Biodiversity and antibiotic resistance profile provide new evidence for a different origin of enterococci in bovine raw milk and feces.

Enterococci are widely distributed in dairy sector. They are commensals of the gastrointestinal tract of animals, thus, via fecal contamination, could reach raw milk and dairy products. The aims of this study were: 1) to investigate the enterococcal diversity in cow feces and milk samples and 2) to evaluate the antibiotic resistance (AR) of dairy-related enterococci and their ability to transfer resistance genes. E. faecalis (59.9%), E. faecium (18.6%) and E. lactis (12.4%) were prevalent in milk, while E. faecium (84.2%) and E. hirae (15.0%) were dominant in bovine feces. RAPD-PCR highlighted a high number of Enterococcus biotypes (45 from milk and 37 from feces) and none of the milk strains exhibited genetic profiles similar to those of feces biotypes. A high percentage of enterococci isolated from milk (71%) were identified as multidrug resistant and resistance against streptomycin and tetracycline were widespread among milk strains while enterococci from feces were commonly resistant to linezolid and quinupristin/dalfopristin. Only E. faecalis strains were able to transfer horizontally the tetM gene to Lb. delbrueckii subsp. lactis. Our results indicated that Enterococcus biotypes from milk and bovine feces belong to different community and the ability of these microorganisms to transfer AR genes is strain-dependent.

Surface layer proteins from Lactobacillus helveticus ATCC® 15009™ affect the gut barrier morphology and function.

Paraprobiotics and postbiotics represent a valid alternative to probiotic strains for ameliorating and preserving a healthy intestinal epithelial barrier (IEB). The present study investigated the effects of surface layer proteins (S-layer) of the dairy strain Lactobacillus helveticus ATCC® 15009™ (Lb ATCC® 15009™), as paraprobiotic, on the morpho-functional modulation of IEB in comparison to live or heat-inactivated Lb ATCC® 15009™ in an in vitro co-culture of Caco-2/HT-29 70/30 cells. Live or heat-inactivated Lb ATCC® 15009™ negatively affected transepithelial electrical resistance (TEER) and paracellular permeability, and impaired the distribution of Claudin-1, a tight junction (TJ) transmembrane protein, as detected by immunofluorescence (IF). Conversely, the addition of the S-layer improved TEER and decreased permeability in physiological conditions in co-cultures with basal TEER lower than 50 ohmcm2, indicative of a more permeable physiological IEB known as leaky gut. Transmission electron microscopy (TEM) and IF analyses suggested that the S-layer induces a structural TJ rearrangement and desmosomes' formation. S-layer also restored TEER and permeability in the presence of LPS, but not of a mixture of pro-inflammatory cytokines (TNF-α plus IFN-γ). IF analyses showed an increase in Claudin-1 staining when LPS and S-layer were co-administered with respect to LPS alone; in addition, the S-layer counteracted the reduction of alkaline phosphatase detoxification activity and the enhancement of pro-inflammatory interleukin-8 release both induced by LPS. Altogether, these data corroborate a paraprobiotic role of S-layer from Lb ATCC® 15009™ as a possible candidate for therapeutic and prophylactic uses in conditions related to gastrointestinal health and correlated with extra-intestinal disorders.

Antibiotic resistance in wild and commercial non-enterococcal Lactic Acid Bacteria and Bifidobacteria strains of dairy origin: An update.

Antibiotic Resistance is a growing concern for public health and global economy. Lactic acid bacteria (LAB) involved in the production of dairy products and commonly present in the agro-zootechnical environment can act as reservoirs of antibiotic resistance genes, acquiring or transferring them to other microorganisms. The review focuses on LAB group of dairy origin (Lactobacillus, Lactococcus, Streptococcus, Leuconostoc, Pediococcus and Weissella) and Bifidobacterium genus, considering its large use in dairy industry. We have analyzed data in the last 25 years, highlighting atypical resistance, genetic traits correlated to antibiotic resistance and their ability to be transmitted to other microorganisms; comparative analysis of resistomes was also considered. Differences were observed among wild strains isolated from different regions because of authorized antibiotic use. Commercial strains belonging to Lactobacillus, Streptococcus and Bifidobacterium currently used for industrial dairy products are frequently resistant to gentamycin, kanamycin, chloramphenicol together with tetracycline. The presence of resistant wild LAB in raw milk products has been significantly reduced as a result of worldwide restrictions on the use of antibiotics in animal husbandry. Transmissible resistances are still present in industrial cultures, despite the great effort of starter industries in the process control and the safety screening of commercial cultures.

Proteolytic Traits of Psychrotrophic Bacteria Potentially Causative of Sterilized Milk Instability: Genotypic, Phenotypic and Peptidomic Insight.

The proteolytic traits of the psychrotrophic strains Pseudomonas poae LP5, Pseudomonas fluorescens LPF3, Chryseobacterium joostei LPR1, Pseudomonas fulva PS1, Citrobacter freundii PS37, Hafnia alvei PS46, and Serratia marcescens PS92 were initially investigated by phenotypic and genotypic approaches. Six strains elicited extracellular proteolytic activity, and five expressed the thermostable AprX or (likely) Ser1 enzymes. Then, the strains were inoculated (104 CFU/mL) in microfiltered pasteurized milk and kept at 4 °C for five days. All of the strains reached 108 CFU/mL at the end of storage and five produced thermostable extracellular proteolytic enzymes. The freshly inoculated samples and the corresponding samples at 108 CFU/mL were batch-sterilized (131 °C, 30 s) and kept at 45 °C up to 100 days. The former samples did not gel until the end of incubation, whereas the latter, containing P. poae, P. fluorescens, C. joostei, C. freundii, and S. marcescens, gelled within a few days of incubation. The thermostable proteolytic activity of strains affected the peptidomic profile, and specific proteolyzed zones of ß-CN were recognized in the gelled samples. Overall, the results confirm some proteolytic traits of psychrotrophic Pseudomonas spp. strains and provide additional insights on the proteolytic activity of psychrotrophic bacteria potentially responsible for sterilized milk destabilization.

Paraprobiotics: A New Perspective for Functional Foods and Nutraceuticals.

Probiotics are live microorganisms that confer health benefits on the host. However, in recent years, several concerns on their use have been raised. In particular, industrial processing and storage of probiotic products are still technological challenges as these could severely impair cell viability. On the other hand, safety of live microorganisms should be taken into account, especially when administered to vulnerable people, such as the elderly and immunodeficient individuals. These drawbacks have enhanced the interest toward new products based on non-viable probiotics such as paraprobiotics and postbiotics. In particular, paraprobiotics, defined as "inactivated microbial cells (non-viable) that confer a health benefit to the consumer," hold the ability to regulate the adaptive and innate immune systems, exhibit anti-inflammatory, antiproliferative and antioxidant properties and exert antagonistic effect against pathogens. Moreover, paraprobiotics can exhibit enhanced safety, assure technological and practical benefits and can also be used in products suitable for people with weak immunity and the elderly. These features offer an important opportunity to prompt the market with novel functional foods or nutraceuticals that are safer and more stable. This review provides an overview of central issues on paraprobiotics and highlights the urgent need for further studies aimed at assessing safety and efficacy of these products and their mechanisms of action in order to support decisions of regulatory authorities. Finally, a definition is proposed that unambiguously distinguishes paraprobiotics from postbiotics.

Assessment of Possible Application of an Atmospheric Pressure Plasma Jet for Shelf Life Extension of Fresh-Cut Salad.

Ready-to-eat salads are very perishable with quality losses within 6-7 days, and the extension of their shelf life is still a challenge. In this work, an atmospheric pressure plasma jet (APPJ) was applied for the surface decontamination of fresh-cut lettuce baby leaves. The APPJ antimicrobial efficiency on the natural microbiota and its impact on some physicochemical attributes of lettuce were evaluated as a function of the treatment duration (0-30 s). Then, the influence of plasma treatment on the salad shelf life was studied, following the growth of aerobic mesophilic bacteria in both untreated and plasma-treated samples during 9 days of storage at 4 °C, together with the plasma-induced changes in physicochemical parameters of lettuce leaves. The APPJ induced a fast (15 s) microbial decontamination (1.3 log10 CFU/g) of the salad surface. Exposure time and salad-plasma plume distance were the parameters that substantially affected the microbial inactivation. APPJ treatment retarded bacterial growth during the refrigerated storage, as plasma-treated samples were noticeably less contaminated than the non-treated ones in the first 3-4 days. No significant effect were observed on electrolyte leakage, pH, and dry matter content in both the set up phase and the shelf life study.

Screening of lactic acid bacteria producing folate and their potential use as adjunct cultures for cheese bio-enrichment.

Lactic acid bacteria (LAB) can be used to increase the folate in foods by in situ fortification. Seventy LAB were screened for their ability to produce folate during growth in de Man, Rogosa and Sharpe/M17 broth. Lactobacillus casei, Lactobacillus plantarum, Lactobacillus paracasei subsp. paracasei, Lactobacillus rhamnosus, Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, Lactococcus lactis subsp. lactis, Enterococcus faecium and Enterococcus lactis were able to synthetize folates in the medium, even if to a different extent. The 47 folate-producing strains were further analyzed by microbiological assay, for total, extra and intracellular folate. Enterococcus faecium VC223 and E. lactis BT161 were able to produce in cultural medium 123,625.74 ± 8.00 ng/ml and 384.22 ± 5.00 ng/ml of folate, respectively. Five strains were further examined for their ability to synthesize folate in cheese. The folate content increased with ripening up to by 54% after 30 d when L. casei VC199 was used and up to 108% and 113% after 60 d, with L. paracasei SE160 and E. lactis BT161 respectively exceeding 100 ng/100g. Results encourage the use of specific LAB to obtain natural folate bio-enriched dairy products improving folate intake.

How we can improve the antimicrobial performances of lactic acid bacteria? A new strategy to control Listeria monocytogenes in Gorgonzola cheese.

This study was aimed to evaluate the effectiveness of two lactic acid bacteria (LAB) cultures (Lactococcus lactis FT27 and Carnobacteroim divergens SCA), lactic acid/sodium lactate (LASL - l-lactic acid 61% (w/w) and L-sodium lactate 21% (w/w)) and their combination against four Listeria monocytogenes biotypes isolated from Gorgonzola cheese. In vitro antilisterial activity showed that the sensitivity to antimicrobials was strain-dependent. Antimicrobial challenge testing was performed on Gorgonzola rinds simulating contamination occurring at the beginning (6 days) and at the end (55 days) of the ripening period, to assess the antilisterial activity of LAB strains and LASL during the subsequent 60 days at 4 °C. LASL showed a higher antilisterial activity than LAB, maintaining the pathogen content below the EC limit (<2.0 log10 CFU/g) for 60 days. A strong listericidal effect was observed combining LAB with LASL (2,8 µL/cm2) Lc. lactis in combination with LASL completely inhibited the two L. monocytogenes strains in the first 50 days, while LASL with C. divergens was more effective in the second part of ripening when the pH raised. Data obtained encourage the use of LASL along with antimicrobial LAB rotation schemes during cheese ripening for the prevention and/or control of the L. monocytogenes on cheese surface.

Does Enterococcus faecalis from Traditional Raw Milk Cheeses Serve as a Reservoir of Antibiotic Resistance and Pathogenic Traits?

Enterococcus faecalis is not only a prevalent species among dairy microbial community but also a well-documented opportunistic pathogen. Food safety should exclude the possibility of consumer exposure to its virulence traits through consumption of dairy products. In this study, an integrated approach based on both phenotypic and genotypic methods was applied to investigate the incidence of antibiotic resistance and pathogenicity potential in 40 E. faecalis isolated from 10 Italian raw milk cheeses over a 13-year period (1997-2009). Among the 14 tested antibiotics, resistance to tetracycline, rifampicin, chloramphenicol, and erythromycin was observed, whereas vancomycin-resistant enterococci were not found. A high incidence (90% of strains) of the tet(M) gene emerged, whereas tet(K), tet(S), tet(L), int, and ermB genes were occasionally amplified (12.5%, 10%, 7.5%, 2.5% and 30%, respectively). No strain was positive for vancomycin-resistant determinants. Among the seven virulence determinants considered, the asa1, gelE, esp, and efaA genes were harbored. No other gene encoding for either different virulence factors (cylA, hyl, and ace) or amino acid decarboxylase activity (hdc, tdc, and odc) was detected. Consequently, E. faecalis isolated from raw milk cheeses does not represent a substantial reservoir of antimicrobial resistance and virulence factors if compared with clinical strains. However, this species occasionally harbors detrimental traits; thus, the possibility that it could be a route for transmission of pathogenic genes through dairy products should never be disregarded.

Enzyme oxidation of plant galactomannans yielding biomaterials with novel properties and applications, including as delivery systems.

New biomaterials from renewable sources and the development of "functionalized biopolymers" are fields of growing industrial interest. Plant polysaccharides represent a valid alternative to traditional synthetic polymers, which are obtained from monomers of fossil, non-renewable origin. Several polysaccharides, either in their natural or chemically/biochemically modified forms, are currently employed in the biomedical, food and feed, and industrial fields, including packaging. Sustainable biochemical reactions, such as enzyme modifications of polysaccharides, open further possibilities for new product and process innovation. In the present review, we summarize the recent progress on enzyme oxidation of galactomannans (GM) from few leguminous plants (performed either with galactose oxidase or laccase) and we focus on the versatile and easily accessible laccase/TEMPO oxidative reaction. The latter causes a steep viscosity increase of GM water solutions and a transition of the gels from a viscous to an elastic form, due to formation of emiacetalic bonds and thus of internal cross-linking of the polymers. Following lyophilization of these hydrogels, stable aerogels can be obtained, which were shown to have good potential as delivery systems (DS) of actives. The active molecules tested and herewith described are polymyxin B, an antibiotic; nisin, an antimicrobial peptide; the enzymes lysozyme, protease and lipase; the mixture of the industrial microbiocides 5-chloro-2-methyl-4-isothiazolin-3-one (CIT) and 2-methyl-4-isothiazolin-3-one (MIT). The advantages of such aerogel systems and the possibilities they open for future developments, including as DS, are described.

Aerogel from chemo-enzymatically oxidized fenugreek gum: an innovative delivery system of isothiazolinones biocides.

Aerogels of chemo-enzymatically oxidized, lyophilized fenugreek gum (EOLFG) were evaluated as "delivery system" (DS) of the microbiocide mix 5-chloro-2-methyl-4-isothiazolin-3-one (CIT) and 2-methyl-4-isothiazolin-3-one (MIT). These biocides have a broad activity spectrum and a low MIC (minimal inhibitory concentration) in the ppm range. They are approved under the EU Biocidal Product Directive for various applications, including cosmetics, are classified as skin sensitizers, and are under increasing scrutiny to limit or eliminate their use.We demonstrate that CIT/MIT can be uptaken into EOLFG aerogel corks by immersion into aqueous solutions of microbiocides, followed by blotting and re-lyophilization to generate "loaded" EOLFG aerogels. Incubation of "loaded" corks in water brings about a slow and almost complete release of the absorbed actives having the same MIC of free biocides against selected bacterial pathogens.This new biomaterial could represent an innovative DS of microbiocides and other actives for a variety of industrial, food, cosmetic, and biomedical applications.

Evaluation of microbial consortia and chemical changes in spontaneous maize bran fermentation.

Sustainable exploitation of agro-industrial by-products has attracted great interest in cereal bran valorization. In this research, a polyphasic approach has been carried out to characterize maize bran at microbiological and chemical level during a sourdough like fermentation process, in order to enhance its technological and nutritional properties. Autochthonous microbiota was isolated at different refreshment steps and subjected to identification and molecular characterization. Fermentation was characterized by a rapid increase in lactic acid bacteria and yeasts, with a co-dominance, at the initial stage, of Weissella spp., Pediococcus spp. and Wickerhamomyces anomalus. At the end of the fermentation, a natural selection was produced, with the prevalence of Lactobacillus plantarum, Lactobacillus brevis and Kazachstania unispora. This is the first time that a specific association between LAB and yeasts is reported, during the maize bran fermentation process. Enzymatic activities related to this microbial consortium promoted a "destructuration" of the fiber fraction, an increase in soluble dietary fiber and a reduction of phytic acid content. Our data also evidenced a noticeable increment in ferulic acid. The results obtained indicate that fermentation processes represent an efficient biotechnological approach to increase nutritional and functional potential of maize bran. Moreover, the characterization of microbiota involved in natural fermentation process will allow the selection of specific biotypes, with appropriate metabolic and enzymatic activities, to conduct "tailored" fermentation processes and improve brans or whole-meal flours from both nutritional and technological points of view.

Prototheca blaschkeae subsp. brasiliensis subsp. nov., isolated from cow milk.

A strain of an achlorophyllic alga, named PR24T, was isolated from cow milk samples from the state of Minas Gerais, Brazil. Based on 18S rDNA, 28S rRNA, D1/D2 region of the LSU rDNA and SSU rRNA gene sequence similarities, this strain was found to be a member of the genus Prototheca and closely related to Protothecablaschkeae SAG2064T. However, the novel strain could easily be distinguished from recognized Prototheca species by internal transcribed spacer, species-specific PCR, single-strand conformation polymorphism-PCR analysis and phenotypic characteristics. The inability to grow in Sabouraud broth at pH 4.0 and the different cellular fatty acid composition clearly distinguished PR24T from the reference strain of P. blaschkeae. The combination of genotypic and phenotypic data indicates that strain PR24T represents a subspecies of P. blaschkeae, for which the name Prototheca blaschkeae subsp. brasiliensis subsp. nov. is proposed. The respective type strain is PR24T (=DSM 103592T=IHEM 26958T).

Efficient milking hygiene reduces bacterial spore contamination in milk.

Clostridia in the milk can lead to late blowing, a cheese defect. Clostridia are ubiquitous, deriving from both the farm environment and the feed ingested by the cows, and are transferred into the milk through faecal contamination. Our aim was to investigate the effect of different in-parlour practices on the content of anaerobic spore-forming bacteria in milk, and to monitor the variation in spore content in the feed and environment. The experiment, conducted in an experimental dairy during autumn, was repeated in exactly the same way for two consecutive years. The experimental design applied three different milking routines in three consecutive 7-d periods: forestripping alone (F); forestripping and post-dipping (F+Post); pre-dipping, wiping, forestripping and post-dipping (Pre+F+Post). Teat skin swabs and samples of feed, faeces, bedding materials and milk were collected for microbiological analyses. The dietary forage of the lactating cows included maize silage, which, in both years, was found to have the highest level of clostridial spore contamination. Pre-dipping with a detergent/emollient solution, and drying with a disposable paper towel, proved much more efficient in reducing spore contamination than forestripping alone, both on the teats (1·30 vs. 2·20 log10 MPN/swab; P < 0·001) and in the milk (1·82 vs. 2·47 log10 MPN/L, P < 0·02), while post-dipping had little influence on spore count. The standard plate count in milk was significantly lower with Pre+F+Post treatment than with F (3·80 vs. 4·51 log10 CFU/mL, P < 0·01). The teat preparation procedure did not influence the lactic acid bacterial levels in the milk, which is very positive in that decreased lactic acid bacterial content can lessen raw milk cheese quality.

"Aerogels of enzymatically oxidized galactomannans from leguminous plants: Versatile delivery systems of antimicrobial peptides and enzymes".

We describe aerogels obtained by laccase/TEMPO-oxidation and lyophilization of galactomannans (GM) from fenugreek, sesbania and guar. Enzymatic oxidation of GM in aqueous solution caused viscosity increase up to fifteen-fold, generating structured, elastic, stable hydrogels, presumably due to establishment of hemiacetalic bonds between newly formed carbonyl groups and free hydroxyl groups. Upon lyophilization, water-insoluble aerogels were obtained, whose mechanical properties and porosity were investigated. Active principles were absorbed into the aerogels from aqueous solutions and, following rinsing, blotting, re-lyophilization, were released in an appropriate medium. The release of the antibiotic polymyxin B was tested against six different Gram-negative bacterial strains, of the antimicrobial peptide nisin against two Gram-positive and of the muraminidase lysozyme against one anaerobic strain. Protease and lipase release in solution from "enzyme loaded" aerogels was monitored by the increase in enzymatic activity. These biomaterials could represent new versatile, biocompatible "delivery systems" of actives for biomedical and industrial applications.

Proteolytic Activity and Production of γ-Aminobutyric Acid by Streptococcus thermophilus Cultivated in Microfiltered Pasteurized Milk.

A set of 191 strains of Streptococcus thermophilus were preliminarily screened for the presence of the genes codifying for cell envelope-associated proteinase (prtS) and for glutamate decarboxylase (gadB) responsible for γ-aminobutyric acid (GABA) production. The growth and proteolytic activity of the gadB-positive strains (9 presenting the prtS gene and 11 lacking it) were studied in microfiltered pasteurized milk. Degradation of both caseins (capillary electrophoresis) and soluble nitrogen fractions (HPLC) and changes in the profile of free amino acids (FAAs; ion-exchange chromatography) were evaluated at inoculation and after 6 and 24 h of incubation at 41 °C. None of the strains was capable of hydrolyzing caseins and ß-lactoglobulin, and only two hydrolyzed part of α-lactalbumin, these proteins being present in their native states in pasteurized milk. Contrarily, most strains were able to hydrolyze peptones and peptides. For initial growth, most strains relied on the FAAs present in milk, whereas, after 6 h, prtS+ strains released variable amounts of FAA. One prtS+ strain expressed a PrtS- phenotype, and two prtS- strains showed a rather intense proteolytic activity. Only five strains (all prtS+) produced GABA, in variable quantities (up to 100 mg/L) and at different rates, depending on the acidification strength. Addition of glutamate did not induce production of GABA in nonproducing strains that, however, unexpectedly were shown to adopt the degradation of arginine into citrulline and ornithine as an alternative acid resistance system and likely as a source of ATP.

Changes in oxidation-reduction potential during milk fermentation by wild lactic acid bacteria.

Oxidation-reduction potential (E h) is a fundamental physicochemical property of lactic acid bacteria that determines the microenvironment during the cheese manufacture and ripening. For this reason the E h is of growing interest in dairy research and the dairy industry. The objective of the study was to perform a comprehensive study on the reduction activity of wild lactic acid bacteria strains collected in different periods (from 1960 to 2012) from Italian dairy products. A total of 709 strains belonging to Lactococcus lactis, Enterococcus durans, E. faecium, E. faecalis and Streptococcus thermophilus species were studied for their reduction activity in milk. Kinetics of milk reduction were characterised by the minimum redox potential (E h7) and time of reaching E h7 (t min), the maximum difference between two measures (Δmax) and the time at which these maximum differences occurred (t*). Broad diversity in kinetic parameters was observed at both species and strain levels. E. faecalis and L. lactis resulted to be the most reducing species, while S. thermophilus was characterised by the lowest reducing power while the greatest heterogeneity was pointed out among E. durans and E. faecium strains. Considering the period of collection (1960-2012) we observed that the more recently isolated strains generally showed less reducing activity. This trend was particularly evident for the species E. durans, E. faecium and L. lactis while an opposite trend was observed in E. faecalis species. Data reported in this research provide new information for a deeper understanding of redox potential changes during milk fermentation due to bacterial growth. Gain knowledge of the redox potential of the LAB cultures could allow a better control and standardisation of cheesemaking process.

Antibiotic Resistance Patterns of Gram-Negative Psychrotrophic Bacteria from Bulk Tank Milk.

Bacterial resistance to antibiotics is a major global health problem and resistance of Pseudomonadaceae and Enterobacteriaceae is a serious concern. We investigated the prevalence of drug-resistance in a total of 80 psychrotrophic strains from bulk milk belonging to Pseudomonas genus (n. 63) and Enterobacteriaceae group (n. 17). All the strains were tested against 16 antibiotics. Pseudomonas were further investigated for their sensitivity against 12 additional antibiotics. Pseudomonas showed a high susceptibility toward fluoroquinolones, aminoglycosides, and piperacillin and, to a lesser extent, to imipenem, ceftazidime, cefepime. Thirty-five out of 63 Pseudomonas strains were susceptible to meropenem, while among antibiotics for which recommended breakpoints are not yet available, 55% of Pseudomonas strains had no inhibition halo in presence of nitrofurantoin, highlighting a resistance toward this drug. The results obtained in this study indicate a high efficiency of fluoroquinolones, chloramphenicol (94%), and kanamycin (76%) for Enterobacteriaceae while a high prevalence of resistant strains was found to ampicillin (13/17). Serratia marcescens is highly susceptible to fluoroquinolones, chloramphenicol, and kanamycin. Moreover, mupirocin seems to be the new antibiotic with the less efficacy for Enterobacteriaceae, with 41% of strains without halo, pointing out an important resistance. Further knowledge on resistance to known and new antibiotics among Pseudomonas species and Enterobacteriaceae of milk origin was acquired.

Development of a triplex real-time PCR assay for the simultaneous detection of Clostridium beijerinckii, Clostridium sporogenes and Clostridium tyrobutyricum in milk.

Clostridium beijerinckii, Clostridium sporogenes and Clostridium tyrobutyricum are considered the leading bacteria implicated in late blowing defects affecting semi-hard and hard cheese production. The aim of this study was to develop a multiplex Real-Time PCR (qPCR) analysis for a rapid and simultaneous detection of C. beijerinckii, C. sporogenes and C. tyrobutyricum, using specific primers respectively targeting the nifH, gerAA and enr genes. The limits of detection in raw milk were 300 CFU/50 mL in the case of C. beijerinckii, 2 CFU/50 mL for C. sporogenes and 5 CFU/50 mL for C. tyrobutyricum spores. The qPCR method was applied to artificially contaminated raw milk samples, and molecular quantification showed good correlation (R(2) = 0.978) with microbiological counting. Our results demonstrate that this method, combined with a DNA extraction protocol optimized for spore lysis, could be a useful tool for the direct quantification of the considered clostridia species.

Characterization of Gram-negative psychrotrophic bacteria isolated from Italian bulk tank milk.

Eighty psychrotrophic bacterial strains, isolated from different northwest Italian bulk tank milks destined for Grana Padano cheese production, were identified by 16S rRNA gene amplification and partial sequence analysis of the rpoB gene. Pseudomonas spp. were the most commonly occurring contaminants, P. fluorescens being the predominant isolated species, along with Enterobacteriaceae, primarily Serratia marcescens. RAPD-PCR was used to study genetic variability and distinguish closely related strains; a high degree of genetic heterogeneity among the strains was highlighted. All the strains were characterized for their ability to produce proteases, lipases and lecithinases at different temperatures (7, 22, and 30 °C). Forty-one of the psychrotrophic strains were positive for all the enzymatic activities. The highest number of positive strains for all the incubation temperatures was found for lipolytic activity (59), followed by proteolytic (31) and lecithinase (28) activities, and the enzymatic traits varied among the Pseudomonas and Enterobacteriaceae strains. The proteolytic psychrotrophic strains were screened for the presence of the aprX gene, coding for a heat-resistant metalloprotease in Pseudomonas spp. The aprX gene was detected in 19 of 63 Pseudomonas strains, and was widespread in the P. fluorescens strains (14/19). PRATICAL APPLICATION: The study provides new data on the enzymatic activity of Gram-negative psychrotrophic bacteria, useful in developing strategies to control the proteo-lipolytic spoilage of raw and processed milk that causes gelation, off-flavors, and loss of sensory quality and shelf life.