Changes of protein oxidation, lipid oxidation and lipolysis in Chinese dry sausage with different sodium chloride curing salt content

Bing Zhao, Huimin Zhou, Shunliang Zhang,?, Xiaoqian Pan, Su Li,Ning Zhu, Qianrong Wu, Shouwei Wang, Xiaoling Qiao, Wenhua Chen

aChina Meat Research Centre, Beijing, 100068, China

bBeijing Academy of Food Sciences, Beijing, 100068, China

ABSTRACT

The effect of sodium chloride (NaCl) curing salt content on protein oxidation, lipid oxidation and lipolysis of Chinese dry sausage was investigated. Two groups Chinese dry sausages with 2% and 4% (m/m) salt content were studied. The degree of protein oxidation increased during the processes in two groups sausages, while the content of phospholipids decreased, neutral lipids and free fatty acids increased. The degree of protein oxidation, lipid oxidation and lipolysis in 4% NaCl content group was higher than those in 2% NaCl content group, while 4% NaCl content group has higher lipase activity. In conclusion, 4% NaCl may facilitate the protein oxidation, lipid hydrolysis and oxidation in Chinese dry sausage, and the protein oxidation had strong correlation with lipid oxidation and lipolysis. The results could provide a basis for improving the technology of industrial production.

Keywords:

Chinese dry sausage

sodium chloride

Protein oxidation

Lipids oxidation

Lipolysis

1. Introduction

Chinese dry sausage is a traditional cured meat product and has been widely accepted by consumers due to its unique flavor and taste. However, in order to get good taste and long shelf life, it is necessary to add high content of NaCl during processing, which is a potential risk for consumers’ health. High sodium diet has been identified as a high disease risk by the Global Burden of Disease(GBD) [1]. Epidemiological studies have shown that there is a positive association between sodium salt intake, blood pressure and hypertension (HBP) [2].

In order to reduce the health risk, the World Health Organization (WHO) had recommended to reduce sodium consumption from 5 g to 2 g daily for adults [3]. Many sodium substitutes were increasingly being used in food. Researchers found that shiitake mushroom extract was a feasible alternative to NaCl for reducing sodium in beef burgers [4]. KCl and MgCl2were popularly incorporated for salamis processing and showed good effects. KCl-added salami had a higher content of free fatty acids, MgCl2-added salami had higher content of free amino acids [5]. However, NaCl cannot be completely replaced by other sodium substitutes, because K+showed a bitter taste, and increased renal burden in patients with diabetes. Low content of NaCl also affected the flavors and taste,due to protein oxidation, lipid oxidation and lipolysis.

The formation of sausage flavors are related to protein oxidation, lipid oxidation and hydrolysis, which is the most remarkable characteristic and important index for quality evaluation. myofibril protein is an important functional protein in meat products, and its oxidation degree is the main indicator of meat quality deterioration [6]. Protein oxidation is a free radical chain reaction which is similar to lipid oxidation, polypeptide backbone and side chains of amino acids are vulnerable to oxidative attack, and formation of protein carbonyls. So during the processing of Chinese dry sausage,there may be some relationships among protein oxidation, lipid oxidation and hydrolysis.

Lipid oxidation and hydrolysis are the chief sources of flavor formation in Chinese dry sausage and produce more than 80% of volatile flavor compounds. While moderate oxidation produces satisfactory flavor during processing, excessive oxidation may bring unpleasant flavor, such as rancidity and nauseous smell,even affected the health of consumers. Therefore, regulating the degree of lipid oxidation is very important in the processing of Chinese dry sausage, which can not only control quality but also protect consumers’ health. Lipid oxidation and hydrolysis are common reactions in the processing of cured meat products [7].Lipid hydrolysis has a promoting effect on lipid oxidation [8].During the processing and storage, although the change in triglycerides contents is not significant, the intramuscular phospholipids undergo significant changes [9]. As the main flavor precursors,intramuscular phospholipids generate polyunsaturated fatty acids(PUFA), which are mainly composed of linolenic acid, linoleic acid and arachidonic acid, through hydrolysis by lipase and microbial activity [10,11]. The unstable double bonds are easy to oxidize,and produce a series of oxidation products including aldehydes,ketones, and alcohols [12]. Detecting the oxidation products at the primary and the secondary stage are the most common methods to evaluate the degree of lipid oxidation [13,14]. The primary lipid oxidation products are usually evaluated by peroxide value (POV) and iodine method [15]. Thiobarbituric acid reaction substance (TBARS)is mainly used to evaluate the degree of lipid secondary oxidation,which mainly reflects the amount of malondialdehyde compounds formed by lipid oxidation, so it is used to evaluate the degree of food oxidation and deterioration [16].

Lipid oxidation of Chinese dry sausage can be affected by some external technological factors, such as processing temperature,time and NaCl content, however, the mechanism of flavor formation in Chinese dry sausage still needs to be elucidated. NaCl is an important component in meat processing, especially for cured meat. NaCl endows good flavor to products, inhibits the growth of microorganisms, promotes the lipid oxidation, and affects the activity of lipoxygenase and lipid hydrolytic enzymes, which correlate with the concentration of sodium chloride [17]. Research studying the effect of different NaCl contents on lipid hydrolysis have different opinions, while some believed that the increase of NaCl content can promote lipid hydrolysis, others found that the effect of NaCl content was not significant [18]. Excessive intake of sodium is suggested to induce hypertension and other cardiovascular diseases [19], thus, it is necessary to examine the effect of NaCl content on lipid oxidation in Chinese dry sausage. However, the influence mechanism of the NaCl content on protein oxidation, fat oxidation and hydrolysis has not been explored.

In this study, the effect of NaCl content on protein oxidation,lipid hydrolysis and oxidation, and the relationship among them during the processing of Chinese dry sausage were examined. It would contribute to the mechanisms of regulating protein and lipid changes by NaCl content and improve the quality of dry sausage products.

2. Material and methods

2.1. Chemicals and reagents

Pork meat was purchased from Beijing Ershang Dahongmeng Meat Food Co., Ltd. NaCl, MgCl2, HCl, ethanol, ethyl acetate,Na2SO4were provided by Sinopharm Chemical Reagent Co., Ltd.Ethylenediaminetetraacetic acid (EDTA), bovine serum protein,Tris, guanidine hydrochloride, glycol-bis-(2-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA) were obtained by Sigma (St. Louis,USA).

2.2. Processing of Chinese dry sausage and sampling

Two different groups of dry sausage, 2% (LS) and 4% (m/m)(HS), were manufactured twice according to traditional techniques in February and July 2018 respectively. The two groups of dry sausage were independently produced with the same ingredients and methods.

The basic Chinese dry sausage mixture included: pork lean meat(80%), pork back fat (20%), sucrose (8 g/kg), glucose (5 g/kg), sodium nitrite (0.05 g/kg), liquor (20 g/kg), soy (20 g/kg), Chinese 5-spice(1 g/kg), sodium ascorbate (2 g/kg), and 2% (LS) or 4% (HS) NaCl. The pork lean meat and back fat were ground into 10- and 5-mm diameter mince with meat grinder separately, and mixed with other ingredients within 5 min under vacuum. The mixed stuffing was kept at 4°C for 24 h and filled into natural hog casings of 30-mm diameter, made into uniform fragments of 20-cm length. The dry sausages were fermented for 2 days at 20°C with 80%–85% relative humidity, then transferred into a drying chamber with 70%–75% relative humidity for 10 days at 10°C. Samples were taken at raw meat, cured meat, fermented for 5 days, and dried for 5 days and 10 days, respectively.

2.3. Protein oxidation determination

2.3.1. Preparation of myofibril protein

Myofibril protein was isolated as described by Xiong et al. [20].10 g of dry sausage was homogenized with 4 vol (m/V) 10 mmol/L phosphate buffer (pH 7.0), containing 0.1 mol/L NaCl, 2 mmol/L MgCl2, 1 mmol/L EGTA, centrifugated at 2000 g for 15 min, Myofibril protein was washed 3 times with 4 vol of the same phosphate buffer, until the washing liquid was clear. Myofibril protein suspension was adjusted to pH 6.0 in the last time of washing. The precipitates were dissolved in the 10 mmol/L phosphate buffer (pH 6.0), containing 0.6 mol/L NaCl, centrifuged at 11000 g for 10 min,the myofibril protein was collected for analyzing immediately.

2.3.2. Protein carbonyls

Protein carbonyls in dry sausages were measured following the procedure by Levine et al. [21]. Aliquots of 100 μL of myofibrillar protein samples reacted with 400 μL of DNPH in 2 N HCl in a micro centrifuge tube and 100 μL of myofibrillar protein samples reacted with 400 μL 2 N HCl (blank) for 1 h at room temperature.Then added 0.5 mL ice cold 20% trichloroacetic acid (TCA) and centrifuged, the supernatant was discarded. Excess DNPH was washed 3 times with 1 mL of ethanol:ethyl acetate (1:1, V/V). The precipitates were dissolved in 1 mL of 6.0 mol/L guanidine hydrochloride(pH 6.5). The absorbance at 370 nm and 280 nm was recorded. The carbonyl content was calculated at 370 nm using an absorption coefficient of 22000 L/mol·cm. The protein content was determined by Lowry method. Carbonyl contents were expressed as nmol/L carbonyl/mg protein.

2.3.3. Protein sulfhydryls

Total free-sulfhydryl groups were determined by reacting with DTNB as described by Ellman [22]. Three aliquots of 1.0 mL myofibrillar protein sample mixed with 4.0 mL urea-SDS solution (pH 7.4), containing 8.0 mol/L urea, 3% SDS, 0.1 mol/L Tris-HCl buffer,then incubated with 1.0 mL DTNB reagent (10 mmol/L DTNB in 100 mmol/L Tris-HCl buffer, pH 7.4) at 25°C for 25 min, the absorbance at 280 nm was recorded. The protein content was determined by Lowry method. Sulfhydryl content was calculated using a molar absorptivity of 11400 L/mol·cm, and expressed as μmol/L sulfhydryl/g protein.

2.4. Determination of neutral lipid, free fatty acids, and phosphatide

Neutral lipid, free fatty acids and phosphatide contents in dry sausages were measured following the procedure by García et al. [23]. Nearly 30–60 mg fat was dissolved in 5 mL 2:1 (V/V)chloroform-methanol solution, and separated with 100 mg of solid phase extraction (SPE) column. The neutral lipid, free fatty acids,and phosphatide were eluted separately using 5 mL chloroformisopropanol solution (2:1, V/V), 5 mL acetic acid-ether solution (2%,m/m), and methanol-HCl solution (9:1, V/V) separately, collected in centrifuge tube separately, dried with nitrogen and weighed.

2.5. Lipase activity determination

2.5.1. Neutral lipase activity

Neutral lipase activity in dry sausages was assayed following the procedure by Motilva et al. [24]. 5 g sample was homogenized with 25 mL 50 mmol/L phosphate buffer (pH 7.5), containing 5 mmol/L EGTA, and stirred for 15 min uniformly at 0°C, centrifuged at 10000 g for 15 min at 4°C, filtered with medium speed filter paper, and the filtrate was collected. The final volume was adjusted to 25 mL with 50 mmol/L phosphate buffer. The samples were taken for crude lipases analysis. Protein content was measured by biuret method. A 100 μL aliquot of crude lipase solution was mixed with 2.8 mL 0.22 mol/L Tris/HCl buffer containing 0.5 g/L (m/V) TritonX-100 (pH 7.5), and 100 μL 1.0 mmol/L 4-methylumbelliferyl-oleate was added as the enzyme substrate. Samples were incubated at 37°C for 30 min and cooled immediately. Fluorescence was detected by fluorescence spectrophotometer at excitation andemission wavelengths 350 and 445 nm, respectively.

2.5.2. Acid lipase activity

Acid lipase activity in dry sausages was measured following the procedure by Vestergaard, Schivazappa & Virgili [25]. A 100 μL aliquot of crude lipase solution was mixed with 2.8 mL 0.1 mol/L phosphate buffer (pH 7.5) 0.8 mg/mL bovine serum protein (BSA)solution and 0.5 g/L (m/V) TritonX-100, then 500 μL 1.0 mmol/L 4-methylumbelliferyl-oleate was added as the enzyme substrate.Samples were incubated at 37°C for 30 min, and the reaction was stopped immediately with 0.5 mL 1 N HCl solution. Fluorescence was detected by fluorescence spectrophotometer, with the excitation and emission wavelengths at 350 nm and 445 nm, respectively.

2.5.3. Phospholipase activity

Phospholipase activity in dry sausages was determined according to Motilva et al. [26]. A 100 μL aliquot of crude lipase solution was mixed with 2.8 mL 0.1 mol/L phosphate buffer (pH7.5),50 mmol/L sodium fluoride, 0.5 g/L TritonX-100 and 0.8 mg/mL BSA solution, then 500 μL 1.0 mmol/L 4-methylumbelliferyl-oleate was added as the enzyme substrate. Samples were incubated at 37°C for 30 min, and the reaction was stopped immediately with 0.5 mL 1 N HCl solution. Fluorescence was detected by fluorescence spectrophotometer, with the excitation and emission wavelengths at 350 nm and 445 nm, respectively.

One unit of activity (U) was defined as the amount of enzyme required to hydrolyze 1 nmol of 4-methylumbelliferone in 1 h at 37°C.

2.6. Determination peroxide value

Peroxide levels in dry sausages were measured following the procedure by Gulgun et al. [27] with little modifications. Exhaustive mashing and mixing of 30 g samples were carried out in 90 mL ether, extracted for 12 h at room temperature, filtered with double filter paper containing moderate Na2SO4.Ether was removed from filtrate completely at 40°C for further analysis. The 2.0 g filtrate was mixed with 30 mL chloroform-acetic acid solution (2:1,V/V) in 250 mL flask, and 1.0 mL saturated KI solution was added,well-distributed and kept in dark for 3 min. Then, 100 mL purified water was added, shaken and titrated with 0.01 mol/L sodium thiosulfate standard solution immediately until the colour turned yellow. Next, 1.0 mL starch-iodide indicator was added and the sample turned blue; the titration was continued until the blue colour disappeared which was the end point of titration. The peroxide value (POV) was calculated as follows: POV (meq/kg) = (V2-V1) ×0.002 × 0.1269 × 100/m, V1is sodium thiosulfate standard titration solution volume titrated by the blank sample (mL), V2is sodium thiosulfate standard titration solution volume titrated by test sample (mL), m is the weight of the test sample (g).

2.7. Analysis of thiobarbituric acid reactive substances value(TBARS)

TBARS values in dry sausages were measured following the procedure by Salih et al. [28] with little modifications. Exactly 5.0 g samples (accurate to 0.01 g) were mixed with 15.0 mL of 20 g/mL trichloroacetic acid solution and homogenized at 10000 r/min for 3 min in an ice bath, deproteinized for 2 h, centrifuged at 2000 g for 10 min at 4°C, the supernatant was filtered with double slow filter paper, and diluted to 50 mL with double-distilled water. A 5.0 mL aliquot of the filtrate was mixed with 5.0 mL 0.02 mol/L 2-thiobarbituric acid solution, allowed to react at 90°C for 30 min,cooled to room temperature immediately, and absorbance was read at 532 nm. Results were expressed as mg malondialdehyde(MDA)/kg sample.

2.8. Lipoxygenase (LOX) enzyme activity determination

LOX activity in dry sausages was measured following the procedure by Gata at al. [29] with little modifications. Exactly 5.0 g samples were weighed and homogenized with 3 vol (m/V)50 mmol/L phosphate buffer (pH 7.0), containing 1.0 mmol/L dithiothreitol, 1.0 mmol/L EDTA at 10000 r/min in an ice bath, and stirred for 30 min uniformly at 0°C. This was then centrifuged at 15000 g for 60 min, the supernatant was filtered with fast filter paper, and the filtrate was collected for analysis. The concentration of protein was detected by the biuret method. The main component of LOX activity analysis, linoleic acid, was prepared as follows: 140 mg linoleic acid was transferred into a 15.0 mL flask and mixed with 5.0 mL deoxygenated double-distilled water containing 180 μL Tween 20, and the pH was adjusted to 9.0 with 2 mol/L NaOH, and diluted to 50 mL with deoxygenated doubledistilled water and kept in a nitrogen environment for further analysis. Then, 200 μL of linoleic acid substrate was mixed with 2.8 mL 50 mmol/L citrate buffer solution (pH 5.5) at 20°C for 1 min,and the absorbance was read at 234 nm immediately, the reading was repeated after 1 min. Then, 100 μL enzyme was added and the absorbance at 234 nm was read again after 1 min. One unit of activity (U) was de fined as increase in absorbance by 1 unit per gram enzyme protein per min at 234 nm.

2.9. Statistical analysis

SPSS 17.0 was used for the majority of data processing. The one-way analyses of variance (ANOVA) was used to determine the significant differences and the P < 0.05 was considered as significant level. Data were presented as means ± standard deviations(SD) of triplicate determinations. The correlation of parameters was evaluated by Pearson correlation.

3. Results and discussion

3.1. Changes in protein oxidation during processing

3.1.1. Changes in protein carbonyls during processing

The formation of protein carbonyls was mainly through three pathways, metal catalytic oxidation, non-enzymatic glycation and complexation of other substances with protein [6]. Metal ions were present in meat naturally, such as Fe2+, which can stimulate the generation of oxygen radicals from oxygen and H2O2, as a free radical chain reaction. The reaction was slow at the beginning, because the activation energy of chain initiation reaction was higher, and then protein oxidation reacted rapidly, carbonyls were produced in the side chain of arginine, lysine, proline and threonine.Adducts which formed by lipid oxidation products and proteins can also form protein carbonylation, such as malondialdehyde and 4-hydroxynonenal [30]. Protein oxidation resulted in changes in protein properties, structures and functions, secondary and tertiary structures of myofibrils proteins, and loss of the solubility and hydrophobicity.

Protein oxidation was an important reaction during processing, and the formation of carbonyl is a significant indicator which mainly produced by fracturing of amino acid side chain [31]. The carbonyl contents of myofibril proteins during processing of Chinese dry sausage were shown in Fig. 1. The carbonyl contents significantly increased (P < 0.05) at different periods. The carbonyl content was only 2.59 nmol/L carbonyls/mg protein in raw meat,close to the reported on dry fermented sausage [6], and showed no significant change after cured both in LS group and HS group. Fermentation was an important procedure for Chinese dry sausage,and the carbonyl content increased rapidly in follow stages in two groups. The carbonyl content reached 28.64 nmol/L carbonyls/mg protein after dried 10 days in HS group, while the value of LS group was only 22.64 nmol/L carbonyls/mg protein, showing the significant lower level (P < 0.05) compared with HS group.

Fig. 1. Changes in protein carbonyls during Chinese dry sausage processing. For the letters A-D, mean values (n = 3) with different letters within the same row are significantly different in different processes. Identical letters in the same row indicate that there is no significant difference in different processes (P < 0.05). For the letters a-b, mean values (n = 3) with different letters within the same column are significantly different (P < 0.05).

3.1.2. Changes in sulfhydryls during processing

The loss of protein sulfhydryls was another important indication which was widely used to measure the degree of protein oxidation in meat products, because cysteine residues showed high susceptibility to oxidation and formed intermolecular disulfide bridges. The sulfhydryl contents during processing of Chinese dry sausage were shown in Fig. 2. Sulfhydryl content of myofibril protein decreased significantly (P < 0.05) at different periods, the protein sulfhydryl content in raw meat was 53.69 μmol/L sulfhydryl/g protein, and showed consistent with other results which studied on Chinesestyle sausage [32]. The sulfhydryls contents of myofibril proteins showed significant different after cured period between HS and LS groups. The sulfhydryls content was only 30.08 μmol/L sulfhydryl/g protein after dried for 10 days in HS group, and significant lower(P < 0.05) than the value of LS group which was 33.03 μmol/L sulfhydryl/g protein.

Fig. 2. Changes in protein sulfhydryls during Chinese dry sausage processing. For the letters A-E, mean values (n = 3) with different letters within the same row are significantly different in different processes. Identical letters in the same row indicate that there is no significant difference in different processes (P < 0.05). For the letters a-b, mean values (n = 3) with different letters within the same column are significantly different (P < 0.05).

3.2. Changes in neutral lipid, free fatty acids, and phosphatide during processing

Lipid oxidation and hydrolysis occurred during the processing and produced new substances, such as neutral lipids, free fatty acids, and phosphatide. The flavor substances in dry sausages mostly came from the lipid oxidation and hydrolysis. The changes of neutral lipid, free fatty acids, and phosphatide could reveal the degree of lipid oxidation and hydrolysis, and affect flavor quality significantly. The neutral lipid contents of dry sausages at different processing periods were shown in Table 1. During the processing, the content of neutral lipids gradually increased and changed markedly (P < 0.05) compared with raw meat in both two groups,which was very similar to the result of dry-cured ham [33]. Compared with the HS group, neutral lipid content was lower in the LS group during the whole processing, indicating that the amount of NaCl affected the content of neutral fat.

Free fatty acids were pivotal products of lipid hydrolysis and oxidation, and their content mainly depended on their own formation and decomposition rate. Phospholipid hydrolysis was also an important way to produce fatty acid. The free fatty acids content at different processing periods were presented in Table 1. The free fatty acids content markedly changed (P < 0.05) during processing, and indicated a faster rate of formation of total free fatty acids than their rate of decomposition. The free fatty acids level of raw meat was 11.25 g/100 g, and finally reached to 22.37 g/100 g rapidly in the LS group. Compared with LS group samples, free fatty acids content exhibited faster changes in the HS group during the whole processing, and finally reached to 26.58 g/100 g.Additionally, there was a significant difference (P < 0.05) in free fatty acids content between the two groups in the same processing.

The phosphatide content from raw meat and sausages at different processing periods were presented in Table 1. The phosphatide content changed noticeably (P < 0.05) during processing. The phosphatide level of raw meat in the LS group decreased quickly from 35.52 g/100 g to 25.88 g/100 g after curing, and finally reached to 15.27 g/100 g after drying for 10 days. The free fatty acids content exhibited faster reduction during the whole processing in HS group compared with LS group samples and reached to 14.00 g/100 g after drying for 10 days in the LS group. During the whole processing, phosphatide content was also significantly different (P < 0.05) between two groups in the same processing,and the result is in agreement with the report of Nevena et al.[34].

Table 1Changes in neutral lipid, free fatty acids and phosphatide content in Chinese dry sausage during processing

Results in Table 1 showed the most significant changes in the curing stage. The free fatty acids increased significantly and provided a large amount of substrates for lipid hydrolysis, which were oxidized to secondary oxidation products in further stages. The neutral lipid in muscle was mainly a relatively stable, monounsaturated fatty acid, but can be hydrolysed by the acidic lipase, thus, this explained the cause for minor fluctuations in the neutral lipid levels here. Phospholipids were the main meat flavor precursors, mainly consist of polyunsaturated fatty acids and saturated fatty acid that were easy to oxidize. Phospholipids were also an important component of cell membrane, thus, being more vulnerable to intracellular lipid hydrolase [35]. This explained the significant decrease in the content of phospholipids.

Table 2The correlation between protein oxidation among lipid oxidation and lipolysis. Note: * represents significantly different values at P < 0.05; ** represents significantly different at P < 0.01.

3.3. Changes in lipase activity during processing

3.3.1. Changes in neutral lipase activity during processing

The hydrolysis of glycerol ester was carried out mainly by neutral lipases. The changes in neutral lipase activity during Chinese dry sausage processing were summarized in Fig. 3. The neutral lipase activity showed significant changes (P < 0.05) during processing, and reached the maximal value after the curing stage, and decreased fast during later stages in both groups. The neutral lipase activity was 5.66 and 7.48 U/g protein in LS and HS groups in curing stage respectively. The samples in the LS group showed significantly lower activity than the samples in the HS group during whole processing at the same stage (P < 0.05), indicated that the amount of NaCl affected the content of neutral lipase activity, and appropriate NaCl concentration contributed towards the increase in the activity of neutral lipase. The results were consistent with those of Vestergaard et al. [25] and Jin et al. [36], who carried out similar assays on the ham and bacon.

3.3.2. Changes in acid lipase activity during processing

The activity of acid lipase during Chinese dry sausage processing was summarized in Fig. 4. The level of neutral lipase activity showed significant changes (P < 0.05) during processing, reached the maximal value after curing stage, and decreased fast during later stages in both groups. The trend was similar to that of the changes in neutral lipase activity. The neutral lipase activity was 4.50 and 8.39 U/g protein in LS group and HS group in cured stage respectively,and were significantly different throughout the processing at same stages (P < 0.05). The results were similar to that of other research results [37], thus, this indicated that appropriate NaCl concentration contributes to increased acid lipase activity, and accelerated speed of lipid and hydrolysis.

Fig. 4. Changes in acid lipase activity during Chinese dry sausage processing. For the letters A-E, mean values (n = 3) with different letters within the same row are significantly different in different processes. Identical letters in the same row indicate that there is no significant difference in different process (P < 0.05). For the letters a-b, mean values(n = 3) with different letters within the same column are significantly different (P < 0.05).

3.3.3. Changes in phospholipase activity during processing

The activity of phospholipase was different from that of lipase,showed poor stability during processing, and greater vulnerability to external factors. The changes in phospholipase activity during Chinese dry sausage processing were summarized in Fig. 5.The level of phospholipase activity showed significant changes(P < 0.05) during processing, and reached the maximal value after drying for 5 days, and decreased fast in later stages in both groups.The phospholipase activity was 3.05 and 3.50 U/g protein in LS group and HS group, respectively, after drying for 5 days, thus indicating that appropriate NaCl concentration contributed to increase in the activity of phospholipase, which was the main endogenous enzyme for free fatty acid formation. The changes in phospholipase activity during processing in two groups correlated with the changes in neutral lipids, free fatty acids, and phosphatide content. The results were in agreement with the results of Muriel ea al. [38] and Motilva et al. [24], but contrary to the results of Jin et al. [36]. The changes in phospholipase activity thus re flect the corresponding changes in free fatty acids and phospholipids contents.

Fig. 5. Changes in phospholipase activity during Chinese dry sausage processing.For the letters A-D, mean values (n = 3) with different letters within the same row are significantly different in different processes. Identical letters in the same row indicate that there is no significant difference in different processes (P < 0.05). For the letters a-b, mean values (n = 3) with different letters within the same column were significantly different (P < 0.05).

The results showed a promoting effect of NaCl on lipase activity,probably because high content of NaCl accelerated fatty acid oxidation. As a protein, an enzyme was easier to dissolve in a certain concentration of NaCl solution than in pure water. Due to diffusional differences, the content of NaCl in muscle was higher than in adipose tissue, but it still may have some beneficial influence on the endogenous enzyme system. Activation of lipase activity by NaCl may also be attributed to water content, water activity and pH value [37].

3.4. Changes in peroxide values during processing

Lipid oxidation was a free radical chain reaction that occurs along the processing. The product of initial oxidation was peroxide, which was very unstable, easily oxidized and produces some small harmful compounds, such as aldehydes, ketones, and so on.Thus, peroxide value was mainly used to evaluate the amount of hydroperoxide produced by lipid oxidation. If the rate of peroxide production was less than their decomposition, peroxide value began to decrease. Generally, peroxide values raise to a certain level and then declines gradually, making the lipid be more harmful.

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The changes in peroxide values during Chinese dry sausage processing were summarized in Fig. 6. The peroxide value brought out violent fluctuations throughout the entire processing and showed an initial rising trend, followed by a decreasing trend later. The peroxide values showed significant changes (P < 0.05) during processing, reached the maximum after fermentation, and decreased fast in the later stages in both groups. The peroxide values were 0.06 and 0.08 meq/kg in LS group and HS group, respectively, after fermentation, indicated that appropriate NaCl concentration promotes formation peroxide formation, leading to increase in the peroxide values, and accelerates the speed of lipid oxidation. There-fore, the peroxide values in HS group remained significantly higher than that in the LS group (P < 0.05).

Fig. 6. Changes in peroxide values during Chinese dry sausage processing. For the letters A-E, mean values (n = 3) with different letters within the same row are significantly different in different processes. Identical letters in the same row indicate that there is no significant difference in different processes (P < 0.05). For the letters a-b, mean values (n = 3) with different letters within the same column are significantly different (P < 0.05).

3.5. Changes in TBARS values during processing

TBARS values evaluated the amount of aldehydes produced by lipids second oxidation and the representative compound was malondialdehyde (MDA). TBARS values also reflected the degree of lipid oxidation [39,40]. The changes in TBARS values during Chinese dry sausage processing were summarized in Fig. 7. The TBARS values brought out the trend which showed an initial rise and decreased later and showed insignificant changes during curing, fermentation and drying for 5 days. TBARS values also showed a similar trend as that of peroxide values, and remained significantly different at same stage between two groups during the whole processing (P <0.05).

Fig. 7. Changes in TBARS values during Chinese dry sausage processing. For the letters A-C, mean values (n = 3) with different letters within the same rowindicate significantly different in different processes. Identical letters in the same row indicate that there is no significant difference in different processes (P < 0.05). For the letters a-b, mean values (n = 3) with different letters within the same column are significantly different (P < 0.05).

Fig. 8. Changes in LOX activity during Chinese dry sausage processing.For the letters A-E, mean values (n = 3) with different letters within the same row are significantly different in different processes. Identical letters in the same row indicate that there is no significant difference in different processes (P < 0.05). For the letters a-b, mean values (n = 3) with different letters within the same column are significantly different (P < 0.05).

The TBARS values showed significant changes (P < 0.05) during processing, reached the maximum value after fermentation, and decreased fast in the later stage in both groups. Normally, the TBARS values reached maximum level later than the maximum level of peroxide if it just occured from the lipid oxidation, because the substances they tested on were different. However, the maximum TBARS and peroxide values appeared at the same time, probably because malondialdehyde could react with free amino acids and other small molecular product as bifunctional group compound during processing [41,42]. Therefore, the TBARS values showed a declining trend during the later stages. The TBARS values from raw meat and sausages at different processing periods were shown in Fig. 5. The TBARS values reached the maximum level of 0.27 and 0.32 mg/100 g in LS and HS groups, respectively, after fermentation.The result agreed with those of Andrés et al. [33] who reported significantly higher TBARS values in the HS group compared with that in LS group (P < 0.05).

3.6. Changes in LOX activity during processing

The LOX activity from raw meat and sausages at different processing periods were presented in Fig. 8. The changes of LOX activity showed similarity to the TBARS values. The results were in accordance with the reports of Fu et al. [43]. The LOX activity in cured meat showed the maximum activity, then decreased rapidly,although the levels were far more in the samples in raw meat.This can be mainly attributed to the structure of LOX, which was a nonheme-iron protein. The catalytic activity of LOX was closely related to the form in which the iron ions existed. LOX was inactive when iron ions was Fe2+[44,45] and got activated when Fe2+was transformed into Fe3+. In raw meat, the iron was present mainly in Fe2+form, rendering LOX in an inactive state, with lower activity.When the hydroperoxides were formed and Fe2+got transformed into Fe3+during curing process, LOX was rapidly activated significantly. The sulfhydryl (SH-groups) can stabilize LOX activity, but hydrogen peroxide oxidized the SH-groups, which leaded to LOX inactivation [46].

3.7. Correlation analysis

The correlation among protein oxidation, lipid oxidation and lipolysis were explored by the Pearson correlation analysis. The results showed that protein oxidation had strong correlation with lipid oxidation and lipolysis. As we know that protein carbonyl and sulfhydryl were the main indicators of protein oxidation, neutral lipid content, free fatty acids content, phosphatide content, peroxide value, and TBARS value can reflect the degree of lipid oxidation and lipolysis. In Table 2, protein carbonyl content showed a significant positive correlation with neutral lipid content, free fatty acids content (P < 0.01) and TBARS value (P < 0.05), and showed a significant negative correlation correlated with sulfhydryl content and phosphatide content (P < 0.01). Sulfhydryl content was positively correlated with phosphatide content (P < 0.01), and negatively correlated with protein carbonyl content, neutral lipid content, free fatty acids content, TBARS value (P < 0.01). It demonstrated that the higher level of protein oxidation, the more contents of neutral lipid content, free fatty acids content and TBARS value were detected.We also found that neutral lipid content, free fatty acids content,phosphatide content, neutral lipase activity, Acid lipase activity,phospholipase activity peroxide value, TBARS value, showed significant correlations with each other (P < 0.05), indicated that there was strong correlation between lipid oxidation and lipid hydrolysis.

4. Conclusion

Declaration of Competing Interest

The authors declare that they have no conflict of interest.

Authorship

Bing Zhao, designed the study, performed the experimental work and analyzed the data. Huimin Zhou contributed in the experiments of Lipase activity test. Shunliang Zhang designed the study,Xiaoqian Pan and Su Li contributed towards data analysis, Shouwei Wang, Xiaoling Qiao and Whenhua Chen directed the experiments.All revised the manuscript and approved the final version.

Acknowledgments

This study was financially supported by National Key R&D Program of China (grant No. 2017YFD0400105) , Natural Science Foundation of Beijing Municipality (grant No. 6192009) and Fengtai science and technology new star (grant No. KJXX201902) .