Genotypic diversity and antagonistic activities of enterococci isolated from pastırma

Abstract

The biodiversity of enterococci from pastırma (a traditional Turkish dry-cured meat product) by genotypic identification and the antagonistic activities of strains were investigated. Pastırma samples taken from 20 different small-scale factories were subjected to microbiological and physicochemical analysis. A hundred enterococci isolates were identified by 16S rRNA gene sequence analysis. To determine antagonistic activity of strains, Listeria monocytogenes and Staphylococcus aureus were used. The lactic acid bacteria and Micrococcus/Staphylococcus counts were ≥ 6 log cfu/g in 55% of the samples and 75% of the samples, respectively. Enterobacteriaceae was generally below the detectable level (< 2 log cfu/g). The enterococci count was higher than 6 log cfu/g in 30–35% of the samples, depending on the medium used. The enterococci isolates (100 isolates) were identified as E. faecium (80 strains), E. faecalis (19 strains) and E. hirae (1 strain) in genotypic identification. The nine E. faecium strains showed antagonistic activity against L. monocytogenes in the well diffusion test. In contrast, in the same antagonistic activity test, all of the strains had no antagonistic activity against S. aureus. Further studies could be planned to characterize E. faecium strains that show antagonistic activity against L. monocytogenes.

Introduction

Pastırma is a traditional Turkish dry-cured meat product that is made from whole muscle and/or muscles of certain parts of beef or water buffalo carcasses. In the production, there are many stages such as curing, pressing and drying. The production period of pastırma may take up to four weeks depending on the size of muscle/muscles used and process conditions. The important properties of the product are that no heat treatment or smoking is applied during its production. Another property of the product is the coating with a paste called çemen which consists of flour ground from fenugreek (Trigonella foenum graecum), mashed fresh garlic and red pepper (Kaban 2013). Lactic acid bacteria and coagulase negative staphylococci are two important microorganism groups having an importance in the production of pastırma (Kaban 2013; Kaya and Kaban 2016). Yeast is another microorganism group taking place in the pastırma microbiota and their number may show high variability (Kaya and Kaban 2016).

Lactic acid bacteria count shows a great variation due to the fact that pastırma production is carried out with traditional methods. The count of lactic acid bacteria changing between 10² and 10⁸ cfu/g had been detected in pastırma (Kaban 2013; Özdemir et al. 1999). However, the studies on isolation and identification of these microorganisms from pastırma are too limited (Özdemir et al. 1999; Dinçer and Kıvanç 2012; Öz et al. 2017). In these studies, enterococci could either not be isolated or isolated in a low ratio, since the mediums used for isolation purposes are not specific to enterococci.

Enterococci are belonging to the lactic acid bacteria group. They are the Gram-positive, catalase-negative and facultative anaerobic cocci. Enterococci are highly tolerant to diverse environmental conditions. They are able to grow in the presence of 6.5% NaCl, at pH 9.6 and at temperatures ranging from 10 to 45 °C and could also survive heat-treatment at 60 °C for 30 min (Franz et al. 2003). Enterococci are ubiquitous bacteria in the gastrointestinal tract of both the humans and the animals. They are also found in the soil, surface waters, plants and vegetables (Pesavento et al. 2014). These bacteria produce L-lactic acid from the hexoses through the homofermentative lactic acid fermentation (Franz et al. 2003).

Enterococci play an important role in the production of various traditional fermented products (Franz et al. 2003). These microorganisms could survive and also grow in the fermented sausages with high pH (Talon et al. 2007). In addition, it is reported that they could be used for the control of some foodborne pathogens due to the production of bacteriocin (Franz et al. 2003, 2001; Callewaert et al. 2000; Hugas et al. 2003). However, it is known that some enterococci cause to infections (Foulquie Moreno et al. 2006). On the other hand, the findings that the enterococci isolated from many foods including the meat products, especially E. faecium and E. faecalis have very low pathogenic potential compared to the clinical strains have increased the interest in enterococci (Hugas et al. 2003). Some Enterococcus members are used as probiotics, and as additives for silage and dietary food supplements. In addition, it has been reported that some Enterococcus strains are used as a starter culture or protective culture in the food industry (Žugić Petrović et al. 2020).

The aim of the study was to determine the biodiversity of enterococci from pastırma obtained from 20 different small factories by genotypic identification and the antagonistic activities of strains. The microbiological and physicochemical properties of pastırma samples were also investigated.

Materials and methods

Material

Pastırma samples taken from 20 different small-scale factories applying traditional methods were used as the material in the study.

pH and moisture analysis

10 g of samples was weighed and mixed in 100 ml distillated water, and then it was homogenized with Ultra-Turrax for 1 min and pH value was determined using pH-meter. Moisture content was determined at 105 °C until the weight remains constant (Gökalp et al. 1999).

Microbiological analysis

25 g of samples was mixed with 225 ml sterile physiological water (0.85 NaCl%, Merck) and then homogenized in Stomacher (Lab Stomacher Blander 400-BA 7021, Sewardmedical, England). After that, the serial dilutions were prepared using sterile physiological water (0.85% NaCl) from this homogenate. For enumeration of lactic acid bacteria and Enterobacteriaceae, de Man Rogosa Sharpe (MRS, Merck, Darmstadt, Germany) Agar and Violet Red Bile Dextrose (VRBD, Merck) Agar were used, respectively. The numbers were determined after the incubation for 2 days at 30 °C under anaerobic conditions. Micrococcus/Staphylococcus were enumerated on Mannitol Salt Phenol Red (MSA, Merck) Agar, incubation was carried out for 2 days at 30 °C in aerobic conditions (Baumgart et al. 1993). Dichloran Rose Bengal Chloramphenicol (DRBC, Merck) Agar was used in the determination of yeast and mold number. The number was determined after the incubation under aerobic conditions for 3–5 days at 25 °C (Mislivic et al. 1992).

For the enumeration of Enterococcus, three different [Enterococcus Agar (Merck), KF Streptococcus Agar (Merck) and Slanetz-Bartley Agar (Oxoid)] mediums were used. After incubation for 48 h at 37 °C, typical colonies were enumerated. At least five colonies were selected from each selective medium and purified by streaking on BHI agar. Isolates were subjected to verification tests Gram (+, catalase, bile-esculin, growth at 6.5% NaCl, PYR (L-pyrrolidonyl-β-naphthylamide) hydrolysis, gas production from glucose and the growth at pH 9.6 (Ertekin 2016).

Genotypic identification of enterococci isolates

A hundred enterococci isolates giving positive result in the verification tests were subjected to genotypic identification. The colonies growing on BHI agar were taken to the microcentrifuge tubes, and total genomic DNA was extracted from pure culture according to Barış (2009). The sequence analysis was conducted in Macrogen Inc. The primers 27F (5’-AGAGTTTGATCMTGGCTCAG -3’) and 1492R (5’- TACGGYTACCTTGTTACGACTT-3’) were used for the extension reactions. ABI PRISM 3730XL automatic DNA sequencer (http://www.macrogen.com) was used in the sequence analysis. The results obtained were compared to the previously deposited sequences with the use of the Basic Local Alignment Search Tool (BLAST, http://www.ncbi.nlm.nih.gov/blast) program from the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov) database via the internet. In the study, these sequences determined were assigned Genbank database under accession numbers KU291269-KU291280, KU321604-KU321635 and KU359782-KU359837. These strains with the accession number are preserved in Food Engineering laboratories of Atatürk University (Erzurum, Türkiye) and Munzur University (Tunceli, Türkiye).

Antagonistic activity tests

Genotypically identified 100 strains were tested for their antagonistic activities against L. monocytogenes ATCC 7644 and S. aureus ATCC 25923. Agar spot and well diffusion tests given by Schillinger and Lücke (1989) were used to determine the antagonistic activity of the strains.

Results and discussion

pH value and moisture content

The percentage frequency distributions of pH value and moisture content in pastırma samples were given in Figs. 1 and 2, respectively. The pH value of pastırma should not fall below 5.5 due to the sensory properties (Kaban 2013). As can be seen in Fig. 1, pH value in only 20% of the samples was under 5.5. According to the Turkish Food Codex Communiqué on Meat and Meat Products, pH value in pastırma must not exceed 6.00 (Anonymous 2019). In the present study, pH value was above 6 only in three samples (15%) (Fig. 1). It has been assumed that this result is probably due to the raw material or the very intensive proteolysis during production (Kaya and Kaban 2016). On the other hand, in another study conducted on samples of pastırma obtained from the market, the pH was found to range between 5.29 and 6.65 (Öz et al. 2017). As can be seen Fig. 2, moisture content was below 50% in 35% of the pastırma samples, 60% of them had a moisture content of 50–55%. The moisture content was found to be over 55% only in one sample. According to the results, pastırma showed a great variation in terms of moisture content. The pH value and moisture content of the samples can vary greatly due to the fact that pastırma is made using traditional methods. In general, pastırma is characterized by a pH value of 5.5–6.0. At same time, it is categorized as an intermediate moisture food, and its production does not include heating or smoking processes (Kaya et al. 2022). Curing, pressing, drying and covering with çemen are the important process stages in the production of pastırma. The drying stage is applied after curing and pressing as well as covering with çemen stages. In the traditional pastırma processing, the drying temperature applied after the curing is generally about 15 °C. In the drying stages applied after the pressing and covering with çemen, the temperature can be around 20 °C and even slightly higher. The moisture content and water activity decrease as the process progresses (Kaban 2013).

Fig. 1.

The percentage frequency distribution of pH values in pastırma samples

Fig. 2.

The percentage frequency distribution of moisture contents in pastırma samples

Microbiological properties

The percentage frequency distributions of lactic acid bacteria, Micrococcus/Staphylococcus, Enterobacteriaceae and yeast-mold counts were shown in Fig. 3. In this study, the count of lactic acid bacteria in 50% of the samples and the count of Micrococcus/Staphylococcus in 55% of the samples varied between 6 and 8 log cfu/ g (Fig. 3). Lactic acid bacteria and Micrococcus/Staphylococcus are two important microorganism groups in pastırma. The count of Micrococcus/Staphylococcus was generally higher than the lactic acid bacteria count. In other studies conducted on pastırma, it was also reported that the count of Micrococcus/Staphylococcus is higher than the count of lactic acid bacteria (Özdemir et al. 1999; Öz et al. 2017). On the other hand, yeast is another microorganism group in the pastırma microbiota and their counts may show high variability depending on the manufacturing conditions (Kaya and Kaban 2016). In the present study, in 55% of the pastırma samples, the yeast-mold count was less than 4 log cfu/g, and the count was below the detectable level in 4 samples (20%) (Fig. 3). Enterobacteriaceae count was generally found below the detectable level (Fig. 3). The members of Enterobacteriaceae, which are sensitive to low pH and a_w values, cannot show any growth under pastırma production conditions (Kaban 2013). However, as detected in this study, high counts of Enterobacteriaceae were also found in a study conducted on pastırma by Öz et al. (2017).

Fig. 3.

The percentage frequency distribution of lactic acid bacteria, Micrococcus/Staphylococcus, Enterobacteriaceae and yeast-mold counts

The percentage frequency distribution of Enterococcus count obtained from three different media was shown in Fig. 4. In both Enterococcus agar and Slanetz- Bartley agar, Enterococcus count changed between 2 and < 4 log cfu/g in 35% of the samples, 4 and < 6 log cfu/g in 30% of them and 6 and < 8 log cfu/g in the remaining 35% of them. In the enumerating on KF Streptococcus agar, while the Enterococcus count was below the detectable level in 10% of the samples, the count changed between 2 and < 4 log cfu/g in 45%. According to the results, Enterococcus count in pastırma may show a great variation. In contrast, it was reported that the Enterococcus count in pastırma has changed between 10² and 10⁴ cfu/g in 75% of the samples (Özdemir et al. 1999). Enterococci can be widely found in the animal products such as fermented sausage and cheese as well as the vegetables and plants (Lebreton et al. 2014). The count of Enterococcus may change between 10² and 10⁸ cfu/g depending on the type of the product in traditionally fermented sausages (Santos et al. 2017). It was previously reported that traditional sausages with high pH value are suitable for survival of enterococci. Moreover, enterococci can growth in these meat products (Hugas et al. 2003).

Fig. 4.

The percentage frequency distribution of Enterococcus counts obtained from three different agars

Genotypic identification of the Enterococcus isolates

A hundred enterococci isolates were identified by 16S rRNA sequencing analyses, and 80 of 100 strains were identified as E. faecium (80%), 19 of them as E. faecalis (19%) and 1 of them as E. hirae (1%) (Table 1). The ecology of enterococci in foods is highly related to the product properties. Enterococci can be a part of microbiota in traditional fermented products. In a study conducted on the isolation/identification of enterococci from different foods such as fermented sausage, ham, ground meat and cheese, E. faecalis (72%) was determined as the predominant species, followed by E. faecium (13%) and E. durans/E. hirae (6%) (Peters et al. 2003). However, in the present study, E. faecium was determined as predominant species. Similarly result has been reported by Martin et al. (2009) in traditional fermented sausages. In another study, 51.7% of the isolates obtained from the fermented sausage were identified as E. faecalis, 44.8% as E. faecium and 3.4% as E. gallinarum (Jahan et al. 2013). Their ability to survive in food and the environment as well as their sensitivity to pH changes and high salt concentrations play an important role in the evaluation of these microorganisms as indicator microorganisms (Klein 2003). However, as in pastırma, enterococci arise as a part of the microbiota. For this reason, it is not true to accept the enterococci as an indicator of only the insufficiency hygienic quality in the dry cured meat products such as pastırma (Kaya and Kaban 2016).

Table 1.

The results of genotypic identification of Enterococcus spp. isolated from pastırma

Strain	Accession number	Identity (%)	Strain	Accession number	Identity (%)
E. faecium EA1-2-1	KU291269	100	E. faecalis EA12-2-3	KU359787	100
E. faecium EA1-2-5	KU291270	100	E. faecium S12-2-5	KU359788	99
E. faecium S1-2-1	KU291271	99	E. faecium KF12-2-1	KU359789	100
E. faecium EA2-2-2	KU291272	100	E. faecalis KF12-2-5	KU359790	100
E. faecium EA2-2-4	KU291273	100	E. faecium EA13-3-2	KU359791	100
E. faecium S2-2-3	KU291274	94	E. faecium EA13-3-4	KU359792	99
E. faecium KF2-2-2	KU291275	100	E. faecium S13-4-2	KU359793	100
E. faecalis EA3-4-4	KU291276	100	E. faecium S13-4-6	KU359794	99
E. faecium EA3-4-5	KU291277	100	E. faecium KF13-4-5	KU359795	99
E. faecium S3-4-3	KU291278	99	E. faecium EA14-3-1	KU359796	99
E. faecium KF3-3-1	KU291279	100	E. faecium EA14-3-3	KU359797	99
E. faecium KF3-3-5	KU291280	100	E. faecium EA14-3-5	KU359798	99
E. faecium EA4-2-3	KU321604	99	E. faecium S14-3-3	KU359799	99
E. faecium EA4-2-4	KU321605	97	E. faecium S14-3-4	KU359800	99
E. faecium S4-2-2	KU321606	99	E. faecium S14-3-5	KU359801	100
E. faecium S4-2-3	KU321607	100	E. faecium EA15-3-8	KU359802	99
E. faecium KF4-2-3	KU321608	99	E. faecium EA15-3-10	KU359803	99
E. faecium EA5-2-1	KU321609	100	E. faecium S15-2-1	KU359804	99
E. faecium EA5-2-3	KU321610	100	E. faecium S15-2-4	KU359805	100
E. faecium EA5-2-4	KU321611	99	E. faecium EA16-4-3	KU359806	95
E. faecium S5-2-1	KU321612	100	E. faecium EA16-4-5	KU359807	99
E. faecium KF5-2-2	KU321613	100	E. faecium S16-4-1	KU359808	99
E. faecium EA6-5-2	KU321614	99	E. faecium KF16-4-1	KU359809	99
E. faecalis EA6-5-5	KU321615	100	E. faecium EA17-5-4	KU359810	100
E. faecium S6-5-4	KU321616	99	E. faecium EA17-5-6	KU359811	99
E. faecium KF6-5-3	KU321617	100	E. faecium S17-5-4	KU359812	99
E. faecium KF6-5-4	KU321618	99	E. faecium KF17-5-6	KU359813	99
E. faecalis EA7-4-2	KU321619	100	E. faecalis KF17-5-10	KU359814	99
E. faecalis EA7-4-3	KU321620	99	E. faecalis EA18-2-1	KU359818	99
E. faecalis S7-4-2	KU321621	99	E. faecalis S18-2-1	KU359815	100
E. faecalis KF7-4-3	KU359817	100	E. faecalis KF18-2-3	KU359819	99
E. faecium EA8-5-1	KU321622	100	E. faecalis KF18-2-5	KU359816	98
E. faecium EA8-5-5	KU321623	100	E. faecium EA19-5-3	KU359820	99
E. faecium S8-5-2	KU321624	100	E. faecium EA19-5-4	KU359821	99
E. faecium KF8-5-4	KU321625	99	E. faecium EA19-5-6	KU359822	99
E. faecium EA9-2-1	KU321626	99	E. faecium S19-5-10	KU359823	100
E. faecalis EA9-2-4	KU321627	100	E. faecium KF19-5-2	KU359824	99
E. faecium S9-2-1	KU321628	99	E. faecium KF19-5-3	KU359825	98
E. faecium KF9-2-1	KU321629	100	E. faecium EA20-5-2	KU359826	100
E. faecalis EA10-3-1	KU321630	100	E. faecium EA20-5-3	KU359827	100
E. faecalis EA10-3-5	KU321631	99	E. faecium EA20-5-4	KU359828	100
E. faecalis S10-3-2	KU321632	100	E. faecium EA20-5-6	KU359829	99
E. faecalis S10-3-3	KU321633	100	E. faecium EA20-5-7	KU359830	99
E. faecalis KF10-3-1	KU321634	100	E. faecium EA20-5-8	KU359831	99
E. faecium KF10-3-3	KU321635	100	E. faecium EA20-5-9	KU359832	99
E. faecium EA11-3-1	KU359782	99	E. faecium S20-5-2	KU359833	99
E. faecium EA11-3-2	KU359783	99	E. faecium S20-5-5	KU359834	99
E. faecium S11-4-2	KU359784	99	E. faecium S20-5-7	KU359835	100
E. hirae KF11-3-4	KU359785	99	E. faecium KF20-5-2	KU359836	99
E. faecium EA12-2-1	KU359786	99	E. faecium KF20-5-4	KU359837	99

Antagonistic activity results

The antagonistic activities of the identified 100 isolates against L. monocytogenes and S. aureus were determined with agar spot and well diffusion tests (Table 2). According to the obtained results, EA_5-2-1, EA_5-2-3, EA_5-2-4, S_5-2-1, EA_9-2-1, EA_9-2-4, S_9-2-1, KF_9-2-1, S_10-3-2, S_10-3-3, KF_10-3-1 and KF_10-3-3 strains showed positive results only in agar spot test. The EA_4-2-3, EA_4-2-4, S_4-2-2, S_4-2-3, KF_4-2-3, EA_8-5-1, EA_8-5-5, S_8-5-2 and KF_8-5-4 strains had antagonistic activity against L. monocytogenes in both agar spot test and well diffusion test.

Table 2.

The antagonistic activities of the strains against Listeria monocytogenes and Staphylococcus aureus

Strain no	Listeria monocytogenes		Staphylococcus aureus
	Well diffusion	Agar spot	Well diffusion	Agar spot
E. faecium EA5-2-1	−	+	−	−
E. faecium EA5-2-3	−	+	−	−
E. faecium EA5-2-4	−	+	−	−
E. faecium S5-2-1	−	+	−	−
E. faecium EA 9-2-1	−	+	−	−
E. faecium EA9-2-4	−	+	−	−
E. faecium S9-2-1	−	+	−	−
E. faecium KF9-2-1	−	+	−	−
E. faecium S10-3-2	−	+	−	−
E. faecium S10-3-3	−	+	−	−
E. faecium KF10-3-1	−	+	−	−
E. faecium KF10-3-3	−	+	−	−
E. faecium EA4-2-3	+	+	−	−
E. faecium EA4-2-4	+	+	−	−
E. faecium S4-2-2	+	+	−	−
E. faecium S4-2-3	+	+	−	−
E. faecium KF4-2-3	+	+	−	−
E. faecium EA8-5-1	+	+	−	−
E. faecium EA8-5-5	+	+	−	−
E. faecium S8-5-2	+	+	−	−
E. faecium KF8-5-4	+	+	−	−

The nine E. faecium strains showed antagonistic activity against Listeria monocytogenes in the well diffusion test. In contrast, all of the strains had no antagonistic activity against Staphylococcus aureus. However, it has been reported that all of the E. faecium strains isolated from a fermented sausage type had antagonistic activity against Listeria species and S. aureus (Ben-Belgacem et al. 2010). On the other hand, in a study investigating the potential use of two different E. faecium strains as a starter culture in fermented sausage production, it was reported that both strains significantly prevented the growth of Listeria during the fermentation process (Callewaert et al. 2000). In a similar study, it was determined that E. faecium P21 strain isolated from Spanish dry fermented sausage showed antimicrobial activity against L. monocytogenes, S. aureus, Clostridium perfringens and C. botulinum (Herranz et al. 2001). In a study conducted on Italian fermented sausages, bacteriocinogenic E. casseliflavus IM 416K1 (Bac⁺) or its bacteriocin (enterocin 416 K1) showed a significantly reduction in the count of L. monocytogenes (Sabia et al. 2003). Similarly, in a study conducted on a dry fermented sausage type, the effect of enterocin CCM 4231 was examined and it was detected that this bacteriocin caused to a significant reduction in the count of L. monocytogenes (Laukova et al. 1999).

Conclusion

The microbiological and physicochemical properties of pastırma may show differences due to the impacts of the internal and external factors such as raw material selection, curing agent, curing ambient temperature, drying/ripening conditions in the production. The number of the enterococci may rise up to 10⁸ cfu/g in pastırma. This result could be due to the fact that enterococci tolerate the high salt levels and they have a wide temperature range for growth. In pastırma, enterococci are too limited on species basis and two species as E. faecium and E. faecalis come to the forefront. The inhibiting effect of these strains on L. monocytogenes is also an important result. In addition, another important result is that E. faecium and E. faecalis strains isolated from pastırma showed a potential for further investigation.

Abbreviations

ATCC

American Type Culture Collection

BHI

Brain heart infusion agar

BLAST

Basic local alignment search tool

CFU

Colony forming unit

DNA

Deoxyribonucleic acid

DRBC

Dichloran rose Bengal chloramphenicol agar

MRS

De man rogosa sharpe agar

MSA

Mannitol salt phenol red agar

RNA

Ribonucleic acid

PYR

L-pyrrolidonyl-β-naphthylamide

VRBD

Violet red bile dextrose agar

Authors' contributions

ÖE: Formal analysis, investigation, validation, methodology, writing-orginal draft preparation. GK: Formal analysis, investigation, writing-review and editing. MK: Supervision, Writing-review and editing.

Funding

This study was supported by Scientific and Technological Research Council of Turkey (TÜBİTAK) under Project Number: 115O030.

Data availability

Not applicable.

Code availability

Not applicable.

Declarations

Conflict of interest

We declare no conflict of interest.

Ethical approval

Not applicable.

Consent to participate

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Consent for publication

Not applicable.