Challenges associated with spray drying of lactic acid bacteria: Understanding cell viability loss.

Lactic acid bacteria (LAB) cultures used in food fermentation are often dried to reduce transportation costs and facilitate handling during use. Dried LAB ferments are generally lyophilized to ensure high cell viability. Spray drying has come to the forefront as a promising technique due to its versatility and lower associated energy costs. Adverse conditions during spray drying, such as mechanical stress, dehydration, heating, and oxygen exposure, can lead to low LAB cell viability. This reduced viability has limited spray drying's industrial applications thus far. This review aims to demonstrate the operations and thermodynamic principles that govern spray drying, then correlate them to the damage suffered by LAB cells during the spray-drying process. The particularities of spray drying that might cause LAB cell death are detailed in this review, and the conclusion may enhance future studies on ways to improve cell viability.

Technological properties of Lactococcus lactis subsp. lactis bv. diacetylactis obtained from dairy and non-dairy niches.

Lactococcus lactis subsp. lactis bv. diacetylactis strains are often used as starter cultures by the dairy industry due to their production of acetoin and diacetyl, important substances that add buttery flavor notes in dairy products. Twenty-three L. lactis subsp. lactis isolates were obtained from dairy products (milk and cheese) and dairy farms (silage), identified at a biovar level, fingerprinted by rep-PCR and characterized for some technological features. Fifteen isolates presented molecular and phenotypical (diacetyl and citrate) characteristics coherent with L. lactis subsp. lactis bv. diacetylactis and rep-PCR allowed the identification of 12 distinct profiles (minimum similarity of 90%). Based on technological features, only two isolates were not able to coagulate skim milk and 10 were able to produce proteases. All isolates were able to acidify skim milk: two isolates, in special, presented high acidifying ability due to their ability in reducing more than two pH units after 24 h. All isolates were also able to grow at different NaCl concentrations (0 to 10%, w/v), and isolates obtained from peanut and grass silages presented the highest NaCl tolerance (10%, w/v). These results indicate that the L. lactis subsp. lactis bv. diacetylactis isolates presented interesting technological features for potential application in fermented foods production. Despite presenting promising technological features, the isolates must be assessed according to their safety before being considered as starter cultures.

New insights about phenotypic heterogeneity within Propionibacterium freudenreichii argue against its division into subspecies.

Propionibacterium freudenreichii is widely used in Swiss-type cheese manufacture, where it contributes to flavour and eye development. It is currently divided into two subspecies, according to the phenotype for lactose fermentation and nitrate reduction (lac+/nit- and lac-/nit+ for P. freudenreichii subsp. shermanii and subsp. freudenreichii, respectively). However, the existence of unclassifiable strains (lac+/nit+ and lac-/nit-) has also been reported. The aim of this study was to revisit the relevance of the subdivision of P. freudenreichii into subspecies, by confirming the existence of unclassifiable strains. Relevant conditions to test the ability of P. freudenreichii for lactose fermentation and nitrate reduction were first determined, by using 10 sequenced strains, in which the presence or absence of the lactose and nitrate genomic islands were known. We also determined whether the subdivision based on lac/nit phenotype was related to other phenotypic properties of interest in cheese manufacture, in this case, the production of aroma compounds, analysed by gas chromatography-mass spectrometry, for a total of 28 strains. The results showed that a too short incubation time can lead to false negative for lactose fermentation and nitrate reduction. They confirmed the existence of four lac/nit phenotypes instead of the two expected, thus leading to 13 unclassifiable strains out of the 28 characterized (7 lac+/nit+ and 6 lac-/nit-). The production of the 15 aroma compounds detected in all cultures varied more within a lac/nit phenotype (up to 20 times) than between them. Taken together, these results demonstrate that the division of P. freudenreichii into two subspecies does not appear to be relevant.

Pulsed field gel electrophoresis for dairy propionibacteria.

Pulsed field gel electrophoresis (PFGE) is a technique using alternating electric fields to migrate high molecular weight DNA fragments with a high resolution. This method consists of the digestion of bacterial chromosomal DNA with rare-cutting restriction enzymes and in applying an alternating electrical current between spatially distinct pairs of electrodes. DNA molecules migrate at different speeds according to the size of the fragments. Among other things, this technique is considered as the "gold standard" for genotyping, genetic fingerprinting, epidemiological studies, genome size estimation, and studying radiation-induced DNA damage and repair. This chapter describes a PFGE method that can be used to differentiate dairy propionibacteria.

Biodiversity of dairy Propionibacterium isolated from dairy farms in Minas Gerais, Brazil.

Dairy propionibacteria are used as ripening cultures for the production of Swiss-type cheeses, and some strains have potential for use as probiotics. This study investigated the biodiversity of wild dairy Propionibacteria isolates in dairy farms that produce Swiss-type cheeses in Minas Gerais State, Brazil. RAPD and PFGE were used for molecular typing of strains and MLST was applied for phylogenetic analysis of strains of Propionibacterium freudenreichii. The results showed considerable genetic diversity of the wild dairy propionibacteria, since three of the main species were observed to be randomly distributed among the samples collected from different farms in different biotopes (raw milk, sillage, soil and pasture). Isolates from different farms showed distinct genetic profiles, suggesting that each location represented a specific niche. Furthermore, the STs identified for the strains of P. freudenreichii by MLST were not related to any specific origin. The environment of dairy farms and milk production proved to be a reservoir for Propionibacterium strains, which are important for future use as possible starter cultures or probiotics, as well as in the study of prevention of cheese defects.

Selective enumeration of propionibacteria in Emmental-type cheese using Petrifilm™ aerobic count plates added to lithium glycerol broth.

Propionibacteria derived from dairy products are relevant starter cultures for the production of Swiss and Emmental-type cheeses, and the monitoring of which is mandatory for proper quality control. This study aimed to evaluate an alternative procedure to enumerate propionibacteria, in order to develop a reliable and practical methodology to be employed by dairy industries. 2,3,5-triphenyltetrazolium chloride (TTC) inhibitory activity was tested against five reference strains (CIRM 09, 38, 39, 40 and 116); TTC at 0·0025% (w/v) was not inhibitory, with the exception of one strain (CIRM 116). Subsequently, the four TTC-resistant strains, three commercial starter cultures (PS-1, PB-I, and CHOO) and twelve Emmental-type cheese samples were subjected to propionibacteria enumeration using Lithium Glycerol (LG) agar, and Petrifilm™ Aerobic Count (AC) plates added to LG broth (anaerobic incubation at 30 °C for 7 d). Petrifilm™ AC added to LG broth presented high counts than LG agar (P<0·05) for only two reference strains (CIRM 39, and 40) and for all commercial starter cultures. Cheese sample counts obtained by both procedures did not show significant differences (P<0·05). Significant correlation indexes were observed between the counts recorded by both methods (P<0·05). These results demonstrate the reliability of Petrifilm™ AC plates added to LG broth in enumerating select Propionibacterium spp., despite some limitations observed for specific commercial starter cultures.

Enumeration of bifidobacteria using Petrifilm™ AC in pure cultures and in a fermented milk manufactured with a commercial culture of Streptococcus thermophilus.

Bifidobacteria are probiotic microorganisms that are widely used in the food industry. With the aim of using of Petrifilm™ Aerobic Count (AC) plates associated with selective culture media, aliquots of sterile skim milk were inoculated separately with four commercial cultures of bifidobacteria. These cultures were plated by both the conventional method and Petrifilm™AC, using the culture media NNLP and ABC. The cultures were incubated under anaerobiosis at 37 °C for 24, 48 and 72 h. No significant differences (p > 0.05) were observed between the obtained counts at 48 and 72 h. Bifidobacteria counts in ABC were usually higher than in NNLP, independent of the plating method. Subsequently, fermented milk was prepared with a Streptococcus thermophilus strain, and aliquots were inoculated with the same bifidobacteria. Then, the fermented milks were submitted to microbiological analysis for bifidobacteria enumeration using the same culture media and methodologies previously described, incubated under anaerobiosis at 37 °C for 48 h. Again, bifidobacteria counts in ABC were higher than in NNLP, with significant differences for some cultures (p < 0.05). The counts obtained by both methodologies presented significant correlations (p < 0.05). The results indicate the viability of Petrifilm™AC as an alternative method for bifidobacteria enumeration when associated to specific culture media, specially the ABC.