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.

Microbial shifts through the ripening of the "Entre Serras" Minas artisanal cheese monitored by high-throughput sequencing.

Minas Gerais is a Brazilian state known as the largest cheese producer in Brazil. Minas Artisanal Cheese (MAC) is produced in different regions of this Brazilian state using raw cow milk to which a natural starter culture ("pingo") is added. "Entre Serras" is one of these regions, in which the MAC production had decreased (even stopped) for decades until recently, when artisanal cheeses production has been resurrected. Here, we aimed to gain insights on the bacterial diversity of "Entre Serras" MAC. 16S rRNA gene amplicon sequencing was used to assess the bacterial community in cheeses produced by four farms (A, B, C, and D) over 60 days of ripening. Overall, Lactococcus lactis was the predominant species found, regardless of the producer/farm. Enterococcus, Streptococcus, Lactobacillus and Leuconostoc genera were also prevalent in the samples microbiota and their levels varied according to the producer/farm. Cheeses produced by Farms A and B presented high contaminant levels (mainly Enterobacteriaceae and S. aureus), which may be attributed to poor hygiene during cheese production and/or herd health management. Chao1 indices varied significantly when the estimated species richness values of the producers/farms were compared (p < 0.05). A principal coordinate analysis also revealed distinct microbial communities for some farms (p < 0.001). However, no statistical significance was identified when samples were grouped by ripening time. Core microbiota analysis indicated that "Entre Serras" MAC microbiota includes not only LAB, but also spoilage and potentially pathogenic bacteria. We provide the first insights on the bacterial diversity of "Entre Serras" MAC, helping the understanding of the inter-regional microbiological diversity of the samples.

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.

Bacterial diversity of artisanal cheese from the Amazonian region of Brazil during the dry and rainy seasons.

The microbiota from artisanal cheeses produced in the Amazonian region is evaluated. Samples of artisanal cheeses were obtained from markets in Conceição do Araguaia and Redenção (Pará, Brazil) over rainy and dry seasons, and their biodiversity was assessed by culture-dependent and culture-independent methods. Mean counts of lactic acid bacteria (LAB) in cheeses ranged from 7.32 to 8.84 log CFU/g, for both seasons. Members of genera Lactobacillus, Lactococcus, Weissella, Enterococcus, Pediococcus, and Leuconostoc were predominant. The amplification of the 16S rRNA V6-V9 region, followed by a temporal temperature-gradient gel electrophoresis (TTGE) and sequencing of the TTGE bands revealed important differences in the microbial composition variability between samples from the two seasons and among cheese samples analyzed. TTGE showed the presence of microorganisms that are frequently found in cheese, such as L. lactis subsp. lactis, as well as other non-usual species, such as Macrococcus caseolyticus and Corynebacterium variabile. Moreover, TTGE analysis revealed the presence of microorganisms that have been isolated from other types of foods (Paralactobacillus selangorenses) along with some not usually found in foods, such as Exiguobacterium acetylicum, plus the presence of pathogenic microorganisms (Granulicatella elegans and Aerococcus sanguinicola). The present molecular approaches combined with culture-dependent methods provided a more detailed description of the microbial ecology of traditional cheeses from the Amazonian region in northern Brazil.

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.

Interference of different storage temperatures in the dynamics of probiotic Bifidobacterium spp. and Streptococcus thermophilus starter cultures in fermented milk / Interferência de diferentes temperaturas de armazenamento na dinâmica de Bifidobacterium spp. probióticas e cultura starterStreptococcus thermophilus em leite fermentado

The variations of temperature during the cold chain can impair the quality of live foods, such as fermented milks. Probiotic bacteria are commonly added to food to provide the consumer with beneficial effects. Nevertheless, the concentration of probiotic in the end products should be elevated to ensure functionality. The aim of this study was to evaluate the viability of probiotic strains of bifidobacteria and starter strain of Streptococcus thermophilus in fermented milks at storage temperatures of 4 and 10ºC, for a period of 28 days. Commercial cultures of Bifidobacterium spp. were added to milk fermented by Streptococcus thermophillus and stored for 28 days at 4 and 10ºC. During this period, bifidobacteria and S. thermophilluscultures were monitored to check their behavior in the evaluated storage conditions. Viable bifidobacteria and S. thermophillus counts showed no significant variation during storage at 4 and 10ºC (p < 0.05), indicating that both of these conditions are adequate for maintaining their initial concentrations. The results indicate that the storage conditions usually adopted in sale establishments of dairy products are suitable to maintain bifidobacteria and S. thermophillus cultures in fermented milk.(AU)

As variações de temperatura que ocorrem durante a cadeia refrigerada da produção leiteira pode interferir na qualidade de alimentos bioativos, como leites fermentados. Bactérias probióticas são usualmente adicionadas a alimentos, visando a oferecer ao consumidor efeitos benéficos. O objetivo deste trabalho foi avaliar a viabilidade de culturas probióticas de bifidobactérias e de cultura starter de Streptococcus thermophilus em leites fermentados, armazenados nas temperaturas de 4 e 10ºC por um período de 28 dias. Culturas comerciais de Bifidobacterium spp. foram adicionadas ao leite fermentado produzido com Streptococcus thermophillus e estocados por 28 dias a 4 e 10ºC. Nesse período, as culturas de Bifidobacterium spp. e Streptococcus thermophillus foram monitoradas, com o objetivo de verificar seus comportamentos nas temperaturas de estocagem testadas. As contagens de ambos os micro-organismos não apresentaram variação significativa ao longo do período de estocagem a 4 e a 10ºC (p < 0,05), indicando que as duas temperaturas testadas podem oferecer condições adequadas de conservação desse produto. Os resultados obtidos indicaram que as condições de conservação de alimentos usualmente adotadas para produtos lácteos em estabelecimentos comerciais são adequadas para manter as contagens de bifidobactérias e Streptococcus thermophillus em leites fermentados.(AU)

Microbiological safety of Minas Frescal Cheese (MFC) and tracking the contamination of Escherichia coli and Staphylococcus aureus in MFC processing.

Minas Frescal cheese (MFC) is a traditional food produced and consumed in Brazil, characterized by its soft texture, low sodium, and high moisture content. This study characterized the microbiological contamination by coliforms, Escherichia coli and Staphylococcus aureus, in 99 MFC samples obtained in retail sale and produced by three distinct industrial procedures. Dairy processors were selected to investigate the key points of E. coli and S. aureus contamination during cheese processing. MFC samples produced by the addition of lactic culture presented higher counts of coliforms and E. coli, when compared to other samples (p<0.05). MFC samples produced by the addition of rennet alone presented higher counts of S. aureus when compared to other samples (p<0.05). Fourteen of 19 MFC samples produced by the addition of lactic culture presented E. coli counts higher than 5 × 10(2) colon-forming units/g. The processing steps after pasteurization were identified as the main sources of E. coli and S. aureus contamination of MFC. Based on the results, MFC was characterized as a potential hazard for consumers due to the high frequency of samples contaminated with E. coli and S. aureus counts in noncompliance with Brazilian standards for sanitary quality and safety.

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.

Enumeration of starter cultures during yogurt production using Petrifilm AC plates associated with acidified MRS and M17 broths.

The efficiency of Petrifilm AC (3M Microbiology, St. Paul, MN, USA) associated with the broths M17 and de Mann-Rogosa-Sharpe (MRS) at pH 5.4 was evaluated to enumerate Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus during the yogurt production. Commercial and reference strains of these microorganisms were experimentally inoculated in nonfat milk and incubated at 42 degrees C for 4 h for yogurt production. At the moment of inoculation and after incubation, aliquots were collected, submitted to dilution using the broths M17 and MRS at pH 5.4, and plated for Strep. salivarius and Lb. bulgaricus enumeration according ISO 9232 and at Petrifilm AC plates, respectively. M17 plates were incubated at 42 degrees C, and MRS plates were incubated at 35 degrees C under anaerobiosis. After 48 h, the formed colonies were enumerated and the counts were compared by correlation and analysis of variance (P<0.05). In addition, colonies were randomly selected from all plates and characterized according to Gram staining and morphology. The obtained results indicated that Petrifilm AC plates associated to M17 and MRS at pH 5.4 can be considered as a suitable alternative for Strep. salivarius and Lb. bulgaricus enumeration during yogurt production, with slight interferences due to the acidity of MRS at the moment of inoculation, and due to the acidity of yogurt at the end of fermentation process. It was also observed that the MRS at pH 5.4 was not sufficiently selective for Lb. delbrueckii enumeration, despite it is indicated by the official protocol from ISO 9232.