Genomic and functional evaluation of exopolysaccharide produced by Liquorilactobacillus mali t6-52: technological implications.

BACKGROUND: This study explores the biosynthesis, characteristics, and functional properties of exopolysaccharide produced by the strain Liquorilactobacillus mali T6-52. The strain demonstrated significant EPS production with a non-ropy phenotype. RESULTS: The genomic analysis unveiled genes associated with EPS biosynthesis, shedding light on the mechanism behind EPS production. These genes suggest a robust EPS production mechanism, providing insights into the strain's adaptability and ecological niche. Chemical composition analysis identified the EPS as a homopolysaccharide primarily composed of glucose, confirming its dextran nature. Furthermore, it demonstrated notable functional properties, including antioxidant activity, fat absorption capacity, and emulsifying activity. Moreover, the EPS displayed promising cryoprotective activities, showing notable performance comparable to standard cryoprotective agents. The EPS concentration also demonstrated significant freeze-drying protective effects, presenting it as a potential alternative cryoprotectant for bacterial storage. CONCLUSIONS: The functional properties of L. mali T6-52 EPS reveal promising opportunities across various industrial domains. The strain's safety profile, antioxidant prowess, and exceptional cryoprotective and freeze-drying characteristics position it as an asset in food processing and pharmaceuticals.

A critical issue on microbiological cut-off value of ampicillin resistance in Lactiplantibacillus plantarum.

AIM: Comprehensive evaluation of antibiotic susceptibility patterns in Lactiplantibacillus plantarum strains isolated from grape marc, based on genomic and phenotypic assessment. METHODS AND RESULTS: We assessed the antibiotic resistance-susceptibility patterns of 20 L. plantarum strains for 16 antibiotics. Genomes of relevant strains were sequenced for in silico assessment and comparative genomic analysis. Results showed high MIC values for spectinomycin, vancomycin, and carbenicillin, indicating natural resistance to these antibiotics. Besides, these strains revealed MIC values for ampicillin higher than previously established by the EFSA, indicating the possible presence of acquired resistance genes in the genomes. However, genomic analysis by complete genome sequencing did not reveal presence of ampicillin resistance genes. CONCLUSION: Comparative genomic analysis between our strains and other L. plantarum genomes present in the literature showed several substantial genomic differences, and suggested the need to adjust the cut-off value for ampicillin in L. plantarum. However, further sequence analysis will reveal how these strains have acquired antibiotic resistance.

New insights into the raw milk microbiota diversity from animals with a different genetic predisposition for feed efficiency and resilience to mastitis.

The main objective of this study was to assess the microbiota diversity in milk samples collected from Holstein cows with different estimated breeding values for predicted feed efficiency, milk coagulation, resilience to mastitis, and consequently, to study its effects on milk quality. One hundred and twenty milk samples were collected in two seasons (summer and winter) from different commercial dairy farms in the Nord-east of Italy. For each trait, 20 animals divided into two groups of the high (10 cows) and the low (10 cows) were selected to study the microbiota profile using 16S rRNA metabarcoding sequencing. The alpha and beta diversity analysis revealed significant differences between the high and the low groups for feed efficiency and resilience to mastitis, while no significant difference was detected for milk coagulation. Moreover, remarkable differences among the taxa were detected between the two seasons, where the winter was more diverse than summer when applied the Chao1 index. Lastly, the linear discriminant analysis (LDA) effect size (LEfSe) indicated Aerococcus, Corynebacterium, Facklamia, and Psychrobacter taxa with more abundance in the high group of feed efficiency, whereas, in resilience to mastitis, only two genera of Mycoplana and Rhodococcus were more abundant in the low group. In addition, LEfSe analysis between the seasons showed significant differences in the abundance of Bacteroides, Lactobacillus, Corynebacterium, Escherichia, Citrobacter, Pantoea, Pseudomonas, and Stenotrophomonas. These findings indicate that the different genetic predisposition for feed efficiency and resilience to mastitis could affect the raw milk microbiota and, consequently, its quality. Moreover, we found more abundance of mastitis-associated bacteria in the milk of dairy cows with a higher feed efficiency index.

Limosilactobacillus fermentum ING8, a Potential Multifunctional Non-Starter Strain with Relevant Technological Properties and Antimicrobial Activity.

Lactic acid bacteria (LAB) have gained particular attention among different exopolysaccharide-producing microorganisms due to their safety status and effects on human health and food production. Exopolysaccharide-producing LAB play a crucial role in different ways, such as improving texture, mouthfeel, controlling viscosity, and for low-calorie food production. In this study, we isolated a multifunctional strain with good exopolysaccharide production properties. Limosilactobacillus fermentum ING8 was isolated from an Indian traditional fermented milk (Dahi) and evaluated for its safety, enzymatic activity, NaCl resistance and temperature tolerance, milk coagulation, and storage stability. Finally, the complete genome of this strain was sequenced and subjected to safety in silico evaluation and genomic analysis. The results revealed that L. fermentum ING8 possesses relevant technological properties, such as exopolysaccharide production, antimicrobial activity, and galactose utilization. Besides, this strain showed very high stability to storage conditions at refrigeration temperature. In addition, the genomic analysis did not evidence any possible deleterious elements, such as acquired antibiotic resistance genes, virulence genes, or hemolysis-related genes. However, all structural genes related to the galactose operon and EPS production were detected. Therefore, L. fermentum ING8 can be considered a promising multifunctional bacterium to be proposed as non-starter in different types of dairy productions.

Genomic and Phenotypic Evaluation of Potential Probiotic Pediococcus Strains with Hypocholesterolemic Effect Isolated from Traditional Fermented Food.

The use of probiotic microorganisms in food with the aim to confer health benefits to the host is one of the most critical roles of functional foods. Many pediococci bacteria frequently related to the meat environment, have technological properties, and are therefore commercially used as starter in the production of fermented meat products, such as different types of sausages. In this study, different lactic acid bacteria were isolated, identified to the species level, and then evaluated for their safety and functionality as possible probiotics. Different properties, such as resistance to simulated human gastrointestinal conditions, antimicrobial activity, and cholesterol-lowering effects, have been studied. Finally, the complete genome of one strain, namely P. acidilactici IRZ12B, which showed interesting features as a promising probiotic candidate, was sequenced and further studied. The results revealed that IRZ12B possesses interesting probiotic properties, particularly cholesterol-lowering capability and antimicrobial activity. In silico analysis evidenced the absence of plasmids, transmissible antibiotic resistance genes, and virulence factors. We also detected a bacteriocin encoding gene and a cholesterol assimilation-related protein. The phenotypical and genomic outcomes described in this study make P. acidilactici IRZ12B a very interesting cholesterol-lowering potential probiotic strain to be considered for the development of novel non-dairy-based functional foods.

Thermal resistance and high-performance microwave decontamination assessment of Bacillus endospores isolated from food-grade herbal extracts.

Generally, endospore contamination can occur from different sources during product manufacturing in many industries and therefore lower its quality by affecting physicochemical properties and shelf-life. Bacterial endospores can germinate inside the product and produce several enzymes, which can cause several undesirable changes. This study assessed the spores thermal resistance and applied a microwave decontamination technique toward herbal extracts (Tilia tomentosa and Centella asiatica) containing ethanol or glycerol. Based on 16S rRNA analysis, the detected contaminant endospores belonged to different Bacillus species, namely B. subtilis, B. zhangzhouensis, and B. pumilus. The thermal resistance assessment using inoculated endospores in the actual products revealed B. pumilus T2 as the most resistant endospore to the heat treatments tested in both T. tomentosa and C. asiatica extracts. Finally, a high-performance microwave technique was used to decontaminate T. tomentosa extract against the mixture of Bacillus spores. Results from the microwave technique indicate that the increase of temperature from 100°C to 105°C not only decontaminated the product but also could dramatically decrease the effective thermal treatment time (10 times), which can benefit the product quality. The results provided in this study considerably contribute to improving an original decontamination method for products containing glycerol and ethanol with the most negligible effect on product quality.

Chemoprevention of DMH-Induced Early Colon Carcinogenesis in Male BALB/c Mice by Administration of Lactobacillus Paracasei DTA81.

We evaluated the effects of the probiotic candidate Lactobacillus paracasei DTA81 (DTA81) on liver oxidative stress, colonic cytokine profile, and gut microbiota in mice with induced early colon carcinogenesis (CRC) by 1,2-dimethylhydrazine (DMH). Animals were divided into four different groups (n = 6) and received the following treatments via orogastric gavage for 8 weeks: Group skim milk (GSM): 300 mg/freeze-dried skim milk/day; Group L. paracasei DTA81 (DTA81): 3 × 109 colony-forming units (CFU)/day; Group Lactobacillus rhamnosus GG (LGG): 3 × 109 CFU/day; Group non-intervention (GNI): 0.1 mL/water/day. A single DMH dose (20 mg/kg body weight) was injected intraperitoneally (i.p), weekly, in all animals (seven applications in total). At the end of the experimental period, DTA81 intake reduced hepatic levels of carbonyl protein and malondialdehyde (MDA). Moreover, low levels of the pro-inflammatory cytokines Interleukin-6 (IL-6) and IL-17, as well as a reduced expression level of the proliferating cell nuclear antigen (PCNA) were observed in colonic homogenates. Lastly, animals who received DTA81 showed an intestinal enrichment of the genus Ruminiclostridium and increased concentrations of caecal acetic acid and total short-chain fatty acids. In conclusion, this study indicates that the administration of the probiotic candidate DTA81 can have beneficial effects on the initial stages of CRC development.

Microbial Diversity and Nutritional Properties of Persian "Yellow Curd" (Kashk Zard), a Promising Functional Fermented Food.

"Yellow curd" (YC) is one of the most popular homemade Persian fermented foods and is consumed by many people. Notwithstanding, no studies are available to date on its nutritional and microbiological composition. In this study, we examined YC samples obtained from different local markets of Sistan and Baluchestan province, Iran. The results of the chemical analyses revealed a homogenous content of protein (13.71% ± 1.07), lipids (4.09% ± 0.73), and carbohydrates (61% ± 2.13) among the samples. By comparing the average mineral content of YC with yogurt, many relevant differences were detected. Apart from the calcium content, which was similar on average to that of YC, all other minerals tested are present in higher amounts in YC than in yogurt. The analysis of the main sugars present (i.e., lactose, galactose and glucose) highlighted relevant differences among samples, indicating that different YC samples contain natural strains with different capabilities to metabolize sugars. The concentration of galactose in YC samples should be taken into consideration by galactose intolerant people. From the microbiological perspective, the metagenomics analysis revealed that lactic acid bacteria, and particularly the genera Lactobacillus, Pediococcus, and Streptococcus, were dominant in YC. The information provided shows that YC is an interesting base for the preparation of novel functional foods with a good content of beneficial bacteria.

Complete Genome Sequence and Carbohydrates-Active EnZymes (CAZymes) Analysis of Lactobacillus paracasei DTA72, a Potential Probiotic Strain with Strong Capability to Use Inulin.

The whole genome sequence of Lactobacillus paracasei DTA72, isolated from healthy infant feces, is reported, along with the Carbohydrates-Active enZymes (CAZymes) analysis and an in silico safety assessment. Strain DTA72 had previously demonstrated some interesting potential probiotic features, such as a good resistance to gastrointestinal conditions and an anti-Listeria activity. The 3.1 Mb sequenced genome consists of 3116 protein-coding sequences distributed on 340 SEED subsystems. In the present study, we analyzed the fermentation capability of strain DTA72 on six different carbohydrate sources, namely, glucose, fructose, lactose, galactose, xylose, and inulin by using phenotypical and genomic approaches. Interestingly, L. paracasei DTA72 evidenced the best growth performances on inulin with a much shorter lag phase and higher number of cells at the stationary phase in comparison with all the sugars tested. The CAZyme analysis using the predicted amino acid sequences detected 80 enzymes, distributed into the five CAZymes classes. Moreover, the in silico analysis revealed the absence of blood hemolytic genes, transmissible antibiotic resistances, and plasmids in DTA72. The results described in this study, together with those previously reported and particularly the strong capability to utilize inulin as energy source, make DTA72 a very interesting potential probiotic strain to be considered for the production of synbiotic foods. The complete genome data have been deposited in GenBank under the accession number WUJH00000000.

Whole-genome sequence and comparative genome analysis of Lactobacillus paracasei DTA93, a promising probiotic lactic acid bacterium.

The complete genome sequence of Lactobacillus paracasei DTA93, isolated from healthy infant feces is reported, along with in silico genetic analyses of its main features. The 3.02 Mb sequenced genome possesses 2990 protein-coding sequences distributed on 341 SEED subsystems. In previous in vitro studies, this strain demonstrated interesting probiotic properties, anti-cancer activity, and anti-bacterial activity. The whole-genome sequencing allowed to identify DTA93 as L. paracasei and to precisely place it within the L. casei group, since it shows the highest number of orthologous genes/proteins in common with the type strain L. paracasei ATCC 25302T. In silico analyses revealed the absence of potentially negative traits, such as presence of prophages, transmissible antibiotic resistance and virulence factors. The results provided here considerably increased the amount of information available on DTA93 in favor of its possible use in food products as health-promoting culture. The complete genome data have been deposited in GenBank under the accession number VTYT00000000.

Genomic and phenotypic assessments of safety and probiotic properties of Streptococcus macedonicus strains of dairy origin.

In this work, we studied the genomes and characterized some probiotic features of four S. macedonicus strains isolated from dairy environments in Italy that already had indicated some technological potential. The genomes of these strains were sequenced and used for genomic in silico studies. All strains were also evaluated for hemolytic activity, susceptibility to most commonly used antibiotics and probiotic potential, such as resistance to simulated gastrointestinal conditions, bile salts hydrolytic activity and adhesion ability to HT-29 human colorectal adenocarcinoma cells. Results revealed that one strain, namely S. macedonicus 211MA, was found to possess probiotic properties, such as resistance to simulated gastrointestinal conditions as well as adherence capability to human epithelial cells. In silico analyses revealed that S. macedonicus 211MA displayed the least number of single copy genes, genomic islands regions and gene content classified as virulence factors when compared to other S. macedonicus and S. gallolyticus strains. Moreover, the maximum gene content associated with bacterial stress response category and the presence of the opuCABCD operon, not detected in the other strains, were correlated with S. macedonicus 211MA capability to resist to low pH and to show higher adhesion to HT-29 human cells. This is the first report on the presence of opuCABCD operon in S. macedonicus and its possible relation with attachment ability and stress response.

Safety and Stability of Two Potentially Probiotic Lactobacillus Strains After In Vitro Gastrointestinal Transit.

According to FAO and WHO, probiotics are defined as live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. Most probiotic bacteria used today belong to the genera Lactobacillus and Bifidobacterium and are of animal or human origin. The fundamental characteristic routinely evaluated in potential probiotics strains is their limited viability loss during gastrointestinal transit (GIT), but to date, no studies reported whether probiotics, besides viability, still also maintain their beneficial properties intact. To study this aspect, we considered two strains, Lactobacillus rhamnosus DTA 79 and L. paracasei DTA 83, previously characterised for the presence of some probiotic properties, isolated from faeces of 7- to 21-day-old babies. Here, we examined some additional properties, namely antibiotic resistance, resistance to lysozyme, presence of haemolytic activity and inhibition of pathogen biofilm formation. We then tested the effect of in vitro GIT on all these features and our results show evidence that this procedure had in some cases limited and in others no significant effects on them. Additionally, we examined the gastrointestinal resistance of the strains after skim milk fermentation and successive storage of the product for 20 and 40 days at refrigeration temperature, to see whether prolonged storage could weaken cell resistance to GIT. Our results demonstrate that a protracted refrigeration period before in vitro GIT did not affect or influenced very weakly this essential probiotic property.

Draft genome sequence data of Lactobacillus paracasei strain DTA83 isolated from infant stools.

Here the draft genome sequence of Lactobacillus paracasei strain DTA83, isolated from stools of healthy infants in Rio de Janeiro (Brazil), is reported. The 2.8-Mb genome possesses 2825 protein-coding sequences distributed on 330 SEED subsystems. This strain belongs to a set of potentially probiotic Lactobacillus spp. strains used to study genetic factors related to antibiotic resistance after stress conditions, such as simulated gastrointestinal conditions. The complete genome data have been deposited in GenBank under the accession number QRBH00000000, https://www.ncbi.nlm.nih.gov/nuccore/QRBH00000000.

A Cryptic Non-Inducible Prophage Confers Phage-Immunity on the Streptococcus thermophilus M17PTZA496.

Streptococcus thermophilus is considered one of the most important species for the dairy industry. Due to their diffusion in dairy environments, bacteriophages can represent a threat to this widely used bacterial species. Despite the presence of a CRISPR-Cas system in the S. thermophilus genome, some lysogenic strains harbor cryptic prophages that can increase the phage-host resistance defense. This characteristic was identified in the dairy strain S. thermophilus M17PTZA496, which contains two integrated prophages 51.8 and 28.3 Kb long, respectively. In the present study, defense mechanisms, such as a lipoprotein-encoding gene and Siphovirus Gp157, the last associated to the presence of a noncoding viral DNA element, were identified in the prophage M17PTZA496 genome. The ability to overexpress genes involved in these defense mechanisms under specific stressful conditions, such as phage attack, has been demonstrated. Despite the addition of increasing amounts of Mitomycin C, M17PTZA496 was found to be non-inducible. However, the transcriptional activity of the phage terminase large subunit was detected in the presence of the antagonist phage vB_SthS-VA460 and of Mitomycin C. The discovery of an additional immune mechanism, associated with bacteriophage-insensitive strains, is of utmost importance, for technological applications and industrial processes. To our knowledge, this is the first study reporting the capability of a prophage integrated into the S. thermophilus genome expressing different phage defense mechanisms. Bacteriophages are widespread entities that constantly threaten starter cultures in the dairy industry. In cheese and yogurt manufacturing, the lysis of Streptococcus thermophilus cultures by viral attacks can lead to huge economic losses. Nowadays S. thermophilus is considered a well-stablished model organism for the study of natural adaptive immunity (CRISPR-Cas) against phage and plasmids, however, the identification of novel bacteriophage-resistance mechanisms, in this species, is strongly desirable. Here, we demonstrated that the presence of a non-inducible prophage confers phage-immunity to an S. thermophilus strain, by the presence of ltp and a viral noncoding region. S. thermophilus M17PTZA496 arises as an unconventional model to study phage resistance and potentially represents an alternative starter strain for dairy productions.

In vitro Probiotic Potential and Anti-cancer Activity of Newly Isolated Folate-Producing Streptococcus thermophilus Strains.

Most probiotic strains commercially available today are lactic acid bacteria. Within this functional group, Streptococcus thermophilus is a thermophilic species widely used as starter culture for a huge number of dairy products. Besides being rapid acidifiers, many S. thermophilus strains are able to produce and release folate during growth but, unfortunately, they are seriously impaired during passage through the human gastrointestinal tract. In this work, we studied eight S. thermophilus strains isolated from dairy environments in Italy, which already had shown good technological properties, to evaluate their possible probiotic potential and cytotoxicity against cancer cells in vitro. All strains were also evaluated for some health-related properties such as susceptibility to most common antibiotics, hemolytic activity, resistance to simulated gastrointestinal conditions, bile salts hydrolytic activity, production of folate, adhesion to HT-29 human colorectal adenocarcinoma cells and cytotoxic activity against cancer cells and production of biogenic amines. Results revealed that two fast acidifying S. thermophilus strains were found to possess in vitro probiotic properties along with anticancer activity and production of folate. These properties resulted similar and, in some cases, superior to those of Lactobacillus rhamnosus GG, a well-known commercial probiotic strain. These findings encourage further in vivo studies to evaluate the actual health benefits of these strains on the human host.

Comparative Transcriptomic Analysis of Streptococcus thermophilus TH1436 and TH1477 Showing Different Capability in the Use of Galactose.

Streptococcus thermophilus is a species widely used in the dairy industry for its capability to rapidly ferment lactose and lower the pH. The capability to use galactose produced from lactose hydrolysis is strain dependent and most of commercial S. thermophilus strains are galactose-negative (Gal-), although galactose-positive (Gal+) would be more technologically advantageous because this feature could provide additional metabolic products and prevent galactose accumulation in foods. In this study, a next generation sequencing transcriptome approach was used to compare for the first time a Gal+ and a Gal- strain to characterize their whole metabolism and shed light on their different properties, metabolic performance and gene regulation. Transcriptome analysis revealed that all genes of the gal operon were expressed very differently in Gal+ and in the Gal- strains. The expression of several genes involved in mixed acid fermentation, PTS sugars transporter and stress response were found enhanced in Gal+. Conversely, genes related to amino acids, proteins metabolism and CRISPR associated proteins were under-expressed. In addition, the strains showed a diverse series of predicted genes controlled by the transcriptional factor catabolite control protein A (CcpA). Overall, transcriptomic analysis suggests that the Gal+ strain underwent a metabolic remodeling to cope with the changed environmental conditions.

Short communication: Comparison of growth kinetics at different temperatures of Streptococcus macedonicus and Streptococcus thermophilus strains of dairy origin.

Within the genus Streptococcus, S. thermophilus and S. macedonicus are the 2 known species related to foods. Streptococci are widely used as starter cultures to rapidly lower milk pH. As S. macedonicus has been introduced quite recently, much less information is available on its technological potential. Because temperature is an important factor in fermented food production, we compared the growth kinetics over 24 h of 8 S. thermophilus and 7 S. macedonicus strains isolated from various dairy environments in Italy, at 4 temperatures, 30°C, 34°C, 37°C and 42°C. We used the Gompertz model to estimate the 3 main growth parameters; namely, lag phase duration (λ), maximum growth rate (µmax), and maximum cell number at the stationary phase (Nmax). Our results showed significant differences in average growth kinetics between the 2 species. Among the strains tested, 37°C appeared to be the optimal temperature for the growth of both species, particularly for S. macedonicus strains, which showed mean shorter lag phases and higher cell numbers compared with S. thermophilus. Overall, the growth curves of S. macedonicus strains were more similar to each other whereas S. thermophilus strains grew very differently. These results help to better define and compare technological characteristics of the 2 species, in view of the potential use of S. macedonicus in place of S. thermophilus in selected technological applications.

Differences in Carbohydrates Utilization and Antibiotic Resistance Between Streptococcus macedonicus and Streptococcus thermophilus Strains Isolated from Dairy Products in Italy.

Streptococcus thermophilus and S. macedonicus are the only two species of the genus related to food productions so far known. In the present study, eight S. thermophilus and seven S. macedonicus strains isolated from dairy environments in Italy were compared in order to evidence possible species-specific technological characteristics. Their capability to use lactose, galactose, fructose, and glucose, sugars commonly present in foods and two carbohydrates considered as prebiotics, xylose and inulin, along with the respective growth kinetics were studied. Results showed a luxuriant growth on lactose and different behaviors on galactose, glucose, and fructose. No growth on inulin and xylose was recorded, which is a positive feature for strains intended to be used as starter cultures. Growth parameters, namely, λ, µmax, and Nmax, were estimated by using the Gompertz model. Antibiotic resistance to 14 drugs revealed an overall similar behavior between the two species with only a marked difference regarding gentamycin. Antimicrobial activity was also tested against six deleterious bacterial strains, but none of the strains evidenced inhibitory capabilities. The results presented here could be helpful to compare technological potentialities of the two species and to choose strains of the most suitable species for selected microbiological food transformations.

Draft Genome Sequences of Three Virulent Streptococcus thermophilus Bacteriophages Isolated from the Dairy Environment in the Veneto Region of Italy.

Streptococcus thermophilus, a very important dairy species, is constantly threatened by phage infection. We report the genome sequences of three S. thermophilus bacteriophages isolated from a dairy environment in the Veneto region of Italy. These sequences will be used for the development of new strategies to detect and control phages in dairy environments.