PCR–DGGE as a tool for characterizing dominant microbial populations in the Spanish blue-veined Cabrales cheese

The microbial populations of cheese milk and rennet extracts used in the production of traditional, Spanish, blue-veined Cabrales cheese were identified by PCR–DGGE analysis of the V3 region of the bacterial 16S rRNA gene and of the D1 region of the eukaryotic 26S rRNA genes. Ripe cheeses (60 days old) were examined in the same way. The results obtained by this culture-independent technique were compared to others previously obtained by conventional culturing methods. Rennet extracts were dominated by a number of Lactobacillus species, including Lb. plantarum, a non-starter lactic acid bacterium dominant during ripening. Lactococcus lactis was only found in one rennet extract. The cheese milk was clearly dominated by Lactococcus-like bacteria, with Lc. lactis in the greatest number. This bacterium was also dominant in the cheese samples (on both the surface and in the interior), in agreement with results obtained by culturing. The sequences of several bacterial DGGE bands from all samples showed less than 97% homology to known, cultured species. This indicates that unknown species are present in the Cabrales cheese environment and that culture-independent methods are needed to fully characterize this ecosystem.     

Artisanal and experimental Pecorino Siciliano cheese: microbial dynamics during manufacture assessed by culturing and PCR-DGGE analyses

Traditional artisanal Pecorino Siciliano (PS) cheeses, and two experimental PS cheeses were manufactured using either raw or pasteurised ewes' milk with the addition of starter cultures. The bacterial diversity and dynamics of the different cheese types were evaluated both by culturing and characterisation of isolates, and a culture-independent approach based on the 16S ribosomal RNA (rRNA) gene. Following cultivation, artisanal and experimental cheese types showed similar microbial counts, and isolates belonging to Lactococcus lactis, Streptococcus thermophilus, Enterococcus faecalis and Leuconostoc mesenteroides were identified by phenotypic characterisation and comparison of the restriction fragment length polymorphism (RFLP) of the 16S rRNA gene to that of reference species. The culture-independent fingerprinting technique PCR and denaturing gradient gel electrophoresis (DGGE) of V6 to V8 regions of the 16S rRNA gene of samples taken during artisanal PS cheese manufacture, from raw milk to the ripened cheese, indicated relevant shifts in the microbial community structure. The dominance of Streptococcus bovis and Lactococcus lactis species in the traditional artisanal PS was revealed by 16S rRNA gene sequencing. Comparison of DGGE profiles of samples from milk to ripened cheese, derived from artisanal procedure and the two experimental PS cheeses during production showed similar trends with the presence of intense bands in common. Nevertheless, the profiles of several artisanal cheeses from different farms appeared more diverse, and these additional species are probably responsible for the generally superior flavour and aroma development of traditional PS cheese.     

Influence of starter and nonstarter on the formation of biogenic amine in goat cheese during ripening

Two commercial starters were investigated for their potential ability to decarboxylate amino acids during goat cheese ripening. Two batches of goat cheese were produced with identical pasteurized milk but different starter cultures. One of them contained Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris and the other Lactococcus lactis subsp. lactis. The amine contents, microbial counts, proteolysis-related parameters, pH, total solids and salt content were studied in raw materials and cheeses. In raw materials, polyamines were the prevailing amines, whereas the main amines in cheeses were putrescine, tryptamine and, in particular, tyramine (94.59 mg/kg). Aerobic mesophilic microorganisms and Lactococcus counts increased throughout ripening, while Enterobacteriaceae were no longer detectable in cheese after 30 days of ripening. Amine concentration rose during cheese ripening in both batches. Moreover, the decarboxylase activity of microorganisms isolated from samples during cheese ripening was assayed and discussed.     

Microbial dynamics of Castelmagno PDO, a traditional Italian cheese, with a focus on lactic acid bacteria ecology

The dynamics of dominant microflora throughout the manufacture and ripening processes were evaluated in three batches of traditional Castelmagno PDO cheese. Milk, curd and cheese samples, at different stages during cheesemaking, were collected and subjected to culture-dependent and -independent analysis. Traditional plating and genetic identification of lactic acid bacteria (LAB) isolates, and PCR-DGGE analysis of V1 region of 16S rRNA gene were carried out. The collected samples were also monitored by HPLC for the presence of organic acids, sugars and ketones. LAB resulted to be the prevailing microflora in all production stages although enterococci, coagulase-negative cocci and yeasts also showed considerable viable counts probably related to the presence, in the dairy samples analysed, of free short-chain fatty acids detected by HPLC. Lactococcus lactis subsp. lactis was the species most frequently isolated during Castelmagno PDO manufacture, while Lactobacillus plantarum and Lactobacillus paracasei were isolated with the highest frequencies from ripened Castelmagno PDO cheese samples. Occasionally strains of Lactobacillus delbrueckii subsp. lactis, Lactobacillus coryniformis subsp. torquens and Lactobacillus casei were isolated. The results obtained on Castelmagno PDO microflora underlines a partial correspondence between culture-dependent method and DGGE analysis. Thus, in this study, it is highlighted once more the importance to combine molecular culture-independent approaches with classical microbiological methods for the study of complex environmental communities occurring in food matrices.     

Microbiological and chemical characterization of Fiore Sardo, a traditional Sardinian cheese made from ewe's milk

The purpose of this study was to assess the chemical and microbial characteristics of 12 batches of artisanal Fiore Sardo, a protected designation of origin (PDO) hard cheese made from raw ewe's milk without addition of starters, during maturation. High standard deviations were observed for moisture percentage, total solids percentage and NaCl percentage content, possibly owing to differences in manufacturing processes and/or milk composition. Total mesophilic bacteria varied between 10 log₁₀ cfu/g in 48-h-old cheese samples and 3 log₁₀ cfu/g in 9-month-old samples. Total coliforms and staphylococci showed the highest counts at 48 h of ripening then decreased significantly, dropping to levels below 2 log₁₀ cfu/g at 3 months of maturation. Lactic acid bacteria and enterococci were the dominant micro-organisms throughout maturation. They were mainly represented by the species Lactococcus lactis ssp. lactis, Enterococcus faecium, Lactobacillus plantarum and Lactobacillus casei group. Low levels of yeasts were detected throughout the maturation period of the cheese. Debaryomyces hansenii and Kluyveromyces lactis var. lactis were the prevalent yeast species isolated.     

Microbial characterization of Iranian traditional Lighvan cheese over manufacturing and ripening via culturing and PCR-DGGE analysis: identification and typing of dominant lactobacilli

This paper reports on the diversity and dynamics of the dominant microbial populations during manufacturing and ripening of Lighvan, a traditional, starter-free Iranian cheese made from raw ewe and goat’s milk as determined by culturing and PCR-DGGE. Similar dominant populations, composed of Lactococcus lactis and Lactobacillus spp. strains, were found by both techniques. However, discrepancies regarding the identity of the Lactobacillus species were encountered. Lactobacillus curvatus and Lactobacillus sakei proved to be dominant by PCR-DGGE; in contrast, Lactobacillus paraplantarum, Lactobacillus paracasei, Lactobacillus brevis and Lactobacillus plantarum were the majority cultivable organisms. RAPD typing of lactobacilli isolates showed wide genetic diversity among the species. Moreover, strain compositions change over time; L. brevis and L. paraplantarum were dominant in milk and were replaced by L. plantarum and L. paracasei strains as ripening progressed.     

阿尤恩地区自然发酵牛乳中乳酸菌分离鉴定及多样性研究

为了分离、保藏自然发酵乳中乳酸菌菌种,丰富自然发酵乳中乳酸菌多样性信息.本文采用传统的纯培养分离方法和宏基因组16S rRNA基因测序技术对阿尤恩地区自然发酵牛乳的乳酸菌多样性进行研究.纯培养结果表明:5份自然发酵牛乳中共分离出111株乳酸菌,鉴定为5个属10个种,其中Lactococcus lactis,占总分离株的...     

The behavior of selected microorganisms during the manufacture of high moisture Jack cheeses from ultrafiltered milk.

Whole milk was pasteurized and concentrated two times by ultrafiltration. Starter cultures, Lactococcus lactis ssp. cremoris and Lactococcus lactis ssp. lactis, were propagated in either reconstituted skim milk, two times UF retentate, or UF permeate, or a direct vat system was used for the starter culture. The cheese milk was simultaneously inoculated with starter culture and Pseudomonas fragi 4973, Staphylococcus aureus 196E, and Salmonella typhimurium var. Hillfarm. Control whole milk, UF control milk, inoculated whole milk, and inoculated UF milk were made into Monterey Jack cheese using traditional procedures. The process of cheese manufacture was followed by determination of pH, titratable acidity, and microbial population levels. The cheeses were stored for 6 mo and analyzed every month for percentage solids and microbial population levels. Generally, numbers of contaminant microbes increased at a similar rate during manufacture in all cheeses. During the 6-mo ripening period, bacterial starter culture population levels remained high, psychrotrophs declined slowly, Staphylococcus levels remained stable, and Salmonella populations decreased. No Staphylococcus enterotoxin was detected by reverse passive latex agglutination assay.     

Microbiological characterization of artisanal Raschera PDO cheese: analysis of its indigenous lactic acid bacteria

The aim of this research was to study the bacterial populations involved in the production of artisanal Raschera PDO cheese (Italian Maritime Alps, northwest Italy) in order to collect preliminary knowledge on indigenous lactic acid bacteria (LAB). A total of 21 samples of Raschera PDO cheese, collected from six dairy farms located in the production area, were submitted to microbiological analysis. LAB were randomly isolated from M17 agar, MRS agar and KAA plates and identified by combining PCR 16S-23S rRNA gene spacer analysis, species-specific primers and 16S rRNA gene sequencing. Biodiversity of Lactococcus lactis subsp. lactis isolates was investigated by RAPD-PCR. LAB microflora showed the highest count values among all microbial groups targeted. They reached counts of 10(9) colony forming unit (cfu)/g in cheese samples after 3 days of salting and 15 days of ripening. Yeast population also showed considerable count values, while enterococci and coagulase-negative cocci (CNC) did not overcome 10(7)cfu/g. L. lactis subsp. lactis was the species most frequently isolated from Raschera PDO samples at all different production stages while in aged cheeses Lactobacillus paracasei was frequently isolated. RAPD-PCR highlighted that isolates of L. lactis subsp. lactis isolated from Raschera PDO were highly homogeneous.     

Bacterial diversity dynamics of traditional Turkish Ezine Cheese as evaluated by PCR‐DGGE and SSCP analysis

The aim of this study was to evaluate the bacterial ecosystem of milk and Ezine cheese by PCR amplification of the V3 region of the bacterial 16S rRNA gene followed by denaturing gradient gel electrophoresis (DGGE) and by monitoring the bacterial diversity dynamics using PCR single‐strand conformation polymorphism (SSCP) analysis. PCR‐DGGE analysis revealed that 17 different bands and strains belonging to the Lactococcus lactis group and Streptococcus thermophilus were predominant during manufacturing and ripening. SSCP analysis revealed that the bacterial profiles of the two cheese samples were similar.     

Homofermentative lactic acid bacteria of a traditional cheese, Comlek peyniri from Cappadocia region

Comlek peyniri is a typical artisanal cheese in Central Anatolia. This type of cheese was made by using the indigenous lactic acid bacteria (LAB) flora of cow or ewes' milk. Majority of the samples were taken from fresh cheese because the aim was to isolate homofermentative LAB. Initially 661 microbial isolates were obtained from 17 cheese samples. Only 107 were found to be homofermentative LAB. These isolates were selected and identified by using both phenotypic and molecular methods. Phenotypic identification included curd formation from skim milk, catalase test, Gram staining and light microscopy, growth at different temperatures and salt concentrations, arginine hydrolysis, gas production from glucose, and carbohydrate fermentation. Molecular identification was based on the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the 16S rRNA gene-ITS (internally transcribed spacer) region. By combining the phenotypic and molecular identification results, isolates belonging to each of the following genera were determined at species or subspecies level: 54 Lactococcus lactis subsp. lactis, 21 Enterococcus faecium, 3 Ec. faecalis, 2 Ec. durans, 10 Ec. sp., 15 Lactobacillus paracasei subsp. paracasei, and 2 Lb. casei strains. Technological characterisation was also performed by culturing each of the strains in UHT skim milk, and by monitoring pH change and lactic acid production at certain time intervals through the 24 h incubation. Results of the technological characterisation indicated that 33% of the isolates (35 strains) were capable of lowering the pH of UHT milk below 5.3 after 6 h incubation at 30 degrees C. Thirty four of these strains were Lc. lactis subsp. lactis, and only one was an Ec. faecium strain.     

Nisin-producing organisms during traditional 'Fior di latte' cheese-making monitored by multiplex-PCR and PFGE analyses

In this work we studied using different molecular methods the population dynamics of nisin-producing organisms and the persistence of such organisms within a complex ecosystem, 'Fior di latte' cheese, a traditional high-moisture pasta filata cheese. Using the primers targeting the eubacterial 16S-23S rRNA spacer region, together with those amplifying the nisA or nisZ gene, we were able to provide a rapid species identification of the isolates. Inhibitors of Lactococcus lactis subsp. lactis DSM 20481T used as indicator occurred during the whole process of cheese manufacture as a significant part of lactic microflora; however, only 12 among 109 isolates of bacteriocin producers were nisin producers. Amplification of the nisA or nisZ gene, using DNA extracted directly from dairy samples as templates, showed that the nisin structural gene was detected during cheese-making from milk samples up to the end of curd ripening but not in the final cheese. In order to monitor nisin-producing strains during cheese manufacturing, the 12 Lactococcus lactis nis+ strains were analysed by low frequency restriction fragment and PFGE. Nine isolates among the 12 nisin-producers exhibited an unique and distinct DNA banding pattern and are considered to be genetically diverse. The other three isolates from curd after ripening showed the same restriction pattern and could be the same strain. In fact, it was also isolated 2 months after the first analysis of cheese-making of 'Fior di latte'.     

Indigenous raw milk microbiota influences the bacterial development in traditional cheese from an alpine natural park

Nostrano di Primiero is a 6-month ripened cheese produced from raw milk collected in the Paneveggio-Pale di San Martino Natural Park area in the Italian Dolomites. In summer, this cheese is made using milk collected from two different areas, Passo Rolle and Vanoi, in the Paneveggio Natural Park. During the experiment, the milk from the two areas was separately processed, and cheeses were made in the same cheese factory using the same technological process. The microbiota of raw milk and cheeses of the two areas was isolated and the dominant population was monitored by RAPD analysis and identified by 16S rRNA sequence. The milk of the Passo Rolle area was mainly composed of mesophilic strains, thermophilic Streptococcus thermophilus, and low amounts of enterococci were also found; the milk of the Vanoi area was dominated by mesophilic microbiota mostly Lactococcus lactis ssp. cremoris and ssp. lactis and Lactobacillus paracasei ssp. paracasei. The plating of the natural starter culture revealed the presence of a relevant community of thermophilic cocci and lower amounts of enterococci. The dynamic population analysis showed the importance of the natural starter culture in the first 2 days of cheese ripening in both cheeses. Moreover, the large biodiversity observed in the raw milks was also detected in the cheeses during ripening. The Vanoi cheese was dominated by Enterococcus faecium and Streptococcus macedonicus in the first two days and mesophilic 21 Lb. paracasei ssp. paracasei became the most represented population after 15 days of ripening. In the first few days, the Rolle cheese was characterized by being mainly composed of thermophilic S. macedonicus and S. thermophilus and secondarily by mesophilic cocci. During ripening, the microbiota composition changed, and at 15 days, mesophilic lactobacilli were the dominant population, but later, this was mainly composed of mesophilic cocci and lactobacilli. The taxonomical identification by 16S rRNA sequence confirmed a large biodiversity related to raw milk microbiota and only five strains of S. macedonicus, Lactobacillus plantarum, 21 Lb. paracasei ssp. paracasei, Lactobacillus fermentum and E. faecium were detected in both cheeses.     

Formation of biogenic amines in raw milk Hispánico cheese manufactured with proteinases and different levels of starter culture

Two proteinases, a neutral proteinase from Bacillus subtilis and a cysteine proteinase from Micrococcus sp., were used to accelerate the ripening process of raw cow's milk Hispánico cheese, a semihard variety. Two levels (0.1% and 1%) of a commercial starter culture containing Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris were added for cheese manufacture. The influence of both factors, proteinase addition and level of starter culture, on the growth of amino acid-decarboxylating microorganisms and on the formation of biogenic amines during cheese ripening was investigated in duplicate experiments. The population of tyrosine decarboxylase-positive bacteria, which represented less than 1% of the total bacterial population in most cheese samples, and tyrosine decarboxylase-positive lactobacilli was not influenced by proteinase addition or level of starter culture. Tyramine was detected in all batches of cheese from day 30. Its concentration was significantly (P < 0.05) influenced by proteinase addition but not by the level of starter culture and increased with cheese age. After 90 days of ripening, 103 to 191 mg/kg of tyramine was found in the different cheese batches. Histamine was not detected until day 60 in cheese with neutral proteinase and 1% starter culture and until day 90 in the rest of the cheeses. The concentration of this amine did not exceed 20 mg/kg in any of the batches investigated. Phenylethylamine and tryptamine were not found in any of the samples.     

Biodiversity of the microbial community in a Spanish farmhouse cheese as revealed by culture-dependent and culture-independent methods

The microbial diversity within Alberquilla cheese, made from a spontaneously fermented mixture of raw goats' and sheep's milk in the Alpujarra mountains (Granada, south-east Spain), has been studied by the classical culturing method and also by molecular analysis of community DNA. A collection of 206 isolates was obtained from the cheese on different selective/differential media, which were then re-grouped to 52 after randomly amplified polymorphic DNA (RAPD)-PCR analyses. Isolates on Man-Rogosa and Sharpe-agar (MRS), M17-glucose agar and Kenner Fecal (KF)-agar medium were identified by specific PCR or 16S rRNA gene sequencing and belonged mainly to the lactic-acid bacteria group. The predominant genus was Lactobacillus, which accounted for more than 50% of the isolates, the most abundant species being Lactobacillus paracasei, followed by considerably less quantities of Lb. plantarum and Lb. brevis. Other lactic-acid bacteria identified were Pediococcus urinaequi, Leuconostoc pseudomesenteroides, Leuc. mesenteroides, Lactococcus lactis and even the enterococci Enterococcus faecium and E. devriesei. Cluster analyses of RAPD-PCR patterns revealed a high degree of diversity among the lactobacilli. The Gram-negative bacterial strains belonged mainly to Hafnia alvei species. The microbes occurring in Alberquilla cheese were also studied by PCR temporal temperature-gradient gel electrophoresis (TTGE) of the 16S rRNA V3 region and partial 16S rRNA sequencing of the TTGE bands. The results showed a major presence of lactic-acid bacteria closely related to Lc. lactis, Lb. paracasei, Lb. plantarum, Lb. brevis, Lb. acidophilus and Enterococcus sp. The non-lactic-acid bacterium detected was identified as Escherichia coli. All the Enterococcus strains showed great susceptibility to the most clinically relevant antibiotics, harbouring only the virulence gene efaAfm. On the basis of their antimicrobial activity against Listeria monocytogenes we chose two strains of Ln. mesenteroides that produced mesenterocin B105 and mesenterocin Y105, as revealed by PCR techniques.     

Application of Single Strand Conformation Polymorphism --PCR method for distinguishing cheese bacterial communities that inhibit Listeria monocytogenes

The aim of this study was to compare the microbial communities of different cheeses where Listeria monocytogenes either grew or did not grow. For this purpose, (i) isolates from the most inhibitory cheese ecosystem were identified and their ability to produce anti-Listeria substances was determined, (ii) bacterial communities of cheeses with and without L. monocytogenes growth were compared using the Single Strand Conformation Polymorphism method. The study showed SSCP to be an effective tool for differentiating between the bacterial communities of different cheeses manufactured with the same technology. All the cheeses with the lowest L. monocytogenes counts on day 8 were distinguished by the dominance in their SSCP profiles, after amplification of the V2 region of the 16S rRNA gene, of 3 peaks whose nucleotide sequences comigrated with Enterococcus faecium and Enterococcus saccharominimus, Chryseobacterium sp and Corynebacterium flavescens, Lactococcus garvieae and Lactococcus lactis respectively. However, no anti-Listeria compounds were produced under our experimental conditions. These six bacterial species were inoculated, separately or together, into pasteurised milk and their anti-listerial activity in cheese was evaluated. The area of inhibition between the control and trial curves confirmed that L. monocytogenes is inhibited by E. saccharominimus, C. flavescens, L. lactis, L. garvieae and the mixture of all six bacterial strains. Further studies should be performed to determine the metabolites involved in L. monocytogenes inhibition.     

益生菌切达干酪成熟过程中细菌群落的多样性分析

采用选择性培养基和聚合酶链式反应和变性梯度凝胶电泳（polymerase chain reaction-denaturing gradient gelelectrophoresis,PCR-DGGE）技术,研究益生菌切达干酪成熟过程中（6 ℃,180 d）细菌群落构成及益生菌（干酪乳杆菌LC2W）的存活情况。结果表明：SBM和MSE等选择性培养基存在选择专一性不强的缺点,不能客观反映干酪内各种微生物的动态变化;随着切达干酪成熟时间的增加,发酵剂嗜热链球菌和乳酸乳球菌的数量明显下降,而非发酵剂菌群乳杆菌的数量和主要种类呈上升趋势;干酪成熟180 d后,干酪乳杆菌LC2W的存活量仍高于1×108CFU/g。切达干酪能作为干酪乳杆菌LC2W存活的良好载体;PCR-DGGE技术和选择计数法联用更加适合干酪细菌群落结构的分析。     

The fate of indigenous microbiota, starter cultures, Escherichia coli, Listeria innocua and Staphylococcus aureus in Danish raw milk and cheeses determined by pyrosequencing and quantitative real time (qRT)-PCR

The purpose of this work was to study the bacterial communities in raw milk and in Danish raw milk cheeses using pyrosequencing of tagged amplicons of the V3 and V4 regions of the 16S rDNA and cDNA. Furthermore, the effects of acidification and ripening starter cultures, cooking temperatures and rate of acidification on survival of added Escherichia coli, Listeria innocua and Staphylococcus aureus in cheeses at different stages of ripening were studied by pyrosequencing and quantitative real time (qRT)-PCR. A high diversity of bacterial species was detected in raw milk. Lactococcus lactis, Streptococcus thermophilus, Lactobacillus casei and Lactobacillus rhamnosus were the main bacteria detected in raw milk and cheeses. Bacteria belonging to the genera Brevibacterium, Staphylococcus, Escherichia, Weissella, Leuconostoc, Pediococcus were also detected in both 16S rDNA and cDNA obtained from raw milk and cheeses. E. coli, which was added to milk used for production of some cheeses, was detected in both DNA and RNA extracted from cheeses at different stages of ripening showing the highest percentage of the total sequence reads at 7 days of ripening and decreased again in the later ripening stages. Growth of E. coli in cheeses appeared to be affected by the cooking temperature and the rate of acidification but not by the ripening starter cultures applied or the indigenous microbiota of raw milk. Growth of L. innocua and S. aureus added to milks was inhibited in all cheeses at different stages of ripening. The use of 16S rRNA gene pyrosequencing and qRT-PCR allows a deeper understanding of the behavior of indigenous microbiota, starter cultures and pathogenic bacteria in raw milk and cheeses.     

Isolation of lactic acid bacteria from Lighvan cheese, a semihard cheese made from raw sheep milk in Iran

The lactic acid bacteria contributing to Lighvan cheese ripening during the different stages of production were investigated. Isolated strains from different culture media were identified phenotypically to species and subspecies level. In total, 413 strains were isolated from raw milk, 1-day-old cheese and fully ripened cheese. The most abundant species belonged to Enterococcus faecium (87 isolates), Lactococcus lactis ssp. lactis (68 isolates), Enterococcus faecalis (55 isolates) and Lactobacillus plantarum (48 isolates). E. faecium, Lc. lactis and Lb. plantarum were the predominantly isolated strains from ripened cheese. Therefore, they may contribute considerably to the aroma and flavour development of Lighvan cheese.     

Bacterial biodiversity of traditional Zabady fermented milk

The aim of this work was to identify the bacterial biodiversity of traditional Zabady fermented milk using PCR-temporal temperature gel electrophoresis (PCR-TTGE) and PCR-denaturing gradient gel electrophoresis (PCR-DGGE). Most of the identified bacterial species in Zabady samples belonged to lactic acid bacteria (LAB), e.g., Streptococcus thermophilus, Lactococcus garvieae, Lactococcus raffinolactis, Lactococcus lactis, Leuconostoc citreum, Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus johnsonii. Using the culture-dependent and independent methods, the streptococcal and lactococcal groups appeared to be the major bacterial species in Zabady fermented milk, whereas the lactobacilli were the minor ones. The main dominant species was St. thermophilus followed by Lc. garvieae. Other molecular tools, e.g., species-specific PCR assay and cloning and sequencing strategy were used to confirm the TTGE and DGGE results. Lc. garvieae, Lc. raffinolactis, Ln. citreum, and Lb. johnsonii were identified for the first time in this type of Egyptian fermented milk.