The formulations presented in Table 1 were used for the production of the fermented sucuks. After the matured meat and frozen animal fat were minced in a meat grinder (12 mm diameter plate), spices, starter culture (Baktogard FT 120, lactic acid bacteria, and cocci), and sodium nitrite were added and mixed well in a mixer. The resulting sucuk dough was left for pre-ripening for a day at 4±1°C. After pre-ripening, the sucuk dough was thinned by pulling it through the 3 mm plate of the meat grinder.

After the prepared sucuk dough was filled into 28–32 caliber natural beef intestine casings soaked in 5% lactic acid solution, they were tied with cotton threads and hung, and after showering, the sucuk samples were subjected to fermentation in the ripening cabin (rooms with controlled temperature, humidity, and air flow rate) (Figure 1). On the first day, the ripening program was started so that the temperature was 28±1°C and the relative humidity was 90±2%. The temperature and relative humidity were reduced by two units until the fifth day. The ripening process was terminated at the end of the 10th day by keeping the temperature constant at 10±1°C, and the relative humidity was 80±2% after the fifth day. Air flow was kept between 1.5 and 2 m/s in the first 3 days of ripening and was gradually reduced to 0.5 m/s in the following days.

Fat, protein, ash, salt, and moisture contents of fermented sucuks were determined following the AOAC methods (A. of O. A. C. AOAC 1990). The protein content of sucuk samples was determined according to the Kjeldahl method by using 6.25 factor to convert nitrogen to protein. The salt content of the samples was determined according to the Mohr’s titration method. Ten grams of the sucuk sample was homogenized by adding 100 mL of distilled water, and the pH value of the samples was read by dipping the digital pH meter (HANNA H1 2211 pH/ORP METER) probe, calibrated with the appropriate buffer solution, into the mixture (Aro Aro et al., 2010).

Extraction of peptides was performed according to the method described by Mora et al. (2015). After the casing of 5 g of fermented sucuk sample was peeled and shredded, it was homogenized in 25 mL of 0.01 N HCl in a stomacher. The mixture was centrifuged at 10000 g for 20 minutes (4±1°C), then three volumes of ethanol were added to the samples, kept at 4±1°C for 20 hours, deproteinized and centrifuged again (Nüve NF 1200R). The mixture was filtered through a Buhner funnel with Whatman No: 4 filter paper and evaporated under vacuum at 45°C (CLS Scientific CLRC-08C). The aqueous fraction of the mixture was freeze-dried and stored at −20°C until use.

As a combination of the enzymatic hydrolysis methods applied by Pepe et al. (2016) and Parrot et al. (2003), 0.5 g of lyophilized water-soluble extracts was weighed and dissolved in ultrapure water. The pH was adjusted to 2 with 1 M HCl, and a pepsin solution of 0.1 g pepsin/L was prepared in 0.01 M HCl, and then 1 mL of pepsin was added to the extract for 10 mL of water-soluble extract. The first hydrolysis was completed in 24 hours in a shaking water bath at 37°C. Then, the extracts were kept in a water bath at 90°C for 15 minutes to inactivate the pepsin enzyme. After the extracts were cooled, the pH was adjusted to 8 with 0.1 N HCl, and an enzyme solution of 0.5 g trypsin/L was prepared in pure water, and 0.20 mL trypsin was added for 10 mL of water-soluble extract. The hydrolysis process was completed in 24 hours in a shaking water bath at 37°C, and the extracts were kept in a water bath at 90°C for 15 minutes to inactivate the trypsin enzyme. At the end of the period, the hydrolysates were cooled in an ice water bath and then centrifuged at 5,000 g for 10 minutes. Then, the hydrolysates were dried and dissolved in 100 mg/mL acetate buffer (0.02 M and pH = 4) to obtain lipid fractions and filtered through 0.45 and 0.22 μm filters, respectively, and fractions were collected by injecting into HPLC according to Gallego et al. (2018).

Five milliliters of each extract was taken and injected into a column (4.6×250 mm) filled with Sephadex G-25 (Amersham iosciences, Uppsala, Sweden) stationary phase. The separation was performed with the following conditions: mobile phase: 0.01 N tris-HCl buffer solution (pH = 7) at 4°C, flow rate: 1 mL/minute, and detection wavelength: 214, 254, and 280 nm (Shimadzu, Detector: PDA, Column oven temperature: 20 °C). Five milliliter fractions were collected manually by monitoring at 214, 254, and 280 nm. The obtained fractions were lyophilized and dissolved in 2 mL of pure water and stored at −20 °C until further analysis (Gallego et al. 2018).

Antimicrobial activity of peptide fractions obtained from fermented sucuk samples subjected to enzymatic hydrolysis was determined by modifying the well diffusion method applied by Daoud et al. (2005). Microorganisms were incubated in Nutrient Soy Broth at 37°C for 24 hours. A bacterial solution was prepared from bacterial cultures with an absorbance value of 0.5 at 600 nm (density of 106 cfu/mL) in a spectrophotometer (EMC-11-UV). Sterilized nutrient agar (20 mL) was poured into sterile petri dishes, and after the agar solidified, 100 μL of bacterial suspension was planted in the petri dishes by the spreading plate method. The media were left to dry for 5–10 minutes at room temperature. After the drying process, three wells with a diameter of 5 mm were opened on the media with a sterile cork drill, equidistant from each other and from the edges of the petri dish. Peptide fractions were added to the wells (20 μL) and the petri dishes were incubated at 35±1ºC for 24 hours.

Antimicrobial activity was measured by evaluating the diameter of the clear growth inhibition zone (formed zones) (Djabou et al. 2013).

8.0 mm and below: Peptide fraction does not have sufficient antimicrobial effect on the relevant bacteria (-),

DPPH radical scavenging activity, which is an expression of the antioxidant capacity of the isolated peptides, was determined by Wang et al. (2003). Peptide fractions (0.1 mL) were taken and added into vials. Five milliliters of DPPH solution with 0.1 mM concentration was added to each vial, mixed by vortex, and incubated for 20 minutes at 27ºC in a dark environment. At the end of the period, absorbances were read at 517 nm wavelength. Pure methanol was added as a blank solution, and 0.1 mL of pure water was added instead of 0.1 mL of peptide fraction as a control solution. DPPH radical scavenging capacity (%) values, which are a measure of the antioxidant capacity of the extracts, were calculated according to the following formula:

A_control: Absorbance of the control sample

A_sample: Absorbance of test sample

Identification of peptide profiles of fractions by LC-MS/MS was done according to the method applied by Gümüş et al. (2022). Chromatographic separation was performed using an HPLC Agilent 1260 Infinity series (Agilent Technologies, Santa Clara, CA, USA) equipped with a binary pump, online degasser, and autosampler. A Poroshell 120 EC-C18 column (3.0×100 mm, particle size 2.7 µm) (Agilent Technologies) was used to separate the components. The gradient program applied for the mobile phase system consisting of water containing 0.1% formic acid (A) and acetonitrile (B) was as follows: 0–0.5 min 5% B; 0.5–7 minutes 25% B; 7–16 minutes 50% B; 16–23 minutes 75% B; 23–30 minutes 95% B, and 5% B concentration was used for 30–40 minutes to equilibrate the column. The column temperature was kept at 35°C. The injection volume was set to 10 µL, and the flow rate was 0.4 mL/min. Ionization of chromatographic eluates was performed under the conditions specified below using an Agilent 6550 iFunnel high-resolution total mass spectrometer (QTOF-MS) equipped with an Agilent Dual Jet Flow electrospray ionization (Dual AJS ESI) interface operating in negative ion.

Drying gas flow 14.0 L/min; drying gas temperature 290°C; nebulizer pressure 35 psi; sheath gas temperature 400°C; sheath gas flow (nitrogen) 14 L/min and capillary voltage 1000V.

All scan mass spectra were collected in targeted MS/MS mode using a scan range of 50–3200 m/z to initiate MS/MS data collection. During analysis, the collision energy was set to 20 eV. Integration and data evaluation were performed using MassHunter Workstation software.

Peptides identified in fermented sucuk samples were subjected to peptide sequence analysis via the PeptiteRanker program internet server, and the identified peptide sequences were matched with peptide sequences showing antioxidant and antibacterial activities in the BIOPEP-UWM database in the program (Minkiewicz et al., 2019).

Five milliliters of each extract obtained by enzymatic hydrolysis was taken and injected into a column (4.6 × 250 mm) filled with Sephadex G-25 (Amersham iosciences, Uppsala, Sweden) stationary phase. Then, the fractions were monitored at 214, 254, and 280 nm and collected as 5 mL in amber glass bottles (Shimadzu, Detector: PDA, 214, 254, 280 nm, flow rate: 1 mL/min, column oven temperature: 20°C). Fraction could only be taken at 254 nm. Chromatograms obtained on days 0, 1, 3, 5, and 10 of fermentation are given in Figure 2.

When the chromatograms obtained on the zeroth, first, third, fifth, and tenth days of fermentation are examined, it is seen that a single clear peak is obtained at the end of the 30 and 45 minute flow times on all days. With this result, it is thought that the column filled with Sephadex G-25 stationary phase is effective in the separation process. As soon as peaks began to appear, the fractions began to be collected in amber glass bottles, and the collection process was terminated when the peak was completed and returned to a straight line.

It is observed that peptide bonds decrease during fermentation. As a result of bacterial fermentation during sausage (sucuk) production, proteins undergo partial degradation, especially with the activity of lactic acid bacteria, and BAPs are formed. Therefore, it is an expected situation that peptide bonds will decrease because of the breakdown of proteins during the formation of BAPs.

The antimicrobial activities of the peptide fractions obtained as a result of enzymatic hydrolysis on Escherichia coli (ATCC 25922), Salmonella typhimurium (ATCC 14028), Listeria monocytogenes (ATCC 7644), and Lactobacillus pentosus (ATCC 8041) were measured under laboratory conditions and the findings are given in Table 4.

The antimicrobial activities of the fractions were measured by evaluating the diameter of the clear growth inhibition zone (formed zones) (Djabou et al., 2013). According to the table, it was detected that:

BAP fractions do not have sufficient antimicrobial effects on E. coli (ATCC 25922) and L. monocytogenes (ATCC 7644) (-),

There are almost no studies examining the antimicrobial activity of BAPs in fermented sucuks. Verma et al. (2017) evaluated the antimicrobial activity of the protein hydrolyzate they extracted from pig liver by measuring the inhibition zone (mm) and found that the antimicrobial activity was positively correlated with the enzymatic hydrolysis time corresponding to the degree of hydrolysis, regardless of the enzyme type. As a result of the 6-hour hydrolysis, they determined that trypsin hydrolysates showed the highest antibacterial activity against L. monocytogenes (23.39±0.31 mm), it has a moderate antimicrobial effect against Staphylococcus aureus (18.35±0.65 mm) and E. coli (18.17±0.36 mm), and it showed the least antimicrobial effect against Bacillus cereus (16.79±0.35 mm). In this study, it was determined that hydrolysates did not have sufficient antimicrobial effects on E. coli and L. monocytogenes bacteria.

In the determination of antioxidant activities of peptide fractions obtained from fermented sucuk samples subjected to enzymatic hydrolysis on the zeroth, first, third, fifth, and tenth days of fermentation, as stated in the section “Determination of Antioxidant Activity Based on DPPH (1,1-Diphenyl-2-Picrylhydrazyl) Radical Capture Capacity Method”, the DPPH (1,1-Diphenyl-2-Picrylhydrazyl) radical scavenging capacity method was used. The findings obtained are given in Table 5.

In the study, it was found that the peptide fractions obtained on different days of fermentation did not exhibit very strong antioxidant activity, and there was no statistical difference between the antioxidant activities of the fractions obtained on days 0 and 10 of fermentation. It was determined that the highest antioxidant activity was reached on the tenth day (16.18±0.04%), and the antioxidant activities of the fractions of days 0 and 5 and days 5 and 10 were statistically different. In the study carried out to identify peptides with antioxidant and antihypertensive capacity from dry fermented camel sucuks produced by inoculating different starter cultures and applying different ripening times, it has been determined that the maturation process causes an increase in antioxidant and antihypertensive capacity and that fractions belonging to peptides with a molecular weight below 3 kDa show the highest bioactivity (Mejri et al., 2017). Luan et al. (2021), in their study investigating the effect of using Lactobacillus plantarum as a starter culture in traditional Chinese fermented sucuk on antioxidant activity, determined that the scavenging capacity of DPPH free radical increased to 75.14%. In the study, it was interpreted that mechanical disintegration increased becaue of the blade being attached to the head of the meat grinder during the filling of the sucuk dough into the intestines, and this caused excessive degradation of proteins and therefore peptide loss.

Mass spectrometry is one of the leading methods used in identifying peptides and determining their amino acid sequences (Kartal et al., 2023). Peptide identification of the fractions obtained on days 0, 1, 3, 5, and 10 of fermentation was made by LC-MS/MS, and the bioactivities of the peptide sequences determined in mass spectroscopy were scanned using the scanning function of the BIOPEP database. As a result of in silico hydrolysis of actin, collagen, and myosin from meat proteins with pepsin and trypsin enzymes, dipeptides were predominantly formed. Findings obtained from the BIOPEP database are given in Table 6.

According to LC-MS/MS mass spectrometry data of peptide fractions obtained from sucuk samples during 10 days of fermentation, a total of 10 different peptides were identified, including 6 different dipeptides (KD, LK, EL, KP, HL, and IR) and 3 different tripeptides (GPP, GAA, and RHA) with antioxidant activity and 1 tetrapeptide (CIRA) with antimicrobial activity. It was determined that the antimicrobial peptide was formed on the third day of fermentation. Kumari et al. (2022) reported that two antioxidant peptides (AIPPHPYP and IAEVFLITDPK) were identified in the Malaysian fermented fish product pekasam (Loma fish/Thynnichthys thynnoides); the naturally fermented fish product budu produced in Iran contains LDDPVFIH and VAAGRTDAGVH antioxidant peptides. They also reported that fermented meat sauce produced in Japan had high DPPH radical scavenging activity and the presence of QYP, an antioxidant tripeptide. Sun et al. (2022) reported that they successfully isolated and identified six antioxidant peptide sequences (GEHGDSSVPVWSGVN, HLDYYLGK, HLTPWIGK, DTYIRQPW, WDDMEKIWHH, and MYPGIAD) from duck liver protein hydrolyzate using alcalase. Vaštag et al. (2010) found that the antioxidant activity in protein extracts from fermented sucuks was two or three times higher in the final product than in the initial sucuk mixture. Korhonen and Pihlanto (2003) reported that antioxidant peptides generally have short sequences (2–20 amino acids long) and their molecular masses are in the range of approximately 400–3000 Da. When the chromatograms obtained by LC-MS/MS mass spectrometry and the antioxidant peptides identified using the BIOPEP database were examined in the study, it is seen that they mostly consist of two amino acids and their molecular masses are less than 3000 Da. Although peptides are generally more effective as antioxidants, free amino acids can also contribute to the bioactivity profile. Meat and fermented sucuks are important sources of free amino acids with antioxidant activity (Bou et al. 2016).

When the bioactive properties of antioxidant peptides identified using the BIOPEP database were examined in the study, it was determined that there was one tetrapeptide (CIRA) with antimicrobial activity. Peptides hydrolyzed by commercial enzymes from bovine sarcoplasmic protein were defined as GLSDGEWQ, GFHI, DFHING, and FHG by Arihara (2006), and the antimicrobial properties of these peptides were investigated.