Download

ORIGINAL ARTICLE

Metagenomic analysis of microbial diversity in sucuk, a traditional Turkish dry-fermented sausage, and its relationship with organic acid compounds

Ali Soyuçok*

Department of Food Processing, Burdur Food Agriculture and Livestock Vocational School, Burdur Mehmet Akif Ersoy University, Burdur, Türkiye

Abstract

Sucuk is a traditional Turkish fermented meat product that is widely consumed in Türkiye. The aim of this study was to determine the microbial diversity and organic acid profile and to elucidate their mutual relationship. The most abundant phylum in sucuk was Firmicutes, followed by Proteobacteria and Cyanobacteria phyla. The most abundant genera in sucuk were Lactobacillus, Pediococcus, and Staphylococcus. Acetic, lactic, and tartaric acids were found in all sucuk samples. Tartaric and lactic acids were positively correlated with microbial diversity parameters. Furthermore, tartaric acid was found to be an indicator of the presence of a rare genus, while lactic acid was found to be an indicator of a balanced distribution among genus and the dominance of some genus. This study for the first time showed that the microbiota of fermented Turkish sausage will be an important contribution to future studies.

Key words: Metagenomic, Microbiota, Organic acid, Sucuk, Turkish fermented sausage

*Corresponding Author: Ali Soyuçok, Burdur Mehmet Akif Ersoy University, Burdur Food Agriculture and Livestock Vocational School, Department of Food Processing, 15030, Burdur, Türkiye. Email: [email protected]

Academic Editor: Prof. Valentina Alessandria – Università di Torino, Italy

Received: 28 October 2024; Accepted: 16 December 2024; Published: 7 January 2025

DOI: 10.15586/ijfs.v37i1.2865

© 2025 Codon Publications
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0). License (http://creativecommons.org/licenses/by-nc-sa/4.0/)

Introduction

Sucuk is a traditional dry fermented sausage that is widely consumed in Türkiye. It is typically prepared using beef/buffalo/mutton, tail fat, salt, sugar, nitrite/nitrate, as well as garlic, cumin, allspice, black pepper, and red pepper. Sucuk dough is a mixture of these ingredients and is naturally fermented by microorganisms at appropriate temperature and time (Kilic, 2009).

The fermentation process of sucuk can last up to 20 days, with initial temperatures ranging from 12 to 26°C (Kaban and Kaya, 2008). Fermentation process contributes to sucuk’s flavor, aroma, and product qualities. Sucuk is characterized by its taste, which results from the complex interplay between its ingredients and microbial communities involved in fermentation (Liao et al., 2022).

Lactic acid bacteria (LAB) and coagulase-negative staphylococci (CNS) are responsible for sucuk fermentation. Starter cultures are widely used in industrial production to produce the same quality product and to inhibit foodborne pathogens. Commonly used culture mixtures are anaerobic LAB, especially Latilactobacillus, Lactiplantobacillus, Leuconostoc, Pediococcus, Lactococcus, and Enterococcus, while CNS are Staphylococcus xylosus and S. carnosus (Van Reckem et al., 2019).

LAB play a crucial role in decreasing the pH of sucuk by producing lactic acid, which inhibits the growth of spoilage and pathogenic bacteria (Akköse et al., 2023; Kamiloğlu et al., 2019). In addition, CNS contribute to the development of flavor and the overall safety of the product by producing antimicrobial compounds (Akköse et al., 2023; Kesmen et al., 2012). The presence of these bacteria not only improves the microbiological safety of sucuk but also influences its organoleptic properties (Demirel and Gürler, 2018).

According to the new taxonomic classification, the lactobacilli commonly found in fermented meat products are Companilactobacillus, Dellaglioa, Lacticaseibacillus, Lactiplantibacillus, Latilactobacillus, and Paucilactobacillus (Zheng et al., 2020). CNS that are frequently isolated from fermented meat products include S. xylosus, S. saprophyticus, and S. equorum (Kaban and Kaya, 2008). The color and flavor of sausage are affected by CNS owing to their enzymes, such as nitrate reductase, protease, and lipase. (Kaban et al., 2012).

The microbiota of fermented foods can be explained by culturomics and nonculturomic approaches. Nevertheless, culturomics has its drawbacks, as it may not fully represent the entire microbial population because of organisms that are difficult to culture or cannot be cultured at all (Parmar et al., 2018). In culture-based methods, microbial loss of samples occurs due to factors such as the selection of colonies with false morphological characteristics, the use of selective media, or the misinterpretation of biochemical tests.

On the other hand, a nonculturomic method such as next generation sequencing (NGS) provides an in-depth assessment of the microbial population without cultivation. Nonculturomic methods have advantages. NGS can detect microorganisms, especially those that are impossible to culture using traditional methods like researchers and different methodologies. NGS can be the vital key for optimizing fermentation processes and improving product quality (De Filippis et al., 2017).

In recent studies, the microbial diversity of fermented meat products, such as Korean dry-fermented sausages (Kim et al., 2022), Mediterranean spontaneously fermented sausages (Bassi et al., 2022), salami-type dry-fermented sausages from Brazil (Degenhardt et al., 2021), Italian sausages (Franciosa et al., 2021), Fuet fermented sausages (Yang et al., 2022), Felino-type sausages (Ferrocino et al., 2018), artisanal fermented sausages (Barbieri et al., 2021), and salami sausages (Liu et al., 2023) have been identified through metagenomic analysis. However, no metagenomic study has been found on the microbiota of sucuk-dry fermented meat products.

This research aimed to unravel sucuk production processes by explaining the relationship between microbial diversity and organic acid production, ensuring food safety and increasing the final product quality. For that purpose, this study examined the diverse microbial communities found in sucuk through metagenomic analysis and also assessed the organic acid content, which plays a key role in the development of the flavor and preservation of sucuk.

Material and Methods

Samples

Ten traditionally produced and fermented Turkish sucuk (Ts) samples obtained from local markets in Isparta-Türkiye were used in this study. The sucuk produced from beef were aseptically collected in July 2023 and stored at +4°C. Sucuk samples were aseptically divided into sterile containers for analysis.

Methods

DNA extraction

DNA extraction was performed according to the method described by Liu et al. (2004) and modified by Ucak et al. (2022). In summary, 10 g of sucuk sample was homogenized in 90 mL pepton water using ultra turrax (Ika, Germany). One milliliter of homogenate was added to a centrifuge tube and centrifuged (10,000 × g for 5 min at room temperature). The pellet was treated with 0.5 mL 1× TE buffer (containing 10 µg/mL lysostaphin and 4 mg/mL lysozyme) during 18 h at 37°C. After incubation, 10% SDS and 20 mg/mL proteinaz K were added to the lysed pellets. Finally, the extracted DNA was dissolved in 70 µL sterile ultrapure water. DNA was quantified using a Qubit 4 fluorometer (Thermofisher, Finland).

Library preparation

DNA library preparation was conducted according to the library preparation procedure for 16S metagenomic sequencing (Illumina Inc.,CA,USA). The primers containing overhang adapter sequences, forward primer:5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3’, and reverse primer:5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3’ were used for amplicon PCR experiments. In amplicon PCR, the 16S rRNA V3-V4 regions were amplified by KAPA HiFi HS Mix (Roche, Germany). After amplicon PCR, the samples were indexed with dual indexes using the Nextera® XT index Kit v2 Set-A (Illumina). For library preparation, all amplicons and indexed samples were cleaned using AMPure XP beads (Beckman Coulter, USA) on a magnetic rack (DynaMag™-96 Side, Invitrogen, Norway). The ratios of the samples were adjusted to equimolar amounts and subsequently diluted to a final concentration of 35 pmol DNA library. A 20 µL library containing 5% (v/v) PhiX control DNA (Illumina) was then loaded into an iSeq100 v1 cartridge (Illumina).

Metagenomic and bioinformatic analysis

Sequencing was performed using the iSeq100 system (Illumina) pair-end read type and two reads of 151 bp read length. Sequencing data were obtained using the iSeq100 system. The data were analyzed by 16S Metagenomics, V. 1.1.0 software (Illumina) following the manufacturer’s instructions.

The raw sequencing data consisted of 4 million reads generated by Illumina iSeq100. Quality control (QC) was performed using RTA2, where low-quality bases (quality score < Q30) and adapter sequences were removed. After QC, reads passed the filtering criteria and were retained for downstream analysis.

For taxonomic classification, the ribosomal database project (RDP) ensured high-accuracy taxonomic assignments. Reads were aligned and classified using 16S Metagenomics, V. 1.1.0 software (Illumina) with a confidence threshold of 97% for species-level identification. The operational taxonomic units (OTUs) were clustered at 98% identity threshold using DADA2.

All steps were performed according to standard pipelines, and the workflow was validated to ensure reproducibility. Raw data were deposited in NCBI Sequence Read Archive database.

Microbial diversity parameters such as Shannon species diversity index value, Species Richness, Evenness, Chao1 Index, Berger–Parker Dominance Index, Simpson Diversity Index, and Margalef Diversity Index were determined by the Biodiversity Component (BİÇEB) Calculation Software (https://kantitatifekoloji.net/biceb).

Organic acid analysis

Organic acid extraction from the sucuk samples was performed using water. Sucuk 5 g was weighed into a tube, and 25 mL of deionized water was added. After thorough homogenization in water, the tubes were kept in a shaker at 250 rpm for 4 h. After the tubes were centrifuged at 1900 × g for 15 min, the samples were prepared for injection by passing them through a 0.45 micron filter. The organic acid composition was determined using a Shimadzu LC2040 Prominence Brand HPLC system (Tokyo, Japan) with an LC20 AT pump and DAD detector. The mobile phase was 10 mM NH4H2PO4 (pH 2.6, H3PO4), the flow rate was 1 mL/min, the injection volume was 10 μL, and the column temperature was 40°C. Column oven CTO-10ASVp and InertSustain C18 5 µm 250 mm *4.6 mm column were used. The results were calculated using the LC Solution computer package program (Soyuçok, 2022).

Statistical analysis

Principal component analysis (PCA) was performed to determine the relationship between organic acids and microbial diversity in sucuk samples using the Unscrambler software (version 10.4, Oslo, Norway). For PCA, all variables were selected at an equal weight level. The full cross-validation method was used for the validation. Singular value decomposition (SVD) was selected as the model algorithm.

The distinction between bacterial diversity and organic acid compounds in the sucuk samples was visualized using a heatmapper (http://www.heatmapper.ca). The values are between −4(blue) and +4 (yellow) with zero in the middle. The rows are clustered, and the different color scale was used to clearly identify and visualize the different values on the heat map.

Results and Discussion

Microbial diversity

This study focused on species and genera that reached concentrations above 0.5% of OTUs in at least one of the samples. In total, more than 350 OTUs were identified, indicating that the sausage microbiota examined had an extremely rich biodiversity. This diversity demonstrates that microbial species vary significantly not only in numerical terms but also in their relative composition among samples.

The relative abundances of bacterial OTU belonging to the phylum, the class, the order, the family, the genus, and the species levels of sucuk samples were presented in figures. The top phylum classification results are shown in Figure 1A. The dominant phylum in the sucuk samples was Firmicutes, and it was followed by Proteobacteria, Cyanobacteria/Chloroplast, Bacteroidetes, and Actinobacteria. The top class classification results are shown in Figure 1B. In the sucuk samples, bacilli were abundant at the class level. The main order was Lactobacillales in all sucuk samples (Figure 1C). The most abundant bacterial families identified were Lactobacillaceae, Staphylococcaceae, and Leuconostocaceae in sucuk samples (Figure 1D).

Figure 1. The relative abundances by operational taxonomic units. (A) Bacterial diversities at the phylum level in sucuk samples, (B) Bacterial diversities at the class level in sucuk samples, (C) Bacterial diversities at the order level in sucuk samples, (D) Bacterial diversities at the family level in sucuk samples.

At the genus level, Lactobacillus genus was dominant in Ts2, Ts3, Ts6, Ts7, and Ts8 samples (Figure 2). Pediococcus was also the dominant genus in Ts1, Ts4, Ts5, and Ts10 samples. Ts9 was the only sucuk sample in which Lactobacillus and Pediococcus densities were balanced (Figure 2).

Figure 2. Bacterial diversities at the genus level in sucuk samples.

Analysis of bacterial diversity at the genus level in sucuk samples revealed significant variations (Table 1). The Shannon species diversity index fluctuated between 0.186 (Ts8) and 0.978 (Ts10). Richness values showed considerable differences, with Ts3 exhibiting the lowest at 38 and Ts10 the highest at 102. Evenness, which quantifies species distribution uniformity, ranged from 0.045 (Ts8) to 0.217 (Ts5). The Chao1 index, estimating species richness, spanned from 47.43 (Ts3) to 210.10 (Ts10).

Table 1. The bacterial diversity parameters at the genus level in sucuk samples.

Sample Name Shannon species diversity indexa Richnessb Evenness* Chao1 Index Berger–Parker Dominance Index Simpson Diversity Index Margalef Diversity Index MenhinickDiversity
Index
Ts1 0.448 63 0.108 79.50 1.104 0.176 5.580 0.244
Ts2 0.355 60 0.087 110.75 1.082 0.143 5.387 0.251
Ts3 0.543 38 0.149 47.43 1.176 0.264 3.244 0.127
Ts4 0.360 94 0.079 160.11 1.069 0.123 8.391 0.368
Ts5 0.891 61 0.217 76.55 1.342 0.416 5.520 0.266
Ts6 0.760 70 0.179 132.00 1.371 0.410 6.328 0.300
Ts7 0.461 67 0.110 139.50 1.104 0.177 5.827 0.233
Ts8 0.186 62 0.045 108.20 1.026 0.050 5.431 0.226
Ts9 0.868 61 0.211 91.60 1.995 0.529 5.492 0.259
Ts10 0.978 102 0.211 210.10 1.588 0.518 8.807 0.330

*: Evenness= a/ln(b).

The Berger–Parker dominance index demonstrated the lowest dominance in Ts8 (1.026) and the highest in Ts9 (1.995). The Simpson diversity index, measuring the likelihood of two individuals belonging to identical species, varied from 0.050 (Ts8) to 0.529 (Ts9). The Margalef diversity index, which reflects species richness in relation to sample size, ranged between 3.244 (Ts3) and 8.807 (Ts10). In addition, the Menhinick diversity index, another measure of species richness, spanned from 0.127 (Ts3) to 0.368 (Ts4). These results underscore the variability in bacterial composition and richness among the analyzed sucuk samples.

According to diversity parameters, such as Shannon species diversity index, Evenness, Berger–Parker Dominance Index, Simpson Diversity Index, Margalef Diversity Index, and Menhinick Diversity Index, the balanced and highest microbial diversity at the genus level was found in Ts5, Ts6, Ts9, and Ts10 (Table 1). It is seen in Figure 2 that no dominant species was found in the Ts9 sample, and this is supported by Table 1. The Berger–Parker Dominance Index and Simpson Diversity Index and Evenness values of Ts2, Ts4, and Ts8 samples were found to be very weak in terms of microbial diversity, as they had low values (Table 1, Figure 2). The microbial diversity parameter values of Ts1, Ts3, and Ts7 samples were very poor in terms of microbial diversity, as they had moderated values (Table 1).

The dominant bacterial species was P. pentosaceus in Ts1, Ts5, and Ts6 samples (Figure 3). For Ts4, Ts7, Ts9, and Ts10 samples, P. lolii was found to be the major species. Llb. graminis was found to be the dominant species in Ts3 and Ts 8 samples. S. xylosus was only found as a major bacterial species in Ts2 sample (Figure 3).

Figure 3. Bacterial diversities at the species level in sucuk samples.

According to Evenness and Berger–Parker Dominance Index, one species was found to be dominant in Ts1, Ts3, Ts4, and Ts9 sucuk samples, and the species found were P. pentosaceus, Llb. graminis, P. Lolii, and P. pentosaceus, respectively (Figure 3). The balanced and highest microbial diversity at the species level was observed in Ts2, Ts7, and Ts8 samples (Table 2). The microbial diversity parameter values of Ts5, Ts6, and Ts10 samples were poor in terms of microbial diversity, as they were average values (Table 2).

Table 2. The bacterial diversity parameters at the species level in sucuk samples.

Sample Name Shannon species diversity index valuea Richnessb Evenness* Chao1 Index Berger–Parker Dominance Index Simpson Diversity Index Margalef Diversity Index MenhinickDiversity Index
Ts1 0.15 97 0.033 145.23 1.019 0.038 8.717 0.394
Ts2 1.53 83 0.346 143 1.553 0.558 9.955 1.350
Ts3 0.098 66 0.023 102.25 1.013 0.025 6.110 0.323
Ts4 0.22 142 0.044 227.45 1.029 0.056 12.788 0.573
Ts5 0.49 98 0.107 167.46 1.105 0.178 9.103 0.476
Ts6 0.47 109 0.100 208 1.073 0.131 11.338 0.931
Ts7 1.65 118 0.346 194 1.873 0.662 13.275 1.439
Ts8 0.806 90 0.179 168.3 1.160 0.255 9.861 0.987
Ts9 0.273 111 0.058 205 1.042 0.078 10.798 0.681
Ts10 0.56 149 0.112 282 1.152 0.239 13.303 0.572

*: Evenness= a/ln(b).

Traditional sucuk microflora include a variety of microorganisms, especially LAB, molds, and yeasts. These organisms play key roles in sucuk fermentation and ripening. Species such as Lactobacillus, Staphylococcus, and Micrococcus have a significant effect on this process. LAB reduce the pH of sucuk by producing lactic acid and bacteriocins and inhibiting the growth of pathogenic organisms. Simultaneously, it contributes to the emergence of various sensory properties by modifying the raw material (Nazlı et al., 2017). Typically, the pH level in dry fermented sausages ranges from 4.5 to 5.5 (De Mey et al., 2017). Sucuk also falls within this range, with a pH value of 5.4 or lower (Anonymous, 2019). As stated by Özdal (2020), these positive effects of LAB improve the overall quality of sucuks and contribute to improving their taste, color, and texture.

In traditionally fermented meat products, fermentation occurs spontaneously, or the natural microbial flora in the environment is influenced by various parameters, such as raw material, spices, fermentation temperature, and duration (Stavropoulou et al., 2018a). Cinar et al. (2018) reported that Lpb. plantarum was dominant in fermented sausages. In a similar study, Lpb. plantarum strains found in sucuk were reported to have antagonistic activity against S. aureus, Listeria monocytogenes, and Bacillus cereus (Kamiloğlu et al., 2020). It has been suggested that the bacteriocin produced by Llb. curvatus isolated from fermented meat products inhibits L. monocytogenes and is therefore used in the production of fermented meat products (Casaburi et al., 2016). The use of Llb. curvatus, Llb. Sakei, and Lpb. plantarum in fermented meat products reduces the amount of biogenic amines in sucuk (Doğan et al., 2020).

The use of high amounts of salt increases the growth of CNS, but differences in salt levels (2–4%) do not affect the biodiversity of CNS (Charmpi et al., 2020; Van Reckem et al., 2019). The low temperature fermentation process with natural microflora increases the growth of S. equorum, S. Saprophyticus, and S. xylosus, while the high temperature enhances the growth of S. lugdunensis and S. aureus (Charmpi et al., 2020; Stavropoulou et al., 2018b).

In Ts1, Ts3, Ts4, Ts6, Ts8, and Ts9, most of the ASVs were attributed to a single species, and the other samples were characterized by higher biodiversity, with an important diversification in the composition of microbiota. Among LAB, Llb. graminis was the major species (>80% of ASVs in Ts3 and Ts8). The Llb. sakei group divides into four species, renamed after the new taxonomy. These are L. sakei, L. graminis, L. curvatus, and L. fuchuensis (could not be discriminated using 16S rRNA sequence) (Zheng et al., 2020). These two types of microorganisms were expressed as L. sakei/L. graminis together in a study in which microbial diversity in the pastırma was determined using the fingerprint method (Metin and Toy, 2023). Therefore, the results of this study were compared with those of L. sakei reported in the literature. Although LAB species diversity in fermented meat products was limited, L. sakei was predominant during the ripening process, due to the species’ excellent adaptation, competitiveness, and assertiveness in the meat medium (Aquilanti et al., 2016; Janßen et al., 2018; Stavropoulou et al., 2018b). This superiority over other LAB can be attributed to its salt-tolerant and psychrotrophic nature and the use of the arginine deiminase pathway and nucleosides in the meat environment (Fontana et al., 2016).

Organic acid components of sucuk samples

The organic acid compositions are listed in Table 3. Tartaric acid, lactic acid, and acetic acid were found in all sausage samples (Table 3). The lowest level of tartaric acid was found in the Ts1 sample at 3.22 mg/g and the highest level was found in the Ts9 sample at 6.00 mg/g. Lactic acid was lowest in the Ts2 sample at 1.28 mg/g and highest in the Ts9 sample at 3.59 mg/g. The lowest level of acetic acid was found in the Ts1 sample at 0.72 mg/g; the highest level was found in the Ts10 sample at 3.89 mg/g. Oxalic acid was detected in Ts4, Ts5, Ts6, Ts7 and Ts9 at 0.23, 0.17, 1.62, 0.15, and 0.22 mg/g, respectively. Malonic acid was found only in the Ts1 (2.01 mg/g) and Ts2 (2.78 mg/g) samples. Citric acid was highest in Ts1 (1.91 mg/g), followed by Ts5 (0.93 mg/g), Ts2 (0.71 mg/g), Ts3 (0.45 mg/g), and Ts7 (0.41 mg/g). Succinic acid was found in all samples, except Ts1 and Ts2. Propionic acid was found at 27.09, 19.81, 35.50, 39.52, and 35.03 mg/g in Ts1, Ts2, Ts3, Ts4, and Ts5 samples, respectively.

Table 3. Organic acid content of sucuk samples.

Sample Organic acid (mg/g)
Oxalic acid Tartaric
acid
Malonic acid Lactic acid Acetic acid Citric acid Succinic
acid
Propionicacid
Ts1 n.d 3.22 2.01 1.35 0.72 1.91 n.d 27.09
Ts2 n.d 4.01 2.78 1.28 1.03 0.71 n.d 19.81
Ts3 n.d 5.64 n.d 2.02 2.18 0.45 7.63 35.50
Ts4 0.23 4.18 n.d 2.30 2.65 n.d 8.55 39.52
Ts5 0.17 4.34 n.d 3.06 2.65 0.93 2.47 35.03
Ts6 1.62 4.32 n.d 2.43 1.26 n.d 6.07 n.d
Ts7 0.15 3.72 n.d 1.84 3.08 0.41 6.51 n.d
Ts8 n.d 3.68 n.d 2.36 1.60 n.d 6.88 n.d
Ts9 0.22 6.00 n.d 3.59 2.99 n.d 9.27 n.d
Ts10 n.d 5.80 n.d 2.80 3.89 n.d 7.38 n.d

n.d: not detected.

Carbohydrate fermentation in fermented meat products produces organic acids that affect the texture and sensory properties of the final product by lowering its pH. Acid formation depends on the type and concentration of sugars present, sheath diameter, and other technological factors, particularly the type of bacteria (Bangar et al., 2022). During the fermentation and drying/maturation stages, LAB utilize the sugars added to fermented meat products, resulting in the formation of lactic acid, which is the primary organic acid responsible for the decrease in pH of fermented meat products. The use of sugar in these products inhibits pathogenic and spoilage bacteria because of the desired decrease in pH and contributes to the typical organoleptic character of the product (Hwang et al., 2023).

The production of organic acids affects the sensory properties, such as the taste and aroma of sucuk (Laranjo et al., 2019). Fermentation parameters such as temperature, ingredients, and fermentation conditions significantly change microbial diversity, leading to variations in organic acids and thus the overall flavor and preservation characteristics of sucuk (Baka et al., 2011). The presence of specific LAB strains can enhance desirable organic acid production, further contributing to the flavor and safety of the product by inhibiting undesirable microbial growth (Laranjo et al., 2019). Besides lactic acid, other organic acids, including acetic and propionic acids, may also be present in sucuk. These acids can be produced as secondary metabolites during fermentation and contribute to the overall flavor profile of sausages (Nediani et al., 2017).

Organic acids in fermented meat products are typically classified into two groups, with lactic acid, succinic acid, and acetic acid being the desired acids, and citric, malonic, pyruvic, formic, butyric, and propionic acids being the undesirable acids that should not exceed certain levels (Erginkaya, 1993). This classification is based on the fermentation method used, with the desired acids being formed homofermentatively and the undesired acids being formed heterofermentatively (Erginkaya, 1993). Organic acids are formed by the fermentation of sugars, acetic acid is also formed through fatty acid oxidation and alanine catabolism, and propionic and butyric acid are formed through the oxidation of aldehydes (Ravyts et al., 2012). Production temperature, starter cultures, and sugar content are the main factors that affect acid formation during the fermentation of meat products (Halagarda and Wójciak, 2022). In production, it is important to determine the ideal temperature and acidity level by considering product-specific acidity levels and microbial flora.

Relationship between microbial communities and organic acids

Eighty percent of the total variance is explained by PC1 and PC2 (Figure 4A). The percentage of variance explained by PC1 and PC2 was 52% and 28%, respectively. Only oxalic acid is located on the positive PC1 line. All the other loadings were located on the negative PC1 line (Figure 4B). All bacterial genera, such as Psychrobacter, Lactobacillus, Acinetobacter, Streptophyto, Streptococcus, and Bacillus, and some organic acids (lactic acid, acetic acid, and succinic acid) were the variables with the highest contribution to the PC (Figure 4B). A 50% variance was explained by genera, such as Carnobacterium, Leuconostoc, Weisella, and oxalic acid. Thus, these loadings did not contain enough structured variants to be discriminated against in sucuk samples. The loadings in the outer ellipse explained 100% of the variance (Figure 4C).

Figure 4. (A) The effects of PC on total variance. (B) The contribution of loadings to PC 1. (C) Correlation loadings of relationships between organic acid and microbial diversity at the genus level.

Ts2, Ts3, and Ts8 were separated from the other sucuk samples in terms of microbial diversity and organic acid compounds by PC1 (Figure 5). The heatmap also supports these results (Figure 6). The heat map shows two major clusters (Figure 6). The first subunit of the first cluster contained only Ts9. The second subunit of the first cluster consisted of Ts6, a subbranch of Ts2-Ts3, and a subbranch of Ts7-Ts8. The first subunits of the second cluster were the Ts5 and Ts4-Ts1 branches. The second subunit of the second cluster consisted of only Ts10. Characteristic differences in the sucuk samples were revealed using both PCA and heat map.

Figure 5. The results of PC1 versus PC2 of sucuk samples.

Figure 6. Cluster analysis of a heat map showing the relationship between organic acid and microbial diversity at the genus level.

The correlation results between the organic acid and microbial diversity parameters at the species level are given in Table 3. According to the results, the correlation between tartaric acid and richness and Chao1 index was significant at 0.05 level (r=0.646 and 0.691, respectively). The highest correlation between lactic acid and microbial diversity parameters was found in Berger–Parker Dominance Index (r=0.801) at 0.01 level. In addition, lactic acid and evennes (r=0.686), Shannon species diversity index (r=0.693), and Simpson Diversity Index (r=0.735) values were correlated at 0.05 level. NGS was used to identify the microbial structures of sucuks to overcome the limitations in identifying bacterial populations. This study is unique in that it is the first study on sucuk. The presence of abundant bacteria at the phylum, the genus, and the species levels was identified. The abundant phylum was Firmicutes. The most species were Lactobacillus, Pediococcus, and Staphylococcus. P. loli, P. Pentosaceus, and Llb. graminis were the dominant species. Lactic acid, tartaric acid, and acetic acid were commonly found in sucuks. When the relationship between organic acids and microbial diversity parameters was analyzed, it was found that only tartaric acid and lactic acid were positively correlated with some parameters. Tartaric acid was generally correlated with parameters indicating the presence of rare species. Lactic acid, on the other hand, was found to be correlated with the parameters that dominate ordam and indicate a balanced distribution of microbial diversity. It may also suggest that lactic acid increases the imbalance between species in the ecosystem and causes a certain species to dominate over others. Particularly high concentrations of lactic acid may have negative effects on species richness and balance in the ecosystem, as a dominant species may suppress other species or cause them to compete. The comprehensive metagenomic analysis conducted with sucuk can enlighten the microbial structures of the products in different regions of the world and strengthen their applications in the food industry.

Data Availability Statement

Raw sequence data obtained in this study using next generation sequencing were submitted to the NCBI Sequence Read Archive database with BioProject accession number PRJNA1195933.

Authors Contribution

Ali Soyuçok: Conceptualization, data curation, formal analysis, investigation, methodology, software, validation, visualization, and writing original draft. The author read and approved the final manuscript.

Conflicts of Interest

The author declare no conflict of interest.

Funding

None.

Acknowledgements

The author would like to thank the Burdur Mehmet Akif Ersoy University Dairy Products and Technologies Application and Research Center for the use of laboratory facilities.

REFERENCES

Aquilanti L., Garofalo C., Osimani A., and Clementi F. 2016. Ecology of lactic acid bacteria and coagulase negative cocci in fermented dry sausages manufactured in Italy and other Mediterranean countries: an overview. Int. Food Res. J. 23(2):429–445.

Akköse A., Oğraş Ş.Ş., Kaya, M and Kaban G. 2023. Microbiological, physicochemical and sensorial changes during the ripening of sucuk, a traditional Turkish dry-fermented sausage: effects of autochthonous strains, sheep tail fat and ripening rate. Fermentation, 9(6):558. 10.3390/fermentation9060558

Anonymous. 2019. Turkish food codex Communiqu´e on meat, prepared meat mixtures and meat products, No.: Codon Publications 2018/52 (https://www.resmigazete.gov.tr/eskiler/2019/01/20190129-4.htm), Last access date: 10/12/2024.

Baka A.M., Papavergou E.J., Pragalaki T., Bloukas J.G., and Kotzekidou P. 2011. Effect of selected autochthonous starter cultures on processing and quality characteristics of Greek fermented sausages. LWT. 44 (1):54–61. 10.1016/j.lwt.2010.05.019

Bangar S.P., Suri S., Trif M., and Ozogul F. 2022. Organic acids production from lactic acid bacteria: a preservation approach. Food biosci. 46:101615. 10.1016/j.fbio.2022.101615

Barbieri F., Tabanelli G., Montanari C., Dall’Osso N., Šimat V., Smole Možina S., et al. 2021. Mediterranean spontaneously fermented sausages: spotlight on microbiological and quality features to exploit their bacterial biodiversity. Foods. 10(11):2691. 10.3390/foods10112691

Bassi D., Milani G., Belloso Daza M.V., Barbieri F., Montanari C., Lorenzini S., et al. 2022. Taxonomical identification and safety characterization of Lactobacillaceae from Mediterranean natural fermented sausages. Foods. 11(18):2776. 10.3390/foods11182776

Casaburi A., Di Martino V., Ferranti P., Picariello L., and Villani, F. 2016. Technological properties and bacteriocins production by Lactobacillus curvatus 54M16 and its use as starter culture for fermented sausage manufacture. Food Contr. 59, 31–45. 10.1016/j.foodcont.2015.05.016

Charmpi C., Van der Veken D., Van Reckem E., De Vuyst L., and Leroy F. 2020. Raw meat quality and salt levels affect the bacterial species diversity and community dynamics during the fermentation of pork mince. Food microbiol. 89:103434. 10.1016/j.fm.2020.103434

Cinar K., Kaban G., Borekci B.S., Gulluce M., Karadayi M., and Kaya M. 2018. Identification and characterization of lactic acid bacteria isolated from sucuk, a traditional Turkish dry-fermented sausage. J. Biotech. 280S,S61–S62. 10.1016/j.jbiotec.2018.06.198

De Filippis F., Parente E., and Ercolini D. 2017. Metagenomics insights into food fermentations. Microb. Biotechnol. 10(1):91–102. 10.1111/1751-7915.12421

Degenhardt R., Sobral Marques Souza D., Acordi Menezes L.A., de Melo Pereira G.V., Rodríguez-Lázaro D., Fongaro G., et al. 2021. Detection of enteric viruses and core microbiome analysis in artisanal colonial salami-type dry-fermented sausages from Santa Catarina, Brazil. Foods. 10(8):1957. 10.3390/foods10081957

Demirel Y.N., and Gürler, Z. 2018. The effect of natural microbiota on colour, texture and sensory properties of sucuk during the production. Ank. Univ. Vet. Fak. Derg. 65(2):137–143.

De Mey E., De Maere H., Paelinck H., and Fraeye I. 2017. Volatile N-nitrosamines in meat products: potential precursors, influence of processing, and mitigation strategies. Crit. Rev. Food Sci. Nutr. 57(13):2909–2923. 10.1080/10408398.2015.1078769

Doğan Y.N., Lenger Ö.F., Düz M., Doğan I., and Gürler Z. 2020. Effects of wild type lactic acid bacteria on histamine and tyramine formation in sucuk. J. Hellenic Vet. Med. Soc. 71:2553–2558. 10.12681/jhvms.25936

Erginkaya Z. 1993. Fermente sucuklarda organik asit miktarlarının belirlenmesi. Gıda, 18:6

Ferrocino I., Bellio A., Giordano M., Macori G., Romano A., Rantsiou K., et al. 2018. Shotgun metagenomics and volatilome profile of the microbiota of fermented sausages. Appl. Environ. Microbiol. 84(3):2120. 10.1128/AEM.02120-17

Fontana C., Bassi D., López C., Pisacane V., Otero M.C., Puglisi E., et al. 2016. Microbial ecology involved in the ripening of naturally fermented llama meat sausages. A focus on lactobacilli diversity. Int. J. Food Microbiol. 236:17–25. 10.1016/j.ijfoodmicro.2016.07.002

Franciosa I., Ferrocino I., Giordano M., Mounier J., Rantsiou K., and Cocolin L. 2021. Specific metagenomic asset drives the spontaneous fermentation of Italian sausages. Food Res. Int. 144:110379. 10.1016/j.foodres.2021.110379

Halagarda M., and Wójciak K.M. 2022. Health and safety aspects of traditional European meat products. A review. Meat Sci. 184:108623. 10.1016/j.meatsci.2021.108623

Hwang J., Kim Y., Seo Y., Sung M., Oh J., and Yoon Y. 2023. Effect of starter cultures on quality of fermented sausages. Food Sci. Anim. Resour. 43(1):1–9. 10.5851/kosfa.2022.e75

Janßen D., Eisenbach L., Ehrmann M.A., and Vogel R.F. 2018. Assertiveness of Lactobacillussakei and Lactobacillus curvatus in a fermented sausage model. Int. J. Food Microbiol. 285:188–197. 10.1016/j.ijfoodmicro.2018.04.030

Kaban G., and Kaya M. 2008. Identification of lactic acid bacteria and gram-positive catalase-positive cocci isolated from naturally fermented sausage (sucuk). J. Food. Sci. 73(8): 385–388. 10.1111/j.1750-3841.2008.00906.x

Kaban G., Kaya M., and Lücke F.K. 2012. Meat starter cultures. Taylor & Francis. New York.

Kamiloğlu A., Kaban G., and Kaya M. 2019. Effects of autochthonous Lactobacillus plantarum strains on Listeria monocytogenes in sucuk during ripening. J. Food Saf. 39(3):e12618. 10.1111/jfs.12618

Kamiloğlu A., Kaban G., and Kaya M. 2020. Technological properties of autochthonous Lactobacillus plantarum strains isolated from sucuk (Turkish dry-fermented sausage). Braz. J. Microbiol. 51(3):1279–1287. 10.1007/s42770-020-00262-9

Kesmen Z., Yetiman A.E., Gulluce A., Kacmaz N., Sagdic O., Çetin B, et al. 2012. Combination of culture-dependent and culture-independent molecular methods for the determination of lactic microbiota in sucuk. Int. J. Food Microbiol. 153(3):428–435. 10.1016/j.ijfoodmicro.2011.12.008

Kilic B. 2009. Current trends in traditional Turkish meat products and cuisine. LWT. 42(10):1581–1589. 10.1016/j.lwt.2009.05.016

Kim J.H., Lee E.S., Kim B.M., and Oh M.H. 2022. Potential correlation between microbial diversity and volatile flavor compounds in different types of Korean dry-fermented sausages. Foods. 11(20):3182. 10.3390/foods11203182

Laranjo M., Potes M.E., and Elias M. 2019. Role of starter cultures on the safety of fermented meat products. Front. Microbiol. 10:853. 10.3389/fmicb.2019.00853

Liao R., Xia Q., Zhou C., Geng F., Wang Y., Sun Y., et al. 2022. LC-MS/MS-based metabolomics and sensory evaluation characterize metabolites and texture of normal and spoiled dry-cured hams. Food Chem. 371:131156. 10.1016/j.foodchem.2021.131156

Liu D., Ainsworth A.J., Austin F.W., and Lawrence M.L. 2004. Use of PCR primers derived from a putative transcriptional regulator gene for species-specific determination of Listeria monocytogenes. Int. J. Food Microbiol. 91(3):297–304. 10.1016/j.ijfoodmicro.2003.07.004

Liu Y., Cao Y., Yohannes Woldemariam K., Zhong S., Yu Q., and Wang J. 2023. Antioxidant effect of yeast on lipid oxidation in salami sausage. Front. Microbiol. 13:1113848. 10.3389/fmicb.2022.1113848

Metin B., and Toy A. 2023. Dynamics of lactic acid bacteria during pastırma production. JMBFS. 12(4):9071. 10.55251/jmbfs.9071

Nazlı B., Pehlivanoglu H., and Caglar M.Y. 2017. Characteristics of traditional Turkish fermented soudjouk and current situation. Int. J. Vet. Sci. Techno. 1(1):13–019.

Nediani M.T., García L., Saavedra L., Martínez S., Lopez Alzogaray S., and Fadda S. 2017. Adding value to goat meat: biochemical and technological characterization of autochthonous lactic acid bacteria to achieve high-quality fermented sausages. Microorganisms. 5(2):26. 10.3390/microorganisms5020026

Özdal T. 2020. Changes in physicochemical, microbiological and sensory characteristics of traditionally produced Turkish sucuk during ripening and storage: natural or synthetic additives? Gıda. 45(2):329–339. 10.15237/gida.GD20013

Parmar S., Li Q., Wu Y., Li X., Yan J., Sharma V.K., et al. 2018. Endophytic fungal community of Dysphania ambrosioides from two heavy metal-contaminated sites: evaluated by culture-dependent and culture-independent approaches. Microb. Biotechnol. 11(6):1170–1183. 10.1111/1751-7915.13308

Ravyts F., Vuyst L., and Leroy F. 2012. Bacterial diversity and functionalities in food fermentations. Eng. Life. Sci. 12(4):356–367. 10.1002/elsc.201100119

Soyuçok A. 2022. Fermantasyon ve Kurutma Boyunca Tarhana Hamurunda Meydana Gelen Organik Asit Kinetiğinin Belirlenmesi. Van Vet. J. 33(3):130–134. 10.36483/vanvetj.1182691

Stavropoulou D.A, Reckem E.V., Smet S.D., Vuyst L.D., and Leroy F. 2018a. The narrowing down of inoculated communities of coagulase-negative staphylococci in fermented meat models is modulated by temperature and pH. Int. J. Food Microbiol. 274:52–59. 10.1016/j.ijfoodmicro.2018.03.00

Stavropoulou D.A., Filippou P., De Smet S., De Vuyst L., and Leroy F. (2018b). Effect of temperature and pH on the community dynamics of coagulase-negative staphylococci during spontaneous meat fermentation in a model system. Food microbiol. 76:180–188. 10.1016/j.fm.2018.05.006

Ucak S., Yurt M.N.Z., Tasbasi B.B., Acar E.E., Altunbas O., Soyucok A., et al. 2022. Identification of bacterial communities of fermented cereal beverage Boza by metagenomic analysis. LWT. 153:112465. 10.1016/j.lwt.2021.112465

Van Reckem E., Geeraerts W., Charmpi C., Van der Veken D., De Vuyst L., and Leroy F. 2019. Exploring the link between the geographical origin of European fermented foods and the diversity of their bacterial communities: the case of fermented meats. Front. Microbiol. 10:2302. 10.3389/fmicb.2019.02302

Yang P., Zhong G., Yang J., Zhao L., Sun D., Tian Y., et al. 2022. Metagenomic and metabolomic profiling reveals the correlation between the microbiota and flavor compounds and nutrients in fermented sausages. Food Chem. 375:131645. 10.1016/j.foodchem.2021.131645

Zheng J., Wittouck S., Salvetti E., Franz C.M., Harris H., Mattarelli P., et al. 2020. A taxonomic note on the genus Lactobacillus: description of 23 novel genera, emended description of the genus Lactobacillus Beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae. Int. J. Syst. Evol. Microbiol. 70(4):2782–2858. 10.1099/ijsem.0.004107