Metagenomic analysis and antibacterial activity of kefir microorganisms.
adhesion to Caco-2 cells
antibacterial activity
kefir microbiota
metagenomics
probiotic traits
Journal
Journal of food science
ISSN: 1750-3841
Titre abrégé: J Food Sci
Pays: United States
ID NLM: 0014052
Informations de publication
Date de publication:
Jul 2023
Jul 2023
Historique:
revised:
27
04
2023
received:
11
01
2023
accepted:
28
04
2023
medline:
17
7
2023
pubmed:
24
5
2023
entrez:
24
5
2023
Statut:
ppublish
Résumé
The microbiota composition of kefir grain and milk kefir was assessed via a metagenomic approach. Significant microorganisms were isolated and identified using molecular methods. A safety assessment was conducted based on antibiotic susceptibility and blood hemolysis. Probiotic traits such as resistance to gastric tract conditions, surface characteristics, adhesion to intestinal cells, and antibacterial activity were also assessed. Metagenomic analysis revealed that kefir grains are a more stable community with clear dominant species as compared to milk kefir. Lactobacillus kefiranofaciens BDGO-A1, Lactobacillus helveticus BDGO-AK2, and Lactobacillu kefiri strains showed tolerance to acidic pH and the presence of bile salts, adhesion capability to Caco-2 cells, in vitro antibacterial activity, and the production of antibacterial proteins. In the metagenomic analysis, contigs associated with these species showed the presence of genes involved in exporting polyketide antibiotics and bacteriocin production. To fully exploit the potential probiotic properties of these microorganisms to help human health, further investigation is necessary to elucidate the mechanisms behind the biological activity and the genotypic characteristics of the isolated strains.
Identifiants
pubmed: 37222548
doi: 10.1111/1750-3841.16614
doi:
Substances chimiques
Anti-Bacterial Agents
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
2933-2949Subventions
Organisme : NCI NIH HHS
ID : P30 CA016058
Pays : United States
Organisme : NCI NIH HHS
ID : P30 CA016058
Pays : United States
Informations de copyright
© 2023 The Authors. Journal of Food Science published by Wiley Periodicals LLC on behalf of Institute of Food Technologists.
Références
Azizi, N. F., Kumar, M. R., Yeap, S. K., Abdullah, J. O., Khalid, M., Omar, A. R., Osman, M. A., Mortadza, S. A. S., & Alitheen, N. B. (2021). Kefir and its biological activities. Foods, 10(6), 1210. https://doi.org/10.3390/foods10061210
Balouiri, M., Sadiki, M., & Ibnsouda, S. K. (2016). Methods for in vitro evaluating antimicrobial activity: A review. Journal of Pharmaceutical Analysis, 6(2), 71-79. https://doi.org/10.1016/j.jpha.2015.11.005
Blasche, S., Kim, Y., Mars, R. A. T., Machado, D., Maansson, M., Kafkia, E., Milanese, A., Zeller, G., Teusink, B., Nielsen, J., Benes, V., Neves, R., Sauer, U., & Patil, K. R. (2021). Metabolic cooperation and spatiotemporal niche partitioning in a kefir microbial community. Nature Microbiology, 6(2), 196-208. https://doi.org/10.1038/s41564-020-00816-5
Bourrie, B. C. T., Willing, B. P., & Cotter, P. D. (2016). The microbiota and health promoting characteristics of the fermented beverage kefir. Frontiers in Microbiology, 7(May), 1-17. https://doi.org/10.3389/fmicb.2016.00647
Breitwieser, F. P., & Salzberg, S. L. (2020). Pavian: Interactive analysis of metagenomics data for microbiome studies and pathogen identification. Bioinformatics (Oxford, England), 36(4), 1303-1304. https://doi.org/10.1093/BIOINFORMATICS/BTZ715
Cantalapiedra, C. P., Hern̗andez-Plaza, A., Letunic, I., Bork, P., & Huerta-Cepas, J. (2021). eggNOG-mapper v2: Functional annotation, orthology assignments, and domain prediction at the metagenomic scale. Molecular Biology and Evolution, 38(12), 5825-5829. https://doi.org/10.1093/MOLBEV/MSAB293
CFR. Code of Federal Regulations. (2002). Title 21: Food and Drugs (Rev. Apr. 1, 2002). U.S. Government Printing Office.
Cassanego, D., Richards, N., Valente, P., Mazutti, M., & Ramírez-Castrillon, M. (2017). Identification by PCR and evaluation of probiotic potential in yeast strains found in kefir samples in the city of Santa Maria, RS, Brazil. Food Science and Technology, 38, 59-65. https://doi.org/10.1590/1678-457X.13617
Carasi, P., Díaz, M., Racedo, S. M., De Antoni, G., Urdaci, M. C., & Serradell, M. D. L. A. (2014). Safety characterization and antimicrobial properties of kefir-isolated Lactobacillus kefiri. BioMed Research International, 2014, e208974. https://doi.org/10.1155/2014/208974
Carasi, P., Malamud, M., & Serradell, M. A. (2022). Potentiality of food-isolated Lentilactobacillus kefiri strains as probiotics: State-of-art and perspectives. Current Microbiology, 79(1), 1-11. https://doi.org/10.1007/S00284-021-02728-X/FIGURES/2
Chen, H. C., Wang, S. Y., & Chen, M. J. (2008). Microbiological study of lactic acid bacteria in kefir grains by culture-dependent and culture-independent methods. Food Microbiology, 25(3), 492-501. https://doi.org/10.1016/j.fm.2008.01.003
Chen, S., Zhou, Y., Chen, Y., & Gu, J. (2018). fastp: An ultra-fast all-in-one FASTQ preprocessor. Bioinformatics (Oxford, England), 34(17), i884-i890. https://doi.org/10.1093/BIOINFORMATICS/BTY560
Clinical and Laboratory Standards Institute (CLSI). (2015). Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Document M07-A10. CLSI.
Contreras-Martel, C., Martins, A., Ecobichon, C., Trindade, D. M., Matteï, P. J., Hicham, S., Hardouin, P., Ghachi, M. E., Boneca, I. G., & Dessen, A. (2017). Molecular architecture of the PBP2-MreC core bacterial cell wall synthesis complex. Nature Communications, 8(1), 1-10. https://doi.org/10.1038/s41467-017-00783-2
Dallas, D. C., Citerne, F., Tian, T., Silva, V. L. M., Kalanetra, K. M., Frese, S. A., Robinson, R. C., Mills, D. A., & Barile, D. (2016). Peptidomic analysis reveals proteolytic activity of kefir microorganisms on bovine milk proteins. Food Chemistry, 197, 273-284. https://doi.org/10.1016/j.foodchem.2015.10.116
de Almeida Brasiel, P. G., Dutra Medeiros, J., Barbosa Ferreira Machado, A., Schuchter Ferreira, M., Gouveia Peluzio, M. D. C., & Potente Dutra Luquetti, S. C. (2021). Microbial community dynamics of fermented kefir beverages changes over time. International Journal of Dairy Technology, 74(2), 324-331. https://doi.org/10.1111/1471-0307.12759
Diosma, G., Romanin, D. E., Rey-Burusco, M. F., Londero, A., & Garrote, G. L. (2014). Yeasts from kefir grains: Isolation, identification, and probiotic characterization. World Journal of Microbiology and Biotechnology, 30(1), 43-53. https://doi.org/10.1007/s11274-013-1419-9
Dobson, A., O'Sullivan, O., Cotter, P. D., Ross, P., & Hill, C. (2011). High-throughput sequence-based analysis of the bacterial composition of kefir and an associated kefir grain. FEMS Microbiology Letters, 320(1), 56-62. https://doi.org/10.1111/j.1574-6968.2011.02290.x
Du, G., Liu, L., Guo, Q., Cui, Y., Chen, H., Yuan, Y., Wang, Z., Gao, Z., Sheng, Q., & Yue, T. (2021). Microbial community diversity associated with Tibetan kefir grains and its detoxification of Ochratoxin A during fermentation. Food Microbiology, 99, 103803. https://doi.org/10.1016/j.fm.2021.103803
Englerová, K., Nemcová, R., & Mudroňová, D. (2017). The study of the probiotic potential of the beneficial bacteria isolated from kefir grains. Folia Veterinaria, 61(1), 27-37. https://doi.org/10.1515/fv-2017-0005
Ferrè, F., & Clote, P. (2006). DiANNA 1.1: An extension of the DiANNA web server for ternary cysteine classification. Nucleic Acids Research, 34, (Web Server issue) W182-W185. https://doi.org/10.1093/NAR/GKL189
García-Cano, I., Campos-Gómez, M., Contreras-Cruz, M., Serrano-Maldonado, C. E., González-Canto, A., Peña-Montes, C., Rodríguez-Sanoja, R., Sánchez, S., & Farrés, A. (2015). Expression, purification, and characterization of a bifunctional 99-kDa peptidoglycan hydrolase from Pediococcus acidilactici ATCC 8042. Applied Microbiology and Biotechnology, 99(20), 8563-8573. https://doi.org/10.1007/S00253-015-6593-2
García-Cano, I., Rocha-Mendoza, D., Ortega-Anaya, J., Wang, K., Kosmerl, E., & Jiménez-Flores, R. (2019). Lactic acid bacteria isolated from dairy products as potential producers of lipolytic, proteolytic and antibacterial proteins. Applied Microbiology and Biotechnology, 103(13), 5243-5257. https://doi.org/10.1007/s00253-019-09844-6
García-Cano, I., Serrano-Maldonado, C. E., Olvera-García, M., Delgado-Arciniega, E., Peña-Montes, C., Mendoza-Hernández, G., & Quirasco, M. (2014). Antibacterial activity produced by Enterococcus spp. isolated from an artisanal Mexican dairy product, Cotija cheese. LWT-Food Science and Technology, 59(1), 26-34. https://doi.org/10.1016/j.lwt.2014.04.059
Garofalo, C., Osimani, A., Milanović, V., Aquilanti, L., De Filippis, F., Stellato, G., Di Mauro, S., Turchetti, B., Buzzini, P., Ercolini, D., & Clementi, F. (2015). Bacteria and yeast microbiota in milk kefir grains from different Italian regions. Food Microbiology, 49(1), 123-133. https://doi.org/10.1016/j.fm.2015.01.017
Golowczyc, M. A., Gugliada, M. J., Hollmann, A., Delfederico, L., Garrote, G. L., Abraham, A. G., Semorile, L., & Antoni, G. D. (2008). Characterization of homofermentative lactobacilli isolated from kefir grains: Potential use as probiotic. Journal of Dairy Research, 75(2), 211-217. https://doi.org/10.1017/S0022029908003117
González-Orozco, B. D., García-Cano, I., Jimenez-Flores, R., Escobar-Zepeda, A., & Alvarez, V. B. (2022). Metagenomic comparison of kefir grains and milk kefir and identification of antimicrobial proteins. Poster presented at the American Dairy Science Association 2022 conference, Abstr. 86142, 86142.
Gut, A. M., Vasiljevic, T., Yeager, T., & Donkor, O. N. (2019). Characterization of yeasts isolated from traditional kefir grains for potential probiotic properties. Journal of Functional Foods, 58, (April) 56-66. https://doi.org/10.1016/j.jff.2019.04.046
Hamida, R. S., Shami, A., Ali, M. A., Almohawes, Z. N., Mohammed, A. E., & Bin-Meferij, M. M. (2021). Kefir: A protective dietary supplementation against viral infection. Biomedicine and Pharmacotherapy, 133, (November 2020) 110974. https://doi.org/10.1016/j.biopha.2020.110974
Hill, C., Guarner, F., Reid, G., Gibson, G. R., Merenstein, D. J., Pot, B., Morelli, L., Canani, R. B., Flint, H. J., Salminen, S., Calder, P. C., & Sanders, M. E. (2014). The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic. Nature Reviews Gastroenterology & Hepatology, 11(8), 506-514. https://doi.org/10.1038/nrgastro.2014.66
Hossain, M. I., Kim, K., Rahaman Mizan, M. F., Toushik, S. H., Ashrafudoulla, M., Roy, P. K., Nahar, S., Jahid, I. K., Choi, C., Park, S. H., & Ha, S. D. (2021). Comprehensive molecular, probiotic, and quorum-sensing characterization of anti-listerial lactic acid bacteria, and application as bioprotective in a food (milk) model. Journal of Dairy Science, 104(6), 6516-6534. https://doi.org/10.3168/jds.2020-19034
Hurtado-Romero, A., Del Toro-Barbosa, M., Gradilla-Hernández, M. S., Garcia-Amezquita, L. E., & García-Cayuela, T. (2021). Probiotic properties, prebiotic fermentability, and gaba-producing capacity of microorganisms isolated from Mexican milk kefir grains: A clustering evaluation for functional dairy food applications. Foods, 10(10), 2275. https://doi.org/10.3390/foods10102275
Jeong, D., Kim, D. H., Kang, I. B., Kim, H., Song, K. Y., Kim, H. S., & Seo, K. H. (2017). Characterization and antibacterial activity of a novel exopolysaccharide produced by Lactobacillus kefiranofaciens DN1 isolated from kefir. Food Control, 78, 436-442. https://doi.org/10.1016/j.foodcont.2017.02.033
Kazou, M., Grafakou, A., Tsakalidou, E., & Georgalaki, M. (2021). Zooming into the microbiota of home-made and industrial kefir produced in Greece using classical microbiological and amplicon-based metagenomics analyses. Frontiers in Microbiology, 12, 64. https://doi.org/10.3389/FMICB.2021.621069/BIBTEX
Kim, D. H., Chon, J. W., Kim, H., & Seo, K. H. (2015). Modulation of intestinal microbiota in mice by kefir administration. Food Science and Biotechnology, 24(4), 1397-1403. https://doi.org/10.1007/s10068-015-0179-8
Korsak, N., Taminiau, B., Leclercq, M., Nezer, C., Crevecoeur, S., Ferauche, C., Detry, E., Delcenserie, V., & Daube, G. (2015). Short communication: Evaluation of the microbiota of kefir samples using metagenetic analysis targeting the 16S and 26S ribosomal DNA fragments. Journal of Dairy Science, 98(6), 3684-3689. https://doi.org/10.3168/jds.2014-9065
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227(5259), 680-685. https://doi.org/10.1038/227680a0
Leite, A. M. D. O., Miguel, M. A. L., Peixoto, R. S., Rosado, A. S., Silva, J. T., & Paschoalin, V. M. F. (2013). Microbiological, technological and therapeutic properties of kefir: A natural probiotic beverage. Brazilian Journal of Microbiology, 44(2), 341-349. https://doi.org/10.1590/S1517-83822013000200001
Leite, A. M. O., Mayo, B., Rachid, C. T. C. C., Peixoto, R. S., Silva, J. T., Paschoalin, V. M. F., & Delgado, S. (2012). Assessment of the microbial diversity of Brazilian kefir grains by PCR-DGGE and pyrosequencing analysis. Food Microbiology, 31(2), 215-221. https://doi.org/10.1016/j.fm.2012.03.011
Lima, M. d. S. F. d., Souza, K. M. S. d., Albuquerque, W. W. C., Teixeira, J. A. C., Cavalcanti, M. T. H., & Porto, A. L. F. (2017). Saccharomyces cerevisiae from Brazilian kefir-fermented milk: An in vitro evaluation of probiotic properties. Microbial Pathogenesis, 110, 670-677. https://doi.org/10.1016/j.micpath.2017.05.010
Lu, J., Breitwieser, F. P., Thielen, P., & Salzberg, S. L. (2017). Bracken: Estimating species abundance in metagenomics data. PeerJ Computer Science, 2017(1), e104. https://doi.org/10.7717/PEERJ-CS.104/SUPP-5
McMurdie, P. J., & Holmes, S. (2013). phyloseq: An R package for reproducible interactive analysis and graphics of microbiome census data. PLoS One, 8(4), e61217. https://doi.org/10.1371/JOURNAL.PONE.0061217
Metras, B. N., Holle, M. J., Parker, V. J., Miller, M. J., & Swanson, K. S. (2021). Commercial kefir products assessed for label accuracy of microbial composition and density. JDS Communications, 2(3), 87-91. https://doi.org/10.3168/jdsc.2020-0056
Mora, D., Musacchio, F., Fortina, M. g., Senini, L., & Manachini, P. L. (2003). Autolytic activity and pediocin-induced lysis in Pediococcus acidilactici and Pediococcus pentosaceus strains. Journal of Applied Microbiology, 94(4), 561-570. https://doi.org/10.1046/j.1365-2672.2003.01868.x
Nalbantoglu, U., Cakar, A., Dogan, H., Abaci, N., Ustek, D., Sayood, K., & Can, H. (2014). Metagenomic analysis of the microbial community in kefir grains. Food Microbiology, 41, 42-51. https://doi.org/10.1016/J.FM.2014.01.014
Nejati, F., Junne, S., & Neubauer, P. (2020). A big world in small grain: A review of natural milk Kefir starters. Microorganisms, 8(2), 192. https://doi.org/10.3390/microorganisms8020192
Oksanen, J., Blanchet, F. G., Friendly, M., Kindt, R., Legendre, P., McGlinn, D., Minchin, P. R., O'Hara, R. B., Simpson, G. L., Solymos, P., Stevens, M. H. H., Szoecs, E., & Wagner, H. (2018). vegan: Community Ecology Package (2.4-6). https://CRAN.R-project.org/package=vegan
Perpetuini, G., Tittarelli, F., Suzzi, G., & Tofalo, R. (2019). Cell wall surface properties of Kluyveromyces marxianus strains from dairy-products. Frontiers in Microbiology, 10, 79. https://www.frontiersin.org/articles/10.3389/fmicb.2019.00079
Prjibelski, A., Antipov, D., Meleshko, D., Lapidus, A., & Korobeynikov, A. (2020). Using SPAdes De Novo Assembler. Current Protocols in Bioinformatics, 70(1), e102. https://doi.org/10.1002/CPBI.102
Quince, C., Walker, A. W., Simpson, J. T., Loman, N. J., & Segata, N. (2017). Shotgun metagenomics, from sampling to analysis. Nature Biotechnology, 35(9), 833-844. https://doi.org/10.1038/nbt.3935
Raja, H. A., Miller, A. N., Pearce, C. J., & Oberlies, N. H. (2017). Fungal identification using molecular tools: A primer for the natural products research community. Journal of Natural Products, 80, 756-770. https://doi.org/10.1021/acs.jnatprod.6b01085
Rocha-Mendoza, D., Kosmerl, E., Miyagusuku-Cruzado, G., Giusti, M. M., Jiménez-Flores, R., & García-Cano, I. (2020). Growth of lactic acid bacteria in milk phospholipids enhances their adhesion to Caco-2 cells. Journal of Dairy Science, 103(9), 7707-7718. https://doi.org/10.3168/jds.2020-18271
Rosa, D. D., Dias, M. M. S., Grześkowiak, Ł. M., Reis, S. A., Conceição, L. L., & Peluzio, M. D. C. G. (2017). Milk kefir: Nutritional, microbiological and health benefits. Nutrition Research Reviews, 30(1), 82-96. https://doi.org/10.1017/S0954422416000275
Sabir, F., Beyatli, Y., Cokmus, C., & Onal-Darilmaz, D. (2010). Assessment of potential probiotic properties of Lactobacillus spp., Lactococcus spp., and Pediococcus spp. strains isolated from kefir. Journal of Food Science, 75(9), M568-M573. https://doi.org/10.1111/j.1750-3841.2010.01855.x
Seemann, T. (2014). Prokka: Rapid prokaryotic genome annotation. Bioinformatics, 30(14), 2068-2069. https://doi.org/10.1093/BIOINFORMATICS/BTU153
Sirichokchatchawan, W., Pupa, P., Praechansri, P., Am-in, N., Tanasupawat, S., Sonthayanon, P., & Prapasarakul, N. (2018). Autochthonous lactic acid bacteria isolated from pig faeces in Thailand show probiotic properties and antibacterial activity against enteric pathogenic bacteria. Microbial Pathogenesis, 119, (March) 208-215. https://doi.org/10.1016/j.micpath.2018.04.031
Soeryapranata, E., Powers, J. R., & Ünlü, G. (2008). Degradation of αs1-CN f1-23 by aminopeptidase N and endopeptidases E, O, O2, and O3 of Lactobacillus helveticus WSU19 under cheese ripening conditions. International Dairy Journal, 18(2), 178-186. https://doi.org/10.1016/j.idairyj.2007.08.002
Tasteyre, A., Barc, M. C., Karjalainen, T., Bourlioux, P., & Collignon, A. (2002). Inhibition of in vitro cell adherence of Clostridium difficile by Saccharomyces boulardii. Microbial Pathogenesis, 32(5), 219-225. https://doi.org/10.1006/MPAT.2002.0495
Tenorio-Salgado, S., Castelán-Sánchez, H. G., Dávila-Ramos, S., Huerta-Saquero, A., Rodríguez-Morales, S., Merino-Pérez, E., Roa de la Fuente, L. F., Solis-Pereira, S. E., Pérez-Rueda, E., & Lizama-Uc, G. (2021). Metagenomic analysis and antimicrobial activity of two fermented milk kefir samples. MicrobiologyOpen, 10(2), e1183. https://doi.org/10.1002/MBO3.1183
Todorov, S. d., Botes, M., Guigas, C., Schillinger, U., Wiid, I., Wachsman, M. b., Holzapfel, W. H., & Dicks, L. M. T. (2008). Boza, a natural source of probiotic lactic acid bacteria. Journal of Applied Microbiology, 104(2), 465-477. https://doi.org/10.1111/j.1365-2672.2007.03558.x
Vall-Jääskeläinen, S. A., & Palva, A. (2005). Lactobacillus surface layers and their applications. FEMS Microbiology Reviews, 29(3), 511-529. https://doi.org/10.1016/J.FMRRE.2005.04.003
Van Wyk, J. (2019). Kefir: The champagne of fermented beverages. Fermented beverages: Volume 5. The science of beverages (pp. 473-527). Elsevier Inc. https://doi.org/10.1016/B978-0-12-815271-3.00012-9
Verce, M., De Vuyst, L., & Weckx, S. (2019). Shotgun metagenomics of a water kefir fermentation ecosystem reveals a novel Oenococcus species. Frontiers in Microbiology, 10, (March) 479. https://doi.org/10.3389/fmicb.2019.00479
Vieweg, L., Kretz, J., Pesic, A., Kerwat, D., Grätz, S., Royer, M., Cociancich, S., Mainz, A., & Süssmuth, R. D. (2015). The albicidin resistance factor AlbD is a serine endopeptidase that hydrolyzes unusual oligoaromatic-type peptides. Journal of the American Chemical Society, 137(24), 7608-7611. https://doi.org/10.1021/JACS.5B04099/SUPPL_FILE/JA5B04099_SI_001.PDF
Vinderola, C. G., Duarte, J., Thangavel, D., Perdigón, G., Farnworth, E., & Matar, C. (2005). Immunomodulating capacity of kefir. Journal of Dairy Research, 72(2), 195-202. https://doi.org/10.1017/S0022029905000828
Von Wintersdorff, C. J. H., Penders, J., Van Niekerk, J. M., Mills, N. D., Majumder, S., Van Alphen, L. B., Savelkoul, P. H. M., & Wolffs, P. F. G. (2016). Dissemination of antimicrobial resistance in microbial ecosystems through horizontal gene transfer. Frontiers in Microbiology, 7, (Feb) 173. https://doi.org/10.3389/FMICB.2016.00173/BIBTEX
Walsh, A. M., Crispie, F., Kilcawley, K., & Sullivan, O. O. (2016). Microbial Succession and Flavor Production in the Fermented Dairy Beverage Kefir. mSystems, 1(5), 1-16. https://doi.org/10.1128/mSystems.00052-16.Editor
Wang, H., Niu, Y., Pan, J., Li, Q., & Lu, R. (2020). Antibacterial effects of Lactobacillus acidophilus surface-layer protein in combination with nisin against Staphylococcus aureus. LWT, 124, 109208. https://doi.org/10.1016/J.LWT.2020.109208
Wang, X., Xiao, J., Jia, Y., Pan, Y., & Wang, Y. (2018). Lactobacillus kefiranofaciens, the sole dominant and stable bacterial species, exhibits distinct morphotypes upon colonization in Tibetan kefir grains. Heliyon, 4(6), 649. https://doi.org/10.1016/J.HELIYON.2018.E00649
Weisburg, W. G., Barns, S. M., Pelletier, D. A., & Lane, D. J. (1991). 16S ribosomal DNA amplification for phylogenetic study. Journal of Bacteriology, 173(2), 697-703. https://doi.org/10.1128/jb.173.2.697-703.1991
Wood, D. E., Lu, J., & Langmead, B. (2019). Improved metagenomic analysis with Kraken 2. Genome Biology, 20(1), 1-13. https://doi.org/10.1186/S13059-019-1891-0/FIGURES/2
Yang, M., Dutta, C., & Tiwari, A. (2015). Disulfide-bond scrambling promotes amorphous aggregates in lysozyme and bovine serum albumin. The Journal of Physical Chemistry. B, 119(10), 3960-3981. https://doi.org/10.1021/ACS.JPCB.5B00144
Yegin, Z., Zafer Yurt, M. N., Tasbasi, B. B., Acar, E. E., Altunbas, O., Ucak, S., Ozalp, V. C., & Sudagidan, M. (2022). Determination of bacterial community structure of Turkish kefir beverages via metagenomic approach. International Dairy Journal, 129, 105337. https://doi.org/10.1016/J.IDAIRYJ.2022.105337
Zanirati, D. F., Abatemarco, M., Sandes, S. H. D. C., Nicoli, J. R., Nunes, Á. C., & Neumann, E. (2015). Selection of lactic acid bacteria from Brazilian kefir grains for potential use as starter or probiotic cultures. Anaerobe, 32, 70-76. https://doi.org/10.1016/j.anaerobe.2014.12.007
Zheng, Y., Lu, Y., Wang, J., Yang, L., Pan, C., & Huang, Y. (2013). Probiotic Properties of Lactobacillus Strains Isolated from Tibetan Kefir Grains. PLoS One, 8(7), e69868. https://doi.org/10.1371/journal.pone.0069868
Zhou, J., Liu, X., Jiang, H., & Dong, M. (2009). Analysis of the microflora in Tibetan kefir grains using denaturing gradient gel electrophoresis. Food Microbiology, 26(8), 770-775. https://doi.org/10.1016/j.fm.2009.04.009