Microbial diversity and dynamics throughout manufacturing and ripening of surface ripened semi-hard Danish Danbo cheeses investigated by culture-independent techniques.

Microbial successions on the surface and in the interior of surface ripened semi-hard Danish Danbo cheeses were investigated by culture-dependent and -independent techniques. Culture-independent detection of microorganisms was obtained by denaturing gradient gel electrophoresis (DGGE) and pyrosequencing, using amplicons of 16S and 26S rRNA genes for prokaryotes and eukaryotes, respectively. With minor exceptions, the results from the culture-independent analyses correlated to the culture-dependent plating results. Even though the predominant microorganisms detected with the two culture-independent techniques correlated, a higher number of genera were detected by pyrosequencing compared to DGGE. Additionally, minor parts of the microbiota, i.e. comprising <10.0% of the operational taxonomic units (OTUs), were detected by pyrosequencing, resulting in more detailed information on the microbial succession. As expected, microbial profiles of the surface and the interior of the cheeses diverged. During cheese production pyrosequencing determined Lactococcus as the dominating genus on cheese surfaces, representing on average 94.7%±2.1% of the OTUs. At day 6 Lactococcus spp. declined to 10.0% of the OTUs, whereas Staphylococcus spp. went from 0.0% during cheese production to 75.5% of the OTUs at smearing. During ripening, i.e. from 4 to 18 weeks, Corynebacterium was the dominant genus on the cheese surface (55.1%±9.8% of the OTUs), with Staphylococcus (17.9%±11.2% of the OTUs) and Brevibacterium (10.4%±8.3% of the OTUs) being the second and third most abundant genera. Other detected bacterial genera included Clostridiisalibacter (5.0%±4.0% of the OTUs), as well as Pseudoclavibacter, Alkalibacterium and Marinilactibacillus, which represented <2% of the OTUs. At smearing, yeast counts were low with Debaryomyces being the dominant genus accounting for 46.5% of the OTUs. During ripening the yeast counts increased significantly with Debaryomyces being the predominant genus, on average accounting for 96.7%±4.1% of the OTUs. The interior of the cheeses was dominated by Lactococcus spp. comprising on average 93.9%±7.8% of the OTUs throughout the cheese processing. The microbial dynamics described at genus level in this study add to a comprehensive understanding of the complex microbiota existing especially on surface ripened semi-hard cheeses.

Impact of NaCl reduction in Danish semi-hard Samsoe cheeses on proliferation and autolysis of DL-starter cultures.

Reduction of sodium chloride (NaCl) in cheese manufacturing is a challenge for the dairy industry. NaCl has a profound role on microbial development influencing cheese sensory and technological properties. The purpose of this work was to investigate how proliferation, distribution and autolysis of two commercial DL-starter cultures (C1 and C2) used in the production of Danish semi-hard Samsoe cheeses were affected by reduced NaCl levels. Cheeses containing <0.3% (unsalted), 2.3% (reduced-salt) and 3.4% (normal-salted) (w/v) NaCl in moisture were produced and analyzed during 12 weeks of ripening. Lactic acid bacteria (LAB), distribution of bacteria as single cells or microcolonies, their viability in the cheeses and cell autolysis were monitored during ripening, as well as the impact of NaCl content and autolysis on the formation of free amino acids (FAA). Reduction of NaCl resulted in higher LAB counts at the early stages of ripening, with differences between the two DL-starter cultures. The unsalted cheeses produced with C1 had retained a significantly higher number of the initial LAB counts (cfu/g) after 1 and 2 weeks of ripening (i.e. 58% and 71%), compared to the normal-salted cheeses (i.e. 22% and 21%), whereas no significant difference was found between the reduced-salt (i.e. 31% and 35%) and normal-salted cheeses. At the later stages of ripening (i.e. 7 and 11 weeks) NaCl had no significant influence. For cheeses produced with C2, a significant influence of NaCl was only found in cheeses ripened for 7 weeks, where the unsalted and reduced-salt cheeses had retained a significantly higher number of the initial LAB counts (cfu/g) (i.e. 39% and 38%), compared to the normal-salted cheeses (i.e. 21%). In the Samsoe cheeses, bacteria were organized as single cells, in groups of 2-3 cells or in groups of ≥4 cells. During ripening the decrease in the number of viable bacteria was mainly due to a reduction in the number of viable bacteria organized in groups of ≥4 cells. A negative correlation between NaCl content and PepX activity was observed. At the end of ripening the total FAA content was lower in the unsalted cheeses, compared to the reduced- and normal-salted cheeses. In conclusion, NaCl had a significant influence on proliferation of both DL-starter cultures. However, the influence of NaCl on culture development was more pronounced in cheeses produced with DL-starter culture C1. As both texture and taste are parameters known to be affected by the development of the starter culture, the design of starter cultures for reduced NaCl cheeses is recommended.

Multi-stress resistance in Lactococcus lactis is actually escape from purine-induced stress sensitivity.

Multi-stress resistance is a widely documented and fascinating phenotype of lactococci where single mutations, preferentially in genes involved in nucleotide metabolism and phosphate uptake, result in elevated tolerance to multiple stresses simultaneously. In this report, we have analysed the metabolic basis behind this multi-stress-resistance phenotype in Lactococcus lactis subsp. cremoris MG1363 using acid stress as a model of multi-stress resistance. Surprisingly, we found that L. lactis MG1363 is fully resistant to pH 3.0 in the chemically defined SA medium, contrary to its sensitivity in the rich and complex M17 medium. When salvage of purines and subsequent conversion to GTP was permitted in various genetic backgrounds of L. lactis MG1363, the cells became sensitive to acid stress, indicating that an excess of guanine nucleotides induces stress sensitivity. The addition of phosphate to the acid-stress medium increased the stress sensitivity of L. lactis MG1363. It is also shown that high intracellular guanine nucleotide pools confer increased sensitivity to high temperatures, thus showing that it is indeed a multi-stress phenotype. Our analysis suggests that an increased level of guanine nucleotides is formed as a result of an improved conversion of guanosine in the salvage pathway. Based upon our findings, we suggest that L. lactis MG1363 is naturally multi-stress resistant in habitats devoid of any purine source. However, any exogenous purine that results in increased guanine nucleotide pools renders the bacterium sensitive to environmental stresses.

Effect of the gastrointestinal environment on pH homeostasis of Lactobacillus plantarum and Lactobacillus brevis cells as measured by real-time fluorescence ratio-imaging microscopy.

In the present work, an in vitro model of the gastrointestinal tract (GIT) was developed to obtain real-time observations of the pH homeostasis of single cells of probiotic Lactobacillus spp. strains as a measure of their physiological state. Changes in the intracellular pH (pHi) were determined using fluorescence ratio imaging microscopy (FRIM) for potential probiotic strains of Lactobacillus plantarum UFLA CH3 and Lactobacillus brevis UFLA FFC199. Heterogeneous populations were observed, with pHi values ranging from 6.5 to 7.5, 3.5 to 5.6 and 6.5 to 8.0 or higher during passage of saliva (pH 6.4), gastric (pH 3.5) and intestinal juices (pH 6.4), respectively. When nutrients were added to gastric juice, the isolate L. brevis significantly decreased its pH(i) closer to the extracellular pH (pH(ex)) than in gastric juice without nutrients. This was not the case for L. plantarum. This study is the first to produce an in vitro GIT model enabling real-time monitoring of pH homeostasis of single cells in response to the wide range of pH(ex) of the GIT. Furthermore, it was possible to observe the heterogeneous response of single cells. The technique can be used to determine the survival and physiological conditions of potential probiotics and other microorganisms during passage through the GIT.

In situ examination of cell growth and death of Lactococcus lactis.

This study enables in situ studying of the growth and death of a large number of individual cells in a solid matrix. A wild type of Lactococcus lactis and several mutants with varying expression of GuaB was investigated. Large variability in the final size of individual microcolonies arising from clonal cells was observed. However, when growth was averaged over 16 locations in a specimen, the SEM was small and notable differences could be observed between the investigated strains, where mutants with lower expression of GuaB had a slower growth rate. The results show that the slow-growing mutants exhibited a lower fraction of dead cells, which indicate that slow-growing mutants are slightly more robust than the faster-growing strains. The large variability in the final size of individual microcolonies arising from clonal cells was quite surprising. We suggest that the control of the size of a microcolony is, at least partially, related to the actual microcolony depended on phenotypic heterogeneity. These findings are important to consider whenever a solid medium with discrete microcolonies is investigated.

Isolation and identification of the microbiota of Danish farmhouse and industrially produced surface-ripened cheeses.

For studying the microbiota of four Danish surface-ripened cheeses produced at three farmhouses and one industrial dairy, both a culture-dependent and culture-independent approach were used. After dereplication of the initial set of 433 isolates by (GTG)5-PCR fingerprinting, 217 bacterial and 25 yeast isolates were identified by sequencing of the 16S rRNA gene or the D1/D2 domain of the 26S rRNA gene, respectively. At the end of ripening, the cheese core microbiota of the farmhouse cheeses consisted of the mesophilic lactic acid bacteria (LAB) starter cultures Lactococcus lactis subsp. lactis and Leuconostoc mesenteorides as well as non-starter LAB including different Lactobacillus spp. The cheese from the industrial dairy was almost exclusively dominated by Lb. paracasei. The surface bacterial microbiota of all four cheeses were dominated by Corynebacterium spp. and/or Brachybacterium spp. Brevibacterium spp. was found to be subdominant compared to other bacteria on the farmhouse cheeses, and no Brevibacterium spp. was found on the cheese from the industrial dairy, even though B. linens was used as surface-ripening culture. Moreover, Gram-negative bacteria identified as Alcalignes faecalis and Proteus vulgaris were found on one of the farmhouse cheeses. The surface yeast microbiota consisted primarily of one dominating species for each cheese. For the farmhouse cheeses, the dominant yeast species were Yarrowia lipolytica, Geotrichum spp. and Debaryomyces hansenii, respectively, and for the cheese from the industrial dairy, D. hansenii was the dominant yeast species. Additionally, denaturing gradient gel electrophoresis (DGGE) analysis revealed that Streptococcus thermophilus was present in the farmhouse raw milk cheese analysed in this study. Furthermore, DGGE bands corresponding to Vagococcus carniphilus, Psychrobacter spp. and Lb. curvatus on the cheese surfaces indicated that these bacterial species may play a role in cheese ripening.