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Lactococcus lactis Diversity Revealed by Targeted Amplicon Sequencing of purR Gene, Metabolic Comparisons and Antimicrobial Properties in an Undefined Mixed Starter Culture Used for Soft-Cheese Manufacture.

ABSTRACT: The undefined mixed starter culture (UMSC) is used in the manufacture of cheeses. Deciphering UMSC microbial diversity is important to optimize industrial processes. The UMSC was studied using culture-dependent and culture-independent based methods. MALDI-TOF MS enabled identification of species primarily from the Lactococcus genus. Comparisons of carbohydrate metabolism profiles allowed to discriminate five phenotypes of Lactococcus (n = 26/1616). The 16S sequences analysis (V1-V3, V3-V4 regions) clustered the UMSC microbial diversity into two Lactococcus operational taxonomic units (OTUs). These clustering results were improved with the DADA2 algorithm on the housekeeping purR sequences. Five L. lactis variants were detected among the UMSC. The whole-genome sequencing of six isolates allowed for the identification of the lactis subspecies using Illumina® (n = 5) and Pacbio® (n = 1) technologies. Kegg analysis confirmed the L. lactis species-specific niche adaptations and highlighted a progressive gene pseudogenization. Then, agar spot tests and agar well diffusion assays were used to assess UMSC antimicrobial activities. Of note, isolate supernatants (n = 34/1616) were shown to inhibit the growth of Salmonella ser. Typhimurium CIP 104115, Lactobacillus sakei CIP 104494, Staphylococcus aureus DSMZ 13661, Enterococcus faecalis CIP103015 and Listeria innocua CIP 80.11. Collectively, these results provide insightful information about UMSC L. lactis diversity and revealed a potential application as a bio-protective starter culture.

SUBMITTER: Saltaji S

PROVIDER: S-EPMC7278722 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Undefined mesophilic mixed (DL) starter cultures are used in the production of continental cheeses and contain unknown strain mixtures of Lactococcus lactis and leuconostocs. The choice of starter culture affects the taste, aroma, and quality of the final product. To gain insight into the diversity of Lactococcus lactis strains in starter cultures, we whole-genome sequenced 95 isolates from three different starter cultures. Pan-genomic analyses, which included 30 publically available complete genomes, grouped the strains into 21 L. lactis subsp. lactis and 28 L. lactis subsp. cremoris lineages. Only one of the 95 isolates grouped with previously sequenced strains, and the three starter cultures showed no overlap in lineage distributions. The culture diversity was assessed by targeted amplicon sequencing using purR, a core gene, and epsD, present in 93 of the 95 starter culture isolates but absent in most of the reference strains. This enabled an unprecedented discrimination of starter culture Lactococcus lactis and revealed substantial differences between the three starter cultures and compositional shifts during the cultivation of cultures in milk.IMPORTANCE In contemporary cheese production, standardized frozen seed stock starter cultures are used to ensure production stability, reproducibility, and quality control of the product. The dairy industry experiences significant disruptions of cheese production due to phage attacks, and one commonly used countermeasure to phage attack is to employ a starter rotation strategy, in which two or more starters with minimal overlap in phage sensitivity are used alternately. A culture-independent analysis of the lactococcal diversity in complex undefined starter cultures revealed large differences between the three starter cultures and temporal shifts in lactococcal composition during the production of bulk starters. A better understanding of the lactococcal diversity in starter cultures will enable the development of more robust starter cultures and assist in maintaining the efficiency and stability of the production process by ensuring the presence of key bacteria that are important to the characteristics of the product.

Project description:BackgroundCheese ripening is a complex, time consuming and expensive process, which involves the generation of precursors from carbohydrates, proteins and fats and their subsequent conversion into a wide range of compounds responsible for the flavour and texture of the cheese. This study aims to investigate production of cheese aroma compounds outside the cheese matrix that could be applied for instance as food supplements in dairy or non-dairy products.ResultsIn this study, aroma formation by a dairy Lactococcus lactis was analysed as a function of the growth medium [milk, hydrolysed micellar casein isolate (MCI) and chemically defined medium (CDM)] and the cultivation conditions (batch culture, retentostat culture and a milli-cheese model system). In the retentostat cultures, the nutrient supply was severely restricted resulting in low growth rates (~ 0.001 h-1), thereby mimicking cheese ripening conditions in which nutrients are scarce and bacteria hardly grow. In total 82 volatile organic compounds were produced by the bacteria. Despite the use of a chemically defined medium, retentostat cultures had the biggest qualitative overlap in aroma production with the milli-cheese model system (36 out of 54 compounds). In the retentostat cultures, 52 known cheese compounds were produced and several important cheese aroma compounds and/or compounds with a buttery or cheese-like aroma increased in retentostat cultures compared to batch cultures and milli-cheeses, such as esters, methyl ketones, diketones and unsaturated ketones. In cultures on CDM and MCI, free fatty acids and their corresponding degradation products were underrepresented compared to what was found in the milli-cheeses. Addition of a mixture of free fatty acids to CDM and MCI could help to enhance flavour formation in these media, thereby even better resembling flavour formation in cheese.ConclusionsThis study demonstrates that retentostat cultivation is the preferred method to produce cheese flavours outside the cheese matrix by mimicking the slow growth of bacteria during cheese ripening.

Dataset Information

Lactococcus lactis Diversity Revealed by Targeted Amplicon Sequencing of purR Gene, Metabolic Comparisons and Antimicrobial Properties in an Undefined Mixed Starter Culture Used for Soft-Cheese Manufacture.

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