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During the last years, the food industry is working on the replacement of high energy methodologies with more sustainable techniques for the encapsulation of natural preservatives, in order to enhance their effectiveness as food additives. In the present study, nisin, an antimicrobial agent, was encapsulated in essential oil-containing microemulsions. More specifically, rosemary, thyme, oregano, and dittany essential oil-containing microemulsions were formulated to encapsulate nisin enhancing the system's overall antimicrobial activity. The systems were investigated for the interfacial properties and size of the surfactants' monolayer using electron paramagnetic resonance spectroscopy and dynamic light scattering. Subsequently, nisin-loaded microemulsions were tested for their antimicrobial activity against Lactococcus lactis, Staphylococcus aureus, Listeria monocytogenes, and Bacillus cereus, using the well diffusion assay. Finally, this technique was validated by a killing assay. Overall, this study provides important information on the antibacterial activity of nisin-loaded nano-carriers enhanced by essential oils, in relation to the microemulsions' structure.

Limosilactobacillus fermentum ACA-DC 179, a strain with well documented probiotic properties, was included in Greek traditional Kaimaki ice cream produced using two different types of milk, namely plain and lactose-free cow milk. L. fermentum survived throughout the ice cream manufacturing and storage. Viable counts did not decline during storage at −20 °C for 28 days both in plain (approximately 106 cfu g−1) and in lactose-free (approximately 108 cfu g−1) Kaimaki. Acidity and pH remained stable during storage, and, regarding fat and protein content, no variations were determined among samples belonging to the same type of ice cream. Melting characteristics of ice cream slightly altered due to strain addition. Ice cream containing L. fermentum was highly acceptable by the panellists evaluating sensory properties. To conclude, ice cream was proved to be a suitable matrix for L. fermentum ACA-DC 179 regarding the production of a probiotic functional food.

The technological and probiotic potential of lactic acid bacteria isolated from artisanal Greek yoghurt and fermented milks were evaluated. Fifty-three strains were identified by rep-PCR and 16S rDNA sequencing to belong to different Lactobacillus or Enterococcus spp., as well as to Streptococcus thermophilus and Lactococcus lactis. Several strains exhibited promising technological and probiotic properties. Among them, we focused on the production of bioactive peptides with angiotensin-converting enzyme inhibitory (ACE-I) activity during milk fermentation. The majority of strains produced ACE-I peptides when grown in skimmed milk. ACE-I peptides were sometimes sequestered in the original fermented milk sample, but were released and detected following high performance liquid chromatography (HPLC) purification. Mass spectrometry analysis of major peptide peaks in HPLC fractions with ACE-I activity revealed that they derived from the N- or C-terminal of the isracidin peptide region of αS1-casein and two internal peptide fragments, one from β-casein and one from κ-casein.

Probiotic Feta cheese was produced using Propionibacterium freudenreichii subsp. shermanii as an adjunct since it has been shown to induce apoptosis of colon cancer cells in vitro and in vivo through the production of propionate and acetate. Microbiological and physicochemical analysis of the cheese was performed through the 60 d period of ripening. Counts of propionibacteria increased until day 7 and then remained constant until the end of ripening (approximately 9 log cfu g−1). Moreover, throughout ripening the presence of P. freudenreichii subsp. shermanii LMG 16424T was confirmed by 16S rRNA gene sequence analysis, while propionic acid was first detected on day 7 and reached a concentration of 52.1 m m on day 60. The ripened cheese containing P. freudenreichii subsp. shermanii was very well accepted by the sensory evaluation panellists. This is the first time that P. freudenreichii subsp. shermanii was studied as a probiotic adjunct in a white-brined cheese.

Water-in-oil (W/O) microemulsions based on either refined olive oil (ROO) or sunflower oil (SO), distilled monoglycerides (DMG), and ethanol were used as nisin carriers in order to ensure its effectiveness as a biopreservative. This work presents experimental evidence on the effects of ethanol concentration, hydration, the nature of oil, and the addition of nisin on the nanostructure of the proposed inverse microemulsions as revealed by electrical conductivity measurements, dynamic light scattering (DLS), small angle X-ray scattering (SAXS), and electron paramagnetic resonance (EPR) spectroscopy. Modeling of representative SAXS profiles was applied to gain further insight into the effects of ethanol and solubilized water content on the inverse swollen micelles' size and morphology. With increasing ethanol content, the overall size of the inverse micelles decreased, whereas hydration resulted in an increase in the micellar size due to the penetration of water into the hydrophilic core of the inverse swollen micelles (hydration-induced swelling behavior). The dynamic properties of the surfactant monolayer were also affected by the nature of the used vegetable oil, the ethanol content, and the presence of the bioactive molecule, as evidenced by EPR spin probing experiments. According to simulation on the experimental spectra, two populations of spin probes at different polarities were revealed. The antimicrobial effect of the encapsulated nisin was evaluated using the well diffusion assay (WDA) technique against Lactococccus lactis. It was found that this encapsulated bacteriocin induced an inhibition of the microorganism growth. The effect was more pronounced at higher ethanol concentrations, but no significant difference was observed between the two used vegetable oils (ROO and SO).

Artisanal cheeses, in particular those prepared from raw milk, are the most commonly used ecosystems to mine microorganisms with both technological and human-health related potential. In the present study, the microbiota of two Greek artisanal cheeses, the PDO Kalathaki and the non-PDO Melichloro, was explored via classical microbiological analysis. Lactic acid bacteria isolated mainly belonged to the genus Enterococcus (66%) and to a lesser degree to Pediococcus (15%), Leuconostoc (8%), Lactobacillus (6%) and Lactococcus (5%). From the 112 initial isolates, 32 were selected and studied for their probiotic potential, i.e. safety traits, survival under gastrointestinal tract conditions, antimicrobial activity, adhesion, angiotensin I-converting enzyme inhibitory activity and immunomodulation of human monocytes. Two isolates, namely Lactobacillus brevis ACA-DC 1705 and Leuconostoc mesenteroides ACA-DC 1738, exhibited the most promising probiotic potential due to strong angiotensin I-converting enzyme inhibitory activity and anti-inflammatory modulation of immune cells, respectively, and, thus they could be used as adjuncts in novel functional products.

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.

The hypothesis of this research paper was that the physicochemical conditions in a low-pH, high-moisture white brined cheese such as Feta would make para-κ-casein vulnerable to residual chymosin activity during ripening and storage. It was important to address this hypothesis, since cheese para-κ-casein could theoretically be used for the assessment of the origin of cheese milk by means of various analytical methods. Feta cheese was manufactured from sheep milk and from four different mixtures of sheep and goat milk in triplicate. The para-κ-casein of Feta samples taken during 120 d of ripening and storage was estimated by means of cation-exchange HPLC and proteolysis was determined in terms of free amino groups. Despite their similarity, sheep and goat para-κ-casein were efficiently separated and the changes of their chromatographic areas indicated that hydrolysis took place during the first stage of ripening. In accordance to the evolution of free amino groups, para-κ-caseins remained stable thereafter. The hydrolysis pattern was not affected by the composition of the cheese milk mixture and after 120 d at least two thirds of the initial quantity remained intact. Considering the efficient separation of sheep and goat para-κ-caseins and their stability during Feta storage, the same method was used for the evaluation of the percentage of goat milk in the cheese milk. The actual and the estimated percentage of goat milk within the range 0–40 were strongly correlated (R = 0·997, n = 60) and the standard error of estimation was 0·914.

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.

Reverse micelles (RMs) as nanocarriers of nisin were optimized for the highest water and bacteriocin content. RMs formulated with either refined olive oil or sunflower oil, distilled monoglycerides, ethanol, and water were effectively designed. Structural characterization of the RMs was assessed using Electron Paramagnetic Resonance Spectroscopy and Small Angle X-ray Scattering in the presence and absence of nisin. No conformational changes occurred in the presence of nisin for the nanocarriers. To assess efficacy of the loaded systems, their antimicrobial activity against Staphylococcus aureus and Listeria monocytogenes was tested in lettuce leaves and minced meat, respectively. Antimicrobial activity was evident in both cases. Interestingly, a synergistic antimicrobial effect was observed in lettuce leaves and to a lesser extent in minced meat between nisin and some of the nanocarriers' constituents (probably ethanol). Our findings suggest complex interactions that take place when RMs are applied in different food matrices.

Lactobacillus delbrueckii subsp. bulgaricus is widely used in the production of yogurt and cheese. In this study, we present the complete genome sequence of L. delbrueckii subsp. bulgaricus ACA-DC 87 isolated from traditional Greek yogurt. Whole-genome analysis may reveal desirable technological traits of the strain for dairy fermentations.

Lactobacillus delbrueckii subsp. bulgaricus is widely used in the production of yogurt and cheese. In this study, we present the complete genome sequence of L. delbrueckii subsp. bulgaricus ACA-DC 87 isolated from traditional Greek yogurt. Whole-genome analysis may reveal desirable technological traits of the strain for dairy fermentations.

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.

Lactobacillus zymae is a Gram-positive lactic acid bacterium belonging to the Lactobacillus brevis clade. Here, we report the first complete genome sequence of L. zymae ACA-DC 3411, which was isolated from traditional Greek wheat sourdough. Whole-genome analysis may reveal adaptive traits of strain ACA-DC 3411 in the sourdough ecosystem.

Maslinic and oleanolic acids are among the most abundant triterpenic acids found in olive fruits. Both acids are considered to be important bioactive compounds which can confer multiple beneficial health effects to the consumer. In the present work, we have monitored and quantified maslinic and oleanolic acids throughout processing in alkaline-treated green olives (Spanish-style) and in natural green olives of the Conservolea variety that is particularly popular in Greece. Our findings clearly demonstrate that the fast de-bittering process with NaOH treatment in Spanish-style olives has a profound negative effect on the concentration of both acids. This decrease of concentration was more prominent regarding maslinic acid when compared to oleanolic acid. In contrast, the slow de-bittering during natural fermentation of green olives had no effect on the content of maslinic or oleanolic acid. To verify the broad applicability of our observation we have also looked into the natural processing of the Kalamon variety (Greek-style). Our findings were consistent, since once again, natural fermentation did not influence the concentration of both acids.

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.

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.

Milk, a basic component of the human diet, is rich in all kinds of nutrients, such as proteins, carbohydrates, lipids, vitamins, and minerals. These compounds comprise an ideal growth substrate for both harmful and beneficial microorganisms, making milk a perishable raw material. As a result, milk contains numerous microorganisms that originate either from the animal itself, even when the animal is clinically healthy, or from the environment during the collection and storage of milk. The increasing social, economic, and research on non-cow types of milk and its products illustrates the renewed interest in the microbiota associated with these matrices. The present work is an overview of existing knowledge on the microbiota of sheep, goat, buffalo, camel, equine, yak, and human milk and their products. Relevant studies concerning both classical microbiological approaches as well as advanced molecular methods are included.

Streptococcus macedonicus ACA-DC 198 produces the bacteriocin macedocin in milk only under low NaCl concentrations (

Over the past decades the food industry has been revolutionized towards 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.

Streptococcus thermophilus ACA-DC 2 is a newly sequenced strain isolated from traditional Greek yogurt. Among the 14 fully sequenced strains of S. thermophilus currently deposited in the NCBI database, the ACA-DC 2 strain has the smallest chromosome, containing 1,731,838 bp. The annotation of its genome revealed the presence of 1,850 genes, including 1,556 protein-coding genes, 70 RNA genes and 224 potential pseudogenes. A large number of pseudogenes were identified. This was also accompanied by the absence of pathogenic features suggesting evolution of strain ACA-DC 2 through genome decay processes, most probably due to adaptation to the milk ecosystem. Analysis revealed the existence of one complete lactose-galactose operon, several proteolytic enzymes, one exopolysaccharide cluster, stress response genes and four putative antimicrobial peptides. Interestingly, one CRISPR-cas system and one orphan CRISPR, both carrying only one spacer, were predicted indicating low activity or inactivation of the cas proteins. Nevertheless, four putative restriction-modification systems were determined that may compensate any deficiencies of the CRISPR-cas system. Furthermore, whole genome phylogeny indicated three distinct clades within S. thermophilus. Comparative analysis among selected strains representative for each clade, including strain ACA-DC 2, revealed a high degree of conservation at the genomic scale, but also strain specific regions. Unique genes and genomic islands of strain ACA-DC 2 contained a number of genes potentially acquired through horizontal gene transfer events, that could be related to important technological properties for dairy starters. Our study suggests genomic traits in strain ACA-DC 2 compatible to the production of dairy fermented foods.