Detection and vitality of Lactococcus lactis in cheese ripening

(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 μM

95°C 5''

40 cycles

Tuf2 r 0,05 μM

68.7°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

_(CFU/g)*

RNA

Plate counts

_{Log CFU/g}

HIS

_**

Toma di capra

8,47

8,59

0 Toma piemontese

7,68

8,95

1 Pecorino Toscano PDO

7,58

6,51

0 Pecorino fioretto

7,48

7,79

7 Toma stagionata

7,40

7,45

0 Asiago PDO

7,13

6,94

2 Toma di Lanzo

7,06

8,15

0 Toma di Lanzo

7,03

8,60

0 Pecorino di Gallura

6,97

9,48

0 Pecorino Fiore Sardo

6,29

7,57

0 Castelmagno PDO

6,16

5,40

0 Fontina d’ Aosta PDO

6,11

8,59

0 Pecorino romano

5,56

6,88

0 Fontina d’Aosta PDO

5,50

7,33

0 Fontina d’ Aosta PDO

5,39

8,36

0 Castelmagno

5,34

6,58

1 Raschera PDO

5,24

8,01

0 Fontal

5,05

5,48

0 Pecorino sardo PDO

4,96

7,76

0 Toma di Lanzo

4,82

8,92

6 Toma di Lanzo

4,55

8,88

0 Fontina d'Aosta PDO

3,82

6,86

1 Fontina PDO

3,78

6,38

0 Raschera PDO

3,76

6,40

0 Toma piemontese PDO

3,72

8,38

3 Asiago d'Allevo

0,00

8,32

0 Bra tenero PDO

0,00

8,41

0 Pecorino pastore

0,00

5,70

0 Raschera

0,00

8,23

0 Raschera PDO

0,00

9,52

0 Toma di Bra

0,00

7,77

0 Toma d'Oropa

0,00

9,16

3 Toma Piemontese

0,00

9,19

0 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.

starters Y B

Step RNA analysis _(CFU/g)* Plate counts_{Log CFU/g} BULK** HIS-PCR

*** RNA (CFU/g)

Plate counts

Log CFU/g BULK HIS-PCR

Manufactur ing milk _3,87 _7,60 ₊ ₁₀ _6,99 _8,01 ₊ 10 curd _4,98 _8,59 ₊ ₁₀ _7,87 _8,82 ₊ 10 dry _5,34 _9,41 ₊ ₁₀ _7,99 _9,46 ₊ 10 salt _6,70 _10,60 ₊ ₁₀ _8,24 _9,69 ₊ 10 Ripening 7d _7,00 _8,46 ₊ ₁₀ _7,27 _9,18 ₊ 10 15d _6,98 _8,45 ₊ ₁₀ _6,33 _9,02 ₊ 10 30d _6,78 _7,78 ₊ ₁₀ _4,96 _8,75 ₊ 7 60d _4,55 _7,00 ₊ ₉ _4,81 _8,66 ₊ 5 90d _3,07 _7,41 ₊ ₉ _4,81 _8,48 ₊ 5 120d _2,39 _5,78 ₊ ₉ _4,75 _8,81 ₊ 0 150d _1,44 _3,90 ₊ ₈ _4,67 _7,90 _- 0 180d _1,40 _4,08 _- ₀ _3,81 _7,76 _- 0 starters J C

Step RNA (CFU/g) Plate counts_{Log CFU/g} BULK HIS-PCR RNA (CFU/g) Plate counts_{Log CFU/g} BULK HIS-PCR

Manufactur ing milk _5,91 _8,06 ₊ 10 _6,14 _7,85 ₊ ₁₀ curd _6,99 _9,38 ₊ 10 _6,37 _8,66 ₊ ₁₀ dry _7,12 _9,78 ₊ 10 _6,52 _9,51 ₊ ₁₀ salt _6,40 _9,95 ₊ 10 _7,00 _9,78 ₊ ₁₀ Ripening 7d _5,99 _9,29 ₊ 10 _7,08 _9,43 ₊ ₁₀ 15d _5,70 _9,04 ₊ 10 _7,87 _9,47 ₊ ₁₀ 30d _5,65 _7,70 ₊ 8 _8,15 _8,93 ₊ ₁₀ 60d _5,42 _6,30 ₊ 8 _6,78 _7,57 ₊ ₁₀ 90d _4,99 _4,48 ₊ 6 _5,37 _5,85 ₊ ₇ 120d _4,58 _<1000 ₊ 0 _2,52 _7,51 ₊ ₄ 150d _3,41 _4,15 ₊ 0 _2,31 _7,00 ₊ ₀ 180d _3,38 _5,95 _- 0 _1,71 _4,30 ₊ ₀ Starters K W

Step RNA (CFU/g) _{Plate counts}

Log CFU/g

BULK HIS-PCR RNA (CFU/g) Plate counts_{Log CFU/g} BULK HIS-PCR

Manufactur ing milk _3,27 _7,70 ₊ ₁₀ 3,17 _8,40 ₊ ₁₀ curd _4,30 _9,27 ₊ ₁₀ 4,29 _8,93 ₊ ₁₀ dry _6,80 _9,61 ₊ ₁₀ 4,57 _9,46 ₊ ₁₀ salt _6,91 _9,52 ₊ ₁₀ 4,60 _9,77 ₊ ₁₀ Ripening 7d _7,05 _9,20 ₊ ₁₀ 4,46 _9,52 ₊ ₁₀ 15d _7,19 _8,26 ₊ ₁₀ 4,42 _8,41 ₊ ₁₀ 30d _4,09 _8,45 ₊ ₈ 4,35 _8,28 ₊ ₁₀ 60d _3,26 _8,15 ₊ ₆ 3,90 _7,95 ₊ ₁₀ 90d _2,76 _7,48 ₊ ₆ 3,78 _6,00 ₊ ₁₀ 120d _2,71 _8,25 _- ₀ 3,54 _5,00 ₊ ₁₀ 150d _2,63 _8,68 _- ₀ 3,41 _4,90 ₊ ₁₀ 180d _2,60 _8,20 _- ₀ 2,55 _2,90 ₊ ₁₀ starters M X

Step _(CFU/g)RNA Plate counts_{Log CFU/g} BULK HIS-PCR RNA (CFU/g) Plate counts_{Log CFU/g} BULK HIS-PCR

Manufactur ing milk _3,20 _8,57 ₊ ₁₀ _6,13 _6,70 ₊ 10 curd _4,45 _8,99 ₊ ₁₀ _7,24 _7,90 ₊ 10 dry _8,17 _9,45 ₊ ₁₀ _7,76 _9,13 ₊ 10 salt _8,23 _9,43 ₊ ₁₀ _6,40 _8,72 ₊ 10 Ripening 7d _8,00 _9,33 ₊ ₁₀ _5,28 _8,18 ₊ 10 15d _7,99 _8,48 ₊ ₁₀ _3,92 _8,00 ₊ 10 30d _7,54 _7,66 ₊ ₁₀ _3,33 _7,63 ₊ 10 60d _7,41 _7,49 ₊ ₁₀ _3,27 _7,00 ₊ 10 90d _6,98 _8,51 ₊ ₁₀ _3,25 _7,56 ₊ 10 120d _6,04 _8,60 ₊ ₁₀ _2,73 _4,90 ₊ 10 150d _4,646 _7,20 ₊ ₉ _2,65 _3,95 ₊ 10 180d _2,80 _8,40 ₊ ₂ _2,44 _2,36 ₊ 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.