Evaluation of bacterial communities belonging to natural whey starters for Grana Padano cheese by length heterogeneity-PCR

AIMS: To detect bacteria present in controlled dairy ecosystems with defined composition by length-heterogeneity (LH)-PCR. LH-PCR allows to distinguish different organisms on the basis of natural variations in the length of 16S rRNA gene sequences. METHODS AND RESULTS: LH-PCR was applied to depict population structure of the lactic acid bacteria (LAB) species recoverable from Grana Padano cheese whey starters. Typical bacterial species present in the LAB community were evidenced and well discriminated. Small differences in species composition, e.g. the frequent finding of Streptococcus thermophilus and the constant presence of thermophilic lactobacilli (Lactobacillus helveticus, Lact. delbrueckii subsp. lactis/bulgaricus and Lact. fermentum) were reliably highlighted. Specificity of LH-PCR was confirmed by species-specific PCR from total DNA of the cultures. CONCLUSIONS: LH-PCR is a useful tool to monitor microbial composition and population dynamics in dairy starter cultures. When present, non-dominant bacterial species present in the whey starters, such as Strep. thermophilus, can easily be visualized and characterized without isolating and cultivating single strains. A similar approach can be applied to more complex dairy ecosystems such as milk or cheese curd. SIGNIFICANCE AND IMPACT OF THE STUDY: Community members and differences in population structure of controlled dairy ecosystems such as whey starters for hard cheeses can be evaluated and compared in a relative easy, fast, reliable and highly reproducible way.     

The spatial distribution of bacteria in Grana-cheese during ripening

The microbial composition and its spatial distribution of Grana Trentino, a hard Parmesan-like cheese, was determined, from vat milk to cheese. After cutting along the vertical axis of the cheese wheels, three layers were sampled diagonally across the cheese: under the cheese rind, an intermediate section and the cheese core. After two different ripening periods (9 and 18 months), the cheese samples were analysed using traditional culture dependent and culture independent methods. Milk samples were dominated by mesophilic and psychrophilic bacterial counts. Thermophilic bacteria (Lactobacillus helveticus) were found in high amounts in cooked whey and natural whey starter cultures. After 9 months of ripening, lactic acid bacteria (LAB) counts were higher than those after 18 months. Furthermore, the LAB numbers in the cheese core was lower than those under the rind or in the intermediate section. The main LAB species isolated from milk (Lactococcus lactis, Pediococcus pentosaceus, Streptococcus uberis and Lactococcus garvieae) were not found in the corresponding cheeses. Some differences were observed in the species composition among the three cheese sections. Microbiota under the rind and in the intermediate section was similar and dominated by Lactobacillus paracasei and Lactobacillus rhamnosus. The core, after 18 months of ripening, was characterized by a total absence of LAB. In each sample, all LAB were genotypically grouped and the different biotypes were subjected to several technological tests indicating that some non-starter LAB (NSLAB) displayed technological features that are favorable for the production of Grana Trentino cheese.     

The potential of a polyphasic PCR-dGGE approach in evaluating microbial diversity of natural whey cultures for water-buffalo Mozzarella cheese production: bias of culture-dependent and culture-independent analyses

A polyphasic PCR-DGGE approach was used to describe the microbial population occurring in natural whey cultures (NWCs) for water-buffalo Mozzarella cheese production. Total microbial community was assessed without cultivation by analyzing DNA directly extracted from the original samples of NWC. In addition, DNA extracted from bulks of cells formed by harvesting colonies from the serial dilution agar plates of a variety of culture media was used to profile the "cultivable" community. The 16S rDNA V3 region was amplified using DNA from NWC as well as DNA from bulks as templates and the amplicons were separated by DGGE. The microbial entities occurring in NWCs were identified by partial 16S rDNA sequencing of DGGE bands: four lactic acid bacteria (LAB) closest relative of Streptococcus thermophilus, Lactococcus lactis, Lactobacillus delbrueckii and Lactobacillus crispatus were revealed by the analysis of DNA directly extracted from NWC while two other LAB, Lactobacillus fermentum and Enterococcus faecalis, were identified by analyzing DNA from the cultivable community. The developed PCR-DGGE analysis of the "cultivable" community showed good potential in evaluating microbial diversity of a dairy environment: it usefully highlighted the bias introduced by selective amplification when compared to the analysis of the total community from NWC and allowed suitability of media and growth conditions to be evaluated. Moreover, it could be used to complete the culture independent study of microbial diversity to give information on concentration ratios among species occurring in a particular environment and can be proposed for rapid identification of dominant microorganisms in alternative to traditional tools.     

PCR-DGGE fingerprints of microbial succession during a manufacture of traditional water buffalo mozzarella cheese

AIMS: To monitor the process and the starter effectiveness recording a series of fingerprints of the microbial diversity occurring at different steps of mozzarella cheese manufacture and to investigate the involvement of the natural starter to the achievement of the final product. METHODS AND RESULTS: Samples of raw milk, natural whey culture (NWC) used as starter, curd after ripening and final product were collected during a mozzarella cheese manufacture. Total microbial DNA was directly extracted from the dairy samples as well as bulk colonies collected from the plates of appropriate culture media generally used for viable counts of mesophilic and thermophilic lactic acid bacteria (LAB) and used in polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) experiments. The analysis of the DGGE profiles showed a strong influence of the microflora of the NWC on the whole process because after the starter addition, the profile of all the dairy samples was identical to the one shown by the NWC. Simple indexes were calculated for the DGGE profiles to have an objective estimation of biodiversity and of technological importance of specific groups of organisms. LAB grown on Man Rogosa Sharp (MRS) and Rogosa agar at 30 degrees C showed high viable counts and the highest diversity in species indicating their importance in the cheese making, which had not been considered so far. Moreover, the NWC profiles were shown to be the most similar to the curd profile suggesting to be effective in manufacture. CONCLUSIONS: The PCR-DGGE analysis showed that in premium quality manufacture the NWC used as starter had a strong influence on the microflora responsible for process development. SIGNIFICANCE AND IMPACT OF THE STUDY: The molecular approach appeared to be valid as a tool to control process development, starter effectiveness and product identity as well as to rank cheese quality.     

Isolation and identification of lactic acid bacteria from Tarag in Eastern Inner Mongolia of China by 16S rRNA sequences and DGGE analysis

Tarag is a characteristic fermented dairy product with rich microflora (especially lactic acid bacteria), developed by the people of Mongolian nationality in Inner Mongolia of China and Mongolia throughout history. One hundred and ninety-eight samples of Tarag were collected from scattered households in Eastern Inner Mongolia, and total of 790 isolates of lactic acid bacteria (LAB) were isolated by traditional pure culture method. To identify these isolates and analyze their biodiversity, 16S rRNA gene sequences analysis and PCR-DGGE were performed respectively. The results showed that 790 isolates could be classified as 31 species and subspecies. Among these isolates, Lactobacillus helveticus (153 strains, about 19.4%), Lactococcus lactis subsp. lactis (132 strains, about 16.7%) and Lactobacillus casei (106 strains, about 11.0%) were considered as the predominated species in the traditional fermented dairy products (Tarag) in Eastern Inner Mongolia. It was shown that the biodiversity of LAB in Tarag in Inner Mongolia was very abundant, and this traditional fermented dairy product could be considered as valuable resources for LAB isolation and probiotic selection.     

Isolation and identification of cultivable lactic acid bacteria in traditional yak milk products of Gansu Province in China

Various traditional fermented yak milk and raw milk foods could be considered as an abundant resource for obtaining novel lactic acid bacteria (LAB) with unique properties. Eighty-eight samples of yak milk products were collected from Gansu Province in China. Three hundred and nineteen strains of LAB isolated from these samples were identified by phenotypic methods, 16S rRNA gene sequence analysis and PCR-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) technology. Among the isolates, one hundred and sixty-four isolates (51.41% of the total) were classified under Lactobacilli, and one hundred and fifty-five (48.59%) belonged to cocci. All the isolates were classified to six genera (Lactobacillus, Lactococcus, Leuconostoc, Streptococcus, Enterococcus and Weissella) and twenty-one species. Lactobacillus helveticus (87 strains), Leuconostoc mesenteroides subsp. mesenteroides (49 strains), Streptococcus thermophilus (39 strains), Lactobacillus casei (31 strains) and Lactococcus lactis subsp. lactis (19 strains) were considered as the predominant populations in the yak milk products. The results showed that there were abundant genus and species LAB existing in yak milk products in Gansu Province in China. The obtained LAB pure cultures may be a valuable source for further starter selection.     

Animal Rennets as Sources of Dairy Lactic Acid Bacteria

The microbial composition of artisan and industrial animal rennet pastes was studied by using both culture-dependent and -independent approaches. Pyrosequencing targeting the 16S rRNA gene allowed to identify 361 operational taxonomic units (OTUs) to the genus/species level. Among lactic acid bacteria (LAB), Streptococcus thermophilus and some lactobacilli, mainly Lactobacillus crispatus and Lactobacillus reuteri, were the most abundant species, with differences among the samples. Twelve groups of microorganisms were targeted by viable plate counts revealing a dominance of mesophilic cocci. All rennets were able to acidify ultrahigh-temperature-processed (UHT) milk as shown by pH and total titratable acidity (TTA). Presumptive LAB isolated at the highest dilutions of acidified milks were phenotypically characterized, grouped, differentiated at the strain level by randomly amplified polymorphic DNA (RAPD)-PCR analysis, and subjected to 16S rRNA gene sequencing. Only 18 strains were clearly identified at the species level, as Enterococcus casseliflavus, Enterococcus faecium, Enterococcus faecalis, Enterococcus lactis, Lactobacillus delbrueckii, and Streptococcus thermophilus, while the other strains, all belonging to the genus Enterococcus, could not be allotted into any previously described species. The phylogenetic analysis showed that these strains might represent different unknown species. All strains were evaluated for their dairy technological performances. All isolates produced diacetyl, and 10 of them produced a rapid pH drop in milk, but only 3 isolates were also autolytic. This work showed that animal rennet pastes can be sources of LAB, mainly enterococci, that might contribute to the microbial diversity associated with dairy productions.     

Characterization of dominant microbiota of a Ghanaian fermented milk product, nyarmie, by culture- and nonculture-based methods

AIMS: To characterize the predominant micro-organisms in a Ghanaian traditional fermented dairy product, nyarmie, made from cows' milk, using both culture- and nonculture-based methods. METHODS AND RESULTS: Samples of nyarmie were analysed from three production sites in Accra, by determining the counts on selective culture media. The microbial diversity occurring in nyarmie was also evaluated by 16S/18S ribosomal DNA PCR amplification and denaturing gradient gel electrophoresis. Results showed that nyarmie contained lactococci and lactobacilli in the range of 10(8) and 10(10) CFU ml(-1), respectively, and yeasts at around 10(7) CFU ml(-1). The pH ranged between 3.49 and 4.25. The predominant lactic acid bacteria (LAB) in nyarmie were Leuconostocmesenteroides ssp. mesenteroides, Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus, Lact.helveticus, Lact. delbrueckii ssp. lactis and Lactococcus lactis, while Saccharomyces cerevisiae was the predominant yeast species. Lactobacillus delbrueckii ssp. delbrueckii was not detected by cultivation but its predominance was revealed by PCR-DGGE analysis. CONCLUSIONS: The flora in products from different producers varied in the LAB composition present and may result in variations in product quality. SIGNIFICANCE AND IMPACT OF THE STUDY: Development and use of starter cultures for nyarmie may be beneficial in improving the consistency of product quality.     

Identification and characterization of lactic acid bacteria isolated from traditional pickles in Sichuan, China

The pickle, a traditional fermented vegetable product, is popular in Sichuan Province of China. The objective of this study was to investigate the diversity of dominant lactic acid bacteria (LAB) in pickles by analyzing 36 samples collected from 6 different regions in Sichuan Province. The LAB counts in these samples varied from 3.90 to 8.40 log cfu ml^-1. In total, 185 presumptive LAB with Gram-positive and catalase-negative properties were obtained from these samples using MRS agar, and those strains were identified at the species level by physiological tests, 16S rRNA gene sequencing and multiplex PCR assay. The results revealed that all isolates were accurately identified as Enterococcus thailandicus (2 strains), Lactobacillus alimentarius (16 strains), L. brevis (24 strains), L. paracasei (9 strains), L. plantarum (81 strains), L. pentosus (38 strains), L. sakei (8 strains), L. spicheri (1 strain), Leuconostoc lactis (1 strain) and Pediococcus ethanolidurans (5 strains). The predominant LAB in Sichuan pickle was L. plantarum, which were isolated from most samples. The results also demonstrated that different regions in Sichuan Province have complex compositions of LAB species, and such a rich resource of LAB strains provides raw data for further studies involving probiotic strain selection.     

Microbial Activation of Wooden Vats Used for Traditional Cheese Production and Evolution of Neoformed Biofilms

Three Lactococcus lactis subsp. cremoris strains were used to develop ad hoc biofilms on the surfaces of virgin wooden vats used for cheese production. Two vats (TZ) were tested under controlled conditions (pilot plant), and two vats (TA) were tested under uncontrolled conditions (industrial plant). In each plant, one vat (TA1 and TZ1) was used for the control, traditional production of PDO Vastedda della Valle del Belìce (Vastedda) cheese, and one (TA2 and TZ2) was used for experimental production performed after lactococcal biofilm activation and the daily addition of a natural whey starter culture (NWSC). Microbiological and scanning electron microscopy analyses showed differences in terms of microbial levels and composition of the neoformed biofilms. The levels of the microbial groups investigated during cheese production showed significant differences between the control trials and between the control and experimental trials, but the differences were not particularly marked between the TA2 and TZ2 productions, which showed the largest numbers of mesophilic lactic acid bacterium (LAB) cocci. LAB populations were characterized phenotypically and genotypically, and 44 dominant strains belonging to 10 species were identified. Direct comparison of the polymorphic profiles of the LAB collected during cheese making showed that the addition of the NWSC reduced their biodiversity. Sensory evaluation showed that the microbial activation of the wooden vats with the multistrain Lactococcus culture generated cheeses with sensory attributes comparable to those of commercial cheese. Thus, neoformed biofilms enable a reduction of microbial variability and stabilize the sensorial attributes of Vastedda cheese.     

How multiple farming conditions correlate with the composition of the raw cow's milk lactic microbiome

Questionnaires on farming conditions were retrieved from 2129 dairy farms and clustered, resulting in 106 representative raw cow's milk samples analysed in winter and summer. Substantiating the efficiency of our survey, some farming conditions affected the milk physicochemical composition. Culturing identified several species of lactic acid bacteria (LAB) per milk, whose number increased through 16S ribosomal RNA (rRNA) gene sequencing and shotgun metagenome analyses. Season, indoor versus outdoor housing, cow numbers, milk substitutes, ratio cattle/rest area, house care system during lactation, and urea and medium-chain fatty acids correlated with the overall microbiome composition and the LAB diversity within it. Shotgun metagenome detected variations in gene numbers and uniqueness per milk. LAB functional pathways differed among milk samples. Focusing on amino acid metabolisms and matching the retrieved annotated genes versus non-starter lactic acid bacteria (NSLAB) references from KEGG and corresponding to those identified, all samples had the same gene spectrum for each pathway. Conversely, gene redundancy varied among samples and agreed with NSLAB diversity. Milk samples with higher numbers of NSLAB species harboured higher number of copies per pathway, which would enable steady-state towards perturbations. Some farming conditions, which affected the microbiome richness, also correlated with the NSLAB composition and functionality.     

Probiotic characteristics of lactic acid bacteria isolated from kimchi

Aims: The present work was aimed at identifying strains of lactic acid bacteria (LAB) from kimchi, with properties suitable for use as starter cultures in yogurt fermentation. Methods and Results: A total of 2344 LAB strains were obtained from two different sources, one group consisted of commercial LAB strains from kimchi, and the second group consisted of those strains isolated from various types of kimchi. The LAB strains from both groups were screened for resistance to biological barriers (acid and bile salts), and the four most promising strains were selected. Further analysis revealed that KFRI342 of the four selected strains displayed the greatest ability to reduce the growth of the cancer cells, SNU‐C4. The in vivo efficacy of strains in quinone reductase induction assay was evaluated, and the extent of DNA strand breakage in individual cells was investigated using the comet assay. Strain KFRI342 was identified as Lactobacillus acidophilus by 16S rRNA sequence analysis, showed protection against tumour initiation and imparted immunostimulation as well as protection against DNA damage. Conclusions: Strain KFRI342, which showed probiotic characteristics reducing cancer cell growth, could be a suitable starter culture for yogurt fermentation because of its strong acid production and high acid tolerance. Significance and Impact of the Study: This is the first report to describe a bacterium, isolated from kimchi, Lact. acidophilus KFRI342 which has the probiotic characteristics and the acid tolerance needed for its use as a starter culture in yogurt fermentation.     

Lactic acid bacteria and yeasts associated with gowé production from sorghum in Bénin

AIMS: To identify the dominant micro-organisms involved in the production of gowé, a fermented beverage, and to select the most appropriate species for starter culture development. METHODS AND RESULTS: Samples of sorghum gowé produced twice at three different production sites were taken at different fermentation times. DNA amplification by internal transcribed spacer-polymerase chain reaction of 288 lactic acid bacteria (LAB) isolates and 16S rRNA gene sequencing of selected strains revealed that the dominant LAB responsible for gowé fermentation were Lactobacillus fermentum, Weissella confusa, Lactobacillus mucosae, Pediococcus acidilactici, Pediococcus pentosaceus and Weissella kimchii. DNA from 200 strains of yeasts was amplified and the D1/D2 domain of the 26S rRNA gene was sequenced for selected isolates, revealing that the yeasts species were Kluyveromyces marxianus, Pichia anomala, Candida krusei and Candida tropicalis. CONCLUSIONS: Gowé processing is characterized by a mixed fermentation dominated by Lact. fermentum, W. confusa and Ped. acidilactici for the LAB and by K. marxianus, P. anomala and C. krusei for the yeasts. SIGNIFICANCE AND IMPACT OF THE STUDY: The diversity of the LAB and yeasts identified offers new opportunities for technology upgrading and products development in gowé production. The identified species can be used as possible starter for a controlled fermentation of gowé.     

Bacterial and fungal diversity in the starter production process of Fen liquor, a traditional Chinese liquor

Fermented foods and beverages are important parts of human diet. Fen liquor, a Chinese liquor is a fermented beverage that uses a traditional fermentation process. Starters are the main microbial source and also provide nutrients for microorganisms during fermentation. In this study, starters of Fen liquor were produced through a complex traditional fermentation process. To investigate the community structure and the composition of microorganisms in the starter production process, bacterial 16S rRNA and fungal internal transcribed spacer (ITS) regions were sequenced using clone libraries and pyrosequencing, respectively. There was much higher diversity among the bacteria than among the fungi in the starter production process. Bacteria on the surface of the starters belonged mostly to the Lactobacillaceae family, while members of the Bacillacae family were dominant in the interior of the samples that lacked access to air and water. In the fungi population, diversity was high only in the raw material. In all other samples, nearly all of the fungal sequences were from Pichia kudriavzevii, a member of the Saccharomycetaceae family. Nearly all samples showed similar fungal community structures, indicating that there was little change in the fungal community. To the best of our knowledge, this is the first report to reveal the whole process of the starter production of Chinese traditional liquor. The findings obtained in this study provide new insights into understanding the composition of the microbial community during the traditional Chinese liquor starter production process and information about the production process control and monitoring.     

Natural whey starter for Parmigiano Reggiano: culture‐independent approach

Aims: The aim of this work was to obtain a deeper insight into the knowledge of microbial composition of Parmigiano Reggiano natural whey starters through different culture‐independent methods. Methods and Results: Eighteen different Parmigiano Reggiano natural whey starters sampled from three different provinces of this cheese production area and the nonacidified wheys from which they arose have been studied by length heterogeneity polymerase chain reaction (LH‐PCR) and fluorescent in situ hybridization (FISH). A high microbial composition variability between different samples has been observed. Conclusions: Revealing different images of the same community, LH‐PCR and FISH have given a more accurate view of the not well‐known Parmigiano Reggiano whey starter ecosystem. Significance and Impact of the Study: New lights have been shed on Parmigiano Reggiano natural whey starters microbial composition, highlighting how culture‐independent approach could be used and improved to study this and other food ecosystems.     

Epifluorescence and atomic force microscopy: Two innovative applications for studying phage-host interactions in Lactobacillus helveticus

Bacteriophages attacking lactic acid bacteria (LAB) still represent a crucial problem in industrial dairy fermentations. The consequences of a phage infection against LAB can lead to fermentation delay, alteration of the product quality and, in most severe cases, the product loss. Phage particles enumeration and phage-host interactions are normally evaluated by conventional plaque count assays, but, in many cases, these methods can be unsuccessful. Bacteriophages of Lactobacillus helveticus, a LAB species widely used as dairy starter or probiotic cultures, are often unable to form lysis plaques, thus impairing their enumeration by plate assay. In this study, we used epifluorescence microscopy to enumerate L. helveticus phage particles from phage-infected cultures and Atomic Force Microscopy (AFM) to visualize both phages and bacteria during the different stages of the lytic cycle. Preliminary, we tested the sensitivity of phage counting by epifluorescence microscopy. To this end, phage particles of ΦAQ113, a lytic phage of L. helveticus isolated from a whey starter culture, were stained by SYBR Green I and enumerated by epifluorescence microscopy. Values obtained by the microscopic method were 10 times higher than plate counts, with a lowest sensitivity limit of ≥ 6 log phage/ml. The interaction of phage ΦAQ113 with its host cell L. helveticus Lh1405 was imaged by AFM after 0, 2 and 5 h from phage-host adsorption. The lytic cycle was followed by epifluorescence microscopy counting and the concomitant cell wall changes were visualized by AFM imaging. Our results showed that these two methods can be combined for a reliable phage enumeration and for studying phage and host morphology during infection processes, thus giving a complete overview of phage-host interactions in L. helveticus strains involved in dairy productions.