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J Sci Nutr Res 2021; 4 (2): 118-130 DOI: 10.26502/jfsnr.2642-11000066

Research Article

Advancing quality control of food samples by Next Generation Sequencing compared to culture-dependent techniques

Maria-Eleni Dimitrakopoulou, Chrysoula Kotsalou,Venia Stavrou, Apostolos Vantarakis* Department of Medicine, University of Patras, Patra, Greece

*Corresponding Author: Apostolos Vantarakis, Department of Medicine, University of Patras, Patra, Greece

Received: 16 April 2021; Accepted: 27 April 2021; Published: 21 May 2021

Citation: Maria-Eleni Dimitrakopoulou, Chrysoula Kotsalou,Venia Stavrou, Apostolos Vantarakis. Advancing quality control of food samples by Next Generation Sequencing compared to culture-dependent techniques. and Research 4 (2021): 118-130.

Abstract and safety had become an increasing issue even production or processing procedures can result an for quality authorities, food , manufacturers, abundant bacterial diversity isolated from each food producers and consumers as well. In the present work, product, so NGS approaches can be suitable to elucidate food quality control using bacterial identification the whole picture of bacteria populations. NGS analysis isolated from food samples was performed. Greek olives regarding management can be applied in (raw drupes), currants (dry fruits), roe (fish eggs), bacon food industries and therefore quality and safety of food and “feta” were collected for this purpose. The products can be reassured. aim of this study was to investigate the quality of traditional Greek food types and to compare bacterial Keywords: NGS; Bacteria; Food quality; Food safety; population observed of each food matrix according to Quality control analysis method. Therefore, culture based and molecular methods were performed in parallel. Total bacterial 1. Introduction DNA isolated from food samples was verified by PCR Both food safety and food quality consitute essential and PCR-RAPD amplification and then analyzed by need for food products for consumers, food industries, Next Generation Sequencing. Results showed that NGS producers and quality authorities as well. According to analysis can provide an advanced and detailed look at consumers’ requirements regarding food quality, food food microbiota in comparison to culture-dependent safety considered to be crucial [1]. A major and techniques. Climate conditions, environmental factors, important area of challenges regarding food safety, is

Journal of Food Science and Nutrition Research Vol. 4 No. 2 - June 2021. [ISSN 2642-1100] 118 J Food Sci Nutr Res 2021; 4 (2): 118-130 DOI: 10.26502/jfsnr.2642-11000066 microbiological safety of foodstuff [2]. In 2010, 600 total in a food sample, regardless million incidents of foodborne infections were reported matrix’ complexity. Moreover, by metagenomic by WHO which lead to 420,000 deaths [3]. Thus, analysis of a food sample, actions such as recalls due to nowdays, due to the increasing interest of consumers, a foodborn pathogen detection or characterization of food quality systems tend to be more and more viable but non-culturable , can easily be demanding, following new regulations and techniques performed [14]. Food pathogens such as Escherichia [4]. The most popular and common method for food coli (E. coli), (C. jejuni), quality audit is a culture-dependent method, which monocytogenes (L. monocytogenes), Shigella spp., allows analysis of live microbial cells after their spp., Staphylococccus aureus, Vibrio spp, selective isolation from microbial population by Bacillus cereus, or, food spoilage microorganisms such inoculating media [5]. However, this standard method as Acinetobacter spp., Pseudomonas spp., Botrytis spp. of food analysis face up some limitations such as low can be detected in a variety of food products and are sensitivity, low accuracy or time-consuming process responsible for major foodborn illnesses [15-17]. [6,7]. Moreover, due to the presence of viable but non- Therefore, there are publications that already have culturable cells , culture-dependent methods can not be utilized NGS for understanding the ecology of food considered as an appropriate tool for analyse and study fermentation, for biodiversity analysis of spoilage total microorganisms’ population of samples [8]. microorganisms in poultry , or studying microbiota Therefore, culture-independent techniques for food of and soya beans paste [18-21]. The aim of this analysis have been developed. In 1999, for the first study was to examine and characterize bacterial time, culture-independent approaches presented in diversity of five complex and different food types in fermented food analysis, in terms of food terms of food quality and safety aspect. For this [9]. Molecular PCR-based techniques are already used purpose, we compare and evaluate standard culture- to characterize microorganisms in food, regarding dependent methods for bacteria analysis of each food fermentation, spoilage and contamination [10]. As far as matrix with a molecular technique, Next Generation these PCR-based techniques concern, real time-PCR, Sequencing. reverse transcription-PCR, Loop Mediated Isothermal Amplification (LAMP) approaches have currently 2. Materials and Methods widely reported as tools for food quality and safety 2.1 Samples collection issues [11,12]. Notably, a new research tool, known as For this experinment, five greek food products from Next Generation Sequencing, has been widely used in a totally different category of food types and process level variety of fields such as microbiology, diagnostics, were chosen. A raw fruit, a dry fruit, a dairy, an animal forsenics etc. By Next Generation Sequencing method, product and a seafood were examined. In particular, total microbiome of a biological sample can be fresh olives (Olea europaea L.cv Kalamon), dry vine identified and sequenced. In the field of foodomics, products (Corinthian currants), cheese (“feta” PDO), genomic analysis of food samples by NGS considered to smoked bacon and fish eggs (avgotaracho be high accurate, rapid and much more informative Mesolonghiou PDO) were purchased from local compared with traditional culture depenedent producers. Natural Greek black table olives from techniques [13]. Next Generation Sequencing Kalamon variety considered to be the most famous applications offer in depth taxonomic identification of variety and characterized by their high nutritional value

Journal of Food Science and Nutrition Research Vol. 4 No. 2 - June 2021. [ISSN 2642-1100] 119 J Food Sci Nutr Res 2021; 4 (2): 118-130 DOI: 10.26502/jfsnr.2642-11000066 [22]. Corinthian currants(Vitis Vinifera L., var. 2.3 Bacterial DNA extraction-PCR amplification Apyrena) are cultivated in Southern Greece and are Bacterial DNA extraction protocol was performed by essential component in Mediterenian , due to their boiling method as described by Ribiero[26].Bacterial high content [23]. “Feta” is a traditional Genomic DNA extracted from each food sample, was Greek industrial cheese produced by pasteurized sheep’s used as template for PCR amplification. To amplify and goat’s (up to 30%) in well-equipped cheese 240bs from V3 region of 16s rRNA gene, the dairies, using rennin and commercial lactic acid following universal primers: 338F cultures as starters [24]. Bacon from selected fine pork (5′ACTCCTACGGGGGCAGCAG,Sigma,France), pancetta has a rich flavour and it was tradiotionally 518R(5′ATTACCGCGGCTGCTGG, Sigma, France) smoked with beech wood. Avgotaracho Mesolonghiou were used [25]. The PCR reaction was carried out in a is known as “Greek caviar” and has a high commercial final volume of 25μl, containing 100mg DNA template, value, due to its unique aroma and [25]. “Feta” 5μL 5xbuffer C Mg free, 1,5μL MgCl2(25mM), 0,5μL cheese and bacon were processed and packaged, while dNTPs(10mM), 5μL primers(1μΜ) and 0.1μL Taq the three other food products were collected raw. polymerase (Kapa TAq PCR kit) (Sigma, France). PCR amplification was performed using Thermocycler 2.2 Culture-dependent approach (Biorad). The amplification program was carried out as Ten grams of each food sample were homogenized with follows: initial denaturation 95⁰C for 3mins, 30 cycles 90ml sterile Buffer Peptone (10-1 dilution). A of denaturing at 95⁰C for 1min, annealing at 55⁰C for series of 5 fold dilutions were prepared and spread plate 1min, extension at 72⁰C for 1min with a final extension technique was followed on appropriate selective media. at 72⁰C for 10mins. The resulting amplicons of PCR Medium for bacterial growth were chosen according to were visualised by electrophoresis in 2% (w/v) agarose ISO 17025:2017. For detection of Salmonella species 25 gel and were stained with 8μL of GelRed(Biotium). ml of the food sample were diluted with 225 ml of sterile Buffer Peptone Water and mixed well. 2.4 PCR- Random Aplification of Polymorphic DNA Microbiological analysis included enumeration and After verifying the amplicon (240bs) from each food identification of potential pathogens conducted product, extracted DNA were amplified by PCR-RAPD according to standard procedures for the number of in order to obtain information about the bacteria LAB, E coli, Listeria monocytogenes, communities each product. The following primers were Enterobacteriaceae, Staphylococcus, Salmonella and tested for PCR-RAPD amplification: M13: 5’- Total mesophilic flora. Appropriate dilutions were then GAGGGTGGCGGTTCT-3’, 1247:5’-AAGAGCCCGT- enumerated for these bacterial categories. All plates 3’, 1290: 5’- GTGGATGCGA-3’, OPA10: 5’ were incubated under aerobic conditions except from GTGATCGCAG-3’, OPA15: 5’- TTCCGAACCC- MRS and VRBG plates, that need an extra layer after 3’[27-30]. PCR mixtures contained 1x PCR buffer, spread plating, for anaerobic conditions. The mean 5mmoll Mgcl2, 200μmoll-1 dNTPs, 2μmol primer, 1.25 number of colonies counted was expressed as colony Taq polymerase (Life Technology) and 50 ng bacterial forming units (cfu)/gr, according to each ISO barring DNA. PCR amplification was performed with a thermal Salmonella and Listeria, whose boundaries were cycler under following conditions : initial denaturation determined by their presence or absence. at 94⁰C for 2 min, followed by 35 cycles of denaturation at 94⁰C for 1 min, annealing at 45⁰C for 40s, elongation

Journal of Food Science and Nutrition Research Vol. 4 No. 2 - June 2021. [ISSN 2642-1100] 120 J Food Sci Nutr Res 2021; 4 (2): 118-130 DOI: 10.26502/jfsnr.2642-11000066 at 72⁰C for 2 min and final extension at 72⁰C for 10 min. PCR products were separated by electrophoresis 2% 3. Results and Discussion agarose gel and DNA ladder was used as DNA 3.1 Microbial cultures molecular weight marker. Culture-dependet approach as a gold standard method for quality control of food products was performed 2.5 Next Generation Sequencing (Table 1). shows the results from this gold standard Bacteria community profiles of food samples were method according to ISO17025:2017. Listeria assessed by 16S rRNA gene sequencing using the monocytogynes and Salmonella spp. were not detected Illumina technology .More specific, bacterial DNA in any of food products. However, enamuration of total extracted from food samples were sequenced using an mesophilic flora in food products consider to be Illumina MiSeq (Illumina RTA v1.17.28; MCS v2.2). essential, as it is an indicator of products’ production, Sequences observed from these samples were storage or transportation conditions [31]. downloaded from the BaseSpace website.

Food E.coli L.monocyto Salmonella Enterobacte Staphylococccu Lactid Total type genes spp. riaceae s aureus acid mesophilic bacteria flora Olives <10 N.D N.D 2x104 <10 7x103 6x105 Currants <10 N.D N.D 1x102 <10 <10 2x104 Fish <10 N.D N.D 3x107 <10 <10 1x107 eggs Bacon <10 N.D N.D <10 <10 4x103 1x106 Feta <10 N.D N.D 1x101 1,1x101 108 3x108 cheese

3.2 PCR-RAPD 3.3 Next Generation Sequencing PCR-RAPD analysis for each food product was Within each different food type, abundance precentages performed in order to examine whether there was a for dominant OTUs in the species classification are diversity in bacteria communities among samples. presented. For instance, dominant bacteria strain Therefore, five different primers were examined for this identified in fish eggs samples were Acinetobacter purpose. Primer OPA15 resulted the most informative venetianus, while in bacon were Photobacterium PCR-RAPD band pattern profile (Figure 1). phosphoreum. As far as fruits concern, Pantoea agglomerans was detected in olives and Bacilus cereus in currants. Regarding “feta” cheese, appeared to be dominated by Lactococcus sp. (Figures 2-5).

Journal of Food Science and Nutrition Research Vol. 4 No. 2 - June 2021. [ISSN 2642-1100] 121 J Food Sci Nutr Res 2021; 4 (2): 118-130 DOI: 10.26502/jfsnr.2642-11000066

Figure 1: PCR-RAPD analysis of 16srRNA of bacterial DNA isolated from 1.fish eggs 2.olives 3.bacon 4.feta cheese 5.currants

Figure 2: Next generation sequencing analysis of 16srRNA of bacteria communities of fish eggs. Red highlighted : Dominant species Yellow highlighted: Next most abundant species.

Journal of Food Science and Nutrition Research Vol. 4 No. 2 - June 2021. [ISSN 2642-1100] 122 J Food Sci Nutr Res 2021; 4 (2): 118-130 DOI: 10.26502/jfsnr.2642-11000066

Figure 3: Next generation sequencing analysis of 16srRNA of bacteria communities of olive. Red highlighted: Dominant species Yellow highlighted: Next most abundant species

Figure 4: Next generation sequencing analysis of 16srRNA of bacteria communities of currants. Red highlighted: Dominant species Journal of Food Science and Nutrition Research Vol. 4 No. 2 - June 2021. [ISSN 2642-1100] 123 J Food Sci Nutr Res 2021; 4 (2): 118-130 DOI: 10.26502/jfsnr.2642-11000066

Figure 5: Next generation sequencing analysis of 16srRNA of bacteria communities of “feta” cheese. Red highlighted: Dominant species

Journal of Food Science and Nutrition Research Vol. 4 No. 2 - June 2021. [ISSN 2642-1100] 124 J Food Sci Nutr Res 2021; 4 (2): 118-130 DOI: 10.26502/jfsnr.2642-11000066

Figure 6: Next generation sequencing analysis of 16srRNA of bacteria communities of bacon. Red highlighted: Dominant species Yellow highlighted: Next most abundant species

4. Discussion first, gold standard method for quality control of food This study provides a comparison of culture-dependent samples was conducted. From the results obtained, in analysis to NGS survey in order to characterize existing none of these food products examined, Salmonella spp. bacterial population isolated from each food type. At and Listeria Monocytogenes were detected. The total Journal of Food Science and Nutrition Research Vol. 4 No. 2 - June 2021. [ISSN 2642-1100] 125 J Food Sci Nutr Res 2021; 4 (2): 118-130 DOI: 10.26502/jfsnr.2642-11000066 concentration of mesophilic flora ranged from 104 to and at 103-105 concentration produces an emetic 108 in all food samples. Except bacon sample, known as cereulide [41]. As far as “feta” cheese Enterobacteriaceae species were abundant too, in all concerns, Lactococcus sp. apperared in the most reads food products. The highest percentage of LAB bacteria in NGS analysis. The microbial composition of “Feta” (108) was detected in “feta”cheese, as expected. PCR- cheese was dominated by LAB as expected due to their RAPD amplification following, revealed high presence in sheep and goat milk. Moreover, biodiversity in all food samples examined. RAPD Lactococcus sp. is used as a starter culture for acid fingerprinting pattern of currant had less strain variation development during cheese production [42]. In bacon compared with the strain variation obtained from the sample analysis, Photobacterium phosphoreum strain other food products. RAPD-PCR analysis of all food was abundant. Genus Photobacterium belongs to the samples produced patterns with products ranging from family Vibrionaceae and is known as a fish spoilage, as approximately 100 bp to more than 1500 bp. However, it is usually isolated from aquaculture environment. NGS analysis enabled in depth identification of total Recently, Photobacterium phosphoreum was bacteria communities of each food product, at the level characterized as a spoilage bacterium, common in of species.More specific, the dominant precentage of modified packaged meat [43,44]. NGS analysis revealed bacteria species found in fish eggs samples, at 86%, was identification of more existing bacteria in food samples Acinetobacter venetianus. Genus Acinetobacter has examined. According to NGS results obtained, it is been reported as a pathogen bacteria for seafood, such essential need to implement this appoach for food as shrimps, catfsih or snout bream [32-34]. quality control, as the existing bacteria strains identified Acinetobacter species such as A. beijerinckii, A. were emerging pathogens. However, based on Greek gyllenbergii, A. haemolyticus, A. junii, A. parvus, A. standards for food quality and safety predominantly tjernbergiae and A. venetianus, can induce hemolytic followed in , main pathogen reactions and lyse mammalian red blood cells [35,36]. microorganisms responsible for food illnesses are tested Macrococcus micrococcus luteus observed by fish eggs’ by culture-dependent techniques. The existing protocols bacteria analysis, is present due to dietary control of for microbiological audits by standard culture- fishes [37]. The highest precentage of bacteria dependent methods face up some limitations. NGS can communities analysis in olives belongs to Pantoea overcome these limitations and provide detailed look at agglomerans or Enterobacter agglomerans. Pantoea the microorganisms extracted from food samples. agglomerans is a gram-negative aerobic bacillus of the Interestingly, our findings are in agreement with other family Enterobacteriaceae. There are publications that publications , such as NGS analysis for determination of have associated presence of Pantoea agglomerans with fish flesh or milk microbiota, which report the Pseudomonas savastanoi pv. Savastanoi, which is superiority of NGS over culture-dependent methods responsible for olive knot diseace [38,39]. Dominant [45,46]. Moreover, “omics” technologies can have a species detected in currants, were Bacilus cereus. potential impact not only in food quality and safety, but Bacilus cereus strains considerd to be an emerging also in food traceability and authentication. By NGS foodborn pathogen, as it can cause gastrointestinal analysis, data for enhancement of “food traceability” or diseases and is characterised by its spores resistance in “food authentication” issues can be also collected heat and acid [40]. Bacilus cereus , which can be present [47,48]. Nevertheless, NGS application can provide in a variety of foodstuff , is a a gram-positive bacterium information not only regarding food industry but also

Journal of Food Science and Nutrition Research Vol. 4 No. 2 - June 2021. [ISSN 2642-1100] 126 J Food Sci Nutr Res 2021; 4 (2): 118-130 DOI: 10.26502/jfsnr.2642-11000066 for agricultural practices such as sustainability, plant Innovation" (NSRF 2014-2020) and co-financed by diseases and irrigation [49]. NGS approaches can be an Greece and the European Union (European Regional additional tool to obtain information for marine Development Fund). environments, as well [50]. Thus, advancing NGS technologies could address important issues related to a Conflicts of Interest variety of fields. The authors declare no conflict of interest.

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