Sheep Plasma Hydrolysate Inhibits Lipid and Protein Oxidation to Improve Color Stability in Mutton Patties

Abstract

In the present study, sheep plasma (SP) was hydrolyzed with 5000 U alcalase/g protein at 45 °C, pH 11 for 6 hours to prepare sheep plasma hydrolysate (SPH). The effects of SPH on meat color stability and oxidation of lipid and proteins were evaluated in mutton patties. Data obtained showed that addition of 2 % SPH significantly improved the redness (a* value) of mutton patties in storage (P < 0.05), which was comparable to 0.05 % vitamin C. At the same time, 2 % SPH effectively inhibited metmyoglobin formation and oxidation of lipid and proteins in mutton patties. The inhibition of oxidation in meat by SPH was concentration dependent. In summary, this study showed that SPH was antioxidant and 2 % SPH could be used in production to prevent quality deterioration of meat products in storage, providing a new way for high-value utilization of sheep blood.

Introduction

Mutton is favorited by consumers for its unique flavor and nutritional value. With the fast-paced life, the number of meals that fully prepared at home is reducing, instead, the consumption of pre-prepared food and fast food are growing (Michalak et al., 2017; Shah et al., 2014). Prepared meat products are made from meat of livestock or poultry. It is shaped, cured and stored in cold or frozen conditions, and then can be eaten just after simple warm up or heating. Prepared meat products such as mutton patties are usually stored at refrigerated temperatures (2–5 °C). However, lipid oxidation and protein oxidation are major challenges for meat quality during refrigeration storage (Estévez, 2011). It is because lipid and protein in mutton after slaughter are easily susceptible to oxidative damages due to the rapid depletion of endogenous antioxidants after slaughtering.

Lipid peroxidation in meat and meat products occurs primarily during postmortem handling and storage through radical chain reaction mechanism which results in the deterioration of meat quality such as discoloration, off-flavor, drip loss, loss of nutrient value, and decreased shelf-life, which may be hazardous to the health of consumers (Falowo et al., 2014). The procedure of lipid oxidation consists of four stages, including initiation, propagation, termination and formation of the secondary product with the production of free radicals. The rate and extent of lipid oxidation are influenced by many factors such as pH, iron content and antioxidants (Jiang and Xiong, 2016). Muscle is mainly composed of protein. It plays a pivotal role in meat quality formation regarding the contribution of protein to meat sensory, nutritional value and physicochemical properties. Similar to lipid oxidation, protein oxidation in muscle also occurs through a chain of reactive oxygen species (ROS) (Sampath and Nazeer, 2012). The presence of ROS and oxygen has been found giving rise to the modification of amino acid side chain and forming covalent intermolecular cross-links (Lund and Baron, 2010). In the reaction of protein oxidation, protein carbonyl groups are generated through the procedure that ROS attacks the free amino or imino of amino acids, resulting in the production of NH3 and carbonyl derivatives (Pieniazek and Gwozdzinski, 2017). Hence, the increase of carbonyl group can be served as a marker to quantify protein oxidation and damage.

Many ways have been developed to prevent the oxidation of meat products, including the use of antioxidants. Antioxidants are substances that could decelerate the oxidation of easily oxidizable biomolecules at low concentrations such as lipids and proteins in meat products, thus improving shelf life of products by protecting them against deterioration caused by oxidation. Exogenous antioxidants can be used to ensure the quality of products. Synthetic antioxidants such as butylated hydroxyanisole, butylated hydroxytoluene, tert-butylhydroquinone, and propyl gallate have been used as antioxidants in meat and poultry products, but synthetic antioxidants have fallen under scrutiny due to the apprehension about the tox-icological/carcinogenic effects (Naveena et al., 2008). In response to recent demands for natural products, the meat and poultry industry is actively seeking natural solutions to minimize oxidative rancidity and increase products' shelf-life (Serraa et al., 2007). Many natural antioxidants reported to be more active than synthetic antioxidants such as ascorbic acid and sodium ascorbate inhibited lipid oxidation in meat products at 0.025–0.05 % level (Rhee et al., 1997). By-products are potential sources of antioxidants for muscle food preservation and nutritional quality improvement (Correa et al., 2014). Sheep blood is abundant in proteins while millions of tons of sheep blood is produced and discharged each year in the world (Toldrá et al., 2016). The direct output of animal blood to environment is not just a waste of protein resource, more seriously, it causes pollution. Functional peptides can be produced from animal proteins and provide a way for high-value-utilization of animal byproducts (Sampath and Nazeer, 2012). Liu et al. (2009) and Seo et al. (2015) prepared antioxidant peptides from porcine and bovine plasma. It is logical to deduce that antioxidants from animal products could be used to improve sensory and possibly nutritional quality of meat and meat products.

In the present study, sheep plasma (SP) was hydrolyzed by alcalase and the effect of the hydrolysate on lipid and protein oxidation as well as meat color stability was evaluated using mutton patties. The results showed that sheep plasma hydrolysate (SPH) was antioxidant. Addition of SPH to mutton patties effectively inhibited oxidation and improved meat color stability.

Materials and Methods

Animals and sample collection    Five 8 months old sheep (Small Tail Han sheep with 20 ± 2.5 kg carcass weight) were slaughtered at an export meat processing facility in accordance with the requirements of National Standards of PR China (Fresh and frozen mutton carcass, GB/T 9961–2008). The sheep selected in this study were passed inspection and quarantine in accordance with the requirements of Chinese Quarantine and Inspection Service and achieved the slaughter requirements. The blood from these sheep was safe and edible after hygienic collection and proper storage. The longissimus lumborum muscles were collected within 30 min postmortem from both sides of the carcass and shipped to the laboratory in refrigeration (4 °C). Fresh sheep blood was treated with anticoagulants (0.5 % sodium citrate) immediately after collection and shipped to laboratory in refrigeration (4–10 °C) condition. SP was prepared by centrifugation (8000×g, 20min) in a refrigerated centrifuge (Hitachi High-speed Refrigerated Centrifuge Model CR21N, Hitachi Global, Tokyo, Japan) and freeze dried into powder with a lyophilizer (LGJ-25C, Beijing Fourth Ring Scientific Instrument Factory Co., Ltd, Beijing, China), and SP was stored at −20 °C before being used.

Sheep plasma hydrolysate preparation    SPH was prepared as described by Liu et al. (2011) with some modifications. SP powder was dissolved in 0.01 mol/L PBS (phosphate buffered saline, pH 7) to a final concentration of 40 mg/mL. Our team has studied the total antioxidant capacity of various sheep plasma hydrolysates derived from different proteases at different stages (data not shown). The results showed that the total antioxidant capacity of alcalase hydrolyzed SP hydrolysate was significantly higher than that of pepsin, papain, trypsin and other protease hydrolysates. Therefore, alcalase was selected as the protease to hydrolyze SP. Alcalase (5000 U/g protein) (EC:3.4.21.62, endoproteinase from Bacillus licheniformis with an activity of 2 × 10⁵ U/g were purchased from Solarbio, Beijing, China) was added to the reconstituted plasma. After incubation at 45 °C for 6 h with stirring, the hydrolysis was terminated by inactivating enzyme at 90 °C for 10 min. After adjustment of pH to 7, the hydrolysate was centrifuged in a refrigerated centrifuge at 8000×g for 15 min. The supernatant was collected, lyophilized and stored at −20 °C until using. The purpose of this study was to evaluate the function of hydrolysates of sheep plasma. However, in actual production, it is necessary to evaluate its safety (especially microorganism) to ensure the sanitarily safety of SP and SPH.

Total antioxidant capacity of SP and SPH    Total antioxidant capacity of SP and SPH was measured using total antioxidant capacity assay kits (Nanjing Jiancheng biological engineering institute, Nanjing, China). SP and SPH were reconstituted to 40 mg/mL, and 0.1 mL were taken for testing respectively. Results are expressed as U/mL.

SDS-PAGE of SP and SPH    SDS-PAGE was used to illustrate the molecular distribution of SP and SPH. 0.25 µg SP and SPH were loaded onto polyacrylamide gels composed of 20 % separating gel and 4 % stacking gel (acrylamide: N, N-bis-methylene acrylamide = 37.5:1). Electrophoresis was performed at room temperature, with 70 and 110 V voltages for stacking gel and separating gel, respectively.

Analysis of amino acid composition of SP and SPH    Amino acid compositions of SP and SPH were analyzed using a fully automated amino acid analyzer (L-8900, Hitachi Amino Analyzer, Hitachi Global, Tokyo, Japan) according to the method described by Wang et al. (2016) with some modifications. Triplicate samples of SP and SPH were hydrolyzed in a glass tube fitted with a cap with 6 mol/L HCl at 110 °C for 24 h. After filtration through a 0.22 µm membrane, samples were injected onto the amino acid analyzer and detected at 570 nm and 440 nm. Amino acid standard solution was used as internal reference. The content of individual amino acids was expressed as mg/g sample.

Mutton patty preparation    After removing fat and fascia, longissimus lumborum muscles were cut into small cubes, salted with 1.5 % salt (w/w) to exclude the influence of spices (Zhou et al., 2016) and evenly divided into 5 groups. For the three experimental groups, mutton was added with 0.5 %, 1 % or 2 % (w/w) SPH. Distilled water was used as negative control and vitamin C (Vc, 0.05 % w/w mutton) was used as positive control (Mancini et al., 2015). Mutton patties (50 g) were prepared in triplicate, sealed in bags and stored in dark at 4 °C for 0, 3, 6, 9, and 12 days.

Meat color determination    Instrumental color measurement was performed using a Minolta colorimeter CM-600D spectrophotometer (CM-600D, Konica Minolta Sensing Americas Inc., Tokyo, Japan) following the method described by Li et al. (2017). Measurements were performed at three random locations for each patty. To eliminate the influence of the packaging material, the sample PVC film was used to cover the standard white plate for calibration.

Metmyoglobin determination    Metmyoglobin (MetMb) was extracted from patties and quantified according to the method described by Krzywicki (1979) with some modifications. One gram of meat was added with 9 mL 0.04 mol/L potassium phosphate buffer (pH 6.8) and homogenized for 3 × 10 s on ice bath with a homogenizer (Ultra Turrax Disperser S25, IKA labortechik, Königswinter, Staufen, Germany). The homogenate was centrifuged and the supernatant was filtered and collected. Absorbance at 525, 572 and 700 nm was measured using an ultraviolet spectrophotometer (UV-1800, Shimadzu Corporation, Tokyo, Japan). The percentage of MetMb was calculated using formula The percentage of MetMb = [1.395−(A₅₇₂−A₇₀₀)/(A₅₂₅−A₇₀₀]×100

TBARS value determination    Lipid oxidation in meat was assessed by thiobarbituric acid reactive substances (TBARS). TBARS values were measured using malondialdehyde (MDA) assay kits (Nanjing Jiancheng biological engineering institute, Nanjing, China). One gram of meat added with 9 mL anhydrous ethanol was homogenized for 3 × 10 s in ice bath with a homogenizer. The supernatant was collected after the centrifugation (5000 g × 10 min, 4 °C) and filtration for TBARS determination. Results are expressed as nmol malonaldehyde equivalent/mg protein (Muíño et al., 2017).

Protein oxidation determination    Protein oxidation in mutton patties was evaluated by the formation of protein carbonyl group using commercial assay kits (Nanjing Jiancheng biological engineering institute, Nanjing, China) according to Feng et al. (2016). 0.1 gram of meat was added with 1 mL extractive solution and homogenized for 3 × 10 s in ice bath. The homogenate was centrifuged (5000 g × 10 min, 4 °C) and the supernatant was filtered and collected. Supernatant was added with 0.1 mL extract, placed at room temperature for 10 min and then centrifuged (12000 g × 10 min, 4 °C). The supernatant was collected of which 20 µL was used for determining the protein content and the rest was used for protein oxidation determination. Results were expressed as nmol carbonyl group/mg protein.

Statistical analysis    Data are expressed as mean ± standard deviation (n = 3). Data were analyzed using 9.2 Statistical Analysis System package (SAS institute, Cary, USA). The result of instrumental color parameters, MetMb %, TBARS values and protein carbonyl groups were analysed by single factor analysis of variance. Fisher's Protected Least Significant Difference (LSD) test were set at a 5 % significance level to estimate the levels of statistical significance (P < 0.05).

Results and Discussion

Total antioxidant capacity of SP and SPH    Antioxidant property of SP was given after hydrolysis. The total antioxidant capacity of SPH was significantly higher than that of SP (P <0.05), with the value of 10.630 ± 1.022 U/mL and 0.122 ± 0.009 U/mL respectively. The hydrolysate achieved from skipjack tuna (Katsuwonus pelamis) dark muscles by Chi et al. (2015) has the similar function, and might be used for food preservation and medicinal purposes. Hence, SPH is expected to be used as a potential antioxidant in food as well.

SDS-PAGE determination of SP and SPH    The SDS-PAGE image of SP and SPH was shown in Fig. 1. The molecular size of SP protein was above 20.1 kDa. While, the molecular weight distribution of SPH was below 20.1 kDa and had a wide range of distribution indicating most of it was hydrolyzed by alkaline proteases.

Fig. 1.

Image of SP and SPH by SDS-PAGE. SP: Sheep plasma with a concentration of 10 mg/mL; SPH: Sheep plasma hydrolysate with a concentration of 10 mg/mL; Marker: The range of molecular weight is 3.3–30.1 kDa.

Tam et al. (2017) studied the antioxidant capacity of peptide fractions isolated from the Tra Catfish (Pangasius hypophthalmus) by-product-derived proteolysate hydrolyzed by alcalase using ultrafiltration centrifigual devices with 5 distinct molecular-weight cutoffs of 1, 3, 5, 10, 30 kDa and the antioxidant activity of peptide fractions was investigated. The result showed that the < 1 kDa fraction showed the strongest antioxidant acivity and 1–3 kDa fraction was the second. Hence, the molecular weight of the hydrolysates was closely related to the antioxidant activity. Fernanda et al. (2017) studied that the hydrolysis of a flaxseed protein isolate with Alcalase^® was performed as a strategy to generate antioxidant peptides. Therefore, SPH was obtained after enzymatic hydrolysis of alcalase which enhances its antioxidant activity and releases more new peptides.

Amino acid composition of SP and SPH    The amino acid composition of SP and SPH was shown in Table 1. The majority of amino acid concentrations in SPH were significantly higher than those in SP (P < 0.05), indicating that the amino acid content of SP can be increased after enzymatic hydrolysis in each gram of sample. Among them, acidic amino acids such as Asp and Glu and hydrophobic amino acids such as Leu and Gly significantly increased (P < 0.05).The increase of these amino acids may play an important role in improving their antioxidant activity.

Table 1. Amino acid composition of sheep plasma and sheep plasma hydrolysates.

	Relative amount (mg/g sample)
	SP	SPH
Asp	12.42±0.22b	17.70±0.42a
Thr	7.65±0.12b	10.83±0.23a
Ser	7.11±0.15b	10.08±0.19a
Glu	19.51±0.21b	27.79±1.21a
Gly	4.19±0.03b	6.04±0.13a
Ala	6.13±0.11b	8.82±0.89a
Cys	3.76±0.02a	0.43±0.02b
Val	8.00±0.13b	11.40±0.16a
Met	1.25±0.01a	1.84±0.09a
Ile	3.74±0.17b	5.35±0.07a
Leu	11.80±0.21b	16.92±0.93a
Tyr	6.34±0.05b	8.79±0.03a
Phe	5.90±0.07b	8.42±0.56a
Lys	10.44±0.14b	13.10±0.97a
His	3.37±0.08a	4.77±0.16a
Arg	6.58±0.14b	9.06±0.83a
Acidic amino acid1	31.93b	45.49a
Basic amino acid2	20.39b	26.93a

1  Sum of Asp and Glu.

2  Sum of Arg, His and Lys.

Data are expressed as mean±standard deviation (n = 3). Means with different lower case letters in the same row are significantly different (P < 0.05).

Chi et al. (2015) confirmed that smaller molecular size and the presence of hydrophobic and aromatic amino acid residues were the key factors that determined the antioxidant activities of the hydrolysate. The amino acid concentrations increased in per gram of sample after enzymatic hydrolysis, indicating that enzymatic hydrolysis can expose more amino acids of SP. Previous reports showed that alcalase is capable of producing antioxidant peptides from numerous protein sources (Li et al., 2008). A small peptide containing Lys at the N end (Lys-Phe, Lys-Leu, Lys-Tyr and Lys-Asp-Tyr-Pro) showed strong inhibitory activity on linoleic acid peroxidation (Guo et al., 2009). Himaya et al. (2012) reported that hydrophobic amino acids could facilitate the interactions with hydrophobic targets, such as the cell membrane. Moreover, Mendis et al. (2005) reported that the high potency of HGPLGPL as an antioxidant could be resulted from its hydrophobic amino acid constitution, which may facilitate a greater interaction between the peptide and fatty acids. Whereby estimation, SPH may contains multiple peptides, so as to have antioxidant activity. In addition, aspartic acid and glutamic acid are flavored amino acids, which can enhance the umami of the mutton patties.

Meat color    The measured CIE-L*a*b* values were shown in Table 2. L* values of all mutton patties except negative control (C) decreased early stage and then increased afterwards during storage. With the addition of SPH and Vc, the L* values decreased than the values of control group on each day during 0–9 days. L* values of SPH group decreased with the increase of the amount of SPH from 0.5–2 % on each days. Muíño et al. (2017) used valorisation of an extract from olive as a natural antioxidant for meat, in which the L* value of treatment group (100, 200 or 400 mg gallic acid equivalents/kg muscle) showed a tendency to rise first and then decline that was similar to the result of this study. The L* value of 0.5–2 % SPH group rise first and then decline during 3–12 days, as natural antioxidants from other plant sources.

Table 2. Effects of SPH on the color of mutton patties.

Color	Treatment	Storage period (days)
		0	3	6	9	12
L*	C	43.48±0.91Aa	38.87±0.36Ac	38.67±0.99Ac	41.19±0.94Ab	40.03±1.23ABbc
	0.5 % SPH	43.15±0.40Aa	38.63±1.47Ab	39.71±0.70Ab	40.54±2.33Aab	41.22±0.64Aab
	1 % SPH	41.30±1.12Ba	38.05±0.97Ab	38.31±0.87Ab	39.10±2.81Aab	39.58±1.30ABab
	2 % SPH	40.65±1.22Ba	37.99±1.16Ab	39.62±1.13Aab	38.59±0.35Ab	38.46±1.27Bb
	Vc	39.57±1.19Bab	36.39±0.52Ac	38.33±0.46Ab	39.27±0.63Aab	40.49±0.46Aa
a*	C	10.47±0.69BCa	9.68±0.50Cab	9.03±0.60Cb	7.49±0.17Cc	5.90±0.26Cd
	0.5 % SPH	11.21±0.33BCa	10.32±0.61BCb	9.28±0.20Cc	7.64±0.59Cd	6.27±0.37Ce
	1 % SPH	11.27±0.32Ba	10.45±0.23Ba	9.29±0.55Cb	8.09±0.61Cc	6.99±0.55Bd
	2 % SPH	12.57±0.59Aa	11.51±0.05Ab	10.25±0.26Ac	9.8±0.21Bc	8.81±0.29Ad
	Vc	10.31±0.54Ca	11.41±0.24Ab	12.12±0.18Bc	11.18±0.31Ac	7.35±0.34Bd
b*	C	8.93±0.82Bc	9.69±0.55BCb	10.29±0.38Bb	8.59±0.22Ba	10.12±0.50ABd
	0.5 % SPH	10.16±0.21ABc	10.69±0.89ABb	10.82±0.58ABa	8.89±0.78Bb	10.41±0.60Ad
	1 % SPH	9.79±0.85ABc	9.58±0.14Ca	10.00±0.38Bb	9.54±1.04ABb	9.09±0.93Bd
	2 % SPH	10.56±0.80Ac	10.89±0.51Aa	10.84±0.56ABb	10.43±0.08Ab	10.58±0.21Ad
	Vc	9.41±0.61ABc	10.68±0.43ABb	11.16±0.42Aa	10.48±0.56Ab	9.60±0.34ABd

C: control group, mutton patties without any additives; Vc: mutton patties treated with 0.1 % Vitamin C.

Data are expressed as mean ± standard deviation (n = 3). Means with different lower case letters in the same row are significantly different (P < 0.05). Means with different upper case letters in the same column are significantly different (P < 0.05).

The a* values in all treatment groups showed a trend of decrease with the extension of storage time. The a* values of SPH group fell less than the control. The a* values of mutton patties with amount of 2 % additive were significantly higher than the control group, 0.5 % SPH group and 1 % SPH group from day 0 to 12 (P < 0.05). The a* values of mutton patties with Vc increased from day 0 to 6 and then decreased from day 6 to 12. Redness is the most important color parameter for fresh meat, which decreases with the increase of storage time (Olivera et al., 2013). The a* value which consumers concern the most is a mainly manifested by the transformation among myoglobin, oxygenated myoglobin, and metmyoglobin reflecting the myoglobin concentration and its redox state in meat (Mancini and Hunt, 2005). The result showed SPH can postpone the deterioration of meat color and maybe the reason is some peptides in SPH take effect in reacting with oxygen reducing the oxidation of myoglobin (Silvestre et al., 2012). The a* values of 2 % SPH kept falling during storage and the values were significantly higher than the control group at each storage time (P < 0.05), while the a* values of the Vc increased in early storage then decreased. Hence, the effect of 2 % SPH on protecting meat color is more stable than Vc group.

The b* values in all treatment group showed a trend of increasing at first and decreasing later, with the extension of storage time compared with the control group. With the addition of SPH and Vc, the b* value increased compared with the control group on each day. But the influence of b* value on meat quality is not clear, so this study could not confirm whether the change trend is beneficial or not.

MetMb content    Changes of MetMb during storage in mutton patties were shown in Fig. 2. The proportion of MetMb gradually increased with storage time, which was consistent with objective color measurement. The percentage of MetMb of patties added 2 % SPH was significantly lower than of control, patties added 0.5 % and 1 % SPH on days 3, 6 and 12 (P < 0.05), but no difference existed among these treatments on days 0 and 9. The proportion of MetMb in patties with 2 % SPH was even lower than that of positive control added with 0.05 % Vc, showing the antioxidant property of SPH to effectively inhibit myoglobin oxidation.

Fig. 2.

Metmyoglobin in mutton patties during 12-day storage. Control: without SPH; 0.5 % SPH: 0.5 % SPH w/w mutton patties; 1 % SPH: 1 % SPH w/w mutton patties; 2 % SPH: 2 % SPH w/w mutton patties; 0.05 % Vc: 0.05 % Vc w/w mutton patties. Different letters (a–b) indicate significant difference among treatments (P < 0.05). Error bars represent standard deviation

Three forms of myoglobin, in terms of myoglobin, oxygenated myoglobin, and metmyoglobin, are able to be converted into each other, and thus lead to the change of meat color (Gddings, 1974). With the prolongation of storage time, the color of the meat become dark with the increase of MetMb in the muscles. 2 % SPH addition can inhibit the increase of MetMb during storage accord with a higher a* value than other groups.

Lipid oxidation    The determined TBARS values of mutton patties in storage are shown in Fig. 3. At beginning, there was no difference in TBARS values among treatments, showing no difference in lipid oxidation. However, the TBARS values of patties added with SPH or Vc became significantly lower than those of control since day 6, showing SPH and Vc was effective to inhibit lipid oxidation in meat. For the three SPH treatments, the TBARS values were inversely related to the concentrations of SPH, demonstrating that the antioxidant capacity of SPH in meat is concentration dependent. On day 12, the TBARS values was not different between patties added with 2 % SPH and 0.05 % Vc, which were still lower than the threshold value for rancidity perception, showing that 2 % SPH was replaceable to 0.05 % Vc to inhibit lipid oxidation and to prevent rancidity of mutton patties.

Fig. 3.

TBARS values of mutton patties during 12-day storage. Control: without SPH; 0.5 % SPH: 0.5 % SPH g/g mutton patties; 1 % SPH: 1 % SPH w/w mutton patties; 2 % SPH: 2 % SPH w/ w mutton patties; 0.05 % Vc: 0.05 % Vc w/w mutton patties. Different letters (a–b) indicate significant difference among treatments (P < 0.05). Error bars represent standard deviation

TBARS in meat gradually increased with the extension of storage time according to Zhang et al. (2016), which was consistent with this study. MDA was the product of lipid peroxidation, which could lead to deterioration of meat quality during storage. Some natural antioxidants, such as pine bark extract and grape seed extract, have a role in reducing TBARS (Karre et al., 2013). According to Bah et al. (2016), a variety of peptides were identified in the plasma of pig, cattle and sheep which had antioxidant effects. According to previous research, SPH maybe consist of a variety of small molecule peptides some of which contains a variety of hydrophobic amino acids such as hydrophobic Ala, Val, Leu. Hydrophobic amino acids' non-polar aliphatic side chain can facilitate interaction between antioxidant peptides and hydrophobic polyunsaturated fatty acid (Gao et al., 2014). Specifically, which kind of peptide in SPH takes effects needs further study and identification.

Protein oxidation    Protein oxidation in mutton patties was evaluated by carbonyl formation as shown in Fig. 4. Protein carbonylation was not different between treatments on days 0 and 9. On days 3, 6 and 12, 2 % SPH mutton patties and Vc mutton patties showed significantly lower carbonyl group than control mutton patties, showing 2 %SPH was effective to prevent protein oxidation in meat

Fig. 4.

Carbonyl content in mutton patties during 12-day storage. Control: without SPH; 0.5 % SPH: 0.5 % SPH w/w mutton patties; 1 % SPH: 1 % SPH w/w mutton patties; 2 % SPH: 2 % SPH g/g mutton patties; 0.05 % Vc: 0.05 % Vc w/w mutton patties. Different letters (a–b) indicate significant difference among treatments (P < 0.05). Error bars represent standard deviation

The carbonylation of proteins can lead to changes in protein structure, resulting in the loss of their biological functions, resulting in dysfunction of cells and tissues (Zheng et al., 2010). Protein carbonylation alters the structure of proteins, resulting in changes of their physicochemical properties, such as reduction of digestibility and nutritional value. ROS are closely related to the formation of protein carbonyl (Rajapakse et al., 2005). Maybe SPH plays a role of antioxidant, reducing the protein carbonyl content in mutton patties during storage, and inhibiting the process of protein oxidation. Hydrophobic amino acids such as Gln, Asn, Tyr and antioxidant amino acids like His in SPH can give hydrogen atom to free radical, which leads to the slow down or termination of radical chain reaction.

Conclusion

SPH inhibited lipid and protein oxidation in mutton patties, thus inhibiting metmyoglobin formation and improving meat color stability. SPH inhibition of oxidation in meat was concentration dependent. Based on the values of the percentage of MetMb, TBARS and carbonyl group content, 2 % SPH was equivalent to 0.05 % Vc to effectively inhibit oxidation and to prevent rancidity and discoloration of mutton patties in storage, which has feasible application in industrial production.

Acknowledgements    This study was supported by “National Agricultural Science and Technology Innovation Project” and Fundamental Research Funds for Central Non-profit Scientific Institution (Y2016CG12)

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