{"paper_id":"23bdec00-dae4-4035-86ac-94bb0f4015fe","body_text":"Characteristics of Bacterial Diversity and Microbiome of Ryazan Farm Cheese | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Characteristics of Bacterial Diversity and Microbiome of Ryazan Farm Cheese Natalia D. Wackerow-Kouzova, Alexey S. Rozanov, Valery D. Bondar, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7794861/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The composition of the microbiome of artisanal cheeses can be influenced by various factors, including temperature, pH, oxygen availability, and milk quality. The present study provides a comparative analysis of the diversity of cultured lactic acid bacteria from Ryazan farm cheese and its microbiome. To analyze the cultured microflora from cheese and previous self – starter raw milk, the different nutrient media and different cultivation temperatures were used. The bacteria of genera Lacticaseibacillus, Limosilactobacillus, Lactiplantibacillus and Leuconostoc were isolated from milk and cheese. The microbiome diversity of cheese by 16S rRNA encoding gene analysis was higher than the diversity of cultured bacteria and included Staphylococcus eqourum (18,13%), Streptococcus thermophilus (16,79%), Lactiplantibacillus plantarum (16,14), Lacticaseibacillus paracasei ( 14,06%), Lactococcus lactis (11,56%), as dominated species. Thus, the cultivated species belonged to dominated ( Lactiplantibacillus plantarum, Lacticaseibacillus paracasei ), sub-dominated ( Leuconostoc falkenbergense , Leuconostoc mesenteroides ) and small-numbered ( Lacticaseibacillus rhamnosus ) species in the microbiome. It can be explained by LAB antagonistic properties manifested during cultivation and non-selective conditions for representatives of families Streptococcaceae ( Streptococcus, Lactococcus ) and Staphylococcaceae . Representatives of the genus Staphylococcus are often present in artisanal cheeses, and some of them may pose a risk to human health. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Cheese microbiota plays a key role in defining its sensory properties, safety, and overall quality; therefore, its diversity is the subject of active study [ 1 , 2 , 3 , 4 ]. The composition of the microflora is influenced, among other things, by the geographical characteristics of the place of manufacture, temperature, humidity and even the altitude above sea level [ 1 , 2 , 3 ]. Bacteria in artisanal cheese primarily originate from two main sources: the inoculated starter cultures and the indigenous milk microbiota. Microorganisms originating from processing environments are also transferred from production surfaces to cheese surfaces and affect the final microbial composition of product. The study and isolation of lactic acid and other bacteria from the microbiomes of lactic acid products is of great biotechnological importance, since they have been shown to possess a number of beneficial properties [ 5 , 6 , 7 ]. At the same time, some of them may have harmful properties, such as antibiotic resistance and toxins production [ 4 ]. Nowadays there are known more than 150 names of liquid and solid lactic acid products in the world. Finally, in farm production their microbial composition may depend on the region of production, even with the same product name. It is also possible that not all national traditional lactic acid products are described. Russia has traditionally produced more than 60 names such products, although the exact number also is still unknown. The fermented dairy products have always been very diverse and popular in Russia; they have been produced industrially since the 18th century. At 19th century, research began on the effects of fermented dairy products and the lactic acid bacteria (LAB) on human health by N. Postnikov, I. Mechnikov (Nobel Prize winner), A. Dzhogin, S. Botkin and N. Sklifosovsky. Hospitals were opened where lactic acid drinks (kefir, koumiss, yogurt) were used for treatment. However, there are few studies of Russian farm cheese [ 1 , 8 ]. These studies mainly affect regions of cheese origin such as Buryatia and Siberia, Asian part of the Russian Federation [ 1 ]. There is also a cheese study in the North Caucasus Federal District, Kabardino-Balkaria and Stavropol [ 8 ], the Southern Federal District, Kalmykia [ 1 ], the Northwestern Federal District, Arkhangelsk [ 8 ], Far Eastern Federal District, Khabarovsk Territory [ 1 ] and the Central Federal District, Tula [ 8 ]. These are isolated studies that do not reflect the full picture. This work includes a comparative analysis of the microbiome of Ryazan farm cheese and the diversity of cultured lactic acid bacteria isolated from fermented milk and finished cheese. The Ryazan region is one of traditional regions of Central Russia in terms of the production of lactic acid products. Ryazan fermented raw milk is a reservoir of LAB that are not affected by anthropogenic influences, so the study of this product allows us to get an idea of the composition of lactic acid bacteria in dairy products of central Russia that are not related to the food industry. Ryazan traditional cheese, on the contrary, is an example of a microbiological environment that has gone through a traditional technological process. The study of these products makes it possible not only to evaluate the bacterial community of lactic acid products in Central Russia, but also to identify strains of LAB that can subsequently be used to develop starter cultures. Materials and Methods Bacterial Isolation and Culture Conditions Ryazan homemade cheese and previous self – starter raw milk were used as objects for studying microbial communities of fermented dairy products of the Ryazan region, village Hirino. Ryazan farm milk was fermented with native microbiota at a temperature of 25 ° C for 2 weeks. The main object of research in this work is artisanal cheese from the Ryazan region. Since the microorganisms of this product have undergone anthropogenic selection (heat treatment, salting), cheese is a potential source of strains with valuable industrial properties, including resistance to high temperatures and salt, the probiotic properties, as well as the taste of the final product. We used the different cultivation temperatures (25, 32, 40 or 43°C) and the different selective media (MRS, De Man–Rogosa–Sharpe, and YPD, Yeast Peptone Dextrose) to analyze the cultured microflora from cheese and previous self – starter raw milk. When using solid versions of the selected media, they were modified as follows: pasteurized milk \"Agusha\" 3.2% fat content (WBD, Russia) was added to the molten medium. The volume ratio of the microbiological medium and milk was 8:1. This stage provides an additional advantage to lactic acid bacteria (LAB), since they have the ability to metabolize peptides and carbohydrates in milk, including casein. After isolation of pure cultures, their morphological properties, catalase test, and milk fermentation were studied. The strains have been in storage at the Sirius University Collection of Microorganisms (SCM), Russia. Each strain name is followed by the strain origin marker - SCM1m- SCM10m from the self – starter raw milk and the cheese strains SCM11c- SCM22c. PCR and Sequencing DNA of strains was extracted by the conventional phenol-chloroform method [ 9 ]. PCR amplification targeting the 16S rRNA gene, purification and sequencing of PCR products was carried out as described in [ 10 ] with the primers for sequencing to yield an expected amplicon of approximately 1400 bp: F27 (sequence: AGAGTTTGATCMTGGCTCAG) and R1492 (sequence: TACGGYTACCTTGTTACGACTT). The BioMaster HS-Taq PCR-Color kit (Biolabmix, Russia) and the amplifier \"GeneExplorer 96\" (BIOER, China) were used. Metagenome sequencing analyses Bacterial DNA was obtained from this mixture using the \"Quick-DNA Miniprep Plus Kit\" (Zymo Research, USA). A library was prepared from the V4 rrs region sample of the Ryazan cheese metagenomic DNA using the VAHTS Universal Plus DNA Library Prep Kit (Nanjing Vazyme Biotech Co., China ) , on Illumina MiSeq (Illumina, USA). Phylogenetic and Metagenome Analysis A BLAST analysis of the obtained sequences was performed against sequences held in GenBank through the NCBI website ( http://www.ncbi.nlm.nih.gov/ ). Phylogenetic and phylogenomic analyses were performed using software package MEGA version X [ 11 ]. Analysis of the rrs gene was based on sequences received in this study and sequences available in GenBank. Distances for both genes were calculated according to the Kimura's two-parameter model [ 12 ] and for the rrs gene phylogenetic constructions the General Time Reversible (GTR) model of nucleotide substitution [ 13 ] which often fts the rRNA data better also used. Clustering was based on the neighbour-joining [ 14 ] and maximum-likelihood [ 15 ] methods. The bootstrap analysis is commonly based on 1000 resamples and was used to evaluate the topology of the phylogenetic tree [ 16 ]. Sequencing data of cheese microbiome was processed using the program Trimmomatic v0.39 [ 17 ]. Data quality control before and after processing was performed using FastQC v0.12.1 ( http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ ). Improved metagenomic analysis with Kraken 2 was used [ 18 ]. Results and Discussion Characteristics of strains isolated from starter raw milk and cheese The present study provides a comparative analysis of the microbiome of Ryazan farm cheese and the diversity of cultured lactic acid bacteria isolated from fermented milk and finished cheese. The different selective media and different cultivation temperatures were used for strains isolation, as described above. At high temperatures (40 o , 43 o C), the cultures of LAB were isolated only from cheese and were not isolated from fermented milk. A greater variety of LAB is also isolated on the MRS than on the YPD nutrient medium. Systematic study has resulted in the presence of gram-positive catalase-negative species, actively fermented milk. They formed dotted slow-growing colorless or milky colonies. The bacteria of genera Leuconostoc, Lacticaseibacillus, Limosilactobacillus and Lactiplantibacillus (totally 22 strains, 5 species) were isolated from the self – starter raw milk ( Leuconostoc mesenteroides, Lactiplantibacillus plantarum, Lactiplantibacillus pentosus, Leuconostoc falkenbergense ) and the cheese ( Leuconostoc mesenteroides, Lacticaseibacillus paracasei, Lacticaseibacillus rhamnosus, Limosilactobacillus fermentum, Lactiplantibacillus pentosus and Lactiplantibacillus plantarum ). Phylogenetic analysis of representatives of family Lactobacillaceae By phylogenetic analysis of 16S rRNA gene sequences, the isolated strains SCM6m1 and SCM14c were closely related Lactiplantibacillus pentosus , and SCM12c was closely related Lactiplantibacillus plantarum (Fig. 1 ). A BLAST analysis of the sequences revealed the degree of 16S rRNA gene sequence similarity with members of recognized taxa. Thus, the rrs sequence similarity of the strain SCM6m1 (1395 bp) was over 99% with the sequences of the type strains of the species L. paraplantarum , L. brownii , L. nangangensis ,, L. daoliensis , L. pingfangensis , and over 99.5% with the sequences of the type strains of the species L. plantarum , L. pentosus , L. argentoratensis. This indicates the need for a phylogenomic analysis of the isolated strains. The phylogenetic analysis of rrs gene sequences revealed the close relationships the strain SCM16c to Lacticaseibacillus paracasei ; the strains SCM17c, SCM18c and SCM20c - Lacticaseibacillus rhamnosus (Fig. 2 ). The strain SCM19c was close to Limosilactobacillus fermentum by phylogenetic analysis (Fig. 3 ). Previously, representatives of the genera Lacticaseibacillus , Lactiplantibacillus and Limosilactobacillus were considered part of the genus Lactobacillus . Morphology of representatives of the genus Lacticaseibacillus : single and paired bacilli, mesophilic, acid-resistant bacteria, moderately salt-resistant bacteria, lactic acid fermentation is homofermentative, optionally heterofermentative. Lacticaseibacillus ssp. are often found in various cheeses, but they are not used in starter cultures. Probiotic properties are attributed to the genus. Limosilactobacillus is acid-resistant, moderately salt-resistant, moderately thermophilic microorganisms. Representatives of the genus carry out heterofermentative lactic acid fermentation. The genus Limosilactobacillus is notable for its ability to synthesize B vitamins and bacteriocins. It is found in fermented dairy products, and also forms part of the human microflora. Representatives of the genus have probiotic properties. Phylogenetic analysis of representatives of family Leuconostocaceae The strains SCM1m, SCM2m, SCM3m, SCM5m, SCM6m2, SCM8m, SCM9m, SCM11c1, SCM15c, SCM20c1 were closely related by 16S rRNA encoding gene analysis to Leuconostoc mesenteroides ; SCM10m - Leuconostoc falkenbergense (Fig. 4 ). Morphologically, representatives of this genus are represented by elongated cocci forming chains. Bacteria of the genus Leuconostoc are characterized by a mesophilic temperature optimum and moderate tolerance to the presence of salts. The size of bacterial cells is 0.7–1.5 microns. These bacteria are characterized by heterofermentative lactic acid fermentation and mucus production. A number of odorous substances are isolated from Leuconostoc ssp., including acetoin and diacetyl. They are often used as an additional culture in industrial ferments, in particular in combination with the genus Lactococcus . Leuconostoc ssp are often found in butter, soft cheeses, and sauerkraut. In rare cases, Leuconostoc species can cause infectious diseases in humans. Microbiome analysis of farm cheese Ryazan homemade cheese metagenome was studies using 16S rRNA encoding gene (Fig. 5 ), its taxonomic and quantitative structures were ascertained. Staphylococcus eqourum (18,13%), Streptococcus thermophilus (16,79%), Lactiplantibacillus plantarum (16,14%), Lacticaseibacillus paracasei ( 14,06%), Lactococcus lactis (11,56%), as dominant species. Comparative analysis of cultivated microflora and microbiome of farm cheese resulted, that the cultivated species belonged to dominant ( Lactiplantibacillus plantarum, Lacticaseibacillus paracasei ), subdominant ( Leuconostoc falkenbergense , Leuconostoc mesenteroides ), small-numbered ( Lacticaseibacillus rhamnosus ) and rare species ( Leuconostoc pseudomesenteroides ) in the microbial community, but not other dominant – the representatives of genera Staphylococcus, Streptococcus and Lactococcus. It can be explained by antagonistic properties of some LAB manifested during cultivation and non-selective conditions for representatives of families Streptococcaceae ( Streptococcus, Lactococcus ) and Staphylococcaceae , which also observed in microbiome, but were not cultivated. Conclusion Comparative analysis of microbiome of farm cheeses from different Russian regions and over the world Russia has a long and rich culture of consumption of lactic acid products for many centuries. However, their composition has not been sufficiently studied. There are few studies of Russian farm cheese [ 1 , 8 ] that do not reflect the full picture. Ryazan city is located in the center of the European part of Russia, and the closest Tula region in the north of the Central Russian Upland was studied before [ 8 ]. The work showed, that the liquid lactic acid product (prostokvasha) samples dominated by Lactococcus , and the microbiome of cottage cheese was presented by Streptococcus ssp., Lactococcus ssp., Lactobacillus ssp. and Leuconostoc ssp. [ 8 ]. The present study provides a comparative analysis of the diversity of cultured lactic acid bacteria from Ryazan farm cheese and its microbiome. The diversity of the cheese cultured microflora was higher than self – starter raw milk one on selective media. The bacteria of genera Leuconostoc, Lactobacillus, Lacticaseibacillus, Limosilactobacillus and Lactiplantibacillus were isolated from self – starter raw milk and cheese. The microbiome diversity of cheese by 16S rRNA encoding gene analysis was higher as the diversity of cultured cheese LAB and included Staphylococcus eqourum (18,13%), Streptococcus thermophilus (16,79%), Lactiplantibacillus plantarum (16,14%), Lacticaseibacillus paracasei ( 14,06%), Lactococcus lactis (11,56%), as dominated species. Thus, the bacteria of the genera Streptococcus, Lactococcus and Staphylococcus were represented in cheese microbiome as dominant and subdominant, but were not isolated on nutrient media. It can be explained by LAB antagonistic properties manifested during cultivation and non-selective conditions for representatives of families Streptococcaceae ( Streptococcus, Lactococcus ) and Staphylococcaceae . The study and isolation of lactic acid and other bacteria from the microbiomes of lactic acid products is of great biotechnological importance, since they have been shown to possess a number of beneficial properties [ 5 , 6 , 7 ]. The biodiversity of cheeses is usually higher than the diversity of some other lactic products [ 1 ]. The bacterial compositions were largely different across the 4 types of naturally fermented milk at the genus level with predominant genera of Lactococcus (55.42%) and Streptococcus (31.90%) in cheese, Lactobacillus (82.75%) and Streptococcus (13.93%) in koumiss, Lactobacillus (78.44%), Streptococcus (14.08%), and Lactococcus (5.30%) in fermented yak milk, and Lactobacillus (64.69%), Lactococcus (14.62%), Streptococcus (10.29%) and Acetobacter (4.78%) in yogurt. Production starter cultures for cheese making are very diverse: they contain the following species Lactococcus lactis subsp. lactis , Lactococcus lactis subsp. cremoris , Lactococcus lactis subsp. diacetylactis , Streptococcus thermophilus , Lactobacillus delbrueckii subsp. bulgaricus , Lactobacillus delbrueckii subsp. helveticus , Lactobacillus delbrueckii subsp. casei ; sometimes – Penicillium sp . The previous studies shown that across the different product samples only several core genera — Lactobacillus , Streptococcus , and Lactococcus – usually predominating [ 1 ]. Lactobacillus are basic lactic acid bacteria in milk fermentation that degrade the disaccharide lactose, the major carbohydrate of milk. In addition to lactose metabolism, other metabolic properties of the Lactobacillus , such as proteolytic and lipolytic capacities, contribute significantly to the texture, taste, and aroma of fermented milk [ 19 ]. Streptococcus is another important genus that exists stably in naturally fermented milk (ranging from 10 to 30%). Streptococcus thermophilus is one of the most commonly used starter cultures in milk fermentation. Apart from its role in contributing to product texture, taste, and aroma, Strep. thermophilus generally produces bacteriocins that protect the final product from microbial spoilage [ 19 ]. Lactococcus accounted for a high proportion (about 50%) in the investigated cheeses [ 19 ].. These results are in line with some previous studies [ 20 , 21 ]. The major functions of these 3 LAB genera are the production of lactic acid, hydrolysis of casein, lipolysis of fat, and fermentation of citric acid. They have a significant influence on the texture and flavor of naturally fermented milk; thus, Lactobacillus , Streptococcus , and Lactococcus could be defined as the core microbiota in naturally fermented milk. A harsher heating step (35–55°C) that forces more whey from the curd is used to produce hard cheeses, which simultaneously selects for thermophilic bacteria such as Streptococcus . During the study, we noted also that some of the cultivated strains were slow-growing and difficult to maintain in culture. Finally, the dominant types of the cheese microbiome accounted for more than 77% of the abundance and belonged to the species Staphylococcus eqourum, Streptococcus thermophilus, Lactiplantibacillus plantarum , Lacticaseibacillus paracasei and Lactococcus lactis . Among the various representatives of the genus Staphylococcus present in homemade cheese, Staphylococcus aureus can pose a potential threat, which is not only capable of causing human diseases, but is also may be an antagonist of other LAB in cheese production [ 22 ]. Staphylococcus ssp. are often found in farm cheeses, in our case Staphylococcus aureus was a rare species (< 0,1%). Thus, we assume the need for research and selection of optimal conditions for the cultivation of lactic acid bacteria isolated from traditional farm lactic acid products, as well as genomic and phylogenomic analysis of the obtained strains. Abbreviations Metagenome, phylogeny, lactic acid bacteria (LAB), Leuconostoc, Lactobacillus, 16S rRNA encoding gene Declarations Conflict of interest. The authors declare that there is no conflict of interest. Funding This work was supported by the grant of the state program of the «Sirius» Federal Territory «Scientific and technological development of the «Sirius» Federal Territory» (Agreement № 18 − 03 dated 10.09.2024). Author Contribution NDW-K performed the phylogenetic and taxonomic analysis and wrote the main manuscript text. VDB performed microbiological and molecular analyses, ASR designed the work and supervised the work of the master's student. AES designed and supervised the sci work and read and approved the final manuscript version. All authors reviewed the manuscript. References Zhong Z, Hou Q, Kwok L, Yu Z, Zheng Y, Sun Z, Menghe B, Zhang H (2016) Bacterial microbiota compositions of naturally fermented milk are shaped by both geographic origin and sample type. 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TOJSAT 3 (2), https://www.tojsat.net/journals/tojsat/articles/v03i02/v03i02-04 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-7794861\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Short Report\",\"associatedPublications\":[],\"authors\":[{\"id\":527037813,\"identity\":\"af471fbd-65d3-4362-b493-4f7f9bab4e0d\",\"order_by\":0,\"name\":\"Natalia D. 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Rozanov\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Sirius University of Science and Technology\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Alexey\",\"middleName\":\"S.\",\"lastName\":\"Rozanov\",\"suffix\":\"\"},{\"id\":527037815,\"identity\":\"1adc5cfe-c473-4d03-b162-a7a10222d4bd\",\"order_by\":2,\"name\":\"Valery D. Bondar\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Sirius University of Science and Technology\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Valery\",\"middleName\":\"D.\",\"lastName\":\"Bondar\",\"suffix\":\"\"},{\"id\":527037816,\"identity\":\"1a656278-ecee-4b82-b5de-88225a14b126\",\"order_by\":3,\"name\":\"Alexey E. Sazonov\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Sirius University of Science and Technology\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Alexey\",\"middleName\":\"E.\",\"lastName\":\"Sazonov\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2025-10-07 01:23:12\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-7794861/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-7794861/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":98254857,\"identity\":\"678e7fb2-2a9f-4b01-b117-4a49f63ea1fc\",\"added_by\":\"auto\",\"created_at\":\"2025-12-15 17:56:31\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":149283,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eNeighbour-joining phylogenetic tree based on the 16S rRNA gene sequences of genus \\u003cem\\u003eLactiplantibacillus\\u003c/em\\u003e. Bar, 0.05 substitutions per nucleotide position. The maximumlikelihood tree produced by both Kimura's two-parameter [12] и the General Time Reversible (GTR) models [13] showed essentially the same topology (data not shown)\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7794861/v1/16e451f133b1e251e45e2cad.png\"},{\"id\":98435096,\"identity\":\"c33cea13-ee07-41ed-846f-cf0fe5083e8f\",\"added_by\":\"auto\",\"created_at\":\"2025-12-17 16:53:06\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":204754,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eNeighbour-joining phylogenetic tree based on the 16S rRNA gene sequences of genus \\u003cem\\u003eLacticaseibacillus\\u003c/em\\u003e. Bar, 0.02 substitutions per nucleotide position. The Maximum-likelihood tree produced by both Kimura's two-parameter [12] и the General Time Reversible (GTR) models [13] showed essentially the same topology (data not shown)\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7794861/v1/9347d2b18b663347a9b1075c.png\"},{\"id\":98435010,\"identity\":\"3d603653-ef68-411b-98ea-f23d0f47ecf7\",\"added_by\":\"auto\",\"created_at\":\"2025-12-17 16:52:55\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":178002,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eMaximum-likelihood phylogenetic tree of genus \\u003cem\\u003eLimosilactobacillus\\u003c/em\\u003e based on the 16S rRNA gene sequences analysis. Bar, 0.05 substitutions per nucleotide position. The neighbour-joining tree [14] showed essentially the same topology (data not shown)\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7794861/v1/0764d4608cde4ce2dafdeba5.png\"},{\"id\":98435064,\"identity\":\"00ac5b01-5fe4-45f4-be07-e0fedc96ce51\",\"added_by\":\"auto\",\"created_at\":\"2025-12-17 16:53:03\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":121375,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eNeighbour-joining phylogenetic tree based on the 16S rRNA gene sequences of genus \\u003cem\\u003eLeuconostoc\\u003c/em\\u003e. Bar, 0.02 substitutions per nucleotide position. The Maximum-likelihood tree produced by both Kimura's two-parameter [12] и the General Time Reversible (GTR) models [13] showed essentially the same topology (data not shown)\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7794861/v1/39d0da4bde04a4316e37bcea.png\"},{\"id\":98254861,\"identity\":\"dfd4c3f3-8e54-4aaa-a0fc-7a5cccb39d68\",\"added_by\":\"auto\",\"created_at\":\"2025-12-15 17:56:31\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":266111,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eMicrobiome 16S rRNA V4 analysis of Ryazan cheese [18]. The microbiome is represented by dominant (\\u0026gt;10%), subdominant (\\u0026gt;1%), small-numbered (\\u0026gt;0,1%) and rare (\\u0026lt;0,1%) species by abundancy.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7794861/v1/493faf8e206312147ef4a32a.png\"},{\"id\":98775567,\"identity\":\"bac1f5a8-27ab-4183-9ec8-f155729b29b0\",\"added_by\":\"auto\",\"created_at\":\"2025-12-22 12:20:20\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":1479268,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7794861/v1/3fb1ff4a-17b8-46a6-8768-3116be622dba.pdf\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Characteristics of Bacterial Diversity and Microbiome of Ryazan Farm Cheese\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eCheese microbiota plays a key role in defining its sensory properties, safety, and overall quality; therefore, its diversity is the subject of active study [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e]. The composition of the microflora is influenced, among other things, by the geographical characteristics of the place of manufacture, temperature, humidity and even the altitude above sea level [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e]. Bacteria in artisanal cheese primarily originate from two main sources: the inoculated starter cultures and the indigenous milk microbiota. Microorganisms originating from processing environments are also transferred from production surfaces to cheese surfaces and affect the final microbial\\u003c/p\\u003e\\u003cp\\u003ecomposition of product.\\u003c/p\\u003e\\u003cp\\u003eThe study and isolation of lactic acid and other bacteria from the microbiomes of lactic acid products is of great biotechnological importance, since they have been shown to possess a number of beneficial properties [\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e]. At the same time, some of them may have harmful properties, such as antibiotic resistance and toxins production [\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e].\\u003c/p\\u003e\\u003cp\\u003eNowadays there are known more than 150 names of liquid and solid lactic acid products in the world. Finally, in farm production their microbial composition may depend on the region of production, even with the same product name. It is also possible that not all national traditional lactic acid products are described. Russia has traditionally produced more than 60 names such products, although the exact number also is still unknown.\\u003c/p\\u003e\\u003cp\\u003eThe fermented dairy products have always been very diverse and popular in Russia; they have been produced industrially since the 18th century. At 19th century, research began on the effects of fermented dairy products and the lactic acid bacteria (LAB) on human health by N. Postnikov, I. Mechnikov (Nobel Prize winner), A. Dzhogin, S. Botkin and N. Sklifosovsky. Hospitals were opened where lactic acid drinks (kefir, koumiss, yogurt) were used for treatment.\\u003c/p\\u003e\\u003cp\\u003eHowever, there are few studies of Russian farm cheese [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e]. These studies mainly affect regions of cheese origin such as Buryatia and Siberia, Asian part of the Russian Federation [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e]. There is also a cheese study in the North Caucasus Federal District, Kabardino-Balkaria and Stavropol [\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e], the Southern Federal District, Kalmykia [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e], the Northwestern Federal District, Arkhangelsk [\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e], Far Eastern Federal District, Khabarovsk Territory [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e] and the Central Federal District, Tula [\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e]. These are isolated studies that do not reflect the full picture.\\u003c/p\\u003e\\u003cp\\u003eThis work includes a comparative analysis of the microbiome of Ryazan farm cheese and the diversity of cultured lactic acid bacteria isolated from fermented milk and finished cheese. The Ryazan region is one of traditional regions of Central Russia in terms of the production of lactic acid products. Ryazan fermented raw milk is a reservoir of LAB that are not affected by anthropogenic influences, so the study of this product allows us to get an idea of the composition of lactic acid bacteria in dairy products of central Russia that are not related to the food industry. Ryazan traditional cheese, on the contrary, is an example of a microbiological environment that has gone through a traditional technological process. The study of these products makes it possible not only to evaluate the bacterial community of lactic acid products in Central Russia, but also to identify strains of LAB that can subsequently be used to develop starter cultures.\\u003c/p\\u003e\"},{\"header\":\"Materials and Methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eBacterial Isolation and Culture Conditions\\u003c/h2\\u003e\\u003cp\\u003eRyazan homemade cheese and previous self \\u0026ndash; starter raw milk were used as objects for studying microbial communities of fermented dairy products of the Ryazan region, village Hirino. Ryazan farm milk was fermented with native microbiota at a temperature of 25 \\u0026deg; C for 2 weeks.\\u003c/p\\u003e\\u003cp\\u003eThe main object of research in this work is artisanal cheese from the Ryazan region. Since the microorganisms of this product have undergone anthropogenic selection (heat treatment, salting), cheese is a potential source of strains with valuable industrial properties, including resistance to high temperatures and salt, the probiotic properties, as well as the taste of the final product.\\u003c/p\\u003e\\u003cp\\u003eWe used the different cultivation temperatures (25, 32, 40 or 43\\u0026deg;C) and the different selective media (MRS, De Man\\u0026ndash;Rogosa\\u0026ndash;Sharpe, and YPD, Yeast Peptone Dextrose) to analyze the cultured microflora from cheese and previous self \\u0026ndash; starter raw milk. When using solid versions of the selected media, they were modified as follows: pasteurized milk \\\"Agusha\\\" 3.2% fat content (WBD, Russia) was added to the molten medium. The volume ratio of the microbiological medium and milk was 8:1. This stage provides an additional advantage to lactic acid bacteria (LAB), since they have the ability to metabolize peptides and carbohydrates in milk, including casein. After isolation of pure cultures, their morphological properties, catalase test, and milk fermentation were studied.\\u003c/p\\u003e\\u003cp\\u003eThe strains have been in storage at the Sirius University Collection of Microorganisms (SCM), Russia. Each strain name is followed by the strain origin marker - SCM1m- SCM10m from the self \\u0026ndash; starter raw milk and the cheese strains SCM11c- SCM22c.\\u003c/p\\u003e\\u003c/div\\u003e\\n\\u003ch3\\u003ePCR and Sequencing\\u003c/h3\\u003e\\n\\u003cp\\u003eDNA of strains was extracted by the conventional phenol-chloroform method [\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e]. PCR amplification targeting the 16S rRNA gene, purification and sequencing of PCR products was carried out as described in [\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e] with the primers for sequencing to yield an expected amplicon of approximately 1400 bp: F27 (sequence: AGAGTTTGATCMTGGCTCAG) and R1492 (sequence: TACGGYTACCTTGTTACGACTT). The BioMaster HS-Taq PCR-Color kit (Biolabmix, Russia) and the amplifier \\\"GeneExplorer 96\\\" (BIOER, China) were used.\\u003c/p\\u003e\\n\\u003ch3\\u003eMetagenome sequencing analyses\\u003c/h3\\u003e\\n\\u003cp\\u003eBacterial DNA was obtained from this mixture using the \\\"Quick-DNA Miniprep Plus Kit\\\" (Zymo Research, USA). A library was prepared from the V4 \\u003cem\\u003errs\\u003c/em\\u003e region sample of the Ryazan cheese metagenomic DNA using the VAHTS Universal Plus DNA Library Prep Kit (Nanjing Vazyme Biotech Co., China\\u003cb\\u003e)\\u003c/b\\u003e, on Illumina MiSeq (Illumina, USA).\\u003c/p\\u003e\\n\\u003ch3\\u003ePhylogenetic and Metagenome Analysis\\u003c/h3\\u003e\\n\\u003cp\\u003eA BLAST analysis of the obtained sequences was performed against sequences held in GenBank through the NCBI website (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttp://www.ncbi.nlm.nih.gov/\\u003c/span\\u003e\\u003cspan address=\\\"http://www.ncbi.nlm.nih.gov/\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e). Phylogenetic and phylogenomic analyses were performed using software package MEGA version X [\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e]. Analysis of the \\u003cem\\u003errs\\u003c/em\\u003e gene was based on sequences received in this study and sequences available in GenBank. Distances for both genes were calculated according to the Kimura's two-parameter model [\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e] and for the \\u003cem\\u003errs\\u003c/em\\u003e gene phylogenetic constructions the General Time Reversible (GTR) model of nucleotide substitution [\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e] which often fts the rRNA data better also used. Clustering was based on the neighbour-joining [\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e] and maximum-likelihood [\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e] methods. The bootstrap analysis is commonly based on 1000 resamples and was used to evaluate the topology of the phylogenetic tree [\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e].\\u003c/p\\u003e\\u003cp\\u003eSequencing data of cheese microbiome was processed using the program Trimmomatic v0.39 [\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e]. Data quality control before and after processing was performed using FastQC v0.12.1 (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttp://www.bioinformatics.babraham.ac.uk/projects/fastqc/\\u003c/span\\u003e\\u003cspan address=\\\"http://www.bioinformatics.babraham.ac.uk/projects/fastqc/\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e). Improved metagenomic analysis with Kraken 2 was used [\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e].\\u003c/p\\u003e\"},{\"header\":\"Results and Discussion\",\"content\":\"\\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eCharacteristics of strains isolated from starter raw milk and cheese\\u003c/h2\\u003e\\u003cp\\u003eThe present study provides a comparative analysis of the microbiome of Ryazan farm cheese and the diversity of cultured lactic acid bacteria isolated from fermented milk and finished cheese. The different selective media and different cultivation temperatures were used for strains isolation, as described above. At high temperatures (40\\u003csup\\u003eo\\u003c/sup\\u003e, 43\\u003csup\\u003eo\\u003c/sup\\u003eC), the cultures of LAB were isolated only from cheese and were not isolated from fermented milk. A greater variety of LAB is also isolated on the MRS than on the YPD nutrient medium.\\u003c/p\\u003e\\u003cp\\u003eSystematic study has resulted in the presence of gram-positive catalase-negative species, actively fermented milk. They formed dotted slow-growing colorless or milky colonies. The bacteria of genera \\u003cem\\u003eLeuconostoc, Lacticaseibacillus, Limosilactobacillus\\u003c/em\\u003e and \\u003cem\\u003eLactiplantibacillus\\u003c/em\\u003e (totally 22 strains, 5 species) were isolated from the self \\u0026ndash; starter raw milk (\\u003cem\\u003eLeuconostoc mesenteroides, Lactiplantibacillus plantarum, Lactiplantibacillus pentosus, Leuconostoc falkenbergense\\u003c/em\\u003e) and the cheese (\\u003cem\\u003eLeuconostoc mesenteroides, Lacticaseibacillus paracasei, Lacticaseibacillus rhamnosus, Limosilactobacillus fermentum, Lactiplantibacillus pentosus\\u003c/em\\u003e and \\u003cem\\u003eLactiplantibacillus plantarum\\u003c/em\\u003e).\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003ePhylogenetic analysis of representatives of family\\u003c/b\\u003e \\u003cb\\u003eLactobacillaceae\\u003c/b\\u003e\\u003c/p\\u003e\\u003cp\\u003eBy phylogenetic analysis of 16S rRNA gene sequences, the isolated strains SCM6m1 and SCM14c were closely related \\u003cem\\u003eLactiplantibacillus pentosus\\u003c/em\\u003e, and SCM12c was closely related \\u003cem\\u003eLactiplantibacillus plantarum\\u003c/em\\u003e (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). A BLAST analysis of the sequences revealed the degree of 16S rRNA gene sequence similarity with members of recognized taxa. Thus, the \\u003cem\\u003errs\\u003c/em\\u003e sequence similarity of the strain SCM6m1 (1395 bp) was over 99% with the sequences of the type strains of the species L. \\u003cem\\u003eparaplantarum\\u003c/em\\u003e, L. \\u003cem\\u003ebrownii\\u003c/em\\u003e, L. \\u003cem\\u003enangangensis\\u003c/em\\u003e,, L. \\u003cem\\u003edaoliensis\\u003c/em\\u003e, L. \\u003cem\\u003epingfangensis\\u003c/em\\u003e, and over 99.5% with the sequences of the type strains of the species L. \\u003cem\\u003eplantarum\\u003c/em\\u003e, L. \\u003cem\\u003epentosus\\u003c/em\\u003e, L. \\u003cem\\u003eargentoratensis.\\u003c/em\\u003e This indicates the need for a phylogenomic analysis of the isolated strains.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003eThe phylogenetic analysis of \\u003cem\\u003errs\\u003c/em\\u003e gene sequences revealed the close relationships the strain SCM16c to \\u003cem\\u003eLacticaseibacillus paracasei\\u003c/em\\u003e; the strains SCM17c, SCM18c and SCM20c - \\u003cem\\u003eLacticaseibacillus rhamnosus\\u003c/em\\u003e (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003eThe strain SCM19c was close to \\u003cem\\u003eLimosilactobacillus fermentum\\u003c/em\\u003e by phylogenetic analysis (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003ePreviously, representatives of the genera \\u003cem\\u003eLacticaseibacillus\\u003c/em\\u003e, \\u003cem\\u003eLactiplantibacillus and Limosilactobacillus\\u003c/em\\u003e were considered part of the genus \\u003cem\\u003eLactobacillus\\u003c/em\\u003e. Morphology of representatives of the genus \\u003cem\\u003eLacticaseibacillus\\u003c/em\\u003e: single and paired bacilli, mesophilic, acid-resistant bacteria, moderately salt-resistant bacteria, lactic acid fermentation is homofermentative, optionally heterofermentative. \\u003cem\\u003eLacticaseibacillus\\u003c/em\\u003e ssp. are often found in various cheeses, but they are not used in starter cultures. Probiotic properties are attributed to the genus.\\u003c/p\\u003e\\u003cp\\u003e\\u003cem\\u003eLimosilactobacillus is\\u003c/em\\u003e acid-resistant, moderately salt-resistant, moderately thermophilic microorganisms. Representatives of the genus carry out heterofermentative lactic acid fermentation. The genus \\u003cem\\u003eLimosilactobacillus\\u003c/em\\u003e is notable for its ability to synthesize B vitamins and bacteriocins. It is found in fermented dairy products, and also forms part of the human microflora. Representatives of the genus have probiotic properties.\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003ePhylogenetic analysis of representatives of family\\u003c/b\\u003e \\u003cb\\u003eLeuconostocaceae\\u003c/b\\u003e\\u003c/p\\u003e\\u003cp\\u003eThe strains SCM1m, SCM2m, SCM3m, SCM5m, SCM6m2, SCM8m, SCM9m, SCM11c1, SCM15c, SCM20c1 were closely related by 16S rRNA encoding gene analysis to \\u003cem\\u003eLeuconostoc mesenteroides\\u003c/em\\u003e; SCM10m - \\u003cem\\u003eLeuconostoc falkenbergense\\u003c/em\\u003e (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003eMorphologically, representatives of this genus are represented by elongated cocci forming chains. Bacteria of the genus \\u003cem\\u003eLeuconostoc\\u003c/em\\u003e are characterized by a mesophilic temperature optimum and moderate tolerance to the presence of salts. The size of bacterial cells is 0.7\\u0026ndash;1.5 microns. These bacteria are characterized by heterofermentative lactic acid fermentation and mucus production. A number of odorous substances are isolated from \\u003cem\\u003eLeuconostoc\\u003c/em\\u003e ssp., including acetoin and diacetyl. They are often used as an additional culture in industrial ferments, in particular in combination with the genus \\u003cem\\u003eLactococcus\\u003c/em\\u003e. \\u003cem\\u003eLeuconostoc\\u003c/em\\u003e ssp are often found in butter, soft cheeses, and sauerkraut. In rare cases, \\u003cem\\u003eLeuconostoc\\u003c/em\\u003e species can cause infectious diseases in humans.\\u003c/p\\u003e\\u003c/div\\u003e\\n\\u003ch3\\u003eMicrobiome analysis of farm cheese\\u003c/h3\\u003e\\n\\u003cp\\u003eRyazan homemade cheese metagenome was studies using 16S rRNA encoding gene (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e), its taxonomic and quantitative structures were ascertained. \\u003cem\\u003eStaphylococcus eqourum\\u003c/em\\u003e (18,13%), \\u003cem\\u003eStreptococcus thermophilus\\u003c/em\\u003e (16,79%), \\u003cem\\u003eLactiplantibacillus plantarum\\u003c/em\\u003e (16,14%), \\u003cem\\u003eLacticaseibacillus paracasei (\\u003c/em\\u003e14,06%), \\u003cem\\u003eLactococcus lactis\\u003c/em\\u003e (11,56%), as dominant species. Comparative analysis of cultivated microflora and microbiome of farm cheese resulted, that the cultivated species belonged to dominant (\\u003cem\\u003eLactiplantibacillus plantarum, Lacticaseibacillus paracasei\\u003c/em\\u003e), subdominant (\\u003cem\\u003eLeuconostoc falkenbergense\\u003c/em\\u003e, \\u003cem\\u003eLeuconostoc mesenteroides\\u003c/em\\u003e), small-numbered (\\u003cem\\u003eLacticaseibacillus rhamnosus\\u003c/em\\u003e ) and rare species (\\u003cem\\u003eLeuconostoc pseudomesenteroides\\u003c/em\\u003e) in the microbial community, but not other dominant \\u0026ndash; the representatives of genera \\u003cem\\u003eStaphylococcus, Streptococcus\\u003c/em\\u003e and \\u003cem\\u003eLactococcus.\\u003c/em\\u003e It can be explained by antagonistic properties of some LAB manifested during cultivation and non-selective conditions for representatives of families \\u003cem\\u003eStreptococcaceae\\u003c/em\\u003e (\\u003cem\\u003eStreptococcus, Lactococcus\\u003c/em\\u003e) and \\u003cem\\u003eStaphylococcaceae\\u003c/em\\u003e, which also observed in microbiome, but were not cultivated.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\"},{\"header\":\"Conclusion\",\"content\":\"\\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eComparative analysis of microbiome of farm cheeses from different Russian regions and over the world\\u003c/h2\\u003e\\u003cp\\u003eRussia has a long and rich culture of consumption of lactic acid products for many centuries. However, their composition has not been sufficiently studied. There are few studies of Russian farm cheese [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e] that do not reflect the full picture. Ryazan city is located in the center of the European part of Russia, and the closest Tula region in the north of the Central Russian Upland was studied before [\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e]. The work showed, that the liquid lactic acid product (prostokvasha) samples dominated by \\u003cem\\u003eLactococcus\\u003c/em\\u003e, and the microbiome of cottage cheese was presented by \\u003cem\\u003eStreptococcus ssp., Lactococcus ssp., Lactobacillus ssp.\\u003c/em\\u003e and \\u003cem\\u003eLeuconostoc ssp.\\u003c/em\\u003e [\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e].\\u003c/p\\u003e\\u003cp\\u003eThe present study provides a comparative analysis of the diversity of cultured lactic acid bacteria from Ryazan farm cheese and its microbiome. The diversity of the cheese cultured microflora was higher than self \\u0026ndash; starter raw milk one on selective media. The bacteria of genera \\u003cem\\u003eLeuconostoc, Lactobacillus, Lacticaseibacillus, Limosilactobacillus\\u003c/em\\u003e and \\u003cem\\u003eLactiplantibacillus\\u003c/em\\u003e were isolated from self \\u0026ndash; starter raw milk and cheese.\\u003c/p\\u003e\\u003cp\\u003eThe microbiome diversity of cheese by 16S rRNA encoding gene analysis was higher as the diversity of cultured cheese LAB and included \\u003cem\\u003eStaphylococcus eqourum\\u003c/em\\u003e (18,13%), \\u003cem\\u003eStreptococcus thermophilus\\u003c/em\\u003e (16,79%), \\u003cem\\u003eLactiplantibacillus plantarum\\u003c/em\\u003e (16,14%), \\u003cem\\u003eLacticaseibacillus paracasei (\\u003c/em\\u003e14,06%), \\u003cem\\u003eLactococcus lactis\\u003c/em\\u003e (11,56%), as dominated species. Thus, the bacteria of the genera \\u003cem\\u003eStreptococcus, Lactococcus\\u003c/em\\u003e and \\u003cem\\u003eStaphylococcus\\u003c/em\\u003e were represented in cheese microbiome as dominant and subdominant, but were not isolated on nutrient media. It can be explained by LAB antagonistic properties manifested during cultivation and non-selective conditions for representatives of families \\u003cem\\u003eStreptococcaceae\\u003c/em\\u003e (\\u003cem\\u003eStreptococcus, Lactococcus\\u003c/em\\u003e) and \\u003cem\\u003eStaphylococcaceae\\u003c/em\\u003e.\\u003c/p\\u003e\\u003cp\\u003eThe study and isolation of lactic acid and other bacteria from the microbiomes of lactic acid products is of great biotechnological importance, since they have been shown to possess a number of beneficial properties [\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e]. The biodiversity of cheeses is usually higher than the diversity of some other lactic products [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e]. The bacterial compositions were largely different across the 4 types of naturally fermented milk at the genus level with predominant genera of \\u003cem\\u003eLactococcus\\u003c/em\\u003e (55.42%) and \\u003cem\\u003eStreptococcus\\u003c/em\\u003e (31.90%) in cheese, \\u003cem\\u003eLactobacillus\\u003c/em\\u003e (82.75%) and \\u003cem\\u003eStreptococcus\\u003c/em\\u003e (13.93%) in koumiss, \\u003cem\\u003eLactobacillus\\u003c/em\\u003e (78.44%), \\u003cem\\u003eStreptococcus\\u003c/em\\u003e (14.08%), and \\u003cem\\u003eLactococcus\\u003c/em\\u003e (5.30%) in fermented yak milk, and \\u003cem\\u003eLactobacillus\\u003c/em\\u003e (64.69%), \\u003cem\\u003eLactococcus\\u003c/em\\u003e (14.62%), \\u003cem\\u003eStreptococcus\\u003c/em\\u003e (10.29%) and \\u003cem\\u003eAcetobacter\\u003c/em\\u003e (4.78%) in yogurt. Production starter cultures for cheese making are very diverse: they contain the following species \\u003cem\\u003eLactococcus lactis subsp. lactis\\u003c/em\\u003e, \\u003cem\\u003eLactococcus lactis subsp. cremoris\\u003c/em\\u003e, \\u003cem\\u003eLactococcus lactis subsp. diacetylactis\\u003c/em\\u003e, \\u003cem\\u003eStreptococcus thermophilus\\u003c/em\\u003e, \\u003cem\\u003eLactobacillus delbrueckii subsp. bulgaricus\\u003c/em\\u003e, \\u003cem\\u003eLactobacillus delbrueckii subsp. helveticus\\u003c/em\\u003e, \\u003cem\\u003eLactobacillus delbrueckii subsp. casei\\u003c/em\\u003e; sometimes \\u0026ndash; \\u003cem\\u003ePenicillium sp\\u003c/em\\u003e.\\u003c/p\\u003e\\u003cp\\u003eThe previous studies shown that across the different product samples only several core genera \\u0026mdash; \\u003cem\\u003eLactobacillus\\u003c/em\\u003e, \\u003cem\\u003eStreptococcus\\u003c/em\\u003e, and \\u003cem\\u003eLactococcus\\u003c/em\\u003e \\u0026ndash; usually predominating [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e]. \\u003cem\\u003eLactobacillus\\u003c/em\\u003e are basic lactic acid bacteria in milk fermentation that degrade the disaccharide lactose, the major carbohydrate of milk. In addition to lactose metabolism, other metabolic properties of the \\u003cem\\u003eLactobacillus\\u003c/em\\u003e, such as proteolytic and lipolytic capacities, contribute significantly to the texture, taste, and aroma of fermented milk [\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e].\\u003c/p\\u003e\\u003cp\\u003e\\u003cem\\u003eStreptococcus\\u003c/em\\u003e is another important genus that exists stably in naturally fermented milk (ranging from 10 to 30%). \\u003cem\\u003eStreptococcus thermophilus\\u003c/em\\u003e is one of the most commonly used starter cultures in milk fermentation. Apart from its role in contributing to product texture, taste, and aroma, Strep. \\u003cem\\u003ethermophilus\\u003c/em\\u003e generally produces bacteriocins that protect the final product from microbial spoilage [\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e].\\u003c/p\\u003e\\u003cp\\u003e\\u003cem\\u003eLactococcus\\u003c/em\\u003e accounted for a high proportion (about 50%) in the investigated cheeses [\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e].. These results are in line with some previous studies [\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e].\\u003c/p\\u003e\\u003cp\\u003eThe major functions of these 3 LAB genera are the production of lactic acid, hydrolysis of casein, lipolysis of fat, and fermentation of citric acid. They have a significant influence on the texture and flavor of naturally fermented milk; thus, \\u003cem\\u003eLactobacillus\\u003c/em\\u003e, \\u003cem\\u003eStreptococcus\\u003c/em\\u003e, and \\u003cem\\u003eLactococcus\\u003c/em\\u003e could be defined as the core microbiota in naturally fermented milk. A harsher heating step (35\\u0026ndash;55\\u0026deg;C) that forces more whey from the curd is used to produce hard cheeses, which simultaneously selects for thermophilic bacteria such as \\u003cem\\u003eStreptococcus\\u003c/em\\u003e. During the study, we noted also that some of the cultivated strains were slow-growing and difficult to maintain in culture.\\u003c/p\\u003e\\u003cp\\u003eFinally, the dominant types of the cheese microbiome accounted for more than 77% of the abundance and belonged to the species \\u003cem\\u003eStaphylococcus eqourum, Streptococcus thermophilus, Lactiplantibacillus plantarum\\u003c/em\\u003e, \\u003cem\\u003eLacticaseibacillus paracasei\\u003c/em\\u003e and \\u003cem\\u003eLactococcus lactis\\u003c/em\\u003e. Among the various representatives of the genus \\u003cem\\u003eStaphylococcus\\u003c/em\\u003e present in homemade cheese, \\u003cem\\u003eStaphylococcus aureus\\u003c/em\\u003e can pose a potential threat, which is not only capable of causing human diseases, but is also may be an antagonist of other LAB in cheese production [\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e]. \\u003cem\\u003eStaphylococcus\\u003c/em\\u003e ssp. are often found in farm cheeses, in our case \\u003cem\\u003eStaphylococcus aureus\\u003c/em\\u003e was a rare species (\\u0026lt;\\u0026thinsp;0,1%).\\u003c/p\\u003e\\u003cp\\u003eThus, we assume the need for research and selection of optimal conditions for the cultivation of lactic acid bacteria isolated from traditional farm lactic acid products, as well as genomic and phylogenomic analysis of the obtained strains.\\u003c/p\\u003e\\u003c/div\\u003e\"},{\"header\":\"Abbreviations\",\"content\":\"\\u003cp\\u003eMetagenome,\\u0026nbsp;phylogeny,\\u0026nbsp;lactic\\u0026nbsp;acid\\u0026nbsp;bacteria\\u0026nbsp;(LAB), \\u003cem\\u003eLeuconostoc, Lactobacillus,\\u0026nbsp;\\u003c/em\\u003e16S rRNA encoding gene\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cbr\\u003e\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eConflict of interest.\\u003c/strong\\u003e\\u003cp\\u003eThe authors declare that there is no conflict of interest.\\u003c/p\\u003e\\u003c/p\\u003e\\u003ch2\\u003eFunding\\u003c/h2\\u003e\\u003cp\\u003eThis work was supported by the grant of the state program of the \\u0026laquo;Sirius\\u0026raquo; Federal Territory \\u0026laquo;Scientific and technological development of the \\u0026laquo;Sirius\\u0026raquo; Federal Territory\\u0026raquo; (Agreement № 18\\u0026thinsp;\\u0026minus;\\u0026thinsp;03 dated 10.09.2024).\\u003c/p\\u003e\\u003ch2\\u003eAuthor Contribution\\u003c/h2\\u003e\\u003cp\\u003eNDW-K performed the phylogenetic and taxonomic analysis and wrote the main manuscript text. VDB performed microbiological and molecular analyses, ASR designed the work and supervised the work of the master's student. AES designed and supervised the sci work and read and approved the final manuscript version. All authors reviewed the manuscript.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eZhong Z, Hou Q, Kwok L, Yu Z, Zheng Y, Sun Z, Menghe B, Zhang H (2016) Bacterial microbiota compositions of naturally fermented milk are shaped by both geographic origin and sample type. 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J Dairy Sci 99:7832\\u0026ndash;7841. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.3168/jds.2015-10825\\u003c/span\\u003e\\u003cspan address=\\\"10.3168/jds.2015-10825\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eAlegr\\u0026iacute;a A, Szczesny P, Mayo B, Bardowski J, Kowalczyk M (2012) Biodiversity in Oscypek, a traditional Polish cheese, determined by culture-dependent and -independent approaches. Appl Environ Microbiol 78:1890\\u0026ndash;1898. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.1128/AEM.06081-11\\u003c/span\\u003e\\u003cspan address=\\\"10.1128/AEM.06081-11\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eQuigley EM (2013) Gut bacteria in health and disease. Gastroenterol Hepatol (N Y) 9(9):560\\u0026ndash;569\\u003c/span\\u003e\\u003c/li\\u003e\\u003cli\\u003e\\u003cspan\\u003eKo\\u0026iuml;che M, Dilmi Bouras A, Bouchakour H, Drahmoune L (2013) Behavior of Staphylococcus aureus in a cheese produced by local lactic acid bacteria. TOJSAT 3 (2), \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://www.tojsat.net/journals/tojsat/articles/v03i02/v03i02-04\\u003c/span\\u003e\\u003cspan address=\\\"https://www.tojsat.net/journals/tojsat/articles/v03i02/v03i02-04\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-7794861/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-7794861/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eThe composition of the microbiome of artisanal cheeses can be influenced by various factors, including temperature, pH, oxygen availability, and milk quality. The present study provides a comparative analysis of the diversity of cultured lactic acid bacteria from Ryazan farm cheese and its microbiome. To analyze the cultured microflora from cheese and previous self \\u0026ndash; starter raw milk, the different nutrient media and different cultivation temperatures were used. The bacteria of genera \\u003cem\\u003eLacticaseibacillus, Limosilactobacillus, Lactiplantibacillus\\u003c/em\\u003e and \\u003cem\\u003eLeuconostoc\\u003c/em\\u003e were isolated from milk and cheese. The microbiome diversity of cheese by 16S rRNA encoding gene analysis was higher than the diversity of cultured bacteria and included \\u003cem\\u003eStaphylococcus eqourum\\u003c/em\\u003e (18,13%), \\u003cem\\u003eStreptococcus thermophilus\\u003c/em\\u003e (16,79%), \\u003cem\\u003eLactiplantibacillus plantarum\\u003c/em\\u003e (16,14), \\u003cem\\u003eLacticaseibacillus paracasei (\\u003c/em\\u003e14,06%), \\u003cem\\u003eLactococcus lactis\\u003c/em\\u003e (11,56%), as dominated species. Thus, the cultivated species belonged to dominated (\\u003cem\\u003eLactiplantibacillus plantarum, Lacticaseibacillus paracasei\\u003c/em\\u003e), sub-dominated (\\u003cem\\u003eLeuconostoc falkenbergense\\u003c/em\\u003e, \\u003cem\\u003eLeuconostoc mesenteroides\\u003c/em\\u003e) and small-numbered (\\u003cem\\u003eLacticaseibacillus rhamnosus\\u003c/em\\u003e) species in the microbiome. It can be explained by LAB antagonistic properties manifested during cultivation and non-selective conditions for representatives of families \\u003cem\\u003eStreptococcaceae\\u003c/em\\u003e (\\u003cem\\u003eStreptococcus, Lactococcus\\u003c/em\\u003e) and \\u003cem\\u003eStaphylococcaceae\\u003c/em\\u003e. Representatives of the genus \\u003cem\\u003eStaphylococcus\\u003c/em\\u003e are often present in artisanal cheeses, and some of them may pose a risk to human health.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Characteristics of Bacterial Diversity and Microbiome of Ryazan Farm Cheese\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-12-15 17:56:26\",\"doi\":\"10.21203/rs.3.rs-7794861/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"f72222f8-c96e-4e2a-9526-70a2e2c76e15\",\"owner\":[],\"postedDate\":\"December 15th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2025-12-20T06:23:40+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2025-12-15 17:56:26\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-7794861\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-7794861\",\"identity\":\"rs-7794861\",\"version\":[\"v1\"]},\"buildId\":\"XKTyCvWXoU3ODBz1xrDgd\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}