1:1, volume/volume) to remove excess DNPH. The pellets were then dissolved in 1.5 mL of 20 nmol/L sodium phosphate buffer containing 6 mol/L guanidine HCl (Fujifilm Wako Pure Chemicals Corp., Osaka, Japan) (pH 6.5), stirred, and centrifuged for 2 min at 2 100×g to remove insoluble fragments. The protein concentration was calculated from the absorption at 280 nm using bovine serum albumin as the standard.
The amount of carbonyls was expressed as nmol of carbonyl per gram of protein using an absorption coefficient of 21.0 nmol/L/cm at 370 nm for protein hydrazones.
The amount of carbonyls was calculated as follows:
Carbonyls (nmol/ g) = {Absorption at 370 nm / (21 nmol/L/cm × 1 cm)} / protein content (g) ・・・ Eq. 5
Valence of iron evaluation The central pink and superficial non-pink portions were cut out, placed in a PE pouch, and vacuum-packaged for the pink portion. Fe K-edge (7 112 eV) X-ray absorption fine structure (XAFS) measurements were conducted immediately after preparation to avoid sample degeneration. The XAFS measurements were carried out in partial fluorescence yield mode using a silicon drift detector (SDD) at BL5S1 and BL11S2 (hard X-ray XAFS beamlines on Aichi Synchrotron Radiation Center, Seto, Japan), while the lighting in the measurement station was turned off to prevent sample degradation. The measured spectra were analyzed by linear combination fitting (LCF) using the Athena program, part of the Demeter package (Ravel and Newville, 2005). FeO[Fe2+], FeSO4・7H2O[Fe2+], Fe2O3[Fe3+], Fe2 (SO4)3• nH2O[Fe3+] tablets, formed with boron nitride (Fujifilm Wako Pure Chemicals Corp., Osaka, Japan) as binder were prepared and measured as reference samples for the LCFs.
Statistical analysis All experiments were performed independently at least three times. Data are expressed as mean ± standard deviation (SD). A multiple comparison test with the Tukey-Kramer method was used for oxygen-consuming, L*, a*, and b* values, pH, and ORP experiments. Student’s t-test was used to estimate the thiol group and protein carbonyl concentration, and Fe valence experiments. A multiple test with the Dunnett method was also used to compare with day 1 for L*, a*, and b* values. The Tukey-Kramer method, Dunnett method and Student’s t-test were conducted using the statistical software packages GraphPad Prism 7 and Excel, respectively. The significance threshold was set at p < 0.05.
Beef meatball appearance evaluation and color measurement The changes in cross-sectional and surface appearances of beef meatball samples after the retort process are shown in Fig. 1. Compared to the cross-section of the meatballs before the retort process, all the centers of the samples on day 1 after storage exhibited slight pinking at all oxygen concentrations [Fig. 1(A) and 1(B)]. After 1 week of storage, the pinking in the centers of the meatballs stored in 21 % and 50 % oxygen became more pronounced, particularly in the 21 % oxygen-treated samples. Meanwhile, pinking was not observed in the center of the cross-section of the 100 % oxygen-treated meatballs after 1 week of storage, and these samples appeared brown. Additionally, as shown in Fig. 1(C) and 1(D), the surface appearances of 21 %, 50 %, and 100 % oxygen-treated meatballs appeared brown during the storage as compared to the meatballs before the retort process, but the change was not as obvious as that observed in the cross-sections.
The L*, a*, and b*values of the centers of cross-sections of 0 %, 21 %, and 100 % oxygenated meatballs are shown in Table 1. The L*values, which indicate the brightness, were significantly higher in the 100 % oxygenated meatball cross-sections after 8 weeks of storage compared to the other samples (p < 0.05), with the highest L*value recorded after 12 weeks of storage. In addition, the a*values, which indicate redness, were significantly higher in the 21 % oxygenated meatball cross-sections after 4 weeks of storage compared to the other samples (p < 0.05), and the a*value after 12 weeks of storage was the highest during the test. In contrast, the a*values of the 100 % oxygen-treated meatball cross-sections after 2 weeks of storage were significantly lower than those of the 0 % and 21 % oxygen-treated samples (p < 0.05). Moreover, the b*values, which indicate yellowness, of the 100 % oxygenated meatball cross-sections after 8 weeks of storage were significantly higher than those of the other samples (p < 0.05). Compared to the L*, a*, and b* values on day 1 of storage in each meatball sample, the a* values in the 0 % oxygen meatball cross-sections were significantly higher after 8 weeks of storage (p < 0.05). Additionally, the a* values were significantly higher after 2 weeks in the cross-sections of the 21 % oxygen-treated meatballs (p < 0.05). In the 100 % oxygen meatball cross-sections, the L* and b* values were significantly higher after 4 weeks, and the a* values were significantly lower after 1 week, respectively (p < 0.05).
pH, ORP, and oxygen consumption in the tested meatball As shown in Table 2, the pH of the center and outer portions of the cross-sections of the 0 %, 21 %, and 100 % oxygen-treated meatballs remained nearly identical after 12 weeks of storage. Meanwhile, the ORP values of the center and outer portions of the 100 % oxygenated meatballs were significantly higher than those of the 0 % oxygen-treated samples and the center of the 21 % oxygenated meatballs (p < 0.05). Thus, the central portion of the cross-section of the 21 % oxygen-treated samples was found to be in a reduced state compared to the outer portion of the 21 % oxygen-treated samples and the 100 % oxygen-treated meatballs. In addition, oxygen consumption in the tested meatballs after 12 weeks of storage was elevated in a concentration-dependent manner, with significant differences observed between the respective oxygen consumption levels (p < 0.05) (Fig. 2).
Total thiol groups and carbonyl contents in the tested meatball To investigate protein oxidation in the tested meatballs, the levels of thiol groups and carbonyl compounds in extracts from the center and outer portions of the cross-section were evaluated. As shown in Fig. 3(A), the thiol group levels in the 0.6 mol/L KCl extracts from the center of the cross-section were significantly higher than those in the outer portion (p < 0.05). However, the thiol group levels of distilled water extracts from the center of the cross-section were nearly identical to those in the outer portion. In addition, the carbonyl compound levels in the outer portion of the meatball cross-section were significantly elevated compared to those in the center (p < 0.05) (Fig. 3(B)). The effect of ascorbic acid on thiol group levels and carbonyl compounds in the center and outer cross-sections of the meatballs was also measured. Extracts from the ascorbic acid-supplemented meatballs demonstrated no significant differences in the thiol group and carbonyl compound levels between the central and outer portions of the cross-section [Fig. 3(A) and (B)]. Furthermore, pinking of the center of the meatballs was not observed in the cross-sections of meatballs with added ascorbic acid after 12 weeks of storage [Fig. 3(C)].
Changes in cross-sectional and surface appearances of beef meatballs before and after the retort sterilization.
(A) The cross-section after the retort process. (B) The cross-section before the retort process. (C) The surface after retort. (D) The surface before retort. To the products, 1 % NaCl (w/w) was added to the ground beef weight. The oxygen concentration of the retorted samples is adjusted before the retort process.
| Oxygen concentration | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| 0% | 21% | 100% | |||||||
| Storage | L* | a* | b* | L* | a* | b* | L* | a* | b* |
| Day 1 | 56.8 ± 2.9 | 7.6 ± 0.4 | 14.1 ± 0.1 | 57.6 ± 1.6 | 7.3 ± 0.3 | 14.3 ± 0.1 | 56.0 ± 1.7 | 8.0 ± 0.8 | 14.1 ± 0.5 |
| Week 1 | 58.8 ± 0.8 | 7.4 ± 0.3 | 13.2 ± 0.5 | 58.4 ± 1.2 | 7.9 ± 0.6 | 13.7 ± 0.8 | 57.0 ± 1.5 | 4.5 ± 2.4* | 14.9 ± 1.6 |
| Week 2 | 58.6 ± 0.6 | 7.8 ± 0.6a | 14.6 ± 0.9 | 57.8 ± 0.8 | 10.4 ± 1.5a, * | 14.1 ± 0.4 | 58.0 ± 0.6 | 3.2 ± 2.1b, * | 15.8 ± 0.7 |
| Week 4 | 58.9 ± 1.0 | 8.4 ± 0.1 a | 14.7 ± 0.2ab | 58.7 ± 0.7 | 13.0 ± 1.7b, * | 13.8 ± 0.7b | 61.0 ± 2.8* | 2.2 ± 0.6c, * | 17.1 ± 1.9a, * |
| Week 8 | 58.6 ± 0.9a | 9.2 ± 0.6a, * | 14.8 ± 1.9a | 59.0 ± 0.6a | 14.6 ± 1.1b, * | 13.3 ± 0.7a | 63.5 ± 0.6b, * | 2.6 ± 0.1c, * | 19.1 ± 0.8b, * |
| Week 12 | 58.7 ± 1.8a | 9.3 ± 0.1a, * | 14.2 ± 0.8a | 59.5 ± 1.1a | 16.3 ± 0.4b, * | 14.0 ± 0.2a | 64.9 ± 2.1b, * | 3.2 ± 0.3c, * | 21.2 ± 0.7b, * |
Different letters indicate statistically significant differences (p < 0.05) by the Tukey-Kramer test within the same storage period. The asterisks show the significant differences from Day 1 in the same column (p < 0.05).
Data are presented as mean ± SD (n = 3).
| Oxygen concentration | ||||||
|---|---|---|---|---|---|---|
| 0 % | 21 % | 100 % | ||||
| Cross-section part | center | outside | center | outside | center | outside |
| pH | 5.94 ± 0.04 | 5.93 ± 0.06 | 5.86 ± 0.02 | 5.85 ± 0.02 | 5.68 ± 0.04 | 5.68 ± 0.07 |
| ORP | −122 ± 11b | −76 ± 15bc | −104 ± 18b | −46 ± 26c | 86 ± 53a | 105 ± 33a |
Different letters indicate statistically significant differences (p < 0.05) using the Tukey-Kramer test for the ORP. Data are presented as mean ± SD (n = 3–6).
Oxygen consumption of the tested beef meatballs.
The samples are stored for 12 weeks. Different letters show statistically significant differences ( p < 0.05 by Tukey-Kramer). Error bars represent SD. Data are presented as mean ± SD (n = 3).
Protein oxidation levels in the center and outside of beef meatball cross-sections and the effect of ascorbic acid.
(A) Thiol group levels of the distilled water and 0.6 mol/L KCl solution extracts from the center and outside of the meatball cross-section.
(B) Carbonyl compound levels of phosphate buffer, including 0.6 mol/L NaCl extracts from the center and outside of the meatball cross-section.
(C) Appearance of the cross-section of meatballs adding ascorbic acid. Ascorbic acid is added at 0.5 % (w/w) to the meatball samples in (A), (B), and (C). All tested samples are from 21 % oxygen meatballs after 12 weeks of storage. Error bars represent SD. Data are presented as the mean ± SD (n = 3–6). (*: p < 0.05, by Student’s t-test).
Comparison of iron valence in pinking and non-pinking areas of beef meatball after 12 weeks of storage.
(A) Fe K-edge XANES spectra of pinking and non-pinking areas. FeO and Fe 2 O 3 are references. (B) The ratio of Fe 2+ and Fe 3+ in pinking and non-pinking areas. The LCF was performed for the four components FeO[Fe 2+ ], FeSO 4 ・7H 2 O[Fe 2+ ], Fe 2 O 3 [Fe 3+ ], and Fe 2 (SO 4 ) 3 • nH 2 O[Fe 3+ ] to determine the average valence. Data are presented as mean ± SD. Compared with the Fe 2+ ratio (n = 4). (*: p < 0.01, by Student’s t-test).
Valence of iron In the XAFS spectrum, the region near the absorption edge is referred to as the X-ray absorption near edge structure (XANES). The Fe K absorption edge is at approximately 7 112 eV, and the valence information can be obtained by analyzing the XANES spectra in the range of approximately 100 eV from the absorption edge. The Fe K-edge XANES spectra of the pinking area and non-pinking area under 21 % oxygen are shown in Fig. 4(A). The absorption edge shifts to higher energies with increasing valence. Therefore, the absorption edge of the reference spectrum of FeO[Fe2+] is lower than that of Fe2O3[Fe3+] [Fig. 4(A)]. The absorption edges of the spectra of both the pinking and non-pinking samples were located between the divalent and trivalent reference spectra. Thus, it was inferred that Fe2+ and Fe3+ were present in the samples. In addition, in the pinking area, the absorption edge was located at a lower energy than that in the non-pinking area. LCF was performed using iron oxide and iron sulfate as reference samples to calculate the relative proportions of Fe2+ and Fe3+, as shown in Fig. 4(B). It should be noted that the evaluation of iron valence by the XANES spectrum is the average value of not only heme iron but also all the iron contained in the sample. Furthermore, the valences obtained were relative, as the LCF was performed using iron oxide and iron sulfate as reference samples. The relative proportions of Fe2+ to Fe3+ were 61 ± 7:39 ± 7 for the pinking area and 29 ± 13:71 ± 13 for the non-pinking area. The pinking area had a significantly higher proportion of Fe2+ than the non-pinking area, indicating a more reduced state (p < 0.01).
Retort sterilization is a food preservation technique that addresses the challenges posed by foodborne pathogens and food spoilage bacteria by heating the product to make it microbiologically safe and stable (Yang et al., 2022; Jimenez et al., 2023). However, there have been reports of the degradation of the nutritional value and quality of some foodstuffs by thermal processing, including retort sterilization (Rice and Robinson, 1944; Reddy and Love, 1999). Consumer impressions of meat and meat products are predominantly influenced by coloration, making it one of the most salient factors (Mancini and Hunt, 2005; Li et al., 2018). Meat color is affected by pH (Trout, 1989; Ahn et al., 1990; King and Whyte, 2006; Suman et al., 2016), oxidation and redox state (Suman et al., 2016; Faustman et al., 2023; Stanislawczyk et al., 2023), oxygen (Suman et al., 2016; Faustman et al., 2023), temperature (Stanislawczyk et al., 2023), carbon oxide (Suman et al., 2016; Faustman et al., 2023), zinc (Wakamatsu et al., 2020), nitrite/nitrate (Binkerd and Kolari, 1975; King and Whyte, 2006; Faustman et al., 2023) and the amount of Mb (Faustman et al., 2023). In this study, we investigated the color change in beef meatballs caused by the retorting process with oxygen and its underlying mechanism.
As shown in Fig. 1, the center color of the meatball in the cross-section after thermal processing became increasingly pink in the 21 % and 50 % oxygen-treated meatballs, and corresponding changes in L*, a*, and b*values were also observed (Table 1). Compared to 0 % oxygen meatballs, the a* values of the cross-section of meatballs treated with 21 % and 100 % oxygen were significantly increased and decreased, respectively, after 4 weeks (p < 0.05) (Table 1), suggesting that the presence of oxygen affected the color of meatballs on the retort sterilization and storage times. Also, significant changes in the a* value were observed in both oxygen-treated meatballs and 0 % meatballs compared to day 1 (Table 1). This may be due to the small amount of residual oxygen in the 0 % samples; however, other factors aside from oxygen may also influence the color of the meatballs during retort sterilization and storage. The observed pinking and elevation of a* value resembled the undesirable persistent pinking in beef, which is often considered unsafe owing to undercooking, although the final endpoint temperature indicates a well-done product (Suman et al., 2016). The color of meat is mainly composed of Mb protein, which is converted to heat-denatured MetMb and metmyochromogen during the meat thermal process, resulting in a brown color (Stanisławczyk et al., 2023). Unfortunately, to the best of our knowledge, there have been few studies on beef color changes caused by retort sterilization. Previously, Claus and Jeong (2018) reported that in samples of turkey breast meat heated in a 90°C water bath, the a* value increased when the meat was salted and/or when the raw material was stored for 7 days before heating. In addition, a previous study suggested that the NaCl adding can solubilize myofibrillar proteins, which can potentially interact with heme pigments during heat denaturation to form heme complexes, leading to the development of a pink defect, like the pinking in this study (Ahn and Maurer, 1989). Furthermore, Speroni et al. (2014) reported that 1 % NaCl, the same concentration of the meatballs prepared in this study, caused protein solubilization. Thus, the salt-soluble protein as well as the oxygen in the tested meatball potentially leads to the observed pinking. Also, since the heating with boiled water was conducted immediately after the meatballs were prepared in this study, the observed pinking would differ from the effects of pre-heating time, as previously reported.
It is known that the higher the pH, the stronger the red color (Ahn et al., 1990; Suman et al., 2016; Faustman et al., 2023) due to increased H+ dissociation from amino acid side chains, which enhances the electronegativity of amino acid side chains from globins and other proteins (Ahn et al., 1990; Claus and Jeong et al., 2018; Bea et al., 2020). In this study, there were no differences between the pH at the center of the 21 % meatballs, which showed distinct pinking, and the pH outside of it, and/or in the 0 % and 100 % oxygen meatballs, which showed no remarkable pinking (Table 2). As shown in Fig. 2, the 21 % and 100 % oxygen meatballs appeared to absorb oxygen in an oxygen concentration-dependent manner. Moreover, the ORP value in the pinking center of the meatball cross-section of the 21 % meatballs was significantly lower than that on the outside, which was non-pinking (p < 0.05), and than the ORP value of that of the 100 % oxygen meatballs (Table 2). Additionally, the ORP value in the pinking center of the meatball cross-section of the 21 % meatballs was not significantly different from that of 0 % meatballs. Lower ORP values are known to indicate reducing power, whereas higher ORP values are potentially indicative of oxidation. Thus, it was inferred that the retort-sterilized meatballs absorbed oxygen and shifted from the reduced state to the oxidized state from the outside to the inside in response to the absorbed oxygen level.
The protein oxidation of the 21 % oxygenated meatballs was evaluated by the amount of total thiol groups and carbonyl content. The increased protein oxidation, as measured by the levels of thiol groups and carbonyl compounds in the 21 % oxygenated meatballs, was suppressed by the addition of ascorbic acid, which has antioxidant activity. The pinking of the center of the 21 % oxygenated meatballs was also controlled by the addition of ascorbic acid. Therefore, protein oxidation may play a role in the mechanism of pinking of the center of 21 % oxygenated meatballs. Regarding the thiol group level of the tested meatballs, the thiol groups in the 0.6 mol/L KCl extract from the non-pinking area on the outside were significantly decreased in the pinking area of the center but were not observed in the water extract (Fig. 3A). Moreover, the carbonyl levels in the phosphate buffer containing 0.6 mol/L NaCl extract from the outside were significantly elevated in the center (Fig. 3B). Thus, protein oxidation is thought to occur in salt-soluble proteins related to meat structure, such as myosin and actin, rather than in water-soluble proteins, such as native Mb. Myofibrillar proteins such as myosin and actin are involved in the physicochemical properties of meat and are also adsorptive substrates for flavor compounds such as aldehydes, alcohols, ketones, sulfur, and nitrogen compounds. Moreover, the oxidation of myofibrillar proteins causes changes in the structure and properties of proteins and impairs the flavor and texture of meat products (Zhang et al., 2024). Therefore, it is suggested that the retort-sterilized 21 % oxygen meatballs tested in this study may exhibit reduced flavor and texture quality.
Based on the results of the XANES spectrum, the pinking area on the cross-section of the 21 % oxygen meatball had a higher proportion of Fe2+ than the non-pinking area (p < 0.01) (Fig. 4). It has been suggested that the presence of Fe2+ in meat is associated with red coloration (Suman et al., 2016). Denatured Mb derivatives often exhibit red or pink color in the presence of Fe2+ and brown or gray color in the presence of Fe3+ (Suman et al., 2016; Faustman et al., 2023). In general, the valence state of Fe2+ is preserved under reducing conditions. In addition, according to the ORP results of the meatball cross-section, the pinking area seemed to be in a reduced state (Table 2). In cooked beef, pink or red denatured globin hemochrome (Fe2+) is formed by heat-induced denaturation of ferrous Mb or reduction of globin hemichrome (Faustman et al., 2023). Thus, the pinking area of the meatball cross-section was potentially promoted by the heat-induced denaturation of ferrous Mb and/or reduction of globin hemichrome in the meatball, which might then lead to the formation of denatured globin hemochrome. Further studies are required to clarify the distribution of denatured globin hemochrome in the cross-section of retort-sterilized beef meatballs.
In this study, we investigated the color change in beef meatballs caused by retort sterilization with oxygen and the underlying mechanism. In the meatballs with 21 % oxygen concentration, the color of the center cross-section became remarkably pink during the storage period following retort sterilization. This meat color change seemed to be reported for persistent pinking in beef, which is undesirable. The observed change in meat color was suggested to result from alterations in the oxidation and reduction states of the meat during storage. Further studies are needed to investigate the effects of oxygen concentration in detail, and to clarify the relationships between oxygen concentration, meat color changes, and oxidation-reduction status. Retort sterilization is a method that can be used for the long-term preservation of food products. In addition to oxygen, other factors such as pH are known to influence meat color. These are also needed to investigate the effect of retort sterilization on meat color and to demonstrate the stability of meat color in retort-sterilized meat products.
Acknowledgements The analysis of the iron valence was supported by the Aichi Synchrotron Radiation Center.
Conflict of interest There are no conflicts of interest to declare.
