Exploring the microbial dynamics of an artisanal cheese-making process

Exploring the microbial dynamics of an

Italian artisanal cheese-making process

1

_{Department of Veterinary Science, Gugliasco (TO) Italy}

2

_{Department of Agricultural, Forest and Food science, Grugliasco (TO) Italy}

3

_{Teagasc Food Research Centre, Moorepark, Fermoy, Cork, Ireland}

INTRODUCTION

The metagenomic results highlighted how L. lactis was the prevalent LAB followed by L. mesenteroides, and at

lower numbers, strains of the

Lactobacillus and Enterococcus genera

were detected.

Fig. 1 Average-linkage clustering based on the Spearman distance of milk (green bars), NMSs (brown bars) curds (red bars), 5 days (blue bars) and 15 days cheeses (magenta bars). Rows and columns are clustered by Ward linkage hierarchical clustering.

The 85 L. lactis strains

isolated represented 6

different pulsotypes,

showing intra-species

diversity within the analysed strains that may determine

different phenotypic

characteristics and

technological traits.

L. lactis sp. lactis showed

high pulsotype diversity (5 pulsotypes) compared to L.

lactis sp. cremoris (1 pulsotype).

Fig. 2 Dendogram of pulsed-field gel electrophoresis (PFGE) fingerprints generated by Bionumerics 7,5 software using UPGMA distance matrix method and Pearson correlation.

Isolates Genotype Phenotype

  40°C 4% NaCl Arginine 48 L. lactis subsp.  cremoris + + + 36 L. lactis subsp. lactis + + + 1 L. lactis biovar  diacetylactis + + + L. lactis HP + + + L. cremoris DRC3 - -

-All the strains were able to grow in milk with some exhibiting fast acidification

profiles. The 3 faster strains were isolated from NMS and fresh cheese.

9 isolates from milk showed inhibition zone against L. lactis HP and mass spectrometry identified Lacticin 481 which has a role in L. monocytogenes control.

None of the strains were susceptible to phages c2, 712 and Tuc2009, suggested perhaps the presence of phage resistance mechanisms.

Federica Biolcat

1

_{, Ilario Ferrocino}

2

_{, Maria Teresa Bottero}

1

_{, Alessandra Dalmasso}

1

_{and Olivia McAuliffe}

3

Phenotypic characterization:

• Ability to degrade Arginine, grow in presence of 4% NaCl and at 40°C

• KCA agar test to differentiate L.

lactis biovar diacetylactis

• Technological analysis:

• Ability to grow and acidify milk • Phage robustness

• Anti-microbial production

MATERIALS & METHODS

Metagenomic approach:

• V3-V4 region of 16S rRNA was

sequenced to determine the

microbiota in milk, NMS, curd and cheese from Robiola di Roccaverano production

Robiola di Roccaverano is an artisanal PDO cheese from Piedmont (North West Italy). During its manufacture, natural milk starter (NMS)

prback-slopping technique is added to raw goat’s milk to start the fermentation process. In this study, we examined the microbial diversity in the Robiola di Roccaverano production process, from raw milk to ripened cheese. We also determine the genotypic and phenotypic characteristics of wild Lactococcus

lactis strains isolated, strains that were prevalent in the process, as interest in the application of wild dairy strains of L. lactis is growing in recent years1.

RESULTS & DISCUSSION

The table below reports the result of

species-specific PCR and phenotypic characterization. It is interesting to

note how phenotype and genotype do not correlate.

L. cremoris phenotype lactis was frequently isolated from various nondairy niche suggesting that L. cremoris isolated from this artisanal production were more similar to nondairy strains 2.

Genotypic characterization:

• Pulsed-Field Gel Electrophoresis

(PFGE) was applied to 85 L. lactis

strains isolated and a phylogenetic

analysis was performed with

Bionumerics 7.5

• Species-specific PCR to determine the two L. lactis subps. lactis and subsp. cremoris

Corresponding author:

Federica Biolcati

Dipartimento di Scienze Veterinarie, Largo Braccini 2, Grugliasco (TO) federica.biolcati@unito.it

CONCLUSION

t0 t1 t2 t3 t4 t5 t6 t24h 4 4.5 5 5.5 6 6.5 7 mrs 72.1 m17 34.1 mrs 45.2 mrs 76.5 m17 8b.2 m17 10.1 m17 12.1 milk Time p H

The results showed high biodiversity in L. lactis strains isolated and promising technological proprieties. The interest of dairy industry in new strains with potential application must been investigated in unexplored artisanal production. In fact, indigenous strains could be potential tools to guarantee high fermentation yield and desired final product with specific organoleptic features.

References:

1. Leroy F and De Vuyst L. Lactic acid bacteria as functional starter cultures for the food fermentation industry. Trends Food Sci Technol 2004; 15: 67-78.

2. Cavanagh D et al. Evaluation of Lactococcus lactis isolates from nondairy sources with potential dairy applications reveals extensive phenotype-genotype disparity and implications for revised species. Appl Environm Microbial 2015; 81: 3961-3972