Combined Phenotypic-Genotypic Analyses of the Genus Lactobacillus and Selection of Cultures for Biopreservation of Fermented Food

Abstract

Food waste is an economic and ethical issue and can be reduced for fermented food products with a biopreservation approach to increase shelf-life and avoid the outgrowth of spoilage microorganisms. Thereby protective cultures are applied to produce antimicrobial compounds which inhibit these spoilage organisms. Lactobacillus species occur in various fermented food products and many strains were detected to supply antimicrobial activity against Listeria and other Gram-positive bacteria and a broad spectrum of fungi. Nevertheless, screening procedures and applications in individual foods is not optimized yet. The aim of this thesis was to develop an approach to screen and select tailor-made protective cultures from a strain collection to implement protective cultures in industrial-scale food fermentations and to understand mechanisms contributing to biopreservation activity to increase food product safety. Therefore, 504 Lactobacillus isolates were screened with a novel developed high-throughput antibacterial and antifungal screening approach (Chapter 2). This novel approach is based on microtiter plates and allows to determine 2000 – 5000 antimicrobial interactions per day. A total of 65 antibacterial and 154 antifungal isolates were detected by this novel approach. To better understand antifungal activity in lactobacilli, the complete genome of the salami isolate Lactobacillus plantarum RI-113 was determined using single-molecule real time sequencing (Chapter 3). The strain showed antifungal activity against Trichosporon spp. and Rhodotorula mucilaginosa LME. Additionally, the genomes of 43 Lactobacillus strains belonging to the species L. curvatus, L. fermentum, L. paracasei, L. plantarum, L. rhamnosus, and L. sakei were determined using Illumina MiSeq (Chapter 4). These strains were selected in a phenotypic screening and exhibited an uncommon or unique physiological properties or were regarded as candidate protective cultures. The phenotype of the 504 Lactobacillus isolates was further characterized by a screening for growth limits in modified MRS to mimic conditions in food matrices (Chapter 5). Antibacterial activity based on proteinaceous compounds was determined for 22 isolates with protease digestion of heat-treated supernatant. The in silico screening with the BAGEL3 and antiSMASH3.0 analysis tools proposed potential genes encoding bacteriocins suppressing bacterial growth. Antifungal activity of tested lactobacilli in MRS was determined to be based on organic acid concentration up to 210 mM lactic acid and 78 mM acetic acid. The power of antifungal activity was demonstrated for L. plantarum RI-162. A minimal amount of 1-2 cfu/ml of this strain was able to decrease 5 x 105 cfu/ml Rhodotorula mucilaginosa LME below the detection limit of 100 cfu/ml in a 1-ml co-culture assay within 48 hours. Based on the combination of phenotypic and genotypic screening potential protective cultures for salami and raw milk cheese fermentations were selected. In a small-scale salami fermentation 1 Lactobacillus sakei and 5 Lactobacillus plantarum strains were tested in a laboratory environment. 4 of these 6 cultures reduced the initial Listeria ivanovii DSM 12491T concentration of 105 cfu/g below the detection limit of 100 cfu/g at 18 - 24 °C within 5 days of incubation and all 6 cultures lowered the pH below 5.0 within 2 days. In an industrial-scale salami fermentation, the protective potential of L. sakei RI-409 was tested in a working environment. Strain RI-409 was able to reduce in situ the initial concentration of 6.33 x 105 cfu/g of L. ivanovii DSM 12491T concentration by 96% to 4.33 x 104 cfu/g within incubation for 5 days. In the absence of any starter and the protective culture, the L. ivanovii DSM 12491T concentration increased in the same time to 6.33 x 106 cfu/g. In another approach, we tested Lactobacillus strains to reduce undesired enterococci proliferating in dairy fermentations. In a 1000-L raw milk soft cheese industrial-scale fermentation, L. plantarum strain RI-271 at initial concentrations of 105 cfu/ml in raw milk decreased resident enterococci in the final cheese product by 96% to 2.20 x 105 cfu/g compared to the non-treated cheese reaching up to 6.50 x 106 cfu/g enterococci after 8 days of ripening. Based on our genome sequencing data, the genomic variation within 5 lactobacilli species and the genus Lactobacillus was investigated (Chapter 6). The core- and pan-genome of 98 completely sequenced genomes of the genus Lactobacillus and of 234 whole genome datasets from the Lactobacillus species L. delbrueckii, L. helveticus, L. reuterin, L. rhamnosus and L. plantarum were calculated. The core-genome of the Lactobacillus genus contained 266 genes and the non-closed pan-genome 20’800 genes. The core-genome of the 5 Lactobacillus species ranged between 756 and 1037 genes and the pan-genomes between 3350 and 7610 genes. The heterogeneity of L. plantarum was visible in the genomic variation since it is the only species with a non-closed pan-genome. Clustering according to core- and pan-genome showed similar phylogenetic trees with species clustering together in general. Outliers were analyzed in detail. We revealed that L. casei type strain ATCC 393 (DSM 20011T) clustered next to L. zeae DSM 20178 instead next to the other L. casei and L. paracasei strains, which confirmed other studies defining the phylogenetic outlier position of the L. casei type strain by using phenotypic approaches. Analysis of the genetic functions of the core gene revealed that genes involved in “genetic information processing” are conserved in the core-genome, whereas genes involved in “signaling and cellular processes” are not conserved in the core-genome. Twenty genomes of the type-species Lactobacillus delbrueckii clustered according to the core-genome in three major clades including one clade solely for the subspecies Lactobacillus delbrueckii subsp. bulgaricus and two other mixed subspecies clades. No clade specific ecological single nucleotide polymorphisms (ecoSNPs) were detected. A total of 57 genes affected by horizontal exchange were found in L. delbrueckii clades. We illustrated an approach to implement whole genome sequencing data into a polyphasic approach to classify bacteria. Conclusively, a new approach was established to select protective cultures for fermentation processes to inhibit spoilage outgrowth by biopreservation including a high-throughput antimicrobial screening approach, a phenotypic growth limit screening assay to mimic culture conditions and selection of strains which can grow in specific food matrices and a genotypic screening to detect bacteriocin encoding genes. Based on whole genome analysis the strains can be further characterized and classified and their exchange of genetic material with other strains can be detected. Our approach, successfully applied with Lactobacillus strains, can now be extended to other genera in order to assign tailor-made protective strains for industrial food fermentations to increase food safety.