Relationship between Volatile Organic Compounds and Microorganisms Isolated from Raw Sheep Milk Cheeses Determined by Sanger Sequencing and GC-IMS.

Recently, the interest of consumers regarding artisan cheeses worldwide has increased. The ability of different autochthonous and characterized lactic acid bacteria (LAB) to produce aromas and the identification of the volatile organic compounds (VOCs) responsible for flavor in cheeses are important aspects to consider when selecting strains with optimal aromatic properties, resulting in the diversification of cheese products. The objective of this work is to determine the relationship between VOCs and microorganisms isolated (Lacticaseibacillus paracasei, Lactiplantibacillus plantarum, Leuconostoc mesenteroides and Lactococcus lactis subsp. hordniae) from raw sheep milk cheeses (matured and creamy natural) using accuracy and alternative methods. On combining Sanger sequencing for LAB identification with Gas Chromatography coupled to Ion Mobility Spectrometry (GC−IMS) to determinate VOCs, we describe cheeses and differentiate the potential role of each microorganism in their volatilome. The contribution of each LAB can be described according to their different VOC profile. Differences between LAB behavior in each cheese are shown, especially between LAB involved in creamy cheeses. Only L. lactis subsp. hordniae and L. mesenteroides show the same VOC profile in de Man Rogosa and Sharpe (MRS) cultures, but for different cheeses, and show two differences in VOC production in skim milk cultures. The occurrence of Lactococcus lactis subsp. hordniae from cheese is reported for first time.

Influence of Food Matrices and the Population of Interfering Microorganisms on the Determination of Listeria monocytogenes by Conventional Methods and VIDAS.

In this study, the possible influence of the food matrix and the interfering population of microorganisms on the detection and count of Listeria monocytogenes in three common foods of the Spanish diet (Spanish omelette, fresh cheese and vegetable salad) was determined. Four groups were assayed: one control, two groups with interfering microorganisms (Salmonella Enteritidis, Staphylococcus aureus and Proteus mirabilis) with different levels of L. monocytogenes and a final group only contaminated with L. monocytogenes. The samples were analyzed with the normalized method (UNE-EN ISO 11290:2018) and with an alternative technique (VIDAS). The results show that the presence of interfering microorganisms did not seem to interfere with the determination of L. monocytogenes. Furthermore, the type of food did not seem to influence the determination of L. monocytogenes, but the culture media used showed differences. In fact, regardless of the type of food, the ALOA medium showed higher sensitivity than the other media, with higher recovery in 100% of samples (only for the Spanish omelette in Group B was the result the same as that for PALCAM, -8.11 log cfu/g). The results obtained using the VIDAS were not influenced by any of the factors or conditions used and show 100% efficiency.

Behavior and effect of combined starter cultures on microbiological and physicochemical characteristics of dry-cured ham.

The behaviour of two combined starter cultures and their influence on the microbiological and physicochemical characteristics of dry-cured ham have been evaluated. Three lots of dry-cured hams were tested during processing (0, 9, 48, 74, 112, 142, 166 and 211 days). Lot 1 had no added starter culture. Lot 2 contained a starter culture of Penicillium chrysogenum, Penicillium digitatum, Penicillium nalgiovense, Debaryomyces hansenii, Lactobacillus plantarum, Lactobacillus acidophilus, Pediococcus pentosaceus and Micrococcus varians was and lot 3 had L. plantarum, L. acidophilus, P. pentosaceus and M. varians. The use of a selected starter culture based on a combination of lactic acid bacteria (LAB) and fungal strains with a demonstrated proteolytic activity such as P. chrysogenum and D. hansenii (lot 2) did not affect the main characteristics of dry-cured ham processing, even enhancing some desirable aspects, like its non-protein nitrogen contents. LAB strains were not significantly affected by combining them with fungal starter, and better counts were found with respect to control. A higher thiobarbituric acid reactive substances content was described in lot inoculated only with LAB (lot 3). Potentially pathogenic microorganisms were not detected in any of the lots studied. The starter culture used in lot 2 showed a potential interest for use in dry-cured ham production.

Target identification of volatile metabolites to allow the differentiation of lactic acid bacteria by gas chromatography-ion mobility spectrometry.

The purpose of this work was to study the potential of gas chromatography-ion mobility spectrometry (GC-IMS) to differentiate lactic acid bacteria (LAB) through target identification and fingerprints of volatile metabolites. The LAB selected were used as reference strains for their influence in the flavour of cheese. The four strains of LAB can be distinguished by the fingerprints generated by the volatile organic compounds (VOCs) emitted. 2-butanone, 2-pentanone, 2-heptanone and 3-methyl-1-butanol were identified as relevant VOCs for Lactobacillus casei and Lactobacillus paracasei subsp. paracasei. 2-Butanone and 3-methyl-1-butanol were identified in Lactococcus lactis subsp. lactis and Lactococcus cremoris subsp. cremoris. The IMS signals monitoring during a 24-30h period showed the growth of the LAB in vitro. The results demonstrated that GC-IMS is a useful technology for bacteria recognition and also for screening the aromatic potential of new isolates of LAB.

Differentiation of toxigenic Staphylococcus aureus in staphylococcal isolates from prepared and frozen foods by combined arbitrarily primed polymerase chain reaction and DNA probe.

In prepared and frozen flamenquín and hake fish fingers Staphylococcus aureus as sanitary hazards have been detected. In the present work, a combined method that includes an arbitrarily primed PCR (AP-PCR) and a mixed DNA probe hybridisation designed for the enterotoxigenic genes sea, seb, sec, and sed will be assayed to differentiate enterotoxigenic S. aureus from other staphylococcal species isolated during the processing of prepared and frozen foods. From the protocols tested for the AP-PCR, the highest number of amplification bands showing the best resolution was achieved at 30 degrees C annealing and 35 degrees C extension temperatures. Several staphylococci identified by a biochemical test as S. aureus showed in the AP-PCR analysis different banding patterns to the references S. aureus. The isolates, were investigated by slot blot hybridisation for genes encoding A, B, C, and D staphylococcal enterotoxins to determine their enterotoxigenic potential. Several isolates characterised by the AP-PCR analysis as S. aureus hybridised with the DNA probe mixture. The combined AP-PCR and DNA probe hybridisation assayed was able to differentiate toxigenic S. aureus from other staphylococcal species from prepared and frozen foods. This method could be considered as microbial quality assurance in these products.

Influence of modified atmosphere packaging on the shelf life of prebaked pizza dough with and without preservative added.

The possible effect of different modified atmospheres on the shelf life of prebaked pizza dough, with and without added calcium propionate, was investigated. Three packaging atmospheres were tested: 20% CO2: 80% N2, 50% CO2: 50% N2, 100% CO2, and air (control). Samples were examined daily for visible mold growth and analysed after 2, 8, 17 and 31 days throughout storage (15-20 degrees C and 54-65% relative humidity, RH) for changes in gaseous composition, pH and microbial populations (mesophilic aerobic and anaerobic bacteria, lactic acid bacteria (LAB), and yeasts and molds). Microbiological results showed that molds had a greater sensitivity to CO2 than bacteria and yeasts. Products containing calcium propionate did not show mold growth throughout storage (31 days) when packaged in air or in CO2-enriched atmospheres (20, 50 and 100%). However, in pizza dough without preservative (calcium propionate), mold growth was evident after 7 days, except under 100% CO2 atmosphere (13 days) regardless of the packaging atmosphere. From these results we conclude that the addition of calcium propionate had more and decisive influence on the shelf life extension of prebaked pizza dough.