Protein oxidation and in vitro digestibility of heat-treated fermented sausages: How do they change with the effect of lipid formulation during processing?

J Food Biochem. 2019;43:e13007. wileyonlinelibrary.com/journal/jfbc  

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© 2019 Wiley Periodicals, Inc. Received: 23 May 2019 

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DOI: 10.1111/jfbc.13007

F U L L A R T I C L E

Protein oxidation and in vitro digestibility of heat‐treated

fermented sausages: How do they change with the effect of

lipid formulation during processing?

Burcu Öztürk‐Kerimoğlu

 | Berker Nacak

 | Vasfiye Hazal Özyurt

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Meltem Serdaroğlu

1_{Engineering Faculty, Food Engineering} Department, Ege University, Izmir, Turkey 2_{Engineering Faculty, Food Engineering} Department, Uşak University, Uşak, Turkey 3_{Engineering Faculty, Food Engineering} Department, Near East University, Lefkoşa, Turkey

Correspondence

Meltem Serdaroğlu, Engineering Faculty, Food Engineering Department, Ege University, 35100 Bornova, Izmir, Turkey. Email: meltem.serdaroglu@ege.edu.tr Funding information

The Scientific and Technical Research Council of Turkey (TÜBİTAK), Grant/Award Number: 214-O-181

Abstract

In the present work, it was aimed to evaluate the oxidative stress indicators and in vitro digestibility during the processing of heat‐treated Turkish sausages (sucuk) manufactured using different lipid formulations. The utilization of olive oil in sausage formulations had considerable impacts on proximate composition, pH, and water activity. The increased olive oil content increased primary lipid oxidation products, whereas it decreased the secondary ones. The use of olive oil increased the total car-bonyl content, while it decreased the α-aminoadipic semialdehyde concentration. In general, pepsin, trypsin, and α-chymotrypsin activities of the treatments were simi-lar to each other. The heat treatment during the processing significantly increased most of the oxidation markers. Though strong correlations were recorded between specific oxidation markers, no relationship was detected between oxidation param-eters and in vitro digestibility. The results indicated that the lipid formulation and processing operations had significant impact on chemical and functional properties of heat-treated fermented sausages, within the complex interrelationships between oxidation mechanisms.

Practical applications

The present work pointed out the changes and correlations between specific oxi-dation markers and in vitro digestibility of heat-treated fermented sausages during the production procedure. Oxidation reactions that occur in both proteins and lipids could have drastic impact on overall quality; for this reason, it is of great importance to provide the data for lightening these impacts regarding the product types and production applications. Since heat-treated muscle foods are widely manufactured to meet industrial needs, the data obtained from this research would contribute to understand the effects of formulation and processing operations in the formation of oxidation products and change in digestibility, thereby to pioneer further research on this topic.

K E Y W O R D S

heat-treated Turkish sausage, in vitro digestibility, olive oil, protein oxidation, sucuk, α‐aminoadipic semialdehyde (AAS), γ‐glutamic semialdehyde (GGS)

1 | INTRODUCTION

Traditional food products are considerable determinants of cultural heritage. Since Turkey has a deep-rooted history leading to the development of its rich culinary culture, many types of traditional and regional meat products are manufactured all over the country. Sucuk is one of the most consumed dry fermented Turkish meat products that is mainly produced from beef, water buffalo, and/or mutton meat (Kaban, 2013). The conventional production of sucuk briefly covers: (a) mixing meat with fat, curing agents (salt, nitrite, saccharose, etc.), spices (black pepper, red pepper, cumin, allspice, garlic, etc.), and other ingredients, (b) stuffing the dough into the casings, and (c) fermentation‐ripening processes until a dry or semi‐ dry product is obtained (Bilenler, Karabulut, & Candogan, 2017; Ercoşkun, Tağı, & Ertaş, 2010; Kaban, 2013). Moreover, due to some commercial reasons, such as shortening the fermentation and drying processes (Kaban, 2013), recently some modifications in the manu-facture procedures have been suggested. The heat treatment has become one of the most common modifications which provides the elimination of foodborne pathogen bacteria, extending shelf life, shortening the production time, and reduction of costs (Bilenler et al., 2017; Ercoşkun et al., 2010). Though heat‐treated sucuk is also a fermented meat product like the conventional one, some quality problems in terms of physical, chemical, and sensory parameters could occur especially in connection with the significant reduction of desirable microbial flora (Bilenler et al., 2017; Dalmış & Soyer, 2008), because of the “light” fermentation and ripening period. Moreover, the heat treatment during the processing could promote reactive oxygen species (ROS) and increase the susceptibility of lipid and pro-tein oxidation reactions (Traore et al., 2012).

Lipid and protein oxidation phenomena in muscle foods have been mentioned as the main reasons for the quality deterioration behind the nonmicrobial degradation (Falowo, Fayemi, & Muchenje, 2014; Guyon, Meynier, & de Lamballerie, 2016). For a long time, the studies investigating oxidative mechanisms in meat and meat prod-ucts mainly focused on the lipid oxidation that was considered as the main reaction that impairs the quality of muscle foods (Guyon et al., 2016); despite the fact that the protein oxidation could also lead serious changes in the quality.

In recent years, the protein oxidation of muscle foods has be-come a “trend topic” since the alterations in the proteins could cause many types of quality problems. Pioneer studies on the protein oxi- dation of food science were started with Xiong (2000), who discov-ered the sensibility of meat proteins to oxidative stress, leading to negative effects on the functionality of proteins and eating quality of meats (Estévez & Luna, 2017). Protein oxidation of meat and meat products can be generated directly through ROS or indirectly by sec-ondary products of oxidative stress that modify the amino acid side chains or attack the polypeptide backbone of the protein, leading to the structural changes in protein's primary, secondary, and tertiary structures (Soladoye, Juárez, Aalhus, Shand, & Estévez, 2015; Zhang, Xiao, & Ahn, 2013). These structural modifications could further af-fect functional properties of proteins (solubility, proteolytic activity,

digestibility, gelation, emulsification, and water holding capacity, etc.) which results in decreased amino acid bioavailability and nutri-tional value, besides the formation of potential toxic peptides poses a risk to food safety (Estévez, 2011; Falowo et al., 2014; Zhang et al., 2013). Since the protein oxidation of muscle foods is mentioned to be interconnected with protein degradation and thereby the di-gestibility of proteins by the effect of various mechanisms (Gan et al., 2019; Santé‐Lhoutellier, Engel, Aubry, & Gatellier, 2008), the re-lationship between the oxidation markers and digestibility is an an-other question to be answered.

Recently the demand for low-fat foods has been continuously rising due to the new trends toward a healthier diet. Since muscle foods are one of the richest sources of dietary fats, the substitution of animal fat with functional oils has long been used as an efficient solution for the manufacture of meat products with a healthier lipid profile and improved nutritional quality to meet consumer demands. Of vegetable oils, olive oil has long been considered as one the most important sources of monounsaturated fatty acids that have nu-merous benefits for health (Lurueña‐Martínez, Vivar‐Quintana, & Revilla, 2004), besides it contains a considerable amount of tocoph-erols and phenolic matters acting as antioxidants (Kayaardı & Gök, 2003). However, the effects of the replacement of animal fats by olive oil on some quality characteristics of different fermented meat products have been investigated (Ansorena & Astiasarán, 2004; Kayaardı & Gök, 2003; Muguerza, Ansorena, Bloukas, & Astiasarán, 2003; Muguerza, Gimeno, Ansorena, Bloukas, & Astiasarán, 2001; Utrilla, García Ruiz, & Soriano, 2014), yet no research has addressed protein–lipid oxidation products and digestibility of heat-treated sausages in which beef fat is replaced with olive oil. The hypothe-sis of this study was the modified lipid formulation and processing factors during the heat-treated fermented sausage production that could alter the oxidative stability and hence affect the bioavailabil-ity and other qualbioavailabil-ity markers. Within the light of the data stated above, the aim of this work was to evaluate the protein and lipid oxidation-specific products and in vitro protein digestibility during the processing of heat‐treated Turkish fermented sausages (sucuk) manufactured using different lipid formulations.

2 | MATERIALS AND METHODS

2.1 | Material

The production procedure was handled by the Meat Pilot Plant of Ege University Food Engineering Department (Izmir, Turkey). Postrigor boneless beef (Musculus semitendinosus) and beef fat were supplied from Tesco‐Kipa Co. (Izmir, Turkey). Extra virgin olive oil was purchased from Tariş Co. (Izmir, Turkey), having totally 75.30% monounsaturated fatty acids (MUFAs), 14.40% saturated fatty acids (SFAs), and 10.30% polyunsaturated fatty acids (PUFAs). The lyophi-lized commercial starter culture mixture consisting of Pediococcus acidilactici, Lactobacillus plantarum, and Staphylococcus carnosus was donated by Frutarom Co. (Istanbul, Turkey). Natural casings (D:36 mm, bovine small intestine) and the other compounds were

purchased from the local market of Izmir. All the chemicals used were of analytical grade except the HPLC-grade methanol. Pepsin from porcine gastric mucosa (product number P6887, lyophilized powder, 3200–4500 units/mg protein), α-amylase from porcine pan-creas (product number A3176, type VI‐B, ≥5 units/mg solid), and trypsin from porcine pancreas (product number 7409, lyophilized powder, Type II‐S, 1,000–2,000 units/mg dry solid) were purchased from the Sigma‐Aldrich Company Ltd (Dorset, UK) for the use in the analysis of in vitro protein digestibility. The Zeneer Power I Water Purification System (Human Corp., Songpa‐Gu, Seoul, South Korea) was used for the purification of water.

2.2 | Experimental design

Three different heat-treated fermented sausage formulations were prepared in two batches as follows: The first treatment was the con-trol (C) that was prepared with beef fat as the lipid source. The other two treatments were prepared by replacing 15% (O15) and 30% (O30) of beef fat with olive oil, respectively. Thereby, the lipid for- mulation of C group consisted of 100% beef fat, whilst the lipid for-mulations of O15 and O30 groups consisted of 85% beef fat + 15% olive oil and 70% beef fat + 30% olive oil, respectively.

2.3 | Production of heat‐treated 

fermented sausages

The heat-treated fermented sausage production was carried out according to Zungur, Serdaroğlu, Nacak, and Öztürk (2016). After trimming visible fat and connective tissue of the meat, meat and beef fat were separately minced using a meat grinder (Arnica AA 1,295 Promeat Grande, Turkey) through a 3 mm plate. The treat- ments were weighed to contain a meat:lipid ratio of 5:1. After minc-ing, sodium chloride (2%), saccharose (0.4%), sodium nitrite (0.015%), ascorbic acid (250 ppm), sweet red pepper (1.25%), black pepper (0.5%), cumin (1%), garlic powder (1%), and commercial starter cul-ture (0.02%) were added to per kg of meat. All the ingredients were mixed homogeneously using a hand‐type mixer (Arzum AR1037, Turkey). After that, the sausage mixture was stuffed into casings using a hydraulic filling machine (Alpina‐SG, Switzerland). Samples were placed in a ripening chamber (Daihan Scientific SWGC‐450, South Korea) and conditioned at 23°C and 60% relative humidity (RH) for 2.5 hr for equilibration. After that, samples were fermented at 23°C and 87% RH until the pH value reached 5.6. Samples were then subjected to the heat treatment in a preheated oven (AFOS Mini Kiln, UK) where the ambient temperature was gradually in-creased from 55 to 80°C until the core temperature of the samples reached 68°C. The ambient and core temperatures were controlled using a Data Logger system equipped with probes (PCE Instruments PCE‐T 1,200, Germany). When the heat treatment finished, the sam-ples were immediately cooled using a cold water spray. The samPCE‐T 1,200, Germany). When the heat treatment finished, the sam-ples were finally ripened at 18°C and 72% RH until the moisture content dropped below 50%. During the production period, samples were taken at the beginning of the stuffing (dough), at the end of the heat

treatment, and at the end of the ripening process (final product) for the selected analysis. On each sampling process, three samples from each batch were taken for analysis. A brief visual summary covering the entire production procedure is shown in Figure 1.

2.4 | Methods

2.4.1 | Proximate composition

The proximate composition of the sausage dough and final products was determined by analyzing total moisture (Association of Official Analytical Chemists [AOAC], 2012), protein (LECO nitrogen ana-lyzer, FP528, USA), lipid (Flynn & Bramblett, 1975), and ash contents (AOAC, 2012).

2.4.2 | pH

pH values of the samples were measured using a portable pH meter (WTW pH 330i/SET, Germany). The penetration probe of the pH meter was dipped straight into three different points of the sample and the pH value was recorded after the value was fixed.

2.4.3 | Water activity

The water activity (aw) of the sausages was measured at 25°C using an a_w measurement device (Testo AG 400, Germany). Approximately 5 g of the sample was placed in the standard plastic container of the device and it was then placed with sample in the equipment cham-ber for equilibration. The sample was kept in the chamcham-ber until the reading did not change by more than 0.01 unit and the result was recorded.

2.4.4 | Analysis of lipid oxidation

Peroxide value (PV)

The peroxide value (PV) was determined according to AOAC (2012). The lipid sample (1 g) was homogenized using 30 ml of ace-tic acid:chloroform solution (3:2, v/v) into which 1 ml of saturated potassium iodate solution (KI) was then added. After the addition of 30 ml of distilled water, titration was performed using 0.1 N sodium thiosulfate (Na₂S₂O₃) solution. The PV was expressed as milliequivalents (mEq) of active oxygen per kilogram of lipid in the sample.

Conjugated diene (CD) content

The lipid phase extracted from the samples (Flynn & Bramblett, 1975) was mixed with isooctane and the absorbance was meas-ured at 233 nm (International Union of Pure and Applied Chemistry [IUPAC], 1992). The conjugated diene (CD) content of the samples was calculated from the equations as follows:

C_cd= A₂₃₃ 𝜀× L CD =Ccd×2.525 × 10 4 Sample weight

where A₂₃₃ is the absorbance measured at 233 nm, ε is the extinction coefficient as 2.525 × 104_{, and L is the length of the cell as cm.}

2‐Thiobarbituric acid reactive substances (TBARS) value

The analysis of 2‐thiobarbituric acid reactive substances (TBARS) was carried out according to Witte, Krause, and Bailey (1970). A sample of 20 g was homogenized with 100 ml of 1:1 20% trichlo-roacetic acid (TCA) (w/v) in 2 M phosphoric acid and distilled water. The slurry was then filtered through the Whatman No. 1 filter paper and the volume was completed to 100 ml. After that, 5 ml of the filtrate was mixed with 5 ml of TBA (0.02 M) in a test tube. A blind solution was prepared using 1:1 TCA:distilled water. The tubes were incubated at 80°C for 35 min. Finally, the absorbance was measured using a spectrophotometer (T60 UV‐VIS, PG Instruments, UK) at 532 nm. The TBARS value was calculated by multiplying the absorb-ance by 5.2 to express the concentration as mg malonaldehyde/kg samples.

2.4.5 | Analysis of protein oxidation

Total carbonyl content

The total carbonyl content of sausage samples was analyzed by deri-vatization of 2,4‐dinitrophenylhydrazine (DNPH) according to Oliver, Ahn, Moerman, Goldstein, and Stadtman (1987). A sample of 1 g was homogenized using 10 ml of 0.15 N potassium chloride (KCl) and the

homogenate was divided into two equal parts of 100 µl. Proteins were precipitated in the aliquots by 1 ml of 10% TCA (w/v) and then centrifuged at 5,000 rpm (2,683 g) for 5 min. One pellet was mixed with 1 ml of 2 N hydrochloric acid (HCl), while the other one was mixed with 1 ml of 0.2% DNPH (w/v) in 2 N HCl. After the samples were incubated for 1 hr at room temperature, they were precipitated using 0.8 ml of 10% TCA (w/v). After that, the pellets were washed twice with 1 ml of 1:1 ethanol:ethyl acetate (v/v) and centrifuged at 5,000 rpm for 2 min. Proteins were finally dissolved in 2 ml of 6 M guanidine HCl with 20 mM sodium phosphate buffer (pH 6.5). The samples were once more centrifuged for 10 min at 5,000 rpm. The protein concentration was analyzed by measuring the absorption at 280 nm utilizing bovine serum albumin (BSA) as the standard. The total carbonyl content was calculated as nmol of carbonyl per mg of protein.

α‐aminoadipic semialdehyde (AAS) and γ‐glutamic semialdehyde (GGS)

concentration

The specific markers of protein oxidation (AAS and γ-glutamic sem-ialdehyde (GGS)) were analyzed according to Utrera and Estévez (2013). Standard AAS and GGS were synthesized from N‐α-acetyl-L-lysine and N-α‐acetyl‐L‐ornithine, respectively (Akagawa et al., 2006). For analysis, an aliquot of protein suspension (200 μl) was placed in Eppendorf tubes. Proteins were then precipitated with 2 ml of 10% TCA and centrifuged at 2,000 rpm for 30 min. Samples F I G U R E 1   Production flow chart of heat-treated fermented sausages. Note: Sampling‐I: Before stuffing (sausage dough), Sampling‐II: After heat treatment, and Sampling‐III: After ripening (final product)

were then mixed again with 2 ml of 5% TCA and centrifuged at 5,000 rpm for 5 min. After that, pellets were mixed with 0.5 ml of 250 mM 2‐(N‐morpholino) ethane sulfonic acid (MES) containing 1% sodium dodecyl sulfate (SDS) and 1 mM diethylenetriamine-pentaacetic acid (DTPA), 0.5 ml of 50 mM p‐aminobenzoic acid (ABA) in 250 mM MES, and 0.25 ml of 100 mM sodium cyanoboro-hydride (NaCNBH₃) in 250 mM MES. The mixture was then kept at 37°C in a water bath for 90 min. After that, 0.5 ml of 50% TCA was added and centrifugation was applied at 5,000 rpm for 5 min. Then, pellets were washed two times with 1:1 10% TCA (1 ml) and ethanol‐diethyl ether (1 ml) (v/v) and centrifuged at 5,000 rpm for 5 min. Proteins were hydrolyzed at 110°C for 18 hr using 6 M HCl. Samples were then dried in an oven at 40°C. Hydrolysates were finally mixed with 200 μl of Milli-Q water and filtered through PVDF membrane disk filters (pore size: 0.45 μm) for HPLC analy-sis. Derivatized semialdehydes were injected into HPLC (Agilent Technologies, 1,100 series, Palo Alto, USA) using an Inertsil ODS‐3 RP‐HPLC column (5 μm, 150 × 4.6 mm) and a guard column (10 × 4.6 mm). The flow rate was maintained at 1 ml/min. The eluate was monitored with excitation and emission wavelengths set at 283 nm and 350 nm, respectively. Results were expressed as nmol AAS/g of protein and nmol GGS/g of protein.

2.4.6 | In vitro digestibility

The isolation of myofibrillar proteins and myofibrillar protein digest-ibility was analyzed in vitro according to Santé-Lhoutellier et al. (2008). Proteins were washed using 33 mM glycine buffer (pH 1.8) until the final concentration reached to 0.8 mg/ml. Proteins were then digested first by pepsin for 1 hr at 37°C. The digestion process was stopped by the addition of 15% of TCA at various times. After that, the samples were centrifuged for 10 min at 4,000 g and the content of hydrolyzed peptides in the soluble phase was calculated according to the absorbance read at 280 nm. The proteolysis rate was expressed as optical density units by an hour (∆OD/hour). The nonsoluble parts of the pepsin hydrolyzate were then washed using 33 mM glycine buffer. Proteins were digested for 30 min at 37°C by trypsin and α-chymotrypsin mix. Digestion was then stopped by the

addition of 15% of TCA and finally the rate of proteolysis was deter-mined as described above.

2.4.7 | Statistical analysis

Statistical analysis of the data was performed utilizing SPSS for Windows (version 21.0, IBM, USA) by one‐way analysis of variance (ANOVA) that evaluated the statistical significance of the effect of different product formulations and two‐way ANOVA as a function of product formulation and processing time. The general linear model (GLM) procedure was applied to the data, in which the formulations and processing time were assigned as fixed effects and replication as a random effect. The least square difference (LSD) was used to compare the mean values of formulations and the Duncan's multiple range test was used as a post hoc test to determine the significant differences among means at a 95% confidence interval. A partial Pearson correlation was used to measure the strengths of the as-sociation between lipid and protein oxidation markers and in vitro digestibility of final products at a significance of 0.05 level.

3 | RESULTS AND DISCUSSION

3.1 | Proximate composition

The number of chemical components of the sausage dough and final products are presented in Table 1. Total moisture, lipid, protein, and ash contents of the dough samples were ranging 59.42%–63.10%, 18.46%–18.74%, 15.52%–18.19%, and 2.28%–2.48%, respectively, while the same contents of the final products were ranging 43.08%– 45.66%, 28.98%–32.48%, 19.02%–23.96%, and 2.58%–2.97%, respectively. The dry matter content of all the treatments consider-ably increased due to the water loss during the production. Similarly, Ercoşkun et al. (2010) reported that total protein, fat, ash, and salt contents of sucuk samples gradually increased throughout fermen-tation as a result of drying. The increased amount of olive oil was ef-fective in decreasing the moisture content of the dough (p < .05), in the meantime addition of olive oil at any concentration significantly decreased the moisture content of the final products (p < .05). The

TA B L E 1   Proximate composition of sausage doughs and final products

Treatments* Moisture (%) Lipid (%) Protein (%) Ash (%)

Dough C 63.10a _{± 1.98 18.74 ± 0.87 15.52}b _{± 1.84 2.48}a _{± 0.03} O15 62.74a _{± 1.60 18.73 ± 0.66 17.47}a _{± 1.25 2.32}b _{± 0.02} O30 59.42b _{± 1.66 18.46 ± 0.40 18.19}a _{± 1.62 2.28}b _{± 0.01} Final product C 45.66a _{± 0.28 32.48 ± 0.39 19.02}c _{± 1.21 2.58}c _{± 0.02} O15 43.08b _{± 0.43 30.22 ± 0.30 23.50}b _{± 0.88 2.68}b _{± 0.07} O30 43.81b _{± 0.78 28.98 ± 1.08 23.96}a _{± 1.12 2.97}a _{± 0.03}

Note: Data were presented as the mean values ± standard deviation. a, b, c, ….: Means with a different letter in the same column (for different

materi-als) are significantly different (p < .05).

*C: Heat‐treated fermented sausages formulated with 100% beef fat, O15: Heat‐treated fermented sausages formulated with 85% beef fat + 15% olive oil, and O30: Heat‐treated fermented sausages formulated with 70% beef fat + 30% olive oil as a lipid source.

probable reason for this decrease could be attributed to the water loss of the olive oil treatments during the heat treatment of the samples. Kaban (2013) stated that “heat‐treated sucuk” may be also named as “semi-dry sucuk” because of its moisture content, which at maximum level could be 50% according to Turkish regulations (Turkish Food Codex Communiqué on Meat and Meat Products, 2012). Despite the modification in the lipid formulation of the treat-ments, no significant effect was recorded between the total lipid content of both the dough and final products. This is presumably be-cause all the addition amounts of the lipid source in the treatments were fixed to the same percentage. This result also pointed out that the fat holding capacities of the samples during the heat treatment were similar to each other. In concordance with that, Kayaardı and Gök (2003) recorded the similar lipid content in sucuk samples for-mulated with beef fat or different percentages of olive oil as beef fat replacers. Similarly, Muguerza et al. (2001) stated that though the total added fat in Chorizo (traditional Spanish fermented sausage) mixtures was reduced by olive oil, no significant differences were found in the lipid content of the finished products. Protein contents of both of the dough samples and final products formulated with olive oil were significantly higher than the control treatments for-mulated solely with beef fat (p < .05). This result could be attributed to the lower moisture contents of those samples that led to a pro-portional increase in the protein content. Similar to other dry matter parameters, the ash content of the final products increased after the production. The data showed that the ash content of the final prod-ucts increased by adding olive oil in the lipid formulation (p < .05). Contrary results were reported by Kayaardı and Gök (2003), who found that the incorporation of olive oil as beef fat replacers in Turkish sucuk did not show any significant effect on the ash content of the samples.

3.2 | pH

The characteristics of a fermented meat product are formed as a consequence of overall physical, microbiological, and biochemical reactions that occur in the meat matrix during fermentation and ripening processes (Dalmış & Soyer, 2008). The formation of lactic acid by the activity of lactic acid bacteria is a characteristic change during the production of sausages that result in a decrease of pH value (Kaban, 2013). The changes in the pH values of the treat-ments during the processing are presented in Figure 2. Initial pH values before the stuffing were between 5.92 and 5.94, where all of the values were similar. The measurements indicated that pH values of all the treatments were significantly affected by process-ing steps (p < .05). As expected, pH values dramatically decreased by the fermentation and heat treatment in all groups (p < .05), very likely due to the formation of lactic acid. Dalmış and Soyer (2008) reported that the fastest pH drop was observed during the first two days of production of sucuk by the heat processing method. After the heat treatment, pH values of sausage samples were re-corded between 5.42 and 5.57, where the highest value belonged to the O30 group formulated with the highest amount of olive oil (p < .05). Besides, C and O15 samples had similar pH values. During the ripening period, significant increments in pH values were ob-served in all treatments (p < .05). This increase might be due to the decomposition of acids and production of basic nitrogenous compounds. Final pH values of the samples were recorded be-tween 5.59 and 5.70. Similar to the previous measurement step, O30 samples had the highest pH value among treatments (p < .05). This result could be attributed to the higher protein content of this group that led to a higher alkaline structure of these samples. In contrast, Kayaardı and Gök (2003) reported that the replacement

F I G U R E 2   The pH values of heat-treated fermented sausages during the production. Note: Data were presented as the mean values with standard error bars. a, b, c, ….: Different letters in the same colored columns show the significant difference among treatments (p < .05). X, Y, Z, ….: Different letters in the different colored columns show significant difference among production steps (p < .05)

of animal fat with olive oil had no considerable impact on pH values of sucuk samples.

3.3 | Water activity (a

)

Water activity is an important parameter that has to be controlled during drying of food products since it affects chemical and micro-biological quality. Water activity (aw ) of fermented sausage treat-ments recorded during production is shown in Figure 3. Modifying the formulation of the sausages and manufacture stages was found to have significant impacts on a_w values (p < .05). In the beginning, the dough formulations with olive oil (0.959 and 0.955 for O15 and O30, respectively) had higher a_w values compared to the formulation with beef fat (0.948) (p < .05). The heat treatment and ripening processes led to a reduction in the free water content and consequently concentrate salt in the matrix and thereby sig-nificantly decreased a_w values of all the samples (p < .05). Utrilla et al. (2014) reported similar data who found that aw declined progressively throughout the ripening of venison sausages due to dehydration inherent in the process. The measurements of the present study indicated that particularly the ripening period that was carried out after the heat treatment much affected the drop in a_w values. The values of the final products ranged between 0.813 and 0.837, showing that C samples had the highest aw among sam-ples (p < .05). Bilenler et al. (2017) reported the a_w values of heat-treated sucuk samples as between 0.898 and 0.905. The amount of olive oil added to the lipid formulation did not have a significant effect on aw values. The lower aw values of olive oil treatments might indicate the higher drying rate of these samples. In contrast, Utrilla et al. (2014) reported increased aw values as the proportion of olive oil increased in dry-ripened venison sausages, which might

be due to the utilization of olive oil in an organogel form that sub-stantially reduced the water loss during the process.

3.4 | Lipid oxidation

Lipid oxidation phenomena are one of the main causes for physi-cal, sensory, and nutritional quality deterioration in muscle foods. Since fermented meat products have relatively high lipid content and also could be stored under aerobic conditions where it is exposed to oxygen, the oxidation reactions lead to changes in the sensory quality and generate rancid taste and odor. The lipid oxidation of heat-treated fermented sausages was evaluated by measuring PV, CD content, and TBARS value that is shown in Table 2.

The PV is one of the most common parameters for measuring the primary products of oxidative degradation. Product formulation and production stages had significant effects on the PV of the samples (p < .05). Though initial PV of the samples did not seem to be sig-nificantly affected by the formulation, after both of the heat treat-ment and ripening stages, O30 samples had the highest PV among treatments (p < .05). Thus, the increased olive oil content in the lipid formulation of the sausages strongly influenced the primary prod-ucts of autoxidation. This could be explained by the susceptibility of olive oil added samples to lipid peroxidation due to the higher un- saturated fatty acid profile. As expected, the heat treatment signifi-cantly increased PV of all the samples (p < .05) by the considerable impact of temperature on oxidation reactions. However, ripening had a less remarkable effect on PV compared to the heat treatment. The correlations evaluated between the oxidation parameters of the final products are shown in Table 3. PV results showed a strong neg-ative correlation (r = −.805) with TBARS values (p < .05), pointing out the decrease in primary oxidation products with the increase in F I G U R E 3   Water activity (a_w) of heat‐treated fermented sausages during the production. Note: Data were presented as the mean values with standard error bars. a, b, c, ….: Different letters in the same colored columns show the significant difference among treatments (p < .05). X, Y, Z, ….: Different letters in the different colored columns show significant difference among production steps (p < .05)

secondary ones. However, no significant correlation was detected between PV and CD contents of the samples.

One of the other primary compounds of lipid oxidation are con-jugated dienes, formed after bisallylic hydrogen is uprooted by the reordering of the double bond that causes the generation of a conju-gated double bond, which can be measured spectrophotometrically (Guyon et al., 2016). Initially (dough samples), the treatments with olive oil had a lower CD content compared to C samples (p < .05), but after the heat treatment, the highest CD content was recorded in O15 samples (p < .05). An increased amount of olive oil did not lead to any increment in the CD content, in contrast, no significant dif-ferences were recorded between C and O30 samples after the heat treatment or ripening. The heat treatment was effective to increase the CD content of olive oil added samples (p < .05), but no signifi-cant change was observed in the CD content of C samples with heat treatment. Interestingly, the CD content of C samples significantly increased after-ripening (p < .05), whilst the CD content of the other samples did not. This data showed that the CD content of the formu-lations with more saturated fatty acids tends to remain stable, while the CD content of the formulations with more unsaturated fatty acids tends to rise in the early stages of processing. Nevertheless, the CD content of the final products showed significant correlation with neither lipid oxidation nor protein oxidation products. This might indicate that the CD content has a less determinative effect compared to other oxidation parameters and thus, it should not be solely used as a lipid oxidation marker of meat.

Due to their unstable characters, primary lipid oxidation prod-ucts are further broken down into secondary prodprod-ucts, such as hexanal, 4-hydroxynonenal, and malondialdehyde (Papastergiadis, Mubiru, Van Langenhove, & De Meulenaer, 2012). The analysis of TBARS is the most common method to determine the malonalde-hyde concentration of meat products. Initial TBARS values of the dough samples were similar to each other. In all stages of produc-tion, O30 samples had the lowest TBARS values among samples (p < .05), indicating that increased levels of olive oil in lipid formu-lations had a protective effect to retard the production of lipid ox-idation secondary products. This result could arise from the high amount of phenolic compounds present in olive oil that act as anti-oxidants. Similar to the findings, in a study on high- and reduced-fat Greek fermented sausages formulated with a partial replacement of pork backfat with olive oil, a significant decrease in oxidation was recorded in treatments consisting of olive oil (Muguerza et al., 2003). In another study, it was found that in dry fermented sau-sages, the substitution of pork backfat with preemulsified olive oil was better than storing samples under vacuum to avoid the lipid oxidation (Ansorena & Astiasarán, 2004). Moreover, Kayaardı and Gök (2003) detected that fermented sausages with olive oil were more susceptible to the lipid oxidation compared to sausages with animal fat. Exposure of the samples under different conditions during processing as well as the formulations could account for the differences in those studies.

Regarding the effects of production stages, it was found that the heat treatment led a significant increment on TBARS values of all T A B LE 2  Li pi d a nd p ro te in ox id at io n o f h ea t-tr ea te d f er m en te d s au sa ge s d ur in g t he p ro du ct io n Lip id  o xid at io n Per ox ide v alue (m eq O2 /k g pr od uc t) C on ju ga ted d ien e c on ten t ( μ m ol /g fa t) TB A RS  v alue  (m g  malon di al deh yde /k g  pr od uc t) B ef or e s tu ff in g (dou gh ) A ft er h ea t tr ea tm en t A ft er rip enin g (fi nal pr od uc t) B ef or e s tu ff in g (dou gh ) A ft er h ea t tr ea tm en t A ft er rip enin g (fi nal pr od uc t) B ef or e s tu ff in g (dou gh ) A ft er h ea t tr ea tm en t A ft er rip enin g (fi nal pr od uc t) C 8. 57 a,Y ± 1 .0 0 11 .3 4 c, X ± 0 .1 3 11 .4 9 c, X ± 1 .0 9 2. 55 a, X ± 0.0 2 2. 62 b, X ± 0.0 8 2. 88 b,Y ± 0.0 2 0. 58 Y ± 0.0 8 1. 29 a, X ± 0.0 7 1. 17 a, X ± 0 .1 3 O 15 8. 49 b, Z ± 0 .5 2 11 .9 7 b,Y ± 1 .0 0 12 .56 b, X ± 0 .2 9 1.9 2 b,Y ± 0.0 8 3.1 0 a, X ± 0.0 8 3.1 5 a, X ± 0 .11 0. 49 Y ± 0 .49 1.1 3 b, X ± 0.0 3 0. 90 b,Y ± 0.0 9 O3 0 8. 62 a,Y ± 0 .71 12 .76 a, X ± 0 .2 9 12 .9 9 a, X ± 0 .8 9 1.4 8 c,Y ± 0.0 8 2. 47 b, X ± 0 .1 0 2.7 9 b, X ± 0 .1 6 0. 45 Y ± 0 .4 5 0. 76 c, X ± 0.0 7 0. 51 c,Y ± 0.0 2 Pr ot ei n o xi da tio n To ta l c ar bo ny l c on te nt  (n m ol /m g  pr ot ei n) A A S co nce nt ra tio n ( nm ol /m g p rot ei n) G G S co nce nt ra tio n ( nm ol /m g p rot ei n) B ef or e s tu ff in g (dou gh ) A ft er h ea t tr ea tm en t A ft er rip enin g (fi nal pr od uc t) B ef or e s tu ff in g (dou gh ) A ft er h ea t tr ea tm en t A ft er rip enin g (fi nal pr od uc t) B ef or e s tu ff in g (dou gh ) A ft er h ea t tr ea tm en t A ft er rip enin g (fi nal pr od uc t) C 1. 55 b,Y ± 0 .3 9 2. 69 b, X ± 0 .11 2. 61 b, X ± 0 .1 3 0.4 88 a,Y ± 0 .0 66 0. 51 9 a, X Y ± 0.0 38 0. 53 8 a, X ± 0 .0 26 0. 391 a ± 0.0 09 0. 31 4 ± 0. 05 8 0. 33 9 a ± 0.0 04 O 15 2. 05 ab ,Y ± 0 .2 5 2.9 4 b, X ± 0 .11 2. 83 b, X ± 0 .2 2 0. 45 7 a,Y ± 0.0 23 0. 585 a, X ± 0 .0 15 0. 57 0 a, X ± 0.0 01 0. 30 8 ab ,X ± 0.0 43 0. 32 0 X ± 0.0 80 0. 151 b,Y ± 0.0 11 O3 0 2. 38 a,Y ± 0.0 9 3. 59 a, X ± 0.0 8 3. 53 a, X ± 0 .1 3 0. 35 6 b,Y ± 0 .11 7 0.4 36 b, X ± 0.0 12 0. 43 9 b, X ± 0.0 10 0. 20 4 b,Y ± 0 .0 54 0. 33 7 X ± 0 .0 05 0.1 27 b, Z ± 0.0 09 N ote s: C : H ea t‐ tr ea te d fe rm en te d sa us ag es fo rm ul at ed w ith 1 00 % b ee f f at , O 15 : H ea t‐ tr ea te d fe rm en te d sa us ag es fo rm ul at ed w ith 8 5% b ee f f at + 1 5% o liv e oi l, an d O 30 : H ea t‐ tr ea te d fe rm en te d sa us ag es fo rm ul at ed w ith 7 0% b ee f f at + 3 0% o liv e oi l a s a lip id s ou rc e. D at a w er e pr es en te d as th e m ea n va lu es ± s ta nd ar d de vi at io n. a , b , c , … .: M ea ns w ith a d iff er en t l et te r i n th e sa m e co lu m n ar e si gn ifi ca nt ly d iff er en t ( p < .0 5) . X , Y , Z , … : M ea ns w ith a d iff er en t l et te r i n th e sa m e ro w a re s ig ni fic an tly d iff er en t ( p < .0 5) . ( al l a na ly si s in it se lf) .

the treatments (p < .05). Similar results were obtained by Ercoşkun et al. (2010) who reported that TBARS values of fermented sausages increased significantly after heat treatment, showing that oxida-tive reactions increased during heating. After the ripening period, C samples had similar TBARS values to their values after the heat treatment. Though, significant decrements were recorded in TBARS values of O15 and O30 samples (p < .05), which could be attributed to the decomposition of secondary products and/or interactions of aldehydes with other polymers like proteins and sugars. Moreover, Fuentes, Utrera, Estévez, Ventanas, and Ventanas (2014) underlined that malonaldehydes could undergo nitrosation reactions as a result of residual nitrite present in the product; thus, leading all or part of these aldehydes unreactive for the TBARS analysis. At the end of the production, TBARS values of the final products were recorded between 0.51 and 1.17 mg malonaldehyde/kg sample. Bloukas, Paneras, and Fournitzis (1997) stated that fermented sausages with olive oil caused rancid notes at TBARS values up to 2 mg malonal-dehyde/kg. Thus, it could be concluded that TBARS values of the heat-treated sausages were within the acceptable ranges during pro-cessing. Previously, the negative correlation between TBARS values and PV of the sucuk samples has been mentioned above (Table 3). TBARS values were also recorded to have high correlations with protein oxidation parameters, which would be discussed within the results of the protein oxidation below.

3.5 | Protein oxidation

Protein oxidation has been mentioned as one of the most innova-tive issues in the assessment of muscle foods’ quality since muscle

proteins have a vital role in nutritional, sensory, and physicochemi-cal characteristics (Falowo et al., 2014). As a result of oxidation in proteins, chemical alterations in amino acid side chains and/or in the peptide backbone occur and these alterations could cause changes in the physical characteristics of proteins, such as fragmentation, ag-gregation, decreased susceptibility to proteolysis, loss of solubility, and functionality (Xiong, 2000). For this reason, it is of great impor-tance to determine the protein oxidation markers in meat products to evaluate the oxidative quality.

Effects of lipid formulation and production steps on protein oxi-dation of the samples are presented in Table 2. One of the most no-ticeable modifications in oxidized food proteins has been highlighted as the generation of carbonyl compounds (Estévez, 2011) and the determination of them is commonly done using the DNPH method (Fuentes, Ventanas, Morcuende, Estévez, & Ventanas, 2010). In heat-treated fermented sausages, significant differences in the total carbonyl content (TCC) were recorded among treatments during processing (p < .05). The TCC of the dough samples was between 1.55 and 2.38 nmol/mg protein, O30 samples had higher TCC than C samples (p < .05), indicating that the increased amounts of olive oil in the lipid phase could alter the carbonylation reactions at the very beginning of the production. In the same vein, O30 samples had the highest TCC after the heat treatment and after the ripening compared to other groups (p < .05). This data underlined the con-siderable impacts of modification of fatty acid composition on gen-erating protein carbonyls, in which vegetable oils seems to be more effective to trigger protein carbonylation compared to saturated an- imal fats. In contrast, Fuentes, Utrera et al. (2014) reported that fer-mented sausages formulated with sunflower oil had lower carbonyl TA B L E 3   Correlations evaluated between lipid oxidation, protein oxidation, and in vitro digestibility of heat-treated fermented sausages

PV CD TBARS TCC AAS GGS PS TRY‐αCHY

Lipid oxidation PV Pearson correlation 1.000 −.037 −.805* .768* −.400 −.887* .119 −.484

Sig. (2‐tailed) – .925 .009 .016 .286 .001 .761 .186

CD Pearson correlation −.037 1.000 .246 −.403 .455 −.101 .121 −.185

Sig. (2‐tailed) .925 – .442 .194 .137 .754 .757 .634

TBARS Pearson correlation −.805* .246 1.000 −.831* .567 .658* −.243 .317

Sig. (2‐tailed) .009 .442 – .001 .055 .020 .528 .406

Protein oxidation TCC Pearson correlation .768* −.403 −.831* 1.000 −.613* −.656* .225 −.125

Sig. (2‐tailed) .016 .194 .001 – .034 .021 .561 .749

AAS Pearson correlation −.400 .455 .567 −.613* 1.000 .262 .259 −.078

Sig. (2‐tailed) .286 .137 .055 .034 – .411 .500 .841 GGS Pearson correlation −.887* −.101 .658* −.656* .262 1.000 −.203 .148 Sig. (2‐tailed) .001 .754 .020 .021 .411 – .601 .705 In vitro digestibility PS Pearson correlation .119 .121 −.243 .225 .259 −.203 1.000 .000 Sig. (2‐tailed) .761 .757 .528 .561 .500 .601 – 1.000

TRY‐αCHY Pearson correlation −.484 −.185 .317 −.125 −.078 .148 .000 1.000

Sig. (2‐tailed) .186 .634 .406 .749 .841 .705 1.000 –

Abbreviations: AAS, α‐aminoadipic semialdehyde; GGS, γ‐glutamic semialdehyde; CD, conjugated diene, PS, pepsin; PV, peroxide value; TBARS, thiobarbituric acid reactive substances; TCC, total carbonyl content; TRY‐αCHY, trypsin and α-chymotrypsin.

compounds compared to sausages formulated with different types of animal fats. The difference in TCC from the present study could arise from the behavior of the lipid sources and manufacturing appli-cations. The results showed that though the incorporation of olive oil decreased the TBARS values of the sausages, the same behav-ior was not observed in the TCC of the samples. Indeed, a strong negative correlation was recorded between these parameters (r = −.831) and a positive correlation between TCC and PV (r = .768) (p < .05) (Table 3). Therefore, the TCC of the samples showed an in-crease with the in(p < .05) (Table 3). Therefore, the TCC of the samples showed an in-crease in primary lipid oxidation products, while it showed a decrease with the increase in secondary ones. This result emphasizes the probable interaction between the lipid and protein oxidation mechanisms in the meat matrix. This interaction was previ-ously elaborated by Estévez (2011), who stated that peroxyl radicals (ROO._{) are probable promoters of protein carbonylation. The TCC}

of the samples steadily increased throughout the production period (p < .05), yet the ripening period after the heat treatment did not significantly change the TCC of any groups. Thus, the heat treatment here had the most noticeable effect on the increment of TCC regard- less of the formulation. Ganhão, Morcuende, and Estévez (2010) re-ported that high temperatures could enhance the protein oxidation of burger patties by degrading myoglobin that causes the release of iron, a strong pro-oxidant. Similar results were also reported by Gan et al. (2019), who found that the amount of protein carbonyl significantly increased throughout the roasting stage of traditional Chinese bacon.

Though the DNPH method is frequently utilized to assess pro-tein oxidation, this method has been mentioned to have some in-consistencies (Utrera & Estévez, 2013). Hence the determination of specific carbonyls, namely, AAS and GGS is a way to better understand the exact characteristics of protein oxidation. AAS is an oxidative product generated by the deamination of lysine and GGS is formed by the oxidation of arginine and proline residues from the Maillard reaction (Zhang et al., 2013). Changes in the sau-sage formulation resulted in significant differences in the AAS and GGS concentration of the samples during the production (p < .05) (Table 2). In all production periods, O30 samples had the lowest AAS concentration among treatments (p < .05), though this group was previously recorded to have the highest TCC. Though a similar constant trend was not observed in GGS values, O30 samples had lower GGS concentration than C samples before stuffing (p < .05). No significant differences were found in GGS concentrations of the samples after the heat treatment; however, in final products, the C group was recorded to have the highest GGS concentration (p < .05). Overall, the results indicated that the incorporation of olive oil to the lipid formulation of heat-treated fermented sau-sages was effective to decrease the protein oxidation-specific products during processing, similar to lipid oxidation results. The concordance data was reported by Santé‐Lhoutellier et al. (2008), who found a negative correlation between protein carbonylation and dietary vitamin E level in lamb meat that indicated a protective effect against oxidation. Moreover, Fuentes, Utrera, et al. (2014) suggested that factors like the presence of antioxidant compounds

play a more efficient role in protein carbonylation compared to the modification of lipid composition.

Regarding the impacts of production, it was observed that at the end of ripening, all of the products had higher AAS concentrations compared to the initial values (p < .05). The heat treatment espe-cially had a significant effect on increased AAS concentrations in the samples formulated with olive oil (p < .05). Utrera, Morcuende, and Estévez (2014) explained that the increment of AAS after cooking of beef patties formulated with different fat levels could be due to: (a) an increased sensitivity of pro‐oxidants to lysine residues due to cell disruption, (b) an increased pro‐oxidant concentration arise from dehydration, and (c) the denaturalization of antioxidant enzymes by heat. GGS concentrations, moreover, were similar during the pro-cessing of C samples whilst final GGS concentrations of O15 and O30 samples were significantly lower than initial values (p < .05). Utrera and Estévez (2013) stated that both semialdehydes (AAS and GGS) could undergo further reactions because of their highly reac- tive moieties. Therefore, lower GGS values at the end of the produc-tion could be associated with the degradative moieties. Therefore, lower GGS values at the end of the produc-tion and further reactive moieties. Therefore, lower GGS values at the end of the produc-tions with lipid oxidation products or amino acid residues.

Evaluating the relationships between oxidation markers of final products, it was recorded that both AAS and GGS concen-trations of the sausages had a significant negative correlation with TCC (p < .05). This could be due to the rise in AAS and GGS by the oxidative deamination of lysine, proline, and arginine amino acids, while the decrease in the TCC by the attachment of DNPH to lipid-derived carbonyls such as malondialdehydes (Estévez, 2011; Fuentes, Estévez, Ventanas, & Ventanas, 2014). Though no significant correlation was observed between AAS concentrations and lipid oxidation parameters of the sausages, strong correlations were recorded between GGS concentrations and both PV and TBARS values (p < .05). Fuentes, Ventanas et al. (2010) found that volatile compounds of lipid oxidation were considerably correlated with both AAS and GGS in dry‐cured hams, which supports the probable relationship between lipid and protein oxidation mech-anisms. We recorded that the correlation coefficients were neg-ative between GGS and PV (r = −.887) and positive between GGS and TBARS (r = .658) (p < .05), which indicated that unlike TCC re-sults, GGS concentrations increased with the decrease in primary lipid oxidation products and with the increase in secondary ones. Thus, GGS would be more likely formed after the further decom-position of hydroperoxides and formation of aldehydes. These re-sults showed that GGS values presented more indicative data than AAS values to explain the relationship between lipid and protein oxidation mechanisms. The lack of correlation between AAS con-centration and lipid oxidation features could be due to the ongoing oxidation of AAS which led to the formation of a stable end‐prod-uct called α‐aminoadipic acid (AAA) (Estévez, 2011).

3.6 | In vitro protein digestibility

The amino acid profile of a food product is important to determine its protein nutritive quality; however, the digestion of the protein

into free amino acids and small peptides is the main indicative of the absorption of them by the human body (Hsu, Vavak, Satterlee, & Miller, 1977). Therefore, it is important to evaluate the digestion rate to make a remark about the health profile of a meat product that is rich in proteins. Proteolysis is one of the main degradation reactions that occur during the ripening of fermented meat products, mainly sourced from endogenous or exogenous enzyme originating from microorganisms (Dalmış & Soyer, 2008). Berardo, Claeys, Vossen, Leroy, and De Smet (2015) stated that the pH drop during the fer-mentation of meat products provokes protein denaturation and enhances the activity of some important proteolytic enzymes. The protein digestibility of the heat-treated fermented sausages in terms of pepsin, trypsin, and α-chymotrypsin is shown in Figure 4. Pepsin activities of the samples were similar to each other in all production stages. Likewise, there were no differences in trypsin and α-chymo-trypsin activities of the dough samples and after the heat treatment. Merely in final products, the highest trypsin and α-chymotrypsin ac-tivity were recorded in C samples (p < .05), but since O30 samples had higher activity than O15 samples, the data did not present a clear comment for the change of digestibility in relation with lipid formulation. Moreover, production applications were noted to have significant effects on the pepsin activity (p < .05), especially we recorded considerable decrease by the heat treatment due to the changes in the protein structure. Yet trypsin and α-chymotrypsin ac-tivities seemed to be less affected by production stages.

It has been already mentioned that protein oxidation reactions induce multiple physicochemical changes in meat proteins including the loss of protein functionality, a decrease in the bioavailability of amino acid protein, loss of proteolytic activity, and impaired digest-ibility of oxidized proteins (Estévez, 2011; Estévez & Luna, 2017; Falowo et al., 2014; Guyon et al., 2016). In the present study, though some significant differences in digestibility were recorded by the effect of production, no similar pattern in the digestibility of heat-treated sausages was observed to the oxidation rate of them. Indeed, any significant correlations were recorded between digestibility and

oxidation products of the sausages. Similarly, Santé-Lhoutellier et al. (2008) investigated the links between protein carbonylation and di-gestibility of lamb meat and found that the activities of pepsin and trypsin plus α-chymotrypsin were not significantly correlated with carbonyl content. In a model system study for dry fermented sau-sages, the inhibition of proteolysis did not influence protein oxida-tion, but proteolysis was negatively affected by a decrement in pH values that led to a reduction in the release of amino acids (Berardo et al., 2015). Gan et al. (2019) found that protein carbonylation was linked with proteolysis during the production of traditional Chinese bacon, presumably as a result of cleavage of peptides by proteolysis that favored the protein oxidation. From this viewpoint, the proba-ble relationship between the mechanisms of oxidation reactions and digestibility should be investigated in a broader approach by assess-ing different types of proteolytic enzymes and by considerassess-ing differ-ent periods of the production procedure. In addition, Soladoye et al. (2015) emphasized that the results obtained from in vitro and animal model system studies may differ from each other, so that in vitro analysis may not present the clear effects of protein oxidation on digestibility and thereby the nutritional value of the meat product. Moreover, a critical point was also mentioned by Xue, Huang, Huang, and Zhou (2012), who stated that the level of protein degradation could be different under different degrees of protein oxidation. Hence, the interaction between the digestibility and oxidation mark-ers should be considered throughout the production as well as the storage period of various meat products. A final mark here was the effect of the heat treatment in in vitro digestibility of the sausages. Dalmış and Soyer (2008) underlined that the temperature is the key factor affecting the activity of muscle and proteinases. In their study, they found out that in both traditional and heat-treated sau-sages, there was intense proteolysis during fermentation, but after heating both sarcoplasmic and myofibrillar proteins were degraded due to denaturation. This data indicated the importance of the heat treatment and heat-induced changes on digestibility that could fur-ther lead to modifications in proteins related to oxidation reactions. F I G U R E 4   Pepsin and trypsin&α-chymotrypsin activities of heat-treated fermented sausages during the production. Note: Data were presented as the mean values with standard error bars. a, b, c, ….: Different letters in the same colored columns show the significant difference among treatments (p < .05). X, Y, Z, ….: Different letters in the different colored columns show significant difference among production steps (p < .05)

4 | CONCLUSIONS

In recent years, protein oxidation of muscle foods has become a novel research subject since the alterations in the proteins are di-rectly or indidi-rectly associated with various quality issues. In the present study, the changes in the specific oxidation markers and in vitro digestibility during the production of heat-treated fermented sausages that were prepared with different lipid formulations were evaluated. The results indicated that the utilization of olive oil had significant impacts on chemical, oxidative, and functional quality of sausages during processing. Oxidation markers showed that lipid and protein oxidation mechanisms progress in a connection and in-teract with each other in the meat matrix. It would be remarkable to take a further look into the relationship between these complex oxidation mechanisms in different muscle foods by evaluating the effect of processing conditions or the effect of incorporation of dif-ferent lipid sources. Moreover, the interrelationship between the oxidation of proteins and in vitro digestibility is a subject that still remains unclear. Therefore, the interaction between oxidative stress mechanisms and protein digestibility would need a comprehensive touch to be lightened in the future, due to the considerable impact of digestibility on the bioavailability and nutritional value. Further studies should also concern the incorporation of natural phenolic compounds into meat products and their impacts on specific oxida-tion markers in relaoxida-tionship to the protein funcoxida-tionality.

ACKNOWLEDGMENTS

The authors would like to thank The Scientific and Technical Research Council of Turkey (TÜBİTAK) for funding this study under the project number 214-O-181.

CONFLIC T OF INTEREST

The authors declare that there is no conflict of interest. ORCID

Burcu Öztürk‐Kerimoğlu https://orcid.

org/0000‐0001‐9777‐8510

Berker Nacak https://orcid.org/0000‐0002‐3512‐6231

Vasfiye Hazal Özyurt https://orcid.org/0000‐0003‐2524‐5381

Meltem Serdaroğlu https://orcid.org/0000‐0003‐1589‐971X

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How to cite this article: Öztürk‐Kerimoğlu B, Nacak B, Özyurt VH, Serdaroğlu M. Protein oxidation and in vitro digestibility of heat-treated fermented sausages: How do they change with the effect of lipid formulation during processing? J Food Biochem. 2019;43:e13007. https ://doi. org/10.1111/jfbc.13007