Isolation and characterization of new bacteriophages active against Clostridium tyrobutyricum and their role in preventing the late blowing defect of cheese.

Lytic bacteriophages (phages) offer a great potential as biocontrol agents for spoilage Clostridium tyrobutyricum, responsible for butyric acid fermentation in semi-hard and hard ripened cheeses, resulting in late gas blowing defect. With this aim, we have isolated, identified and characterized new lytic phages of C. tyrobutyricum, and have evaluated their efficacy to control cheese late blowing by adding them to manufacture milk. Silage, soil, milk and cheese from dairy farms were screened for anti-clostridial phages, obtaining 96 isolates active against C. tyrobutyricum. According to host range, source and plaque morphology, we obtained 20 phage profiles, 8 of them (represented by phages FA3, FA21, FA29, FA52, FA58, FA67, FA70 and FA88) showing a wider host range and high quality lysis, which were further characterized. Selected isolates showed a non-contractile tail, belonging to the Siphoviridae family, and were grouped into 3 restriction profiles. Viable phages were detected after storage in sodium-magnesium buffer (SM buffer), skim milk and acidified skim milk (pH 5) for 7 d at 4 °C, 12 °C and 37 °C, although a decline in infectivity was observed in some cases. Good phage survival was also detected during semi-hard cheese manufacture and ripening (60 d), and cheese lactococci counts, pH, dry matter values, and volatile compounds were not affected by phage addition. In semi-hard cheese, phage FA67 impaired the early germination of C. tyrobutyricum spores and caused a significant decrease in clostridial vegetative cells counts at 14 d of ripening, delaying by 2 weeks the consumption of lactic acid, formation of butyric acid and appearance of late blowing symptoms, compared to the spoilt control cheese without the phage. This is the first report on the application of phage to control C. tyrobutyricum in cheese.

Inhibitory activity of aromatic plant extracts against dairy-related Clostridium species and their use to prevent the late blowing defect of cheese.

The aim of the present work was the selection of aromatic plant essential oils (EOs) and/or ethanolic extracts (EEs) to prevent the late blowing defect (LBD) of cheese caused by Clostridium spp. EEs resulted more effective than EOs to inhibit dairy-borne Clostridium spp. in vitro. Savory, hyssop, lavender and tarragon EEs, which showed the lowest minimal inhibitory concentration against Clostridium tyrobutyricum, were selected to study the prevention of LBD caused by this bacterium in cheese. Addition of savory and lavender EEs to cheese milk delayed LBD by 2 weeks, but at the end of ripening these cheeses showed similar clostridial vegetative cells counts, spoilage symptoms and propionic, and butyric acids levels than blown control cheese. Tarragon EE, with the highest content in caffeic acid, also delayed LBD by 2 weeks, but it was more effective to inhibit Clostridium, since cheese with tarragon EE showed minor LBD symptoms, lower vegetative cells count and lower concentrations of propionic and butyric acids than the rest of cheeses made with EEs. This fact could be also attributable to the greater number of antimicrobial terpenes (1,8-cineole, 4-terpineol, α-terpineol, isoelemicin, methyl eugenol, and methyl trans-isoeugenol) detected in this cheese. This is the first report on the application of EEs to control C. tyrobutyricum in cheese.

Volatile Transference and Antimicrobial Activity of Cheeses Made with Ewes' Milk Fortified with Essential Oils.

During the last decades, essential oils (EOs) have been proven to be a natural alternative to additives or pasteurization for the prevention of microbial spoilage in several food matrices. In this work, we tested the antimicrobial activity of EOs from Melissa officinalis, Ocimum basilicum, and Thymus vulgaris against three different microorganisms: Escherichia coli, Clostridium tyrobutyricum, and Penicillium verrucosum. Pressed ewes' cheese made from milk fortified with EOs (250 mg/kg) was used as a model. The carryover effect of each oil was studied by analyzing the volatile fraction of dairy samples along the cheese-making process using headspace stir bar sorptive extraction coupled to gas chromatography/mass spectrometry. Results showed that the EOs contained in T. vulgaris effectively reduced the counts of C. tyrobutyricum and inhibited completely the growth of P. verrucosum without affecting the natural flora present in the cheese. By contrast, the inhibitory effect of M. officinalis against lactic acid bacteria starter cultures rendered this oil unsuitable for this matrix.

Dairy matrix effect on the transference of rosemary (Rosmarinus officinalis) essential oil compounds during cheese making.

BACKGROUND: The use of aromatic plant extracts as ingredients may be compromised owing to low transference and activity lack in food matrixes compared with in vitro trials. Rosemary essential oil (REO) was added to sheep milk to study the transference of its compounds during the cheese-making process and to determine how cheese antimicrobial activity is modified. RESULTS: The volatile characterization of dairy samples was performed using headspace stir bar sorptive extraction coupled to gas chromatography/mass spectrometry (HS-SBSE/GC/MS) so that fat matrix interferences were reduced. This method detected a decrease in volatile recovery concentration of 19.33% when REO was added to milk. A total recovery volatile yield of 62.51% was measured from the initial quantification of milk to cheese, with hydrocarbon volatiles being transferred in a higher ratio (64.88%) than oxygenated ones (58.74%). No effects were observed for REO in fortified cheese on the counts of native flora necessary for ripening processes, but the total inhibition of Clostridium spp. was provoked CONCLUSION: The study of active compound transference during cheese elaboration was achieved. The antimicrobial results in fortified cheeses with REO showed a preventive effect in the case of clostridial species, which are responsible for late cheese blowing.

Mycotoxicogenic fungal inhibition by innovative cheese cover with aromatic plants.

BACKGROUND: The use of aromatic plants and their extracts with antimicrobial properties may be compromised in the case of cheese, as some type of fungal starter is needed during its production. Penicillium verrucosum is considered a common cheese spoiler. The aim of this study was to evaluate the innovative use of certain aromatic plants as natural cheese covers in order to prevent mycotoxicogenic fungal growth (P. verrucosum). A collection of 12 essential oils (EOs) was obtained from various aromatic plants by solvent-free microwave extraction technology, and volatile characterisation of the EOs was carried out by gas chromatography/mass spectrometry. RESULTS: The most effective EOs against P. verrucosum were obtained from Anethum graveolens, Hyssopus officinalis and Chamaemelum nobile, yielding 50% inhibition of fungal growth at concentration values lower than 0.02 µL mL⁻¹. All EOs showed high volatile heterogeneity, with α-phellandrene, pinocamphone, isopinocamphone, α-pinene, camphene, 1,8-cineole, carvacrol and trans-anethole being found to be statistically significant in the antifungal model. CONCLUSION: The use of these aromatic plants as natural covers on cheese can satisfactorily inhibit the growth of some mycotoxicogenic fungal spoilers. Among the volatile compounds present, α- and ß-phellandrene were confirmed as the most relevant in the inhibition.