1. Table 2a, 2b, 2c, 2d. Detection of alive cells of L. lactis throughout manufacturing and ripening of miniature cheeses: comparison of the results obtained by culture-independent and -dependent approaches. Table 2b. Table 2c. Table 2d. XIX WORKSHOP ON THE DEVELOPMENTS IN THE ITALIAN PHD RESEARCH ON FOOD SCIENCE TECHNOLOGY AND BIOTECHNOLOGY Bari, September 24 th -26 th, 2014 Detection and vitality of Lactococcus lactis in cheese ripening Marianna Ruggirello (marianna.ruggirello@unito.it) DISAFA - Dept. of Agricultural, Forest and Food Sciences, University of Turin, Italy Tutor: Dott.ssa Paola Dolci The objective of my PhD project is to assess the presence of metabolically active populations of Lactococcus lactis in ripened cheeses and to understand the possible contribute of this microorganism in the development of unique chemical and sensory characteristics of the final product. ACTIVITIES and MATHERIALS AND METHODS : A1) Optimization of qPCR protocols for L. lactis detection and viability determination: the housekeeping gene tuf (Ulve et al. 2008) was chosen as target and the primers Tuf2f and Tuf2r were used on both target (L. lactis subsp. lactis and L. lactis subsp. cremoris) and non-target lactic acid bacteria commonly found in dairy products. Different primer concentrations and annealing temperatures were tested in qPCR and RT-qPCR experiments to determine the most selective and efficient amplification conditions. Standard curves were constructed by plotting the Ct values against the colony forming units (CFU)/mL and CFU/g, respectively, of the pure culture and the grated cheese inoculated with L. lactis subsp. lactis, evaluated by traditional plating on M17 agar. Correlation coefficients (R 2 ) and the efficiency (E) parameters were calculated according to Rutledge & Coté (2003). A2) Application of the qPCR protocols for the analysis of ripened commercial cheeses to detect metabolically active populations of L. lactis: thirty-three ripened commercial cheeses were analyzed by both qPCR and RT-qPCR and traditional plating. The identification of L. lactis isolates obtained on M17 plates was carried out by L. lactis specific PCR (His-PCR)(Corroler et al. 1998). Culture-independent and –dependent results were compared. A3) Manufacturing and ripening of miniature cheeses to follow L. lactis persistence: miniature cheeses produced according to the protocol described by Shakeel-Ur-Rehman et al. (1998) were inoculated with 8 L. lactis commercial starters, ripened at 8°C vacuum for 180 days and analyzed by both qPCR and RT-qPCR and traditional plating. The plates obtained from the cheese sample 10 -1 dilution were used for bulk formation (Ercolini et al., 2001). One millilitre of bulk cell suspension was collected and submitted to DNA extraction and qPCR for the detection of L. lactis. RESULTS: A1) A2) A3) Amplification conditions Amplification Cycle SsoAdvanced TM SYBR@ Green Supermix 1X (Biorad) 98°C 2' Tuf2 f 0,4 μM95°C 5'' 40 cycles Tuf2 r 0,05 μM68.7°C 30'' Fig. 1: qPCR and RT-qPCR protocols. Figure 2. Standard curves for L. lactis inoculated in cheese matrix [DNA (A) and RNA (B)] and L. lactis pure culture [DNA (C) and RNA (D)]. The fluorescence signal was detected for the subspecies L. lactis subsp. lactis and subsp. cremoris at Ct values of 14.37 (  0.12) and 15.69 (  0.31), respectively; the other lactic acid bacteria species were not amplified within the 40 cycles set in the amplification protocol. The melting curve showed a single peak confirming the high specificity of the protocol optimized. Sample RNA (CFU/g)* Plate counts Log CFU/g HIS ** Toma di capra8,478,590 Toma piemontese7,688,951 Pecorino Toscano PDO7,586,510 Pecorino fioretto7,487,797 Toma stagionata7,407,450 Asiago PDO7,136,942 Toma di Lanzo7,068,150 Toma di Lanzo7,038,600 Pecorino di Gallura6,979,480 Pecorino Fiore Sardo6,297,570 Castelmagno PDO6,165,400 Fontina d’ Aosta PDO6,118,590 Pecorino romano5,566,880 Fontina d’Aosta PDO5,507,330 Fontina d’ Aosta PDO5,398,360 Castelmagno5,346,581 Raschera PDO5,248,010 Fontal5,055,480 Pecorino sardo PDO4,967,760 Toma di Lanzo4,828,926 Toma di Lanzo4,558,880 Fontina d'Aosta PDO3,826,861 Fontina PDO3,786,380 Raschera PDO3,766,400 Toma piemontese PDO3,728,383 Asiago d'Allevo0,008,320 Bra tenero PDO0,008,410 Pecorino pastore0,005,700 Raschera0,008,230 Raschera PDO0,009,520 Toma di Bra0,007,770 Toma d'Oropa0,009,163 Toma Piemontese0,009,190 Table 1. Detection of alive cells of L. lactis in ripened commercial cheeses: comparison of the results obtained by culture-independent and -dependent approach. Selected References Corroler, D, I Mangin, N Desmasures, and M Gueguen. 1998. “An Ecological Study of Lactococci Isolated from Raw Milk in the Camembert Cheese Registered Designation of Origin Area.” Applied and Environmental Microbiology 64 (12): 4729–35. Ercolini D, Moschetti G, Blaiotta G, Coppola S (2001) The Potential of a Polyphasic PCR-DGGE Approach in Evaluating Microbial Diversity of Natural Whey Cultures for Water-Buffalo Mozzarella Cheese Production: Bias of Culture-Dependent and Culture-Independent Analyses. System. Appl. Microbiol. 24, 610–617 Ganesan B, Stuart MR, Weimer BC (2007) Carbohydrate starvation causes a metabolically active but nonculturable state in Lactococcus lactis. Appl Environ Microbiol 73: 2498–2512. Shakeel-Ur-Rehman, McSweeney P.I.H., Fox P.F. 1998. "Protocol for the manufacture of miniature cheeses". Lait 78 (6): 607-620. Ulve, V M, C Monnet, F Valence, J Fauquant, H Falentin, and S Lortal. 2008. “RNA Extraction from Cheese for Analysis of in Situ Gene Expression of Lactococcus Lactis.” Journal of Applied Microbiology 105 (5): 1327–33. *The values, expressed as microbial loads, have been extrapolated from standard curves. ** Number of isolates found belonging to L. lactis species on a total of 12 colonies. startersYB Step RNA analysis (CFU/g)* Plate counts Log CFU/g BULK** HIS- PCR *** RNA (CFU/g) Plate counts Log CFU/g BULKHIS-PCR Manufacturing milk 3,877,60+106,998,01+ 10 curd 4,988,59+107,878,82+ 10 dry 5,349,41+107,999,46+ 10 salt 6,7010,60+108,249,69+ 10 Ripening 7d 7,008,46+107,279,18+ 10 15d 6,988,45+106,339,02+ 10 30d 6,787,78+104,968,75+ 7 60d 4,557,00+94,818,66+ 5 90d 3,077,41+94,818,48+ 5 120d 2,395,78+94,758,81+ 0 150d 1,443,90+84,677,90- 0 180d 1,404,08-03,817,76- 0 startersJC StepRNA (CFU/g) Plate counts Log CFU/g BULKHIS-PCRRNA (CFU/g) Plate counts Log CFU/g BULKHIS-PCR Manufacturing milk 5,918,06+ 10 6,147,85+10 curd 6,999,38+ 10 6,378,66+10 dry 7,129,78+ 10 6,529,51+10 salt 6,409,95+ 10 7,009,78+10 Ripening 7d 5,999,29+ 10 7,089,43+10 15d 5,709,04+ 10 7,879,47+10 30d 5,657,70+ 8 8,158,93+10 60d 5,426,30+ 8 6,787,57+10 90d 4,994,48+ 6 5,375,85+7 120d 4,58<1000+ 0 2,527,51+4 150d 3,414,15+ 0 2,317,00+0 180d 3,385,95- 0 1,714,30+0 StartersKW StepRNA (CFU/g) Plate counts Log CFU/g BULKHIS-PCRRNA (CFU/g) Plate counts Log CFU/g BULKHIS-PCR Manufacturing milk 3,277,70+10 3,17 8,40+10 curd 4,309,27+10 4,29 8,93+10 dry 6,809,61+10 4,57 9,46+10 salt 6,919,52+10 4,60 9,77+10 Ripening 7d 7,059,20+10 4,46 9,52+10 15d 7,198,26+10 4,42 8,41+10 30d 4,098,45+8 4,35 8,28+10 60d 3,268,15+6 3,90 7,95+10 90d 2,767,48+6 3,78 6,00+10 120d 2,718,25-0 3,54 5,00+10 150d 2,638,68-0 3,41 4,90+10 180d 2,608,20-0 2,55 2,90+10 startersMX Step RNA (CFU/g) Plate counts Log CFU/g BULKHIS-PCRRNA (CFU/g) Plate counts Log CFU/g BULKHIS-PCR Manufacturing milk 3,208,57+106,136,70+ 10 curd 4,458,99+107,247,90+ 10 dry 8,179,45+107,769,13+ 10 salt 8,239,43+106,408,72+ 10 Ripening 7d 8,009,33+105,288,18+ 10 15d 7,998,48+103,928,00+ 10 30d 7,547,66+103,337,63+ 10 60d 7,417,49+103,277,00+ 10 90d 6,988,51+103,257,56+ 10 120d 6,048,60+102,734,90+ 10 150d 4,6467,20+92,653,95+ 10 180d 2,808,40+22,442,36+ 10 *The values, expressed as microbial loads, have been extrapolated from standard curves. ** “+”: L. lactis was detected; “-”: L. lactis was not detected. *** Number of isolates found belonging to L. lactis species on a total of 10 colonies. On the basis of RNA analysis, L. lactis was found with loads up to 10 4 CFU/g until the sixth month of ripening. Conversely, in a few late ripened cheese samples, L. lactis was not found on M17 plates. These preliminary data could support the thesis hypothesized by other authors (Ganesan et al., 2007) that L. lactis starter populations are mainly present in viable but not culturable state during cheese ripening and, for this reason, culture-dependent methods are not able to detect their presence and have to be complemented by direct analysis. Twenty-five samples, on a total of thirty-three, resulted positive for the presence of metabolically active cells of L. lactis, as detected by RT-qPCR. The L. lactis loads ranged from 10 3 to 10 8 CFU/g for RNA analysis. The highest values were obtained for some of the Toma, Pecorino and Asiago cheese type samples. On the contrary, eight samples belonging to Asiago, Bra, Pecorino, Raschera and Toma cheese type were negative for L. lactis. The total viable counts on M17 medium ranged from 10 5 to 10 9 CFU/g but only 24 isolates, spread among eight cheese samples, resulted to belong to L. lactis species. The absence or low abundance of L. lactis isolates on M17 could be interpreted as a lack of selectivity of the medium where colony growth is not always related to lactococcal species.