Maria Kazou

Discovering probiotic microorganisms: in vitro, in vivo, genetic and omics approaches

Over the past decades the food industry has been revolutionized toward the production of functional foods due to an increasing awareness of the consumers on the positive role of food in wellbeing and health. By definition probiotic foods must contain live microorganisms in adequate amounts so as to be beneficial for the consumer’s health. There are numerous probiotic foods marketed today and many probiotic strains are commercially available. However, the question that arises is how to determine the real probiotic potential of microorganisms. This is becoming increasingly important, as even a superficial search of the relevant literature reveals that the number of proclaimed probiotics is growing fast. While the vast majority of probiotic microorganisms are food-related or commensal bacteria that are often regarded as safe, probiotics from other sources are increasingly being reported raising possible regulatory and safety issues. Potential probiotics are selected after in vitro or in vivo assays by evaluating simple traits such as resistance to the acidic conditions of the stomach or bile resistance, or by assessing their impact on complicated host functions such as immune development, metabolic function or gut–brain interaction. While final human clinical trials are considered mandatory for communicating health benefits, rather few strains with positive studies have been able to convince legal authorities with these health claims. Consequently, concern has been raised about the validity of the workflows currently used to characterize probiotics. In this review we will present an overview of the most common assays employed in screening for probiotics, highlighting the potential strengths and limitations of these approaches. Furthermore, we will focus on how the advent of omics technologies has reshaped our understanding of the biology of probiotics, allowing the exploration of novel routes for screening and studying such microorganisms.

Complete Genome Sequence of the Dairy Isolate Lactobacillus acidipiscis ACA-DC 1533

ABSTRACT Lactobacillus acidipiscis is a Gram-positive lactic acid bacterium belonging to the Lactobacillus salivarius clade. Here, we present the first complete genome sequence of L. acidipiscis isolated from traditional Greek Kopanisti cheese. Strain ACA-DC 1533 may play a key role in the strong organoleptic characteristics of Kopanisti cheese.

Probiotic Features of Lactic Acid Bacteria Isolated from a Diverse Pool of Traditional Greek Dairy Products Regarding Specific Strain-Host Interactions

The increased consumers’ interest on the positive role of food in wellbeing and health underscores the need to determine new probiotic microorganisms. Triggered by the fact that artisanal food products can be a valuable source of novel probiotic strains, 106 lactic acid bacteria, all isolated from traditional Greek dairy products, namely Feta, Kasseri, Xynotyri, Graviera, Formaela, Galotyri, and Kefalotyri cheeses as well as yogurt and milk, were studied for probiotic properties. Based on their survival at pH 2.5 and their stability in the presence of bile salts, 20 strains were selected for further analysis. These strains exhibited diverse susceptibility to commonly used antibiotics, while none was hemolytic. Seven out of the 20 strains produced functional bile salt hydrolases in vitro. The only antimicrobial activity detected of Streptococcus thermophilus ACA-DC 26 against the oral pathogen Streptococcus mutans LMG 14558T was attributed to compound(s) of proteinaceous nature. Two Lactobacillus plantarum strains, namely ACA-DC 2640 and ACA-DC 4039, displayed the highest adhesion according to a collagen-based microplate assay and by using ΗΤ-29 and Caco-2 cells. Co-cultivation of THP-1 cells with selected strains indicated a tendency for anti-inflammatory modulation by Lactobacillus plantarum ACA-DC 2640 as well as Streptococcus thermophilus ACA-DC 26 and ACA-DC 170, as shown by an increase in IL10 mRNA levels. Moreover, milk cell-free supernatants of Lactobacillus plantarum ACA-DC 2640 and ACA-DC 4039 exhibited strong angiotensin I-converting enzyme inhibition. To conclude, new isolates presenting interesting probiotic features were described and should be further investigated as health-promoting factors.

Engineered strains of Streptococcus macedonicus towards an osmotic stress resistant phenotype retain their ability to produce the bacteriocin macedocin under hyperosmotic conditions.

Streptococcus macedonicus ACA-DC 198 produces the bacteriocin macedocin in milk only under low NaCl concentrations (<1.0%w/v). The thermosensitive plasmid pGh9:ISS1 was employed to generate osmotic stress resistant (osmr) mutants of S. macedonicus. Three osmr mutants showing integration of the vector in unique chromosomal sites were identified and the disrupted loci were characterized. Interestingly, the mutants were able to grow and to produce macedocin at considerably higher concentrations of NaCl compared to the wild-type (up to 4.0%w/v). The production of macedocin under hyperosmotic conditions solely by the osmr mutants was validated by the well diffusion assay and by mass spectrometry analysis. RT-PCR experiments demonstrated that the macedocin biosynthetic regulon was transcribed at high salt concentrations only in the mutants. Mutant osmr3, the most robust mutant, was converted in its markerless derivative (osmr3f). Co-culture of S. macedonicus with spores of Clostridium tyrobutyricum in milk demonstrated that only the osmr3f mutant and not the wild-type inhibited the growth of the spores under hyperosmotic conditions (i.e., 2.5%w/v NaCl) due to the production of macedocin. Our study shows how genetic manipulation of a strain towards a stress resistant phenotype could improve bacteriocin production under conditions of the same stress.

Probiotics and Prebiotics: An Overview on Recent Trends

The ban of antibiotics at subtherapeutic levels as growth promoters in animals, along with the emerging challenges and prospects deriving from the use of probiotics and prebiotics in humans, opened the era of similar applications in the field of animal nutrition. Probiotics and prebiotics as novel additives in animal feeding aiming at host-protecting functions, which can maintain animal health and welfare, create fascinating options in favor of both animals and consumers of products of animal origin. In recent years, a considerable amount of knowledge regarding the potential of probiotics and prebiotics as supplements at prophylactic and therapeutic level has been accumulated. However, several bottlenecks are still observed, not only at the science, technology, and application level but also at the regulatory level. In this chapter, we summarize the current knowledge, along with the future trends on the applications of probiotics and prebiotics in animals and more specifically in polygastric, monogastric, and aquatic animals, pets, and bees. Moreover, we discuss the respective regulatory frame and its limitations.

Comparative Genomics of Lactobacillus acidipiscis ACA-DC 1533 Isolated From Traditional Greek Kopanisti Cheese Against Species Within the Lactobacillus salivarius Clade

Lactobacillus acidipiscis belongs to the Lactobacillus salivarius clade and it is found in a variety of fermented foods. Strain ACA-DC 1533 was isolated from traditional Greek Kopanisti cheese and among the available L. acidipiscis genomes it is the only one with a fully sequenced chromosome. L. acidipiscis strains exhibited a high degree of conservation at the genome level. Investigation of the distribution of prophages and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) among the three strains suggests the potential existence of lineages within the species. Based on the presence/absence patterns of these genomic traits, strain ACA-DC 1533 seems to be more related to strain JCM 10692T than strain KCTC 13900. Interestingly, strains ACA-DC 1533 and JCM 10692T which lack CRISPRs, carry two similar prophages. In contrast, strain KCTC 13900 seems to have acquired immunity to these prophages according to the sequences of spacers in its CRISPRs. Nonetheless, strain KCTC 13900 has a prophage that is absent from strains ACA-DC 1533 and JCM 10692T. Furthermore, comparative genomic analysis was performed among L. acidipiscis ACA-DC 1533, L. salivarius UCC118 and Lactobacillus ruminis ATCC 27782. The chromosomes of the three species lack long-range synteny. Important differences were also determined in the number of glycobiome related proteins, proteolytic enzymes, transporters, insertion sequences and regulatory proteins. Moreover, no obvious genomic traits supporting a probiotic potential of L. acidipiscis ACA-DC 1533 were detected when compared to the probiotic L. salivarius UCC118. However, the existence of more than one glycine-betaine transporter within the genome of ACA-DC 1533 may explain the ability of L. acidipiscis to grow in fermented foods containing high salt concentrations. Finally, in silico analysis of the L. acidipiscis ACA-DC 1533 genome revealed pathways that could underpin the production of major volatile compounds during the catabolism of amino acids that may contribute to the typical piquant flavors of Kopanisti cheese.

Whole-Genome Sequence of the Cheese Isolate Lactobacillus rennini ACA-DC 565

ABSTRACT In this study, we present the first complete genome sequence of Lactobacillus rennini ACA-DC 565, a strain isolated from a traditional Greek overripened Kopanisti cheese called Mana. Although the species has been associated with cheese spoilage, the strain ACA-DC 565 may contribute to the intense organoleptic characteristics of Mana cheese.