Biodiversity in Lactobacillus helveticus strains present in natural whey starter used for Parmigiano Reggiano cheese

AIMS: Lactobacillus helveticus is the dominant microflora of the natural whey starters used for Parmigiano Reggiano cheese making. The aim of this work was to study the biodiversity of different strains of Lact. helveticus present in six cultures and to compare them with strains of the same species previously isolated from natural whey cultures used for Grana Padano and Provolone cheeses. METHODS AND RESULTS: Twenty different biotypes of Lact. helveticus strains were identified combining the results deriving from SDS-PAGE of cell surface proteins and PCR fingerprinting using M13 as a primer. The biotypes were present in varying amounts in the six natural whey starters and the biodiversity was demonstrated not only within the whey cultures, but also between the whey cultures. CONCLUSIONS: Lact. helveticus strains isolated from Parmigiano Reggiano whey cultures analysed by PCR M13, SDS-PAGE and RFLP were distinguishable from Lact. helveticus strains of different dairy origin, namely Grana Padano and Provolone natural whey starters. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of different Lact. helveticus biotypes seems to be related to the specific ecosystem of cheese making and may be considered as one of the elements contributing to the typicality of Parmigiano Reggiano cheese.     

Use of RAPD and 16S rDNA sequencing for the study of Lactobacillus population dynamics in natural whey culture

The development of communities of the thermophilic microflora of natural whey culture for Parmigiano Reggiano cheese production was studied by means of molecular techniques. RAPD analysis facilitates the identification of the Lactobacillus strains involved in this microbial association and permitted the study of population dynamics during two cycles of whey fermentation. Analysis of RAPD fingerprints revealed the presence of four biotypes that dominate the whey fermentation process. Sequence analysis of 16S rDNA demonstrated that the strains isolated from whey belong to Lact. helveticus and Lact. delbrueckii ssp. lactis species.     

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.     

Bile salt and acid tolerance of Lactobacillus rhamnosus strains isolated from Parmigiano Reggiano cheese

This study aimed to compare phenotypic and genetic characteristics of Lactobacillus rhamnosus strains isolated at the end of the ripening of Parmigiano Reggiano cheese and to investigate an important prerequisite of probiotic interest, such as the capability to survive at low pH and in presence of bile salts. The use of API 50 CH, RAPD-PCR analysis and species-specific PCR allowed to ascertain the identity of 63 L. rhamnosus strains. Three L. rhamnosus strains isolated from Parmigiano Reggiano cheese, L. rhamnosus ATCC 7469T and the commercial strain L. GG were assayed to estimate the resistance to various stress factors reproducing in vitro some conditions of the gastro-intestinal environment such as low pH and different amounts of bile salts and acids. The behaviour of almost all the tested strains isolated from Parmigiano Reggiano cheese resulted analogous to that showed by L. GG.     

Fluorescence microscopy for studying the viability of micro-organisms in natural whey starters

AIMS: The aim of this work was to study the viability and cultivability of microbial populations of different natural whey starters and to evaluate their resistance to thermal treatments (such as exposure to high or low temperatures). METHODS AND RESULTS: Twenty-three natural whey starters for Grana Padano cheese were investigated and subsequently pH measurement, plate count agar using Man-Rogasa-Sharpe (MRS) pH 5.4 agar and whey agar medium (WAM) were performed using these samples. LIVE/DEAD BacLight bacterial viability kit was used. Total count and viability of all the 23 samples were high and similar to each other (CV 20%). However, the cultivable population was lower in terms of cfu ml(-1) and number of cells per millilitre than the viable fraction and highly variable, although its count value was higher in WAM than in MRS pH 5.4. The heating (60 degrees C for 5 min and 54 degrees C for 1 h) and freezing (-20 and -80 degrees C) treatments affected the cultivability and viability of the microbial population. CONCLUSIONS: This study demonstrated the effectiveness of LIVE/DEAD BacLight bacterial viability kit, which has already been used to evaluate bacterial populations, in investigating microbial viability in a complex ecosystem such as a natural whey starter. Significance and Impact of the Study: The aim of this study was to quantify the presence of damaged nonviable bacterial cells in natural whey starters. The Thoma Glass is a useful method to obtain fluorescence microscopy counts to evaluate the technological performance of natural whey starters.     

Biodiversity among Lactobacillus helveticus strains isolated from different natural whey starter cultures as revealed by classification trees

Lactobacillus helveticus is a homofermentative thermophilic lactic acid bacterium used extensively for manufacturing Swiss type and aged Italian cheese. In this study, the phenotypic and genotypic diversity of strains isolated from different natural dairy starter cultures used for Grana Padano, Parmigiano Reggiano, and Provolone cheeses was investigated by a classification tree technique. A data set was used that consists of 119 L. helveticus strains, each of which was studied for its physiological characters, as well as surface protein profiles and hybridization with a species-specific DNA probe. The methodology employed in this work allowed the strains to be grouped into terminal nodes without difficult and subjective interpretation. In particular, good discrimination was obtained between L. helveticus strains isolated, respectively, from Grana Padano and from Provolone natural whey starter cultures. The method used in this work allowed identification of the main characteristics that permit discrimination of biotypes. In order to understand what kind of genes could code for phenotypes of technological relevance, evidence that specific DNA sequences are present only in particular biotypes may be of great interest.     

Interactions among thermophilic lactobacilli during growth in cheese whey

The starters used in the Parmesan cheese technology are whey-cultured, mixed strain cultures of thermophilic rod lactic acid bacteria. In mixed strain cultures, interactions among different strains can result in both stimulatory or inhibitory effects on microbial growth rate and metabolic activity. These effects, studied by the use of conductance, were observed for individual strains of thermophilic lactobacilli, which were grown in whey and in the presence of cell-free supernatant fluids from whey cultures of other strains. The importance of whey as a growth medium on the microbial composition of the whey cultures is discussed.     

Segregated flux balance analysis constrained by population structure/function data: The case of PHA production by mixed microbial cultures

In this study we developed a segregated flux balance analysis (FBA) method to calculate metabolic flux distributions of the individual populations present in a mixed microbial culture (MMC). Population specific flux data constraints were derived from the raw data typically obtained by the fluorescence in situ hybridization (FISH) and microautoradiography (MAR)‐FISH techniques. This method was applied to study the metabolic heterogeneity of a MMC that produces polyhydroxyalkanoates (PHA) from fermented sugar cane molasses. Three populations were identified by FISH, namely Paracoccus sp., Thauera sp., and Azoarcus sp. The segregated FBA method predicts a flux distribution for each of the identified populations. The method is shown to predict with high accuracy the average PHA storage flux and the respective monomeric composition for 16 independent experiments. Moreover, flux predictions by segregated FBA were slightly better than those obtained by nonsegregated FBA, and also highly concordant with metabolic flux analysis (MFA) estimated fluxes. The segregated FBA method can be of high value to assess metabolic heterogeneity in MMC systems and to derive more efficient eco‐engineering strategies. For the case of PHA‐producing MMC considered in this work, it becomes apparent that the PHA average monomeric composition might be controlled not only by the volatile fatty acids (VFA) feeding profile but also by the population composition present in the MMC. Biotechnol. Bioeng. 2013; 110: 2267–2276. © 2013 Wiley Periodicals, Inc.     

Analysis of Microbial Composition in Acid Whey for Dairy Fan Making in Yunnan by Conventional Method and 16S rRNA Sequencing

Dairy fan is a traditional milk product made by adding ‘acid juice’ and heating up. It has been consumed by people of minority ethnic region in Yunnan Province of China for over 1,000 years. In order to improve the quality of dairy fan for further industrial-scale production, we investigated the microbial composition, especially Lactic Acid Bacteria (LAB), by conventional method and 16S rRNA sequences analysis in acid whey, which was the starter culture for traditional dairy fan making. Twenty samples of acid whey were collected from Yunnan. Ninety-one phylotypes of LAB were isolated from these samples and identified by conventional method and 16S rRNA sequencing analysis. The results showed that all isolates belonged to three genera and nine different species. Moreover, Lactobacillus helveticus was the predominant population among these samples, mesophilic and thermophilic LAB could be considered as the major microbial composition of acid whey in the Yunnan Province of China. This paper systematically studied the LAB composition of acid whey, which may be valuable for designing starter culture for dairy fan production.     

Abundance of Zetaproteobacteria within crustal fluids in back‐arc hydrothermal fields of the Southern Mariana Trough

To extend knowledge of subseafloor microbial communities within the oceanic crust, the abundance, diversity and composition of microbial communities in crustal fluids at back‐arc hydrothermal fields of the Southern Mariana Trough (SMT) were investigated using culture‐independent molecular techniques based on 16S rRNA gene sequences. Seafloor drilling was carried out at two hydrothermal fields, on‐ and off‐ridge of the back‐arc spreading centre of the SMT. 16S rRNA gene clone libraries for bacterial and archaeal communities were constructed from the fluid samples collected from the boreholes. Phylotypes related to Thiomicrospira in the Gammaproteobacteria (putative sulfide‐oxidizers) and Mariprofundus in the Zetaproteobacteria (putative iron‐oxidizers) were recovered from the fluid samples. A number of unique archaeal phylotypes were also recovered. Fluorescence in situ hybridization (FISH) analysis indicated the presence of active bacterial and archaeal populations in the fluids. The Zetaproteobacteria accounted for up to 32% of the total prokaryotic cell number as shown by FISH analysis using a specific probe designed in this study. Our results lead to the hypothesis that the Zetaproteobacteria play a role in iron oxidation within the oceanic crust.     

Characterization of starter lactic acid bacteria from the Finnish fermented milk product viili

Aims: Phenotypic and molecular methods were used to identify and compare the strain composition of three industrial dairy starters used for the manufacture of viili. Methods and Results: Preliminary differentiation was made by phenotypic methods. Genotypic differentiation was carried out using polymerase chain reaction (PCR) and further characterization at strain level by pulsed‐field gel electrophoresis (PFGE). The isolates could be assigned as acid‐producing Lactococcus lactis strains of both lactis and cremoris subspecies, and aroma producers, identified as L. lactis subsp. lactis biovar diacetylactis and Leuconostoc mesenteroides. PCR analysis discriminated between the lactococcal subspecies, and cluster analysis of the digestion patterns of PFGE analysis revealed different genotypes in each subspecies. Each Leuconostoc‐genotype seemed to be specific to only a single starter mix. Conclusions: The work proved that in addition to L. lactis subsp. lactis biovar diacetylactis and Leuc. mesenteroides subsp. cremoris, commercial viili starters of traditional origin may contain (i) only L. lactis subsp. cremoris, (ii) both L. lactis subsp. cremoris and L. lactis subsp. lactis as a minority, and – as a new discovery – (iii) only L. lactis subsp. lactis. Significance and Impact of the Study: The results obtained give an overview of the microbial population of viili starters and can be exploited in the development of optimized starter cultures for industrial‐scale manufacture of viili.     

Metabolic and proteomic adaptation of Lactobacillus rhamnosus strains during growth under cheese‐like environmental conditions compared to de Man,Rogosa, and Sharpe medium

The aim of this study was to demonstrate the metabolic and proteomic adaptation of Lactobacillus rhamnosus strains, which were isolated at different stages of Parmigiano Reggiano cheese ripening. Compared to de Man, Rogosa, and Sharpe (MRS) broth, cultivation under cheese‐like conditions (cheese broth, CB) increased the number of free amino acids used as carbon sources. Compared with growth on MRS or pasteurized and microfiltrated milk, all strains cultivated in CB showed a low synthesis of d,l ‐lactic acid and elevated levels of acetic acid. The proteomic maps of the five representative strains, showing different metabolic traits, were comparatively determined after growth on MRS and CB media. The amount of intracellular and cell‐associated proteins was affected by culture conditions and diversity between strains, depending on their time of isolation. Protein spots showing decreased (62 spots) or increased (59 spot) amounts during growth on CB were identified using MALDI‐TOF‐MS/MS or LC‐nano‐ESI‐MS/MS. Compared with cultivation on MRS broth, the L. rhamnosus strains cultivated under cheese‐like conditions had modified amounts of some proteins responsible for protein biosynthesis, nucleotide, and carbohydrate metabolisms, the glycolysis pathway, proteolytic activity, cell wall, and exopolysaccharide biosynthesis, cell regulation, amino acid, and citrate metabolism, oxidation/reduction processes, and stress responses.     

Spread and variability of the integrase gene in Lactobacillus delbrueckii ssp. lactis strains and phages isolated from whey starter cultures

AIMS: To determine the presence, diffusion and variability of the integrase (int) gene in Lactobacillus delbrueckii ssp. lactis isolated from natural whey starters used for the production of Italian hard cheeses. METHODS AND RESULTS: A PCR-based protocol aimed to amplify an internal fragment of the int gene was optimized taking into account phage genome sequences available from public databases. Thirty-seven of the 39 strains tested showed the presence of the putative int gene. Southern blot hybridization experiments confirmed data obtained by PCR. The presence of the putative int gene was observed also in 20 of 23 Lact. delbrueckii ssp. lactis lytic phages isolated from the same starter cultures used to isolate strains. Phylogenetic analysis of partial int gene revealed a high similarity both within and between strain- and phage-derived sequences. Sixty per cent of the int-positive strains resulted inducible with mitomycin C, and two of them released active phage particles. CONCLUSIONS: Our preliminary findings seem to suggest that an important number of Lact. delbrueckii ssp. lactis strains associated with the whey starters are lysogenic. SIGNIFICANCE AND IMPACT OF THE STUDY: Further contribution to obtain a clearer picture of the complex relationship between thermophilic lactic acid bacteria phage and host in whey starters for Italian, hard-cooked cheeses.     

A combination of direct viable count and fluorescence in situ hybridization for specific enumeration of viable Lactobacillus delbrueckii subsp.bulgaricus and Streptococcus thermophilus

Aims: We have developed a direct viable count (DVC)‐FISH procedure for quickly and easily discriminating between viable and nonviable cells of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains, the traditional yogurt bacteria. Methods and Results: direct viable count method has been modified and adapted for Lact. delbrueckii subsp. bulgaricus and Strep. thermophilus analysis by testing different times of incubation and concentrations of DNA‐gyrase inhibitors. DVC procedure has been combined with fluorescent in situ hybridization (FISH) for the specific detection of viable cells of both bacteria with specific rRNA oligonucleotide probes (DVC‐FISH). Of the four antibiotics tested (novobiocin, nalidixic acid, pipemidic acid and ciprofloxacin), novobiocin was the most effective for DVC method and the optimum incubation time was 7 h for both bacteria. The number of viable cells was obtained by the enumeration of specific hybridized cells that were elongated at least twice their original length for Lactobacillus and twice their original size for Streptococcus. Conclusions: This technique was successfully applied to detect viable cells in inoculated faeces. Significance and Impact of the Study: Results showed that this DVC‐FISH procedure is a quick and culture‐independent useful method to specifically detect viable Lact. delbrueckii subsp. bulgaricus and Strep. thermophilus in different samples, being applied for the first time to lactic acid bacteria.     

Inventory of non starter lactic acid bacteria from ripened Parmigiano Reggiano cheese as assessed by a culture dependent multiphasic approach

The objective of this study was to investigate microbial species diversity and strain complexity of the cultivable non starter lactic acid bacteria (NSLAB) occurring in 31 ripened Parmigiano Reggiano (PR) cheeses. Dereplication of 127 lactobacilli isolates by (GTG)(5)-PCR fingerprinting yielded a total of 51 genotypes. Phylogenetic relatedness of all the genotypes with known Lactobacillus species was determined by a novel combined amplified 16S rDNA restriction analysis (16S-ARDRA), species-specific PCR assays and 16S rRNA gene sequencing. The species Lactobacillus rhamnosus and Lactobacillus paracasei comprise the largest portions of the cultivable NSLAB community in PR cheese, with an inter-individual diversity ranging from one to four dominant genotypes per sample. Lactobacillus casei, Lactobacillus harbinensis and Lactobacillus fermentum species were also detected at low frequency. The data showed differences in cultivable NSLAB population, with an overall decrease in diversity and complexity from early to advanced stages of ripening. Finally the de-replicated collection of genotypes resulting from this work is the bases for further functional screening.     

Evidence for exopolysaccharide production by Oenococcus oeni strains isolated from non‐ropy wines

Aims: The aim of this study was to assess the exopolysaccharide (EPS) production capacities of various strains of Oenococcus oeni, including malolactic starters and strains recently isolated from wine . Methods and Results: Fourteen O. oeni strains displaying or not (PCR check on genomic DNA) the gtf gene generally associated with β‐glucan formation and ropiness were grown on grape juice medium, dialysed MRS‐derived medium or synthetic medium. The soluble polysaccharides (PS) remaining in the culture supernatant were alcohol precipitated, and their concentration was quantified by the phenol‐sulfuric method. Most of the O. oeni strains studied produced significant amounts of EPS, independently of their genotype (gtf+ or gtf?). The EPS production was not directly connected with growth and could be stimulated by changing the growth medium composition. The molecular weight distribution analysis and attempts to determine the PS chemical structure suggested that most strains produce a mixture of EPS. Conclusion: Oenococcus oeni strains recently isolated from wine or cultivated for many generations as a malolactic starter are able to produce EPS other than β‐glucan. Significance and Impact of the Study: These EPS may enhance the bacteria survival in wine (advantage for malolactic starters) and may contribute to the wine colloidal equilibrium.     

Yeast populations associated with the artisanal cheese produced in the region of Serra da Canastra, Brazil

The aim of this work was to describe the yeast populations present during the manufacturing of Minas cheese of the region of Serra da Canastra, Minas Gerais state, Brazil. Canastra cheese is produced from raw cow’s milk at the farmhouse level using artisanal procedures and natural whey cultures as starters. Samples from 10 farms were studied, and they included: raw milk, natural starter, cheese curd before salting and cheese after 5 days of ripening. The most frequent yeasts in whey, curd and cheese were Debaryomyces hansenii, Kluyveromyces lactis, Kodamaea ohmeri and Torulaspora delbrueckii. Many yeast isolates were able to produce proteases, lipases and β-galactosidades. Production of these enzymes by yeasts in the cheese would contribute to the development of the characteristic flavor and smell during the ripening process.     

Microbial diversity and dynamics of a groundwater and a still bottled natural mineral water

The microbial abundance and diversity at source, after bottling and through 6 months of storage of a commercial still natural mineral water were assessed by culture‐dependent and culture‐independent methods. The results revealed clear shifts of the dominant communities present in the three different stages. The borehole waters displayed low cell densities that increased 1.5‐fold upon bottling and storage, reaching a maximum (6.2 × 10⁸ cells l^?1) within 15 days after bottling, but experienced a significant decrease in diversity. In all cases, communities were largely dominated by Bacteria. The culturable heterotrophic community was characterized by recovering 3626 isolates, which were primarily affiliated with the Alphaproteobacteria, Betaproteobacteria and Gammaproteobacteria. This study indicates that bottling and storage induce quantitative and qualitative changes in the microbial assemblages that seem to be similar as revealed by the two sample batches collected on 2 consecutive years. To our knowledge, this is the first study combining culture‐independent with culture‐dependent methods, and repeated tests to reveal the microbial dynamics occurring from source to stored bottled water.