Effects of hydrolyzed eggshell membrane and hydrolyzed egg white on the coloration of cured cooked sausages

Abstract

This study aimed to investigate the effects of hydrolyzed eggshell membrane (HESM) and hydrolyzed egg white (HEW) on the reddening of cured meat products, focusing on their reducing properties. HESM significantly enhanced meat redness of the model sausage, as indicated by the increased redness (a*) values observed compared with those obtained with treatments using HEW, egg white (EW), and control samples. Although HEW improved redness relative to EW and control samples, its efficacy was less than that of HESM. Additionally, 70 % ethanol-extract of HESM effectively reduced Fe³⁺ and lowered the oxidation-reduction potential. The HESM extract also significantly facilitated the formation of nitrosyl myoglobin (NOMb) in myoglobin solutions. However, this effect was significantly lower than that of the maleimide-treated HESM extract, indicating that the thiol groups in HESM are crucial for NOMb formation. Therefore, HESM enhances the coloration of cured meat products by the reducing potential of its thiol groups.

Introduction

Consumer purchasing choices are strongly influenced by the appearance of meat and meat products, with color serving as a key, intuitive indicator of quality (Li et al., 2018). The characteristic pink color of cured meats primarily results from the use of curing agents, specifically nitrites and nitrates , during processing. These agents not only contribute to visual appeal but also play a crucial role in enhancing food safety by inhibiting bacterial growth and preventing oxidation (Parthasarathy and Bryan, 2012). The formation of this pink coloration is primarily attributed to the concentration and redox state of heme pigments, particularly myoglobin (Bekhit and Faustman, 2005; Faustman et al., 2010). Nitrosyl myoglobin (NOMb), a complex of myoglobin and nitric oxide (NO), is the primary pigment responsible for the distinct pink hue in cured meat products (Møller et al., 2003). However, the generation of NO from and requires reducing agents, such as ascorbic acid, to facilitate their conversion (Honikel, 2008; Waga et al., 2017). These agents not only facilitate NO formation but also mitigate the risk of residual , which could otherwise lead to the formation of carcinogenic N-nitrosamines and other harmful compounds in the final product (Wang et al., 2015).

Annually, over one trillion eggs are consumed worldwide, leading to a significant increase in egg-related waste (Park et al., 2016). Egg by-products, particularly the eggshell membrane (ESM)—a protein-rich layer between the eggshell and egg white—are typically discarded as waste. However, ESM has been utilized in various applications, including wound healing (Maeda and Sasaki, 1982), cell culture (Wong et al., 1984), and the adsorption of heavy metals (Suyama et al., 1994; Park et al., 2016). Despite its high protein content, ESM is rarely used in food processing because of its strong adhesion to eggshells and low solubility. Whereas, hydrolysis has been demonstrated to improve the functional characteristics of animal-derived proteins, with hydrolyzed peptides exhibiting greater bioactivity, such as antioxidant and angiotensin I converting enzyme inhibitory activities (Bhat et al., 2015; Takeda et al., 2023a; Takeda et al., 2020). Moreover, the research has been conducted on the functionality of hydrolyzed egg white (EW) and their application in food processing (Abeyrathne et al., 2013). Thus, the application and value of egg hydrolysate peptides, including those from ESM and EW, have been widely studied (Liu et al., 2018). Also, the hydrolyzed eggshell membrane (HESM) powder holds promise for use in food processing.

Previous studies on the application of egg products in meat processing have predominantly focused on EW, demonstrating improvements in palatability, quality, and nutritional benefits (Akatsuka, 1984; Lee et al., 2016; Razi et al., 2023). Moreover, the potential of ESM for meat products was previously reported by Redbotten et al. (2022). The aim of this study was to investigate the potential of HESM in enhancing the reddish coloration of cured meat products, particularly focusing on its reducing properties. This study also seeks to compare the effects of HESM powder and hydrolyzed egg white (HEW) powder on meat coloration and investigate the mechanism by which they influence color enhancement in myoglobin solutions, the principal protein responsible for meat color.

Materials and Methods

Materials  Ground pork for the test was purchased from a local butcher shop (Sagamihara, Kanagawa, Japan). The HESM (EMlastic; product code 28888), HEW (EP-1; product code: 13565), and EW (ST; product code: 59397) powders were prepared by Kewpie Co. Ltd., and these products were used in all the experiments in this study.

Preparation of 70 % ethanol-extracts  To prepare 70 % ethanol-extracts of HESM, HEW, and EW, each was suspended in distilled water at a concentration of 10 % (weight/volume). Next, ethanol was added to achieve a final ethanol concentration of 70 % (volume/volume). The suspensions were kept overnight at 4 °C in the dark and centrifuged at 2 500 ×g for 5 min at 4 °C. Subsequently, the supernatants were filtered through No. 5A filter paper (ADVANTEC, Tokyo, Japan), and each filtrate was dried using a rotary evaporator (N-1300; EYELA, Tokyo, Japan). The dried samples were suspended in distilled water, frozen at −80 °C, and lyophilized using a lyophilizer (FDU-1200; EYELA).

Sausage model preparation  The sausage model was prepared as described previously (Takeda et al., 2023b). Briefly, 2 % sodium chloride and 30 ppm sodium nitrite were added to ground pork and mixed on ice using a mortar. Subsequently, HESM, HEW, and EW were added at concentrations of 1.0, 2.5, or 5.0 % (weight per weight of the mixed ground pork) and mixed thoroughly on ice using a mortar. After mixing, the samples were packed in sanitary plastic bags and heated in a water bath (Thermometer SD; TAITEC, Koshigaya, Japan) at 75 °C for 20 min. The positive control sample was prepared by mixing 0.1 % (w/w) sodium ascorbate with ground pork.

Meat color measurement  The color of the sausage model was evaluated using a spectrophotometer (MINOLTA CM-3500; Konica Minolta Sensing, Inc., Tokyo, Japan) configured with a D65 light source, reflectance rejection, and a 10° field of view for reflectance measurement. The results are expressed as lightness (L*), redness (a*), and yellow (b*) values.

Fe³-reducing activity and oxidation-reduction potential measurements  The 70 % ethanol-extracts of HESM, HEW, and EW were suspended in distilled water, and their Fe³-reducing ability was measured as described by Ferreira et al. (2007). The reducing capacity was expressed as the Trolox equivalent value µmol/L) based on a calibration curve prepared with Trolox. Additionally, oxidation-reduction potential (ORP) values were measured using an ORP sensor (OR-101S; Kasahara Chemical Instruments Corp., Saitama, Japan) equipped with a pH/ORP meter KP-10F (Kasahara Chemical Instruments Corp.).

Preparation of myoglobin solution for the NOMb forming assay  The myoglobin solution for the NOMb forming ratio of tested samples were prepared as described by Takeda et al., (2023b). Briefly, 0.4 mL of 0.625 % myoglobin (Nacalai Tesque, Inc., Kyoto, Japan) solution was mixed with 2.0 mL of each tested sample. Nitrogen gas was bubbled in the solution on ice for 15 min and in the headspace of the tubes for 5 min. The tubes were then sealed and 0.1 mL of 0.25 % NaNO₂ solution was added anaerobically to the sample solution. The sample solution was then incubated in a water bath at 75 °C. The NOMb formation ratio was evaluated as described below.

Maleimide modification and measurement of thiol group concentration  Lyophilized HESM 70 % ethanol-extract was dissolved in 0.1 mol/L tris(hydroxymethyl)aminomethane buffer (pH 7.0) to prepare 700 mL of 10 mg/mL (i.e., 7.0 g HESM 70 % ethanol-extract containing). Subsequently, 77.8 mL of 1.0 mol/L maleimide (Combi-Blocks Inc., San Diego, CA, USA) solution was prepared (i.e., 7.55 g maleimide containing). The buffer dissolved the HESM 70 % ethanol-extract and the maleimide solution were mixed and incubated at approximately 25 °C with stirring for 1 h. The mixed solution was kept overnight at −80 °C and lyophilized. The resulting lyophilized powder was designated as maleimide-treated HESM 70 % ethanol-extract. Owing to the presence of maleimide, the ratio of HESM 70 % ethanol-extract in the final maleimide-treated lyophilized sample is 48 %. This is calculated as follows: HESM 70 % ethanol-extract is 7.0 g (approximately 48 %) and maleimide is 7.55 g (approximately 52 %) in total. Thus, the sample of maleimide-treated HESM 70 % ethanol-extract was used to adjust to the content of HESM 70 % ethanol-extract for the experiment. Thiol group concentration was measured as described by Ahhmed et al. (2019). 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 buffer (pH 6.8) containing 2 % sodium dodecyl sulfate, 48 % urea, and 0.29 % ethylenediaminetetraacetic acid. The sample was suspended in distilled water, and 0.5 mL of this suspension was mixed with 2.5 mL of tris(hydroxymethyl)aminomethane buffer (50 mmol/L, pH 6.8) and 20 µL of 0.4 % 5,5′-Dithiobis(2-nitrobenzoic acid) solution. The mixture was maintained in the dark at approximately 25 °C for 1 h and subsequently centrifuged at 2 500 ×g for 7 min. The absorbance of the supernatant was measured at 412 nm using a spectrophotometer (UV-1800, Shimadzu, Kyoto, Japan), and the thiol group concentration was calculated using the following formula:

  

Nitrosyl myoglobin forming activity measurement  NOMb forming ratio for the tested sausage model and the tested myoglobin solution were measured as described previously by Sakata (1999) and Takeda et al. (2023b). Nitrosyl heme dye level was determined using the modified 75 % acetone extraction method, and the total heme pigment concentration was determined using the 75 % acetone-0.7 % hydrochloric acid extraction method. The absorbances of the nitrosyl and total heme dyes were measured at 395 and 383 nm, respectively. The evaluation was based on the ratio of total heme pigment content to nitrosyl heme pigment content. The coloration ratio was calculated using the following formula:

  

Statistical analysis  The data are expressed as the mean ± standard deviation (SD). All data were analyzed using GraphPad PRISM 5.01 (GraphPad Software, Inc., Boston, MA, USA) for a one-way analysis of variance followed by Tukey’s test or Student’s t-test. Statistical significance was set at p < 0.05.

Results and Discussion

Meat color of sausage models with HEMP and HEW  Sausages containing HESM and HEW powders exhibited a red color compared to the control sausages, respectively (Fig. 1). Notably, sausages containing 5 % HESM displayed the most intense redness, with both the 2.5 % and 5 % HESM sausages achieving a red coloration similar to that of the sodium ascorbate-added sausages. Additionally, the meat color of sausages containing HEW appeared redder than that of sausages containing EW at the same concentration. The L*, a*, and b* values of the sausages are shown in Table 1. The a* values of sausages containing HESM was higher than those of control sausages. In particular, a* values increased with the addition of 1 % and 2.5 % HESM, and 5 % HESM sausages exhibited a* values equivalent to those 2.5 % HESM sausages. The a* values for 1 % and 2.5 % HESM sausages were significantly higher than those for HEW and EW sausages at the same concentrations (p < 0.05). In addition, the a* values for 5 % HESM and 5 % HEW sausages were significantly higher than those for 5 % EW sausages. Those a* values and trends in the reddish appearance across sausage surfaces appeared consistent. The NOMb formation ratios in the tested sausages were also assessed (Table 1). The L* values for 5 % EW sausages were also significantly higher than those for 5 % HESM and HEW sausages. No significant differences were observed in the b* values in all sausages. The NOMb formation ratio for 1 % HESM sausage was significantly higher than that for HEW and EW sausages at the same concentration. Similarly, the NOMb forming ratios of 2.5 % and 5.0 % HESM, and 2.5 % and 5.0 % HEW sausages were significantly higher than those of the same levels of EW sausages, respectively (p < 0.05).

Fig. 1

Visual color of the sausage model treated with hydrolyzed ESM (HESM), hydrolyzed egg white (HEW), egg white (EW) and sodium ascorbate. All products were added to ground pork weight per weight, respectively. Control is the sausage model just added 2 % NaCl and 30 ppm NaNO₂.

Table 1. Measurement of L*, a*, b* values and NOMb forming ratio in the tested model sausages.

	Concentration (% W/W)	L*	a*	b*	NOMb forming ratio (%)
Control	—	60.78 ± 2.70	3.89 ± 0.51	9.89 ± 0.38	27.79 ± 0.31
Sodium ascorbate	0.1%	58.40 ± 1.36	6.42 ± 0.29	7.62 ± 0.87	52.71 ± 1.14
HESM	1.0%	56.80 ± 1.82	5.20 ± 0.28a	8.71 ± 0.96	45.28 ± 3.48a
	2.5%	57.57 ± 2.19	7.52 ± 0.59a	8.20 ± 0.96	56.60 ± 6.89a,
	5.0%	54.86 ± 1.54b	7.64 ± 0.30a	8.49 ± 0.46	51.77 ± 5.08a
HEW	1.0%	59.31 ± 1.11	3.88 ± 0.43b	8.55 ± 1.36	24.12 ± 4.57b
	2.5%	58.36 ± 3.15	3.80 ± 0.52b	6.89 ± 0.69	38.32 ± 6.16a
	5.0%	57.13 ± 2.92b	5.25 ± 1.14a	7.11 ± 1.02	54.40 ± 8.72a
EW	1.0%	56.93 ± 2.14	4.17 ± 0.16b	9.16 ± 0.69	29.08 ± 3.83b
	2.5%	59.86 ± 3.13	3.93 ± 0.53b	8.15 ± 0.94	25.82 ± 3.04b
	5.0%	63.00 ± 1.34a	3.33 ± 0.46b	6.79 ± 0.48	20.58 ± 2.13b

*p < 0.05 vs each value of the sample at the same concentration using one-way analysis of variance followed by Tukey’s test.

A previous study demonstrated that the ESM promoted a* value in emulsion-type meat products such as sausages, and the addition of 1.5 % ESM resulted in an approximate 25 % increase in the a* value in cooked samples compared to those without ESM (Rødbotten et al., 2022). Although this study could not prepare a sausage model using the ESM, the addition of 1.5 % and 2.5 % HESM increased the a* values of control sausages by approximately 33 % and 48 %, respectively (control: 3.89, 1.0 % HESM: 5.20, and 2.5 % HESM: 7.52).

Those results indicate that HESM would increase the reddish appearance of the cured sausage as compared to that of ESM. Moreover, compared to a previous study, the reddening effect of the appearance of the sausage model added HESM in this study was also clearly more effective than that of whey enzymatic degradation products (Takeda et al., 2023b). The addition of 5.0 % HEW also increased a* values compared with those observed for the control and 5.0 %> EW sausages. Given that HESM and HEW are prepared by hydrolyzing ESM and EW, they contain many peptides derived from ESM or EW. Therefore, the hydrolyzed peptides are believed to have an effect on the color development of the meat color. The main pigment in cured and cooked meat products is nitrosyl hemochromogen (Pegg and Shahidi, 1996; Sun et al., 2009). During the curing process, is reduced and converted to NO before cooking, binding to myoglobin and subsequently initiating a NOMb-formation reaction (Honikel, 2008). In the present study, the NOMb formation ratio for sausages containing 2.5 % and 5 % HESM and 5 % HEW was comparable to that of the sodium ascorbate positive control, suggesting that HESM and HEW enhance meat color by accelerating the reaction of myoglobin to NOMb. In the previous study, reducing activities of hydrolyzed whey contributed to the coloration of cured sausages (Takeda et al., 2023b). Thus, HESM and HEW were also thought to promote coloration through their reducing effects.

Reducing activities of 70 % ethanol-extracts of HESM and HEW  The Fe³⁺-reducing activities and ORP values of 70 % ethanol-extracts of HESM and HEW were assessed (Table 2). All extracts demonstrated Fe³⁺-reducing activities expressed as Trolox equivalents in a dose-dependent manner. The HESM 70 % ethanol-extract exhibited significantly higher activity than the HEW 70 % ethanol-extract at the same concentration (p < 0.05). In addition, ORP values, which indicate the oxidizing or reducing power of a solution were measured. Both the 70 % ethanol-extracts of HESM and HEW exhibited decreased ORP values in a dose-dependent manner. Notably, the ORP value for the HESM 70 % ethanol-extract was significantly lower than that of the HEW 70 % ethanol-extract at the same concentration (p < 0.05). Thus, the tested 70 % ethanol-extracts of HESM and HEW have the reducing effects, in particular, the HESM 70 % ethanol-extract was suggested to have a stronger reducing effect than that of HEW 70 % ethanol-extract.

Table 2. Reducing activities of 70 % ethanol extracts of HESM and HEW.

	HESM 70 % ethanol extract			HEW 70 % ethanol extract
Concentration (mg/mL)	10	25	50	10	25	50
Fe3+ reduction Trolox equivalent value (µmol/L)	49.70 ± 1.90*	102.80 ± 1.90*	174.70 ± 4.90*	19.70 ± 2.00	44.30 ± 2.50	77.80 ± 4.00
ORP value (mV)	71.0 ± 5.7*	31.4 ± 6.9*	8.0 ± 3.9*	113.4 ± 7.5	82.6 ± 5.8	59.0 ± 8.6

* p < 0.05 vs. the value of HEW 70 % ethanol extract at the same concentration using Student’s t-test.

Data are presented as mean ± SD (n = 3–5)

It is known that hydrolysis of animal food resources or by-products yields bioactive peptides such as antioxidant peptides derived from their proteins (Liu et al., 2015; Bechaux et al, 2019; Zhu et al, 2022b). Thus, in this study, the 70 % ethanol-extracts of HESM and HEW were prepared to obtain low molecular weight substances such as bioactive peptides, and these extracts were used for the experiments. The 70 % ethanol-extracts of HESM and HEW demonstrated reducing activity, as indicated by Fe³⁺ reduction and reduced ORP values, which seems to consist with previously reported reducing and/or antioxidant activities (Davalos et al., 2004; Shi et al., 2014; Zhu et al., 2022a). Several studies have isolated peptides with antioxidant activity from HESM and HEW (Liu et al., 2015; Zhu et al., 2022b). Additionally, ESM is constituted of structural proteins including collagens and cysteine-rich eggshell membrane proteins (Ahmed et al., 2017). Cysteine is known as a reducing amino acid since the thiol group in its side chain has a reducing effect. Thus, the reducing effect of HESM might be attributed to the thiol groups derived from cysteine in the ESM. Moreover, the reducing effect of HESM was potentially caused by hydrolysis of ESM, resulting in low molecular weight substances such as peptides with newly exposed thiol groups.

Effects of 70 % ethanol-extracts of HESM and HEW on coloration in myoglobin solution  The effects of 70 % ethanol-extracts of HESM and HEW on NOMb formation in myoglobin solutions were investigated. Both extracts promoted NOMb formation after the incubation (Fig. 2A). The HESM 70 % ethanol-extract yielded a NOMb formation ratio similar to that of ascorbic acid, with notably greater coloration enhancement than that of HEW. The concentration-dependent effects of HESM 70 % ethanol-extract on the ratio of NOMb formation after 5 min of incubation are shown in Figure 2B; the 25 mg/mL and 50 mg/mL samples of HESM 70 % ethanol extracts promoted approximately 80 % and 90 % NOMb formation, respectively, which is consistent with the increased redness observed on sausage surfaces with HESM (Fig. 1). Thiol concentrations in the HESM 70 % ethanol and maleimide-treated extracts were 256.8 µmol/g and 107.8 µmol/g, respectively. The NOMb formation ratio of the maleimide-treated HESM 70 % ethanol-extract was significantly lower than that of the untreated HESM 70 % ethanol-extract at 50 mg/mL (p < 0.05).

Fig. 2

NOMb-forming ratio by the 70 % ethanol-extracts of HESM, HEW, and maleimide-treated HESM in the myoglobin solution. (A) shows the time course of the NOMb forming ratio of each sample. (B) shows the NOMb forming ratio of each sample at 5 min after incubation. Error bars represent SD. Data are presented as mean ± SD (n = 3). Different superscript letters indicate statistically significant differences (p < 0.05, by one-way analysis of variance followed by Tukey’s test).

* Since the ratio of HESM 70 % ethanol-extract in the final maleimide-treated lyophilized powder is 48 %, 104 mg/mL of the maleimide-treated HESM sample was subjected for the experiment.

Thiol groups in low-molecular-weight fractions are suggested to stimulate NOMb formation during meat processing (Tinbergen, 1974). The significantly decreased NOMb formation ratio of maleimide-treated HESM 70 % ethanol-extracts suggests that the observed NOMb formation and model sausage reddening-promoting effects of HESM in this study were attributed to the presence of thiol groups. Although inferior to the 70 % ethanol HESM extract, the 70 % ethanol HEW extract also showed reddening and increased NOMb formation in the sausage model (Fig. 1 and Table 1). In a previous report, L-Lysine/L-arginine/L-cysteine amino acids promoted color development by NaNO₂ in meat processing; color development caused from the antioxidation of these amino acids (Ning et al., 2019). The amino acid compositions of 70 % ethanol-extracts of HESM and HEW were meausred (Table S1), and the lysine concentration of HEW 70 % ethanol-extract was higher than that of HESM 70 % ethanol-extract. The thiol group-independent peptides demonstrated to the color-promoting effect of hydrolyzed whey (Takeda et al., 2023b). Thus, the promotion of NOMb formation and coloration effect of HEW on model sausage observed in this study may involve a mechanism different from that of HESM. In addition, the peptides derived from HEW potentially have reducing effects independent of thiol groups. Further studies of HEW 70 % ethanol-extracts are needed to isolate and identify peptides that promote NOMb formation or to investigate other coloration mechanisms in the cured sausages.

Conclusions

This study demonstrated that HESM and HEW promoted reddening in a sausage model, particularly highlighting the reddening-promoting effect of HESM. These effects are likely attributed to the reducing properties of HESM and HEW, which copromote NOMb formation in sausages. The coloration-promoting effect of HESM could be attributed to the content of thiol groups in the low molecular weight substances. Meanwhile, the coloration-promoting effect of HEW may cause from the other reducing peptides. Given that both HESM and HEW are egg products with high nutritional value, this study demonstrates their potential as useful components for improving the functional properties and overall quality of meat products. Future studies on the effects of HESM and HEW on the characteristics of meat products other than the coloration are warranted.

Acknowledgements  The authors would like to thank Mr. Yugo Isshiki and Mr. Akira Meo (Department of Animal Science and Biotechnology, School of Veterinary Medicine, Azabu University) for their technical assistance and Editage (www.editage.com) for English language editing.

Conflict of interest  This study was funded by Kewpie Co. Ryou SASAHARA and Takahisa SUZUKI, they are an employee of Kewpie Co.

Abbreviations

ESM

eggshell membrane

EW

egg white

HESM

hydrolyzed eggshell membrane

HEW

hydrolyzed egg white

NOMb

nitrosyl myoglobin

nitrates

nitrites

NO

nitric oxide

ORP

oxidation-reduction potential

SD

standard deviation

Supplemental data

Table S1. Amino acid compositions of 70 % ethanol-extracts of HESM and HEW

Amino acid	Concentration (g / 100g)
	HESM 70 % ethanol-extract	HEW 70 % ethanol-extract
Arginine	5.00	4.56
Lysine	2.26	5.01
Histidine	2.86	1.98
Phenylalanine	1.17	4.46
Tylosine	1.25	3.54
Leucine	3.70	7.16
Isoleucine	2.50	4.13
Methionine	3.03	2.97
Valine	5.39	5.76
Alanine	2.36	5.32
Glycine	4.41	3.23
Proline	7.17	3.38
Glutamic acid	8.86	11.4
Serine	3.61	5.78
Threonine	3.86	3.86
Aspartic acid	5.73	8.51
Tryptophan	2.59	1.18
Cystine	4.52	1.66

Amino acid compositions of 70 % ethanol-extracts of HESM and HEW were measured. Amino acids except for cystine and tryptophan were determined after acid-hydrolysis of those extract samples by an automated amino acid analyzer. The samples were hydrolyzed following performic acid oxidation and cysteic acid were determined by analyzer and converted to cystine. Tryptophan was determined by an HPLC method after alkaline hydrolysis.

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