Benedetta Bottari

Application of FISH technology for microbiological analysis: current state and prospects

In order to identify and quantify the microorganisms present in a certain ecosystem, it has become necessary to develop molecular methods avoiding cultivation, which allows to characterize only the countable part of the microorganisms in the sample, therefore losing the information related to the microbial component which presents a vitality condition, although it cannot duplicate in culture medium. In this context, one of the most used techniques is fluorescence in situ hybridization (FISH) with ribosomal RNA targeted oligonucleotide probes. Owing to its speed and sensitivity, this technique is considered a powerful tool for phylogenetic, ecological, diagnostic and environmental studies in microbiology. Through the use of species-specific probes, it is possible to identify different microorganisms in complex microbial communities, thus providing a solid support to the understanding of inter-species interaction. The knowledge of the composition and distribution of microorganisms in natural habitats can be interesting for ecological reasons in microbial ecology, and for safety and technological aspects in food microbiology. Methodological aspects, use of different probes and applications of FISH to microbial ecosystems are presented in this review.

Invited review: Microbial evolution in raw-milk, long-ripened cheeses produced using undefined natural whey starters

The robustness of the starter culture during cheese fermentation is enhanced by the presence of a rich consortium of microbes. Natural starters are consortia of microbes undoubtedly richer than selected starters. Among natural starters, natural whey starters (NWS) are the most common cultures currently used to produce different varieties of cheeses. Undefined NWS are typically used for Italian cooked, long-ripened, extra-hard, raw milk cheeses, such as Parmigiano Reggiano and Grana Padano. Together with raw milk microbiota, NWS are responsible for most cheese characteristics. The microbial ecology of these 2 cheese varieties is based on a complex interaction among starter lactic acid bacteria (SLAB) and nonstarter lactic acid bacteria (NSLAB), which are characterized by their different abilities to grow in a changing substrate. This review aims to summarize the latest findings on Parmigiano Reggiano and Grana Padano to better understand the dynamics of SLAB, which mainly arise from NWS, and NSLAB, which mainly arise from raw milk, and their possible role in determining the characteristics of these cheeses. The review is presented in 4 main sections. The first summarizes the main microbiological and chemical properties of the ripened cheese as determined by cheese-making process variables, as these variables may affect microbial growth. The second describes the microbiota of raw milk as affected by specific milk treatments, from milking to the filling of the cheese milk vat. The third describes the microbiota of NWS, and the fourth reviews the knowledge available on microbial dynamics from curd to ripened cheese. As the dynamics and functionality of complex undefined NWS is one of the most important areas of focus in current food microbiology research, this review may serve as a good starting point for implementing future studies on microbial diversity and functionality of undefined cheese starter cultures.

Dynamics of whole and lysed bacterial cells during Parmigiano-Reggiano cheese production and ripening.

ABSTRACT Microbial succession during Parmigiano-Reggiano cheesemaking was monitored by length heterogeneity PCR (LH-PCR), considering the intact and lysed cells at different stages of cheese production and ripening. When starter species underwent autolysis, species coming from milk were able to grow. For the first time, the LH-PCR technique was applied to study a fermented food.

Antibiotic Resistance of Lactobacilli Isolated from Two Italian Hard Cheeses

One hundred forty-one lactobacilli strains isolated from natural whey starter cultures and ripened Grana Padano and Parmigiano Reggiano cheeses were tested for their susceptibility to 13 antibiotics, in particular, penicillin G, ampicillin, amoxicillin, oxacillin, cephalotin, cefuroxime, vancomycin, gentamicin, tetracycline, erythromycin, clindamycin, co-trimoxazole, and nitrofurantoin. The strains belonged to the species Lactobacillus helveticus, L. delbrueckii subsp. lactis, L. rhamnosus, and L. casei. The strains of the first two species were isolated from whey starter cultures, and the strains of the last two species were from the ripened cheeses. Significant differences among the strains in their antibiotic resistance were found in relation to the type of cheese and, especially, the strains from Parmigiano Reggiano were more resistant to gentamicin and penicillin G. The strains isolated in the ripened cheese were generally more resistant than those isolated from natural whey starter cultures; in particular, significant differences regarding oxacillin, vancomycin, cephalotin, and co-trimoxazole were observed. Finally, no significant difference in relation to the type of cheese was found among the thermophilic lactobacilli isolated from whey cultures, while the facultatively heterofermentative lactobacilli isolated from Parmigiano Reggiano showed higher resistance toward gentamicin and penicillin G than did the same species isolated from Grana Padano.

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.

New developments in the study of the microbiota of raw-milk, long-ripened cheeses by molecular methods: the case of Grana Padano and Parmigiano Reggiano

Microorganisms are an essential component of cheeses and play important roles during both cheese manufacture and ripening. Both starter and secondary flora modify the physical and chemical properties of cheese, contributing and reacting to changes that occur during the manufacture and ripening of cheese. As the composition of microbial population changes under the influence of continuous shifts in environmental conditions and microorganisms interactions during manufacturing and ripening, the characteristics of a given cheese depend also on microflora dynamics. The microbiota present in cheese is complex and its growth and activity represent the most important, but the least controllable steps. In the past, research in this area was dependent on classical microbiological techniques. However, culture-dependent methods are time-consuming and approaches that include a culturing step can lead to inaccuracies due to species present in low numbers or simply uncultivable. Therefore, they cannot be used as a unique tool to monitor community dynamics. For these reasons approaches to cheese microbiology had to change dramatically. To address this, in recent years the focus on the use of culture-independent methods based on the direct analysis of DNA (or RNA) has rapidly increased. Application of such techniques to the study of cheese microbiology represents a rapid, sound, reliable, and effective way for the detection and identification of the microorganisms present in dairy products, leading to major advances in understanding this complex microbial ecosystem and its impact on cheese ripening and quality. In this article, an overview on the recent advances in the use of molecular methods for thorough analysis of microbial communities in cheeses is given. Furthermore, applications of culture-independent approaches to study the microbiology of two important raw-milk, long-ripened cheeses such as Grana Padano and Parmigiano Reggiano, are presented.

Diversity and dynamic of lactic acid bacteria strains during aging of a long ripened hard cheese produced from raw milk and undefined natural starter.

The aim of this study was to explore diversity and dynamic of indigenous LAB strains associated with a long ripened hard cheese produced from raw milk and undefined natural starter such as PDO Grana Padano cheese. Samples of milk, curd, natural whey culture and cheeses (2nd, 6th, 9th and 13th months of ripening) were collected from 6 cheese factories in northern Italy. DNA was extracted from each sample and from 194 LAB isolates. tRNA(Ala)-23S rDNA-RFLP was applied to identify isolates. Strain diversity was assessed by (GTG)5 rep-PCR and RAPD(P1)-PCR. Finally, culture-independent LH-PCR (V1-V2 16S-rDNA), was considered to explore structure and dynamic of the microbiota. Grana Padano LAB were represented mainly by Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus delbrueckii, Lactobacillus helveticus and Pediococcus acidilactici, while the structure and dynamic of microbiota at different localities was specific. The strength of this work is to have focused the study on isolates coming from more than one cheese factories rather than a high number of isolates from one unique production. We provided a valuable insight into inter and intraspecies diversity of typical LAB strains during ripening of traditional PDO Grana Padano, contributing to the understanding of specific microbial ecosystem of this cheese.

Survey on the community and dynamics of lactic acid bacteria in Grana Padano cheese

Grana Padano (GP) is a Protected Designation of Origin cheese made with raw milk and natural whey culture (NWC) that is characterised by a long ripening period. In this study, six GP productions were considered in order to evaluate the trend of microbial dynamics and compare lactic acid bacteria (LAB) population levels in cheeses during the entire cheese-making process. To reach this goal, for each GP production, samples of vat raw milk, NWC and cheeses at 48h, 2, 6, 9 and 13 months were subjected to plate counts and direct counts by fluorescence microscopy, as well as amplicon length heterogeneity-PCR (LH-PCR). Statistical analysis was applied to the results and ecological indices were estimated. It was demonstrated that the LAB able to grow in the cheese-environment conditions could arise from both raw milk and NWC. Starter lactobacilli (SLAB) from NWC were the main species present during acidification, and non-starter LAB (NSLAB), mainly from milk but also from NWC, were able to grow after brining and they dominated during ripening. The peak areas of LH-PCR profiles were used to determine ecological indices during manufacture and ripening. Among cheese ecosystems with different ageing times, diversity, Evenness and Richness were different, with highest bacterial growth and diversity occurring in cheese ripening at 2 months. At this time point, which seemed to be a crucial moment for GP microbial evolution, cell lysis of both SLAB and NSLAB was also observed. Sampling modality and statistical analysis gave greater significance to the results used to describe the microbiological characteristics of a cheese recognised worldwide.

Development of a multiplex real time PCR to detect thermophilic lactic acid bacteria in natural whey starters.

A multiplex real time PCR (mRealT-PCR) useful to rapidly screen microbial composition of thermophilic starter cultures for hard cooked cheeses and to compare samples with potentially different technological properties was developed. Novel primers directed toward pheS gene were designed and optimized for multiple detection of Lactobacillus helveticus, Lactobacillus delbrueckii, Streptococcus thermophilus and Lactobacillus fermentum. The assay was based on SYBR Green chemistry followed by melting curves analysis. The method was then evaluated for applications in the specific detection of the 4 lactic acid bacteria (LAB) in 29 different natural whey starters for Parmigiano Reggiano cheese production. The results obtained by mRealT-PCR were also compared with those obtained on the same samples by Fluorescence in Situ Hybridization (FISH) and Length-Heterogeneity PCR (LH-PCR). The mRealT-PCR developed in this study, was found to be effective for analyzing species present in the samples with an average sensitivity down to less than 600 copies of DNA and therefore sensitive enough to detect even minor LAB community members of thermophilic starter cultures. The assay was able to describe the microbial population of all the different natural whey starter samples analyzed, despite their natural variability. A higher number of whey starter samples with S. thermophilus and L. fermentum present in their microbial community were revealed, suggesting that these species could be more frequent in Parmigiano Reggiano natural whey starter samples than previously shown. The method was more effective than LH-PCR and FISH and, considering that these two techniques have to be used in combination to detect the less abundant species, the mRealT-PCR was also faster. Providing a single step sensitive detection of L. helveticus, L. delbrueckii, S. thermophilus and L. fermentum, the developed mRealT-PCR could be used for screening thermophilic starter cultures and to follow the presence of those species during ripening of derived dairy products. A major increase in understanding the starter culture contribution to cheese ecosystem could be harnessed to control cheese ripening and flavor formation.

Study of the bacterial diversity of foods: PCR-DGGE versus LH-PCR

The present study compared two culture-independent methods, polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) and length-heterogeneity polymerase chain reaction (LH-PCR), for their ability to reveal food bacterial microbiota. Total microbial DNA and RNA were extracted directly from fourteen fermented and unfermented foods, and domain A of the variable regions V1 and V2 of the 16S rRNA gene was analyzed through LH-PCR and PCR-DGGE. Finally, the outline of these analyses was compared with bacterial viable counts obtained after bacterial growth on suitable selective media. For the majority of the samples, RNA-based PCR-DGGE revealed species that the DNA-based PCR-DGGE was not able to highlight. When analyzing either DNA or RNA, LH-PCR identified several lactic acid bacteria (LAB) and coagulase negative cocci (CCN) species that were not identified by PCR-DGGE. This phenomenon was particularly evident in food samples with viable loads<5.0 Logcfug-1. Furthermore, LH-PCR was able to detect a higher number of peaks in the analyzed food matrices relative to species identified by PCR-DGGE. In light of these findings, it may be suggested that LH-PCR shows greater sensitivity than PCR-DGGE. However, PCR-DGGE detected some other species (LAB included) that were not detected by LH-PCR. Therefore, certain LH-PCR peaks not attributed to known species within the LH-PCR database could be solved by comparing them with species identified by PCR-DGGE. Overall, this study also showed that LH-PCR is a promising method for use in the food microbiology field, indicating the necessity to expand the LH-PCR database, which is based, up to now, mainly on LAB isolates from dairy products.