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 48 h, 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.     

Population dynamics of lactobacilli in Grana cheese

A survey on the presence, microbial diversity, and population dynamics of lactobacilli in Grana cheese is presented. Evolution of thermophilic rod lactic acid bacteria within the first two days from cheese making and during ripening was different according to different bacterial groups, which were selectively enumerated and identified by molecular methods. Species-specific microbial counts indicated prevalence ofLactobacillus helveticus in both the whey starter and the cheese at moulding, and ofLactobacillus delbrueckii subsp.lactis in cheese after two months of ripening. In more advanced ripening, a decrease of total thermophilic lactobacilli and an increase of mesophilic lactobacilli (mostly belonging toLactobacillus casei/paracasei andLactobacillus rhamnosus) was observed. PCR fingerprinting of lactobacilli, which was performed by PCR-fingerprinting, indicated a marked microbial heterogeneity within theLactobacillus spp. populations, which enabled strain (or group)-specific fingerprints to be observed.     

Preliminary characterization of lactic acid bacteria isolated from Zlatar cheese

AIMS: Isolation, characterization and identification of lactic acid bacteria (LAB) from artisanal Zlatar cheese during the ripening process and selection of strains with good technological characteristics. METHODS AND RESULTS: Characterization of LAB was performed based on morphological, physiological and biochemical assays, as well as, by determining proteolytic activity and plasmid profile. rep-polymerase chain reaction (PCR) analysis and 16S rDNA sequencing were used for the identification of LAB. PCR analysis was performed with specific primers for detection of the gene encoding nisin production. Strains Lactobacillus paracasei subsp. paracasei, Lactobacillus plantarum, Lactobacillus brevis, Lactococcus lactis subsp. lactis, Enterococcus faecium and Enterococcus faecalis were the main groups present in the Zlatar cheese during ripening. CONCLUSIONS: Temporal changes in the species were observed during the Zlatar cheese ripening. Mesophilic lactobacilli are predominant microflora in Zlatar cheese. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study we determined that Zlatar cheese up to 30 days old could be used as a source of strains for the preparation of potential starter cultures in the process of industrial cheese production. As the Serbian food market is adjusting to European Union regulations, the standardization of Zlatar cheese production by using starter culture(s) based on autochtonous well-characterized LAB will enable the industrial production of this popular cheese in the future.     

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.     

Whey valorisation: A complete and novel technology development for dairy industry starter culture production

Whey is the major by-product of the dairy industry, produced in large quantities and usually disposed off causing major environmental pollution, due to its high organic load that makes treatment cost prohibitive. This paper comprises a contribution on the valorisation of this high polluting liquid waste of the dairy industry, based on research for the production of novel dairy starter cultures using whey as raw material. Starter cultures are used for cheese ripening in order to: (i) accelerate ripening, (ii) improve quality and (iii) increase shelf-life. The developed technology involves biomass production from whey followed by thermal drying of cultures. Specifically, Kluyveromyces marxianus, Lactobacillus bulgaricus and kefir yeasts were thermally dried, and their efficiency in lactose and milk whey fermentations was studied. The most suitable culture regarding its technological properties was kefir, which was used for cheese ripening in freeze-dried and thermally dried form. Besides the reduction of production cost, which is an essential requirement for the food industry, the use of thermally dried kefir displayed several other advantages such as acceleration of ripening, increase of shelf-life, and improvement of hard-type cheese quality.     

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.     

Physicochemical and microbiological characteristics of Kulek cheese made from raw and heat-treated milk

The effect of heat treatment and commercial starter culture utilization on the physicochemical and microbiological properties of Kulek cheese made from raw milk with or without starter culture and heated milk with starter culture were investigated during ripening. Titratable acidity (TA) was the highest in cheeses made from heated milk while total solids (TS), salt, and fat were the highest in cheeses made from raw milk. The heat treatment significantly decreased the counts of coliforms and Enterobacteriaceae in cheeses. At the beginning of the ripening period, cheeses manufactured from heated milk with starter exhibited significantly higher counts of lactococci and proteolytic organisms and lower counts of lactobacilli than the other cheeses. After the first day, raw milk cheeses without starter showed higher microbiological counts than the others. In fresh cheeses, Lactococcus was the main lactic acid bacterium, with Lc. lactis lactis being predominant. Lactobacillus plantarum and Lactobacillus paracasei paracasei dominated at the later stages of the ripening.     

Influence of ripening container on the lactic acid bacteria population in Tulum cheese

The objective of the present study was to investigate the influence of container material (plastic or goat-skin bag) on the growth of lactic acid bacteria in Tulum cheese during 9 months of ripening. The lactic acid bacteria in Tulum cheeses were periodically counted on MRS and M17 agars throughout ripening. Results showed that the highest counts of lactic acid bacteria on MRS or M17 were observed at the beginning of ripening and their counts decreased during later stages of ripening. The cheese samples ripened in plastic bags exhibited higher numbers of LAB on MRS and M-17 agars than those ripened in goat-skin bags. A total of 112 strains of lactic acid bacteria were isolated from Tulum cheeses ripened in plastic or goat-skin bags during ripening. The lactic acid bacteria present in the cheese were classified by Microbial Identification System (MIS) based on a comparison of the fatty acid methyl ester profiles. Different species including Enteroccocus, Lactobacillus, Streptococcus, Lactococcus and Pediococcus genera were found in unripened cheese. As ripening proceeded, the species Streptococcus and Lactococcus disappeared and the percentages of the species Enterococcus was unchanged in both containers. There were slight differences between the cheeses ripened in plastic or goat-skin bags in terms of the profiles of lactic acid bacteria isolated. Some species including L. brevis, L. mesenteroides subsp. dextranicum, P. damnosus and E. mundtii were isolated only in the cheeses ripened in plastic bags; however, L. coryniformis and L. malafermentans were isolated only in the cheeses ripened in goat-skin bags at 6 or 9 months of ripening. Also the numbers of E. faecalis isolates were higher in the cheeses ripened in plastic containers than cheeses ripened goat-skin bags at the 6 or 9 months of ripening. The results showed that Lactobacillus and Enterococcus were the predominant species in matured Tulum cheeses in both ripening containers. It seemed possible to produce Tulum cheese with similar characteristics from both the containers used.     

Microbiological profile in Serra ewes' cheese during ripening

The microflora of Serra cheese was monitored during a 35 d ripening period at three different periods within the ewe's lactation season. After 7 d ripening, the numbers of micro-organisms reached their maximum, and lactic acid bacteria (LAB) and coliforms were the predominant groups. Pseudomonads were not detected after 1 week of ripening. At all stages of ripening, cheeses manufactured in spring exhibited the lowest numbers of LAB and yeasts, whereas cheeses manufactured in winter showed the lowest numbers of coliforms and staphylococci.
Leuconostoc lactis was the most abundant LAB found in Serra cheese whereas Enterococcus faecium and Lactococcus lactis spp. lactis exhibited the highest decrease in percentage composition. Numbers of both Leuc. mesenteroides and Lactobacillus paracasei tended to increase throughout ripening. The most abundant coliform was Hafnia alvei. Klebsiella oxytoca was found in curd but declined in number during ripening. Staphylococcal flora of curd was mainly composed of Staphylococcus xylosus, Staph. aureus and Staph. epidermidis. Staphylococcus xylosus was the major species found at the end of ripening. Pseudomonas fluorescens , was the only Pseudomonas species isolated from the curd. Although a broad spectrum of yeasts were found in Serra cheese, Sporobolomyces roseus was the most abundant yeast isolated.     

Variability of bacterial biofilms of the "tina" wood vats used in the ragusano cheese-making process

Ragusano cheese is a "protected denomination of origin" cheese made in the Hyblean region of Sicily from raw milk using traditional wooden tools, without starter. To explore the Ragusano bacterial ecosystem, molecular fingerprinting was conducted at different times during the ripening and biofilms from the wooden vats called "tinas" were investigated. Raw milks collected at two farm sites, one on the mountain and one at sea level, were processed to produce Ragusano cheese. Raw milk, curd before and after cooking, curd at stretching time (cheese 0 time), and cheese samples (4 and 7 months) were analyzed by PCR-temporal temperature gel electrophoresis (PCR-TTGE) and by classical enumeration microbiology. With the use of universal primers, PCR-TTGE revealed many differences between the raw milk profiles, but also notable common bands identified as Streptococcus thermophilus, Lactobacillus lactis, Lactobacillus delbrueckii, and Enterococcus faecium. After the stretching, TTGE profiles revealed three to five dominant species only through the entire process of ripening. In the biofilms of the two tinas used, one to five species were detected, S. thermophilus being predominant in both. Biofilms from five other tinas were also analyzed by PCR-TTGE, PCR-denaturating gradient gel electrophoresis, specific PCR tests, and sequencing, confirming the predominance of lactic acid bacteria (S. thermophilus, L. lactis, and L. delbrueckii subsp. lactis) and the presence of a few high-GC-content species, like coryneform bacteria. The spontaneous acidification of raw milks before and after contact with the five tinas was followed in two independent experiments. The lag period before acidification can be up to 5 h, depending on the raw milk and the specific tina, highlighting the complexity of this natural inoculation system.     

Use of RAPD-PCR and TTGE for the evaluation of biodiversity of whey cultures for Grana Padano cheese

AIMS: This work was carried out in order to evaluate the microbial diversity of whey cultures collected from different Grana Padano cheese plants in Veneto region (north-east Italy) by means of RAPD-PCR and Temporal Temperature Gradient Gel Electrophoresis (TTGE) analysis. METHODS AND RESULTS: Lactobacillus helveticus was the dominant species among isolated thermophilic lactobacilli. RAPD-PCR with primers M13 and D8635 resulted a suitable method for typing Lact. helveticus at strain level. Thirteen different Lact. helveticus biotypes were detected in the seven whey cultures studied with one biotype present in all the whey cultures. Besides Lact. helveticus, Lact. delbrueckii subsp. lactis was the main microbial species detected by TTGE. CONCLUSIONS: RAPD-PCR resulted very useful in studying Lact. helveticus biodiversity; furthermore, TTGE analysis allowed to detect the dominant thermophilic microflora characteristic of Grana Padano cheese whey cultures. IMPACT OF THE STUDY: By the combined used of RAPD-PCR and TTGE it could be possible to follow the behaviour in strain or species composition of whey cultures during time.     

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.     

The effect of a commercial starter culture addition on the ripening of an artisanal goat's cheese (Cameros cheese)

The evolution of physicochemical parameters, and the most important microbial groups, were determined for the following three batches of 'Cameros' goat's milk cheese during ripening: Batch R elaborated with raw milk, Batch RS elaborated with raw milk and with the addition of a starter culture, and Batch PS elaborated with pasteurized milk and with the addition of the same culture. No differences in total solids (TS) or in the content of NaCl, fat and total nitrogen (expressed as percentages of TS) were found during the ripening. The pH, fat acidity and non-protein nitrogen (NPN, expressed as a percentage of TN) showed significant differences between the batches. The inoculated batches showed the fastest drop in pH at the beginning of the ripening period, but the cheeses of Batch R showed a higher degree of lipolysis and proteolysis. The addition of a starter influenced the microbiological quality of the cheeses. Differences in the counts of Enterobacteriaceae and faecal coliforms were found between Batches R and RS after 15 days. Staphylococcus aureus increased in number during the early period of ripening and attained a population above 6 log cfu g-1 in Batch R in the period from 5 to 10 days. However, enterotoxins were not detected in this Batch. Batch R showed lower values of lactic acid bacteria at the beginning of the ripening period, but no significant differences were found between batches in the period from 5 to 15 days of ripening. At the beginning of the ripening, Lactococcus was the main lactic acid bacteria, with L. lactis lactis being predominant. After 15 days, the lactic acid bacteria counts decreased in the three batches, especially in the cheeses of Batch PS (only 2.2 log cfu g-1 was found at 60 days), as lactococci (the only lactic acid bacteria present in Batch PS) are incapable of growing under the conditions found in cheeses at the end of their ripening period. At this time, Lactobacillus was the predominant genus in Batches R and RS, with L. plantarum predominant. No lactococci were found from day 30 in Batch R and from day 40 in Batch RS. The cheeses of Batch RS received the most favourable scores from the tasting panel for all attributes judged: cut appearance, colour, aroma, taste, texture and general acceptance.     

Presence of peptidase activities in different varieties of cheese

Lactic acid bacteria (LAB) counts, PepX activity towards H-Phe-Pro-beta NA, and aminopeptidase activity towards H-Arg-beta NA. HCl, H-Lys-beta NA, H-Leu-beta NA, H-Pro-beta NA, H-Glu-beta NA derivatives have been evaluated in 32 commercial samples of cheese, one processed cheese, and one yoghurt. The presence of intracellular exo-peptidase activities in cheese extracts free from bacterial cells was detected, even after 1 year of ripening. An inverse ratio between the presence of viable lactic microflora and peptidase activity in the cheese extracts was observed. The importance of LAB starter exo-peptidases in the degradation of casein oligopeptides, and the key role of autolysis in the release of peptidases in the cheese, are discussed.     

Antimicrobial activity of lactic acid bacteria isolated from Tenerife cheese: initial characterization of plantaricin TF711, a bacteriocin-like substance produced by Lactobacillus plantarum TF711

AIMS: The screening and initial characterization of bacteriocins produced by lactic acid bacteria (LAB) from raw Tenerife goats' cheese with possible application as biopreservatives or ripening accelerators for Tenerife cheese. METHODS AND RESULTS: One hundred and eighty LAB of the genera Lactobacillus (95), Leuconostoc (64) and Lactococcus (21) isolated from raw Tenerife goats' cheese were screened for the production of antimicrobial substances. Lactobacillus plantarum TF711, which had the broadest spectrum of antimicrobial activity, was selected for further characterization. The antimicrobial compound was determined as a proteinaceous substance, as it was sensitive to proteases. The bacteriocin-like substance, which we called plantaricin TF711, was active against the Gram-positive bacteria Bacillus cereus, Clostridium sporogenes and Staphylococcus aureus; and against the Enterobacteriaceae Shigella sonnei and Klebsiella pneumoniae. It was stable to heat and to treatment with surfactants and organic solvents. Highest antimicrobial activity was found between pH 1 and 9. Plantaricin TF711 exhibited primary metabolite kinetics, a bacteriostatic mode of action and a molecular mass of c. 2.5 kDa as determined by tricine SDS-PAGE. CONCLUSIONS: Lact. plantarum TF711 produces a low molecular mass bacteriocin-like compound with a wide spectrum of activity and interesting technological properties (thermostability, good pH stability and stability against surfactants and organic solvents). SIGNIFICANCE AND IMPACT OF THE STUDY: Plantaricin TF711 was found to have potential for use as a biopreservative in the food industry.     

Growth and survival of E. coli O157:H7 during the manufacture and ripening of a smear-ripened cheese produced from raw milk

AIMS: The behaviour of Escherichia coli O157:H7 was studied during the manufacture and ripening of a smear-ripened cheese produced from raw milk. METHODS AND RESULTS: Cheese was manufactured on a laboratory scale using milk (20 l) inoculated with E. coli O157:H7, and enumeration was carried out using CT-SMAC. From an initial level of 1.52 +/- 0.03 log cfu ml-1 in the milk (34 +/- 2 cfu ml-1), the numbers increased to 3.4 +/- 0.05 log cfu g-1 in the cheese at day 1. During ripening, the numbers decreased to <1 cfu g-1 and <10 cfu g-1 in the rind and core, respectively, after 21 days, although viable cells were detected by enrichment after 90 days. The presence of E. coli O157:H7 in the cheese was confirmed by latex agglutination and by multiplex PCR. CONCLUSION: The results indicate that the manufacturing procedure encouraged substantial growth of E. coli O157:H7 to levels that permitted survival during ripening and extended storage. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of low numbers of E. coli O157:H7 in milk, destined for raw milk cheese manufacture, could constitute a threat to the consumer.     

Resident lactic acid bacteria in raw milk Canestrato Pugliese cheese

AIMS: Investigation of the autochthonous lactic acid bacteria (LAB) population of the raw milk protected designation of origin Canestrato Pugliese cheese using phenotypic and genotypic methodologies. METHODS AND RESULTS: Thirty phenotypic assays and three molecular techniques (restriction fragment length polymorphism, partial sequencing of the 16S rRNA gene and recA multiplex PCR assay) were applied to the identification of 304 isolates from raw milk Canestrato Pugliese cheese. As a result, 168 of 207 isolates identified were ascribed to genus Enterococcus, 25 to Lactobacillus, 13 to Lactococcus and one to Leuconostoc. More in details among the lactobacilli, the species Lactobacillus brevis and Lactobacillus plantarum were predominant, including 13 and 10 isolates respectively, whereas among the lactococci, Lactococcus lactis subsp.cremoris [corrected] was the species more frequently detected (seven isolates). CONCLUSIONS: Except for the enterococci, phenotypic tests were not reliable enough for the identification of the isolates, if not combined to the genotype-based molecular techniques. The polyphasic approach utilized allowed 10 different LAB species to be detected; thus suggesting the appreciable LAB diversity of the autochthonous microbial population of the Canestrato Pugliese cheese. SIGNIFICANCE AND IMPACT OF THE STUDY: A comprehensive study of the resident raw milk Canestrato Pugliese cheese microbial population has been undertaken.     

Microbial analysis of raw cows’ milk used for cheese-making: influence of storage treatments on microbial composition and other technological traits

Raw milk used to produce Grana cheese was subjected to several treatment regimes, including varying temperatures and storage times. Milk from morning and evening milking were transferred to a dairy factory separately (double delivery) or together (single delivery), after storage at the farm for 12 h; in the former case, milk was stored at 12 or 8°C, whereas, in the latter, it was kept at ambient temperature or 18°C. Values of pH of the vat milk were lower for milk samples kept at room temperature, while other physico-chemical parameters and rheological characteristics tested did not show significant differences linked to the different storage temperatures of milk used for “Grana Trentino” cheese production. Total microorganisms and several microbial groups (psychrotrophic bacteria, coliforms, mesophilic and termophilic lactic acid bacteria, including enterococci, pseudomonads and clostridia) were detected and quantified by classical (plate count and most probable number) techniques, after each technological treatment for a total of 212 milk and cream samples. The application of a culture-independent microbiological strategy, consisting of denaturing gradient gel electrophoresis, allowed the recognition of several bacterial genera and species.     

Two 2[5H]-Furanones as Possible Signaling Molecules in Lactobacillus helveticus

Two 2[5H]-furanones, in association with medium-chain fatty acids, were released in whey by Lactobacillus helveticus exposed to oxidative and heat stresses. This species plays an important role in cheese technology, particularly for Swiss-type cheeses and Grana cheese. Moreover, it significantly contributes to cheese ripening by means of an early autolysis and the release of enzymes during processing. Experimental evidence of the involvement of the two 2[5H]-furanones, detected by a gas chromatography-mass spectrometry/solid-phase microextraction technique, in the autolysis phenomenon has been obtained. Zymograms performed by using renaturing sodium dodecyl sulfate-polyacrylamide gels were used to detect the bioactivity of the supernatants containing the two furanones on fresh cells of the same strain. In addition to bands corresponding to known autolysins, new autolysins were detected concomitant with the exposure of Lactobacillus helveticus to the supernatants, which can be regarded as conditioned media (CM), and to a commercial furanone, 5-ethyl-3-hydroxy-4-methyl-2[5H]-furanone (HEMFi), having spectral data similar to those of the newly described 2[5H]-furanones. Morphological changes were observed when fresh cells were exposed to CM containing the two 2[5H]-furanones and HEMFi. The two furanones produced by Lactobacillus helveticus, which met a number of criteria to be included in cell-cell signaling molecules, have a presumptive molecular mass lower than those of already known 3[2H]-furanones having an autolytic activity and being produced by gram-negative bacteria. Moreover, they present a different chemical structure with respect to the furanones already identified as products of Lactococcus lactis subsp. cremoris or to those identified in some cheeses with Lactobacillus helveticus as a starter culture.