Exploring microbial diversity in Kermanshah province’s Kermanshahi oil through DGGE and sequencing analysis

Abstract

Background

Ghee, known as “roghane heiwâni,” or “Kermanshahi oil” is a traditional fermented butter-like product highly esteemed for its nutritional value. Ghee is prepared using traditional methods and has substantial potential as a reservoir of probiotic microorganisms. Previous research delved into isolating and identifying lactic acid bacteria (LAB) in Kermanshahi through culture and PCR sequencing. This study seeks to elucidate the microbial profiles and diversity within Kermanshahi using culture, Denaturing Gradient Gel Electrophoresis (DGGE), and sequencing methodologies.

Methods

Twenty samples of Kermanshahi oil were meticulously gathered from diverse locales across Kermanshah province. These samples were cultivated under specialized conditions in MRS and M17 environments spanning 24 to 72 h. Following DNA extraction, amplification of the 16SrRNA gene sequences was performed, culminating in sequencing for conclusive identification of the isolates. Furthermore, the DGGE technique was directly employed to separate and identify various species present in the oil samples utilizing bioinformatics software.

Results

Sequencing outcomes revealed a diverse array of microorganisms among the isolates, with Lactobacillus constituting 43%, Streptococcus comprising 27.6%, Enterococcus at 4.61%, and yeasts at 7.6%. Other species exhibited lower frequencies, encompassing Rhizobium, Bacillus coagulans, and Staphylococcus hominis.

Conclusions

The isolation of a diverse spectrum of probiotic microorganisms underscores their potential utility in the realm of industrial dairy product production. These findings allude to the possibility of integrating these valuable microorganisms, which have historically been associated with traditional products, into the contemporary dairy industry. As consumer interest in probiotic-enriched products surges, the insights gained from this study pave the way for harnessing the benefits of Kermanshahi-derived probiotics.

Background

Kermanshahi oil, known as “roghane heiwâni,” is a cherished traditional butter-like product renowned for its distinct flavor and exceptional nutritional value, enjoyed not only in Iran but also in countries like Turkey and India [1]. Among the repertoire of traditional offerings, Kermanshahi traditional oil stands out as a potential haven for harboring probiotic species. This oil, a staple in the diets of certain Iranian regions, holds the promise of delivering resilient probiotics capable of enduring high temperatures and challenging conditions, thus contributing to the development of functional foods [2].

The production process of Kermanshahi oil is briefly mentioned below. After producing local yogurt with local starters, it is placed in containers called Mashk, and the local butter is separated. Then, by heating the local butter, its oil is separated and stored in appropriate containers [3]. The local starters used for the preparation of yogurt are different.

The utilization of beneficial microorganisms, particularly lactic acid bacteria (LAB), has garnered attention for their pivotal role in supporting human health and well-being. Over the past few decades, the exploration of probiotics has yielded transformative breakthroughs in treating gastrointestinal disorders linked to dysbiosis, restoring equilibrium within the gut microbiota [4]. Clinical trials have substantiated the advantageous effects of probiotics, extending their potential to ameliorate a spectrum of ailments including Alzheimer’s, Parkinson’s, diabetes, obesity, cancer, hypertension, and liver disorders [5, 6]. The growing global demand for functional foods fortified with probiotics underscores their newfound popularity, driven by both enhanced palatability and their potential to confer health benefits without accompanying side effects [7, 8]. Criteria such as acid and bile salt tolerance, digestive enzyme resistance, adhesion to mucosal and epithelial cells, as well as antimicrobial capabilities, serve as litmus tests to discern the functional prowess of lactic acid bacteria (LAB) [9, 10]. Notably, the treasure trove of probiotics isn’t confined to dairy products alone; it encompasses a diverse array of microorganisms culled from traditional dairy offerings [11].

Leading LAB genera, including Lactobacillus, Lactococcus, Bifidobacterium, Streptococcus, and Enterococcus, wield substantial influence, with species like L. fermentum, L. rhamnosus, L. acidophilus, S. thermophilus, and E. faecium assuming positions of prominence due to their pronounced probiotic potential [12].

In the quest to unravel the intricate microbial tapestry within traditional products, innovative methods have come to the fore. Culture-independent techniques, exemplified by denaturing gradient gel electrophoresis (DGGE), furnish unprecedented insights by discerning a plethora of microbial species through the isolation of DNA fragments with uniform lengths and distinct sequences [13]. In this landscape, the amalgamation of culture-based techniques, molecular methodologies, and sequencing technologies becomes indispensable for isolating and identifying microbial enclaves endowed with unique attributes [14].

This study embarks on an uncharted expedition, channeling its focus toward a reservoir that has remained relatively unexplored—LAB within traditional oil. While endeavors to extract probiotics from dairy stalwarts like milk, cheese, curd, and yogurt have proliferated, the realm of traditional oil has witnessed limited scrutiny. It is predicted that due to the presence of bacteria at all stages of the process of Kermanshahi oil, these bacteria will be present in Kermanshahi oil, and by consuming an appropriate amount of these bacteria, consumers will benefit from their advantages.

In light of this underexplored terrain, our present study endeavors to uncover the microbial tableau ensconced within animal oil, unraveling its composition through a trifecta of culture, DGGE, and sequencing methods.

With the horizon of scientific inquiry broadening, this study aims to extend the boundaries of knowledge by uncovering the hidden microbial gemstones harbored within traditional oil, thus shedding light on a novel avenue of probiotic exploration.

Methods

Samples

Twenty oil samples, each weighing 100 g, were meticulously collected from diverse regions within Kermanshah province between the months of January and April 2020. These samples were sourced from non-industrial and non-commercial settings and were prepared utilizing traditional methods.

Culture conditions

Initially, oil samples were subjected to a 15-minute incubation in a Bain-Marie set at 45°C. Subsequently, 1 ml of each sample was combined with 4 ml of both MRS broth and M17 broth. The resulting mixtures were vortexed and incubated in a Bain-Marie at 56 °C for 30 min. To induce anaerobic conditions in the MRS broth, the samples were treated within an Anoxomat system with an atmosphere comprising 0.2% O₂ and 80% N₂. The MRS and M17 broths were incubated at 37°C for 24 to 72 h. Cultured samples from the MRS broth were then inoculated onto MRS agar containing 0.05% L-cysteine, while samples from the M17 broth were added to M17 agar. All samples Incubation of at 37°C for 24 to 72 h, with MRS agar incubated anaerobically and M17 agar aerobically. Following growth, gram staining, oxidase, and catalase tests were performed.

DNA extraction

For bacterial and fungal DNA extraction, two distinct methods were employed. Bacterial DNA was extracted using the Gene-spin™ DNA extraction kit (Cat.NO.17045) from South Korea, followed by storage at -20˚C. Fungal DNA extraction was conducted using a boiling method as outlined below.

For yeast colonies, approximately 100 µl of lysis buffer was employed. The microtube containing the mixture was vortexed for 5 s before being subjected to boiling at 100 ˚C for 15 min. Subsequently, 100 µl of 3 M Sodium acetate was added to the mixture and vortexed, after which it was allowed to rest on an icepack for 1 h. Following this, the samples underwent centrifugation at 12,000 rpm for 5 min. The supernatant was transferred to a new microtube, and an equal volume of isopropanol was added. After vortexing, the samples were placed at -20˚C for 30 min before centrifugation at 10,000 rpm for 15 min. The supernatant was removed, and 200 µl of 99% ethanol was introduced to the pellet. Another centrifugation was carried out for 5 min at 10,000 rpm, with the supernatant discarded. Subsequently, 200 µl of 75% ethanol was added for further dehydration, followed by another centrifugation at 10,000 rpm for 5 min. The samples were then placed under a laboratory hood to facilitate alcohol evaporation and sample drying for 20 min, after which 50 µl of distilled water was added.

Polymerase chain reaction (PCR)

PCR was conducted using a Thermal cycler (Bio-Rad, Singapore) for a total of 30 cycles. Each cycle consisted of a final volume of 25 µl, which included 8.5 µL of Master Mix 2X (Amplicon, Denmark), specific primers at a concentration of 10pmol/µl (Bioneer, Korea), and 2 µL (20ng) of Template DNA derived from bacterial isolates. Three pairs of primers were employed for PCR, targeting the universal 16SrRNA gene, specific primers for Streptococcus thermophilus, and specific primers for the fungal gene. To facilitate subsequent DGGE analysis, a GC-clamp was integrated at the commencement of the 16SrRNA forward primer (Table 1). The PCR protocol encompassed initial denaturation at 92°C for 2 min, denaturation at 92°C for 45 s, annealing at 52°C for the universal 16SrRNA gene, 59°C for the fungal gene, and 50°C for the Streptococcus thermophilus 16SrRNA gene, each for 45 s. Extension at 72°C for 1 min, culminating in a final extension at 72°C for 10 min. The PCR products were visualized through electrophoresis on a 1.5% agarose gel using a Bio-Rad electrophoresis apparatus, applying a voltage of 85 for 45 min. Gel visualization was achieved using a Gel documentation device.

Table 1 Sequence of primers

Denaturing gradient gel electrophoresis (DGGE)

Polyacrylamide gels with a concentration of 6% wt/vol were prepared, featuring denaturants at two concentrations: 0% and 100%. The latter comprised 7 M urea and 40% formamide. The polymerization of the gel was facilitated using ammonium persulfate and TEMED. Subsequent electrophoresis was conducted for 5 h and 30 min at 110 V and 60˚C. Staining of the gel was performed using Safe Stain, and the resulting bands were scrutinized using a Gel documentation device. Appropriate and well-defined bands were excised from the gel. The bands, along with PCR products derived from bacteria isolated through the culture method, were dispatched to Takapozist Co. for sequencing.

Bioinformatical and statistical analysis

Bioinformatics analysis involved aligning the sequences obtained from 16SrRNA gene sequencing using the Bioedit bioinformatics program and Clustal Omega software. The Blast software available through NCBI was then employed to identify probiotic species, with the subsequent results subjected to analysis using SPSS22 statistical software (Schematic Fig. 1).

Schematic Fig. 1

Collecting oil from different parts of Kermanshah province. 1 A: Culture on both media, 1B: Aerobic and anaerobic incubation, 1 C: Early diagnosis using biochemical tests and staining, 1D: DNA extraction from pure colony. 2 A: Direct extraction of DNA from oil, 2B: PCR using 16s-DGGE, 2 C: Running on a DGGE, 2D: DNA extraction from the gel. E: Sequencing, F: Edit and BLAST, G: Identification of bacterial species

Results

Isolate identification through phenotypic tests

Among the 20 samples examined, 13 and 16 cases exhibited growth on MRS and M17 agar, respectively. Of these, 17 cases were identified as gram-positive bacilli, 5 as gram-positive cocci, and 9 as yeast. Catalase and oxidase tests were conducted for bacilli and cocci, revealing negative catalase activity across all isolates, except for a single case.

PCR amplification and detection

Utilizing the 16SrRNA gene as the target, PCR was performed on DNA extracted from both oil samples and cultured microorganisms. Amplified bands of 450 base pairs were visualized. For yeast detection, specific primers ITS1 and ITS2 were employed, while S. thermophilus was targeted using specific primers. Among the 9 identified yeast, 5 displayed the desired band within the 270 bp range. Notably, electrophoresis of the Streptococcus thermophilus-specific gene yielded no discernible bands (Figs. 1 and 2).

Fig. 1

Electrophoresis results of PCR product for 16SrRNA gene; L: Ladder, C: negative control, 1–10: samples. The desired band for this gene was shown about 450 bp

Fig. 2

Electrophoresis results of PCR product for ITS gene; L: Ladder, C: negative control, 1–8: samples. The desired band for this gene was shown in the range of 270 bp

PCR-DGGE and sequencing analysis

The 16SrRNA gene was amplified using a GC-clamp primer, and the resulting DGGE pattern is depicted in Fig. 3; Table 2. Examination of Table 2 revealed that samples denoted as “N” and “A” exhibited the highest and lowest isolate counts, totaling 12 and 4, respectively (Fig. 3).

Fig. 3

Polyacrylamide gels in DGGE method; directly extracted DNA from oil samples after amplified by PCR for 16SrRNA gene were Electrophoresed

Table 2 The number of bands from each sample using DGGE method

Probiotic species Identification via Culture and sequencing

Sequence alignment of PCR products obtained from cultured bacteria was performed using BLAST software, elucidating a range of probiotic species within the Kermanshahi oil samples. Notably identified were Enterococcus faecium, Lacticaseibacillus rhamnosus, Lactiplantibacillus plantarum, Lactobacillus fermentum, Lactobacillus buchneri, Lactobacillus parabuchneri, Lacticaseibacillus paracasei, Bacillus coagulans, Acinetobacter baumannii, Staphylococcus hominis, and Kluyveromyces marxianus yeast (Table 3).

Table 3 Species isolated from Kermanshahi oil samples by culture method and sequencing

Probiotic species Identification through PCR-DGGE and sequencing

Sharp bands from the polyacrylamide gels post-DGGE were excised and subjected to sequencing. Blast analysis validated the presence of probiotic bacteria, including Streptococcus thermophiles, Lactobacillus acidophilus, Lactobacillus helveticus, and Rhizobium cellulosilyticum. Notably, Streptococcus thermophiles were consistently present across all examined samples (Table 4).

Table 4 Species isolated from oil samples by DGGE method and sequencing

Comparison of Probiotic species isolation methods

The probiotic species isolated based on the 16SrRNA gene through both culture and PCR-DGGE methods are detailed in Tables 5 and 6. Intriguingly, Streptococcus thermophiles, absent in the culture-based approach, were universally detected through DGGE. Additionally, Rhizobium cellulosilyticum was exclusively identified through the DGGE method. Conversely, the culture method led to the identification of Enterococcus faecium, Bacillus coagulans, Staphylococcus hominis, Acinetobacter baumannii, and Kluyveromyces marxianus yeast. However, DGGE demonstrated a greater diversity of isolates, albeit with a broader variety of species captured through the culture method (Table 6).

Table 5 Species isolated from oil samples by both DGGE and culture method and sequencing

Table 6 Genera isolated from oil sample by both DGGE and culture method and sequencing

Discussion

Fermented milk and dairy products have long been recognized for their substantial contribution to human diets, offering a rich source of lactic acid bacteria (LAB). LAB genera such as Lactobacillus, Lactococcus, Pediococcus, Enterococcus, and Streptococcus are acknowledged as generally safe (GRAS) microorganisms, either occurring naturally in products or intentionally added [15]. The multifaceted activities of LABs encompass ameliorating lactose intolerance, bolstering natural resistance to gastrointestinal infections, and exerting potential benefits in cancer prevention and skin health [16]. An additional favorable attribute of LABs is their role in cholesterol reduction [17]. Numerous reports highlight the cholesterol-assimilating capabilities of LABs, with Lactobacillus acidophilus and Lactobacillus fermentum SM-7 being exemplars [18, 19]. Lactobacillus casei, Lactobacillus plantarum, Lactobacillus paracasei, Lactococcus lactis, and Enterococcus faecium have been identified as harboring hypocholesterolemia effects [19]. The mechanisms underpinning LAB-mediated cholesterol reduction encompass enzymatic inhibition, facilitated excretion of bile salts, reduced absorption, and cholesterol assimilation [17]. Noteworthy studies by Kim et al. demonstrated the cholesterol-lowering potential of the LRCC5307 strain from kimchi [20], while Hatice Alog et al. highlighted specific LAB strains in butter as cholesterol modulators [21]. Despite such advancements, exploration into the microbiota of traditional oils has remained relatively unexplored.

The limitations of culture-dependent methodologies in strain classification have prompted the emergence of various molecular tools for LAB characterization [22]. Among these, PCR-denaturing gradient gel electrophoresis (PCR-DGGE) stands out as a potent technique for diversifying strains [23]. Our study encompassed two distinct approaches - culture and PCR-16SrDNA/DGGE - to unravel the strain diversity within this product. Similar to other studies employing culture [15, 24] and PCR-DGGE [25] techniques to profile microbial diversity, our focus was on obtaining insights into bacterial diversity composition. Consequently, band identification was not pursued. The outcomes of our investigation underscore the presence of both LABs and yeasts within the microbial community, with each sample exhibiting a distinct microbial profile. Remarkably, the culture-independent method unveiled a higher tally of identified bacteria compared to the culture-based approach. This observation can be attributed to factors such as the lower pH resulting from fermentation and the duration of incubation in the culture method, which may be suboptimal for certain bacterial strains. Culture-independent methods detected L. acidophilus, L. helveticus, S. thermophilus, and Rhizobium cellulosilyticum, which remained elusive through cultivation. Conversely, the culture-dependent approach yielded isolates like Enterococcus faecium, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus buchneri, Lactobacillus parabuchneri, Lactobacillus paracasei, Bacillus coagulans, Acinetobacter baumanni, and Staphylococcus hominis. Notably, S. thermophilus emerged as the predominant species, aligning with its pivotal role in the dairy industry [26] Furthermore, the differential profile of isolated LAB species was apparent. While Lactobacillus casei, L. fermentum, L. plantarum, L. helveticus, and L. rhamnosus generally exhibit modest lipolytic activity due to enzymes like esterase that catalyze acyl ester chain hydrolysis, contributing to lipolysis [27].

Intriguingly, the use of both direct PCR-DGGE of total community DNA and culture-dependent techniques revealed divergent microbial community descriptions in fermented cassava dough [28]. The presence of Kluyvermyces marxianus yeast isolated through culture underscores its potential role as a starter organism, potentially influencing LAB growth dynamics. Co-cultivation studies highlight the possible synergistic effects between LABs and Candida species [29]. Although dissimilarities were observed in microbial profiles obtained through the two methods, a shared profile trend emerged across most samples. Notably, the PCR-DGGE method demonstrated a superior capacity to unravel microbial diversity within each sample compared to the culture-based approach. The predominant constituents of the community predominantly comprised LABs.

The origin of organisms in Kermanshah oil is two things. One of them is the primary starters used in the production process of yogurt, but these organisms should be resistant to heat such as S. thermophiles. Another source of these organisms could be from the organisms in Mashk, which are used for the processing and storage of Kermanshahi oil.

Collectively, the utilization of both methods facilitated the semi-quantitative identification of a diverse array of bacteria and yeast species. Enhanced strategies, encompassing diverse culture media, sample dilutions, and varied incubation temperatures, are crucial for optimizing bacterial community cultivation. Furthermore, the choice of sampling strategy significantly influences outcomes. Future endeavors should prioritize assessing the functional attributes of the most promising isolates in this product, thereby bolstering safety evaluations and unlocking the full potential of these described probiotic species for both human and animal nutrition.

Conclusions

In this study, different species of probiotic microorganisms were isolated using methods culture, DGGE, and sequencing and realized that due to their useful and valuable benefits, they are used in the production of industrial dairy products, which nowadays have been greatly increased and have been significantly replaced by traditional products. Therefore, by using locally selected starters and controlling the production process, Kermanshahi oil containing useful organisms can be produced in the appropriate number as a functional food.