Table_2_Zooming Into the Microbiota of Home-Made and Industrial Kefir Produced in Greece Using Classical Microbiological and Amplicon-Based Metagenomics Analyses.XLSX

Kefir is a high nutritional fermented dairy beverage associated with a wide range of health benefits. It constitutes a unique symbiotic association, comprising mainly lactic acid bacteria, yeasts, and occasionally acetic acid bacteria, which is strongly influenced by the geographical origin of the grains, the type of milk used, and the manufacture technology applied. Until recently, kefir microbiota has been almost exclusively studied by culture-dependent techniques. However, high-throughput sequencing, alongside omics approaches, has revolutionized the study of food microbial communities. In the present study, the bacterial, and yeast/fungal microbiota of four home-made samples (both grains and drinks), deriving from well spread geographical regions of Greece, and four industrial beverages, was elucidated by culture-dependent and -independent analyses. In all samples, classical microbiological analysis revealed varying populations of LAB and yeasts, ranging from 5.32 to 9.60 log CFU mL–1 or g–1, and 2.49 to 7.80 log CFU mL–1 or g–1, respectively, while in two industrial samples no yeasts were detected. Listeria monocytogenes, Salmonella spp. and Staphylococcus spp. were absent from all the samples analyzed, whereas Enterobacteriaceae were detected in one of them. From a total of 123 isolates, including 91 bacteria and 32 yeasts, Lentilactobacillus kefiri, Leuconostoc mesenteroides, and Lactococcus lactis as well as Kluvyeromyces marxianus and Saccharomyces cerevisiae were the mostly identified bacterial and yeast species, respectively, in the home-made samples. On the contrary, Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, and Lacticaseibacillus rhamnosus along with Debaryomyces hansenii and K. marxianus were the main bacterial and yeast species, respectively, isolated from the industrial beverages. In agreement with the identification results obtained from the culture-dependent approaches, amplicon-based metagenomics analysis revealed that the most abundant bacterial genera in almost all home-made samples (both grains and drinks) were Lactobacillus and Lactococcus, while Saccharomyces, Kazachstania, and Kluvyeromyces were the predominant yeasts/fungi. On the other hand, Streptococcus, Lactobacillus, and Lactococcus as well as Kluvyeromyces and Debaryomyces dominated the bacterial and yeast/fungal microbiota, respectively, in the industrial beverages. This is the first report on the microbiota of kefir produced in Greece by a holistic approach combining classical microbiological, molecular, and amplicon-based metagenomics analyses.

开菲尔（Kefir）是一种高营养发酵乳饮料，与多种健康益处相关。其具有独特的共生联合体结构，主要由乳酸菌（lactic acid bacteria）、酵母菌构成，偶含醋酸菌；该菌群组成受开菲尔粒的地理来源、所用乳类类型及生产工艺的显著影响。此前，开菲尔微生物群的研究几乎完全依赖培养技术。然而，高通量测序（high-throughput sequencing）与组学方法的结合，彻底革新了食品微生物群落的研究范式。本研究针对源自希腊广泛分布地理区域的4份自制开菲尔样品（含开菲尔粒与饮品）及4份工业级开菲尔饮品，通过依赖培养与非依赖培养的分析方法，解析其细菌与酵母菌/真菌微生物群。所有样品的经典微生物学分析显示，乳酸菌与酵母菌的菌落数分别为5.32~9.60 log CFU·mL⁻¹或g⁻¹、2.49~7.80 log CFU·mL⁻¹或g⁻¹；其中2份工业样品未检出酵母菌。所有分析样品中均未检出单核细胞增生李斯特菌（Listeria monocytogenes）、沙门氏菌属（Salmonella spp.）与葡萄球菌属（Staphylococcus spp.），仅1份样品检出肠杆菌科（Enterobacteriaceae）。本研究共获得123株分离株，包括91株细菌与32株酵母菌。自制样品中主要鉴定出的细菌物种为开菲乳杆菌（Lentilactobacillus kefiri）、肠膜明串珠菌（Leuconostoc mesenteroides）与乳酸乳球菌（Lactococcus lactis），酵母菌则以马克斯克鲁维酵母（Kluvyeromyces marxianus）与酿酒酵母（Saccharomyces cerevisiae）为主。与之相反，工业饮品中分离得到的主要细菌物种为嗜热链球菌（Streptococcus thermophilus）、德氏乳杆菌保加利亚亚种（Lactobacillus delbrueckii subsp. bulgaricus）与鼠李糖乳杆菌（Lacticaseibacillus rhamnosus），酵母菌则以汉逊德巴利酵母（Debaryomyces hansenii）与马克斯克鲁维酵母为主。与依赖培养方法的鉴定结果一致，基于扩增子的宏基因组学分析显示，几乎所有自制样品（开菲尔粒与饮品）中丰度最高的细菌属为乳杆菌属（Lactobacillus）与乳球菌属（Lactococcus），而优势酵母菌/真菌则为酿酒酵母属（Saccharomyces）、Kazachstania属与克鲁维酵母属（Kluvyeromyces）。另一方面，工业饮品的细菌与酵母菌/真菌微生物群分别以链球菌属（Streptococcus）、乳杆菌属、乳球菌属，以及克鲁维酵母属与德巴利酵母属（Debaryomyces）为主。本研究是首次采用结合经典微生物学、分子生物学与基于扩增子的宏基因组学的整体性研究方法，针对希腊生产的开菲尔微生物群开展的相关报道。