Fermented Dairy Research Articles

Characterization and Genomic Analysis of Phage asccφ28, a Phage of the Family Podoviridae Infecting Lactococcus lactis

Bacteriophage asccphi28 infects dairy fermentation strains of Lactococcus lactis. This report describes characterization of asccphi28 and its full genome sequence. Phage asccphi28 has a prolate head, whiskers, and a short tail (C2 morphotype). This morphology and DNA hybridization to L. lactis phage P369 DNA showed that asccphi28 belongs to the P034 phage species, a group rarely encountered in the dairy industry. The burst size of asccphi28 was found to be 121 +/- 18 PFU per infected bacterial cell after a latent period of 44 min. The linear genome (18,762 bp) contains 28 possible open reading frames (ORFs) comprising 90% of the total genome. The ORFs are arranged bidirectionally in recognizable functional modules. The genome contains 577 bp inverted terminal repeats (ITRs) and putatively eight promoters and four terminators. The presence of ITRs, a phage-encoded DNA polymerase, and a terminal protein that binds to the DNA, along with BLAST and morphology data, show that asccphi28 more closely resembles streptococcal phage Cp-1 and the phi29-like phages that infect Bacillus subtilis than it resembles common lactococcal phages. The sequence of this phage is the first published sequence of a P034 species phage genome.

Phenotypic identification and technological properties of lactic acid bacteria isolated from three breeds dromedary raw milks in south Algeria

A total of 9 samples of individual dromedary raw milks from N'ajjer (3 samples), Targui (3 samples) and Reguibi (3 samples) breeds were collected from 3 camels nomad herd in south Algeria and were analysed for bacterial load. A total of 23 isolates of lactic acid bacteria were obtained and characterized; 12 isolates were cocci and 11 isolates were facultatively heterofermentative lactobacilli. Lactic acid bacteria were identified on the basis of phenotypic characters as Lactococcus lactis subsp. lactis, Enterococcus faecalis, Enterococcus faecium, Enterococcus durans, Lactobacillus paracasei subsp. paracsei, Lactobacillus plantarum and Lactobacillus rhamnosus. Lactococci, enterococci and lactobacilli showed proteolytic activity and were found that differ in their acidifying activities. Proteolytic and autolytic activities were generally higher for most lactobacilli compared to other isolates and none of the isolates produced biogenic amines under the conditions tested. A number of the 23 lactic acid bacteria isolates from Algerian dromedary milks that were characterized here showed properties suggest that they are good candidates for camels milk processing or other dairy fermentation process.

Phenotypic identification and technological properties of lactic acid bacteria isolated from three breeds dromedary raw milks in south Algeria

A total of 9 samples of individual dromedary raw milks from N’ajjer (3 samples), Targui (3 samples) and Reguibi (3 samples) breeds were collected from 3 camels nomad herd in south Algeria and were analysed for bacterial load. A total of 23 isolates of lactic acid bacteria were obtained and characterized; 12 isolates were cocci and 11 isolates were facultatively heterofermentative lactobacilli. Lactic acid bacteria were identified on the basis of phenotypic characters as Lactococcus lactis subsp. lactis, Enterococcus faecalis, Enterococcus faecium, Enterococcus durans, Lactobacillus paracasei subsp. paracsei, Lactobacillus plantarum and Lactobacillus rhamnosus. Lactococci, enterococci and lactobacilli showed proteolytic activity and were found that differ in their acidifying activities. Proteolytic and autolytic activities were generally higher for most lactobacilli compared to other isolates and none of the isolates produced biogenic amines under the conditions tested. A number of the 23 lactic acid bacteria isolates from Algerian dromedary milks that were characterized here showed properties suggest that they are good candidates for camels milk processing or other dairy fermentation process.

Identification and Antimicrobial Susceptibility of Lactic Acid Bacteria from Retail Fermented Foods

One important safety criterion of using lactic acid bacteria (LAB) in food applications is to ensure that they do not carry transferable antimicrobial resistance (AR) determinants. In this study, 63 LAB belonging to six genera, Streptococcus, Lactobacillus, Lactococcus, Enterococcus, Leuconostoc, and Pediococcus, were recovered from 28 retail fermented food products in Maryland, identified to species with 16S–23S rRNA spacer PCRs, and characterized for antimicrobial susceptibility against eight antimicrobials. Besides intrinsic resistance to ciprofloxacin or vancomycin in some lactobacilli, tetracycline resistance was observed in two Streptococcus thermophilus isolates from one cheese and one sour cream sample and was associated with the presence of a nonconjugative tet(S) gene. The results indicated a low level of AR among naturally occurring and starter LAB cultures in fermented dairy and meat products in the United States; therefore, the probability for foodborne LAB to serve as reservoirs of AR is low. Further studies involving a larger sample size are needed to assess the potential risk of AR gene transfer from LAB in fermented food products.

Molecular organization of plasmid pER13 in Streptococcus thermophilus

Molecular features of the 4139-bp plasmid pER13 found in the dairy fermentation bacterium Streptococcus thermophilus ST113 include five open reading frames (ORFs). ORF1, ORF2 and ORF3 encode proteins for transcriptional repression (CopG), replication (RepB) and mobilization (Mob) that share homology with corresponding proteins of the pMV158 plasmid family, while ORF4 and ORF5 encode putative proteins with unspecified functions. Sequence homologies shared with plasmids found in group B and group D streptococci imply the possibility for genetic exchange with the food-grade S. thermophilus. The structural features of pER13 may be useful in designing strategies for gene transfer in lactic fermentation bacteria.

Detection of bacteriophage-infected cells of Lactococcus lactis by using flow cytometry.

Bacteriophage infection in dairy fermentation constitutes a serious problem worldwide. We have studied bacteriophage infection in Lactococcus lactis by using the flow cytometer. The first effect of the infection of the bacterium is a change from cells in chains toward single cells. We interpret this change as a consequence of a cease in cell growth, while the ongoing cell divisions leave the cells as single cells. Late in the infection cycle, cells with low-density cell walls appear, and these cells can be detected on cytograms of light scatter versus, for instance, fluorescence of stained DNA. We describe a new method for detection of phage infection in Lactococcus lactis dairy cultures. The method is based on flow cytometric detection of cells with low-density cell walls. The method allows fast and early detection of phage-infected bacteria, independently of which phage has infected the culture. The method can be performed in real time and therefore increases the chance of successful intervention in the fermentation process.

Safety assessment of dairy microorganisms: The Enterococcus genus

Unlike most of lactic acid bacteria, the Enterococcus genus is not considered “generally recognized as safe” (GRAS). Safety assessment for enterococci remains controversial. While enterococci are considered “positive" or useful in cheese technology, isolates of this genus have emerged as opportunistic pathogens for humans. Thus these bacteria have the paradoxical position of being useful in dairy fermentations, but also potentially dangerous. The aim of this review is to summarize both the positive and negative traits of enterococci that illustrate the controversial nature of this bacterial genus. According to food safety assessment guidelines, we propose a case-by-case evaluation of each potential technological strain and suggest several lines of research before using enterococci in fermented food products.

Comparative Sequence Analysis of Plasmids from Lactobacillus delbrueckii and Construction of a Shuttle Cloning Vector

While plasmids are very commonly associated with the majority of the lactic acid bacteria, they are only very rarely associated with Lactobacillus delbrueckii, with only four characterized to date. In this study, the complete sequence of a native plasmid, pDOJ1, from a strain of Lactobacillus delbrueckii subsp. bulgaricus was determined. It consisted of a circular DNA molecule of 6,220 bp with a G+C content of 44.6% and a characteristic ori and encoded six open reading frames (ORFs), of which functions could be predicted for three-a mobilization (Mob) protein, a transposase, and a fused primase-helicase replication protein. Comparative analysis of pDOJ1 and the other available L. delbrueckii plasmids (pLBB1, pJBL2, pN42, and pLL1212) revealed a very similar organization and amino acid identities between 85 and 98% for the putative proteins of all six predicted ORFs from pDOJ1, reflecting a common origin for L. delbrueckii plasmids. Analysis of the fused primase-helicase replication gene found a similar fused organization only in the theta replicating group B plasmids from Streptococcus thermophilus. This observation and the ability of the replicon to function in S. thermophilus support the idea that the origin of plasmids in L. delbrueckii was likely from S. thermophilus. This may reflect the close association of these two species in dairy fermentations, particularly yogurt production. As no vector based on plasmid replicons from L. delbrueckii has previously been constructed, an Escherichia coli-L. delbrueckii shuttle cloning vector, pDOJ4, was constructed from pDOJ1, the p15A ori, the chloramphenicol resistance gene of pCI372, and the lacZ polylinker from pUC18. This cloning vector was successfully introduced into E. coli, L. delbrueckii subsp. bulgaricus, S. thermophilus, and Lactococcus lactis. This shuttle cloning vector provides a new tool for molecular analysis of Lactobacillus delbrueckii and other lactic acid bacteria.

Putting microbes to work: Dairy fermentation, cell factories and bioactive peptides. Part II: Bioactive peptide functions

A variety of milk-derived biologically active peptides have been shown to exert both functional and physiological roles in vitro and in vivo, and because of this are of particular interest for food science and nutrition applications. Biological activities associated with such peptides include immunomodulatory, antibacterial, anti-hypertensive and opioid-like properties. Milk proteins are recognized as a primary source of bioactive peptides, which can be encrypted within the amino acid sequence of dairy proteins, requiring proteolysis for release and activation. Fermentation of milk proteins using the proteolytic systems of lactic acid bacteria is an attractive approach for generation of functional foods enriched in bioactive peptides given the low cost and positive nutritional image associated with fermented milk drinks and yoghurt. In Part II of this review, we focus on examples of milk-derived bioactive peptides and their associated health benefits, to illustrate the potential of this area for the design and improvement of future functional foods.

Putting microbes to work: Dairy fermentation, cell factories and bioactive peptides. Part I: Overview

A variety of milk-derived biologically active peptides have been shown to exert both functional and physiological roles in vitro and in vivo, and because of this are of particular interest for food science and nutrition applications. Biological activities associated with such peptides include immunomodulatory, antibacterial, anti-hypertensive and opioid-like properties. Milk proteins are recognized as a primary source of bioactive peptides, which can be encrypted within the amino acid sequence of dairy proteins, requiring proteolysis for release and activation. Fermentation of milk proteins using the proteolytic systems of lactic acid bacteria (LAB) is an attractive approach for generation of functional foods enriched in bioactive peptides given the low cost and positive nutritional image associated with fermented milk drinks and yoghurt. In this review, we discuss the exploitation of such fermentation towards the development of functional foods conferring specific health benefits to the consumer beyond basic nutrition. In particular, in Part I, we focus on the release of encrypted bioactive peptides from a range of food protein sources, as well as the use of LAB as cell factories for the de novo generation of bioactivities.

Influence of the Dairy Environment on Gene Expression and Substrate Utilization in Lactic Acid Bacteria1, ,

Lactic acid bacteria (LAB) are widely used for the industrial production of fermented dairy products and form a group of related low-GC-content gram-positive bacteria. The major species used in dairy manufacturing are Lactobacillus, Lactococcus, Streptococcus, and Leuconostoc. Traditionally most are applied as starter cultures for dairy fermentations or used as probiotic cultures, delivered in dairy vehicles. The appearance of the genomes of Lactococcus lactis, Bidifobacterium longum, Lactobacillus plantarum, L. johnsonii, L. acidophilus, 2 strains of Streptococcus thermophilus, and pending completion of many draft genomic sequences, is now promoting in-depth investigation into the comparative genetic content of LAB. Moreover, whole-genome transcriptional arrays are quickly revealing critical genes/operons that are coordinately expressed and the impact of environmental factors on expression of multiple gene sets. Comparative genomics between multiple genomes is providing insights into genes that are important in metabolic, physiological, and functional roles for different LAB in the environments they inhabit, ranging from the gastrointestinal tract to milk and acidified dairy products.

Technologically important properties of lactic acid bacteria isolated from raw milk of three breeds of Algerian dromedary ( Camelus dromedarius )

A total of 9 samples of individual dromedary raw milks from N’ajjer (3), Targui (3) and Reguibi (3) breeds were collected from 3 camels nomad herd in south Algeria and were analysed for bacterial load. A total of 23 strains of lactic acid bacteria were isolated, out of which 12 strains were cocci and 11 strains were facultatively heterofermentative lactobacilli. Lactic acid bacteria were identified on the basis of phenotypic characters as Lactococcus lactis subsp. lactis, Enterococcus faecalis, Enterococcus faecium, Enterococcus durans, Lactobacillus paracasei subsp. paracasei, Lactobacillus plantarum and Lactobacillus rhamnosus. Whole cells of lactococci, enterococci and lactobacilli showed proteolytic activity and were found to differed in terms of their acidifying activities. Proteolytic and autolytic activity were generally higher for most lactobacilli compared to other isolates and none of the strains produced biogenic amines in the method applied. A wide variety of this 23 lactic acid bacteria strains isolated from Algerian dromedary milks that showed potentially important properties suggest that they are good candidate for camels milk processing or other dairy fermentation process.

Expression of the heat shock gene clpL of Streptococcus thermophilus is induced by both heat and cold shock

BackgroundHeat and cold shock response are normally considered as independent phenomena. A small amount of evidence suggests instead that interactions may exist between them in two Lactococcus strains.ResultsWe show the occurrence of molecular relationships between the mechanisms of cold and heat adaptations in Streptococcus thermophilus, a lactic acid bacterium widely used in dairy fermentation, where it undergoes both types of stress. We observed that cryotolerance is increased when cells are pre-incubated at high temperature. In addition, the production of a protein, identified as ClpL, a member of the heat-shock ATPase family Clp A/B, is induced at both high and low temperature. A knock-out clpL mutant is deficient in both heat and cold tolerance. However lack of production of this protein does not abolish the positive effect of heat pre-treatment towards cryotolerance.ConclusionDual induction of ClpL by cold and heat exposure of cells and reduced tolerance to both temperature shocks in a clpL mutant indicates that the two stress responses are correlated in S. thermophilus. However this protein is not responsible by itself for cryotolerance of cells pre-treated at high temperature, indicating that ClpL is necessary for the two phenomena, but does not account by itself for the relationships between them.

Application of reverse transcriptase PCR-based T-RFLP to perform semi-quantitative analysis of metabolically active bacteria in dairy fermentations

A method consisting of reverse transcriptase (RT)-PCR amplification of 16S rRNA from the total microbial community, coupled with T-RFLP, was optimized for semi-quantitative characterization of the metabolically active population in defined strain cultures of Lactococcus lactis ssp. lactis and Leuconostoc citreum, two mesophilic lactic acid bacteria (LAB) species routinely used in cheese manufacture. The set of PCR primers selected efficiently amplified the 16S rRNA from both bacterial species. The digestion of the PCR products with DdeI yielded different terminal restriction fragments (T-RFs) for each species. Nevertheless, additional T-RFs due to formation of chimeric molecules and pseudo-T-RFs derived from partly single-stranded 16S rRNA amplicons were observed in both species, although in minor amounts. Twenty PCR cycles were determined as the optimum to minimize the presence of artifactual fragments and to avoid underestimation of populations due to the saturation effect on DNA quantification caused by a PCR product excess. T-RFLP analysis showed a good repeatability when applied to mixed dairy cultures. Dynamics of two defined mixed starters consisting of a L. lactis ssp. lactis strain and a L. citreum strain were studied by this method and results compared to those obtained by a culture-dependent technique. The data indicated the suitability of T-RFLP to perform semi-quantitative analyses of microbial populations. Some slight differences could be explained by the presence of metabolically active cells that could not be detected by colony counting. RT-PCR-based T-RFLP can be an alternative to classical methods in order to study dynamics of metabolically active populations in relatively simple microbial ecosystems, such as defined dairy starter cultures.

Assessment of microbial population dynamics during yoghurt and hard cheese fermentation and ripening by DNA population fingerprinting

The application of Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis of the small subunit ribosomal gene to examine the bacterial dynamics of relatively fast dairy fermentation processes is illustrated. All individual starter strains yielded characteristic terminal restriction fragments (TRFs). Growth independent T-RFLP analysis was used to identify and follow the dynamics of streptococci, lactobacilli, lactococci and propionibacteria in yoghurt and hard cheese production while samples had been stored frozen until analysis. Starter cultures could be differentiated and followed during production and ripening of hard cheese (Gouda type and Maasdam) as well as production of yoghurt. T-RFLP analysis enabled the characterization of bacterial population structure and dynamics between short time spans, i.e., within hours. Eight samples taken at different times within 5.5 h yoghurt fermentation revealed shifting relative signal intensities in TRF peaks between streptococci and lactobacilli, indicating a (semi) quantitative relation of the TRFLP signal with the actual numbers of bacteria.

Flavour formation by lactic acid bacteria and biochemical flavour profiling of cheese products

Flavour development in dairy fermentations, most notably cheeses, results from a series of (bio)chemical processes in which the starter cultures provide the enzymes. Particularly the enzymatic degradation of proteins (caseins) leads to the formation of key-flavour components, which contribute to the sensory perception of dairy products. More specifically, caseins are degraded into peptides and amino acids and the latter are major precursors for volatile aroma compounds. In particular, the conversion of methionine, the aromatic and the branched-chain amino acids are crucial. A lot of research has focused on the degradation of caseins into peptides and free amino acids, and more recently, enzymes involved in the conversion of amino acids were identified. Most data are generated on Lactococcus lactis, which is the predominant organism in starter cultures used for cheese-making, but also Lactobacillus, Streptococcus, Propionibacterium and species used for surface ripening of cheeses are characterised in their flavour-forming capacity. In this paper, various enzymes and pathways involved in flavour formation will be highlighted and the impact of these findings for the development of industrial starter cultures will be discussed.

Diversity of L-leucine catabolism in various microorganisms involved in dairy fermentations, and identification of the rate-controlling step in the formation of the potent flavour component 3-methylbutanal.

Various microorganisms, belonging to the genera Lactococcus, Lactobacillus, Streptococcus, Leuconostoc, Bifidobacterium, Propionibacterium, Brevibacterium, Corynebacterium and Arthrobacter, used in dairy fermentations such as cheese making, were analysed for their potential to convert leucine into flavour components, most notably 3-methylbutanal. A large variation between and within species was observed for various enzyme activities involved in the conversion pathway, e.g. transaminases, alpha-hydroxy acid dehydrogenase and alpha-keto acid decarboxylase. In particular, alpha-keto acid decarboxylase activity-leading to 3-methylbutanal-was found to be present in only two of the strains tested. It is proposed that this activity is rate-controlling in the conversion pathway leading to the flavour compound 3-methylbutanal.

ADSA Foundation Scholar Award—An Integrated Science-Based Approach to Dairy Food Safety: Listeria monocytogenes as a Model System

Transmission of food- and milkborne pathogens often involves complex interactions among the pathogen, the environment, and one or multiple host species. A complete understanding of these interactions is critical to allow the development of science-based, effective intervention strategies for foodborne infectious diseases. This article summarizes our studies on the transmission, ecology, pathogenesis and population genetics of Listeria monocytogenes, which we have used as model for a food- and milkborne pathogen that infects multiple hosts and also has considerable ability to survive and multiply in nonhost environments. Application of molecular subtyping tools in conjunction with phenotypic characterization of selected strains has allowed us to define distinct L. monocytogenes subtypes and clonal groups that appear to differ in relevant phenotypic characteristics that may affect their abilities to be transmitted through food systems. For example, a genetic group designated as lineage I has been shown to be not only more common among human listeriosis cases than among animal cases, but lineage I strains also appear to show an increased in vitro ability to spread intracellularly from host cell to host cell. These findings are consistent with the fact that while genetically diverse strains may be classified to one bacterial species, these strains often differ from one another in important genetic and phenotypic characteristics. I thus propose that evolutionary- and molecular subtyping-based definitions of bacterial subtypes and clonal groups will provide critical insight into the microbial ecology of dairy food systems, including not only foodborne pathogens, but also organisms important for dairy fermentation and spoilage.

Characterisation of technologically proficient wild Lactococcus lactis strains resistant to phage infection

The aim of this work was to establish whether Lactococcus lactis strains isolated from spontaneous dairy fermentations exhibited useful milk-processing capabilities and resistance to bacteriophage infection in order to be used as components in starter formulations. The 33 out of 100 isolates of L. lactis, originated from farmhouse cheeses, were found to be resistant to a collection of 34 phages belonging to the c2 and 936 groups. Six of the isolates were discarded as potential starters because they were lysogenic and other five because they produced tyramine. Plasmid and chromosomal profiles of the 22 remaining isolates allowed their classification into 16 different strains. All of these were good lactic acid producers from lactose, moderately proteolytic and, in eight cases, diacetyl production from citrate was observed. The mechanism(s) leading to the phenotype of phage resistance was identified for all the strains used in this study. Inhibition of adsorption was the most frequent one, although genetic determinants for some abortive infection systems were also detected ( abiB, abiG and abiI). Frequently, more than one mechanism was present in the same strain. One of the strains, L. lactis IPLA542, was selected as a model starter for pilot fermentations. It clotted milk normally both in the absence and in the presence of phage at concentrations that completely abolished the process when promoted by a phage-susceptible strain.

The role of processing and matrix design in development and control of microstructures in dairy food production—a survey

New options to process dairy products by means of new single unit operations or combinations thereof are presented. The innovative possibilities discussed in this paper mainly refer to novel ways of influencing product microstructures or textures as perceived during consumption, by means of contributions of single molecule fractions of complex systems derived from membrane fractionation techniques. In addition, options to integrate physiologically active components which also have a structure forming effect in fermented or other dairy products are highlighted. Alternatives derived from thermal, mechanical and enzymatic processing and from fractionation techniques by means of membranes are presented which can be exploited for fractionation and compositional matrix design of dairy products.