Metabolic Gene-Targeted Monitoring of Non-Starter Lactic Acid Bacteria during Cheese Ripening
Abstract
Long ripened cheeses, such as Grana Padano (GP), a Protected Designation of Origin (PDO) Italian cheese, harbor a viable microbiota mainly composed of non-starter lactic acid bacteria (NSLAB), which contribute to the final characteristics of cheese. The NSLAB species Lactobacillus rhamnosus, Lb. casei and Lb. paracasei are frequently found in GP, and form a closely related taxonomic group (Lb. casei group), making it difficult to distinguish the three species through 16S rRNA sequencing. SpxB, a metabolic gene coding for pyruvate oxidase in Lb. casei group, was recently used to distinguish the species within this bacterial group, both in pure cultures and in cheese, where it could provide an alternative energy source through the conversion of pyruvate to acetate. The aim of this work was to study the evolution of the metabolically active microbiota during different stages of GP ripening, targeting 16S rRNA to describe the whole microbiota composition, and spxB gene to monitor the biodiversity within the Lb. casei group. Furthermore, activation of pyruvate oxidase pathway was measured directly in cheese by reverse transcription real time PCR (RT-qPCR). The results showed that Lb. casei group dominates throughout the ripening and high-throughput sequencing of spxB allowed to identify four clusters inside the Lb. casei group. The dynamics of the sequence types forming the clusters were followed during ripening. Pyruvate oxidase pathway was expressed in cheese, showing a decreasing trend over ripening time. This work highlights how the composition of the microbiota in the early manufacturing stages influences the microbial dynamics throughout ripening, and how targeting of a metabolic gene can provide an insight into the activity of strains relevant for dairy products.
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Studies on long-ripened cheeses have shown that the counts of starter LAB (SLAB) can remain high for several months during cheese ripening, although different species and biotypes may succeed over time.
“Studies on long-ripened cheeses have shown that the counts of starter LAB (SLAB) can remain high for several months during cheese ripening, although different species and biotypes may succeed over time [73,88,89].”
From Microbiota of Cheese Ecosystems: A Perspective on Cheesemaking (2025) · cites this paper as [88]
This large population of bacteria represents a significant reservoir of enzymatic activity because their breakdown and the release of intracellular enzymes are crucial factors in cheese ripening.
“This large population of bacteria represents a significant reservoir of enzymatic activity because their breakdown and the release of intracellular enzymes are crucial factors in cheese ripening [18,69,73,88,90,91,92,93,94,95,96,97,98].”
From Microbiota of Cheese Ecosystems: A Perspective on Cheesemaking (2025) · cites this paper as [88]
In the 6-month-old samples, other minority species, such as L. fermentum, L. crispatus, S. thermophilus and Lc. lactis, were also present.
“In the 6-month-old samples, other minority species, such as L. fermentum, L. crispatus, S. thermophilus and Lc. lactis, were also present, as observed in other long-ripened, raw milk cheeses manufactured with similar technology (Alessandria et al., 2016; Levante et al., 2017).”
From The Interrelationship Between Microbiota and Peptides During Ripening as a Driver for Parmigiano Reggiano Cheese Quality (2020) · cites this paper as Levante et al., 2017
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- Authors
- Levante, A., De Filippis, F., La Storia, A., Gatti, M., Neviani, E., Ercolini, D., & Lazzi, C.
- Affiliations
- University of Parma (IT)
- University of Naples Federico II (IT)
- Corresponding author
- Levante, Alessia
- ORCID
- Venue
- International Journal of Food Microbiology
- Publisher
- Elsevier BV
- Published
- 2017-09
- Volume
- 257
- Pages
- 276-284
- ISSN
- 0168-1605 (ISSN-L)
- OpenAlex
- W2731573909
- Cited by
- 35
- References
- 50
- Citations by year
- 2026: 2 · 2025: 3 · 2024: 1 · 2023: 4 · 2022: 4 · 2021: 4 · 2020: 6 · 2019: 8 · 2018: 3
- FWCI
- 5.23
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- International Journal of Food Microbiology (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Consorzio per la Tutela del Formaggio Grana Padano
- Topics
- Life Sciences › Agricultural and Biological Sciences › Food Science › Probiotics and Fermented Foods
- Life Sciences › Agricultural and Biological Sciences › Food Science › Fermentation and Sensory Analysis
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gut microbiota and health
- Keywords
- non-starter lactic acid bacteria, cheese ripening, Grana Padano, Lactobacillus casei group, pyruvate oxidase, cheese microbiota, Lactobacillus rhamnosus, Lactobacillus paracasei, species discrimination, microbial dynamics
- MeSH terms
- Lacticaseibacillus paracasei, Base Sequence, Cheese, Italy, Lacticaseibacillus casei, Pyruvate Oxidase, RNA, Ribosomal, 16S, Sequence Analysis, DNA, Biodiversity, Lacticaseibacillus rhamnosus, High-Throughput Nucleotide Sequencing, Real-Time Polymerase Chain Reaction
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