miRNAs: possible regulators of toll like receptors and inflammatory tumor microenvironment in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is ranked as the third most commonly diagnosed cancer and the third cause of cancer related deaths. CRC is greatly attributed to genetic and epigenetic mutations and immune dysregulation. Tumor aberrant expression of Toll-like Receptors (TLRs) can contribute to tumorigenesis. Recent studies suggested that microRNAs act as direct ligands of TLRs altering their expression and signaling pathways. AIM: To prove our concept that specific miRNA mimics may act as antagonists of their specific toll like receptors inhibiting their expression that could limit the release of pro-inflammatory and pro-tumorigenic cytokines leading to apoptosis of tumor cells. METHODS: From public microarray databases, we retrieved TLRs and miRNAs related to CRC followed by in silico docking of the selected miRNA ligands into the TLRs. Clinical validation after co-immunoprecipitation of TLRs and their interacting miRNA ligands was done. Expression of TLRs 1, 7,8 was determined by ELISA while miRNAs was measured by RT-qPCR. In addition, microRNA mimics of the down regulated miRNAs were transfected into human CRC cell lines. RESULTS: Our data demonstrate that TLRs 1, 7, 8 are up regulated in CRC compared to controls. Further, three miRNAs (-122, -29b and -15b) are relatively downregulated, while 4 miRNAs (-202, miRNA-98, -21 and -let7i) are upregulated in CRC patients compared to those with benign tumor and healthy controls. Transfection of down regulated miRNA mimics into CRC cell lines resulted in a significant reduction of the number and viability of cells as well as down regulating the expression of TLRs 1, 7 and 8 with ultimate reduction of downstream effector IL6 protein, suggesting that these miRNAs are negative regulators of carcinogenesis. CONCLUSION: MicroRNAs could act as antagonistic ligands of TLRs limiting the inflammatory tumor microenvironment.

Real-time PCR methods for identification and stability monitoring of Bifidobacterium longum subsp. longum UABl-14 during shelf life.

Bifidobacterium longum subsp. longum UABl-14™ is an important probiotic strain that was found to support digestive health. Here we present the development and validation of real-time PCR methods for strain-specific identification and enumeration of this important strain. The identification method was evaluated for specificity using 22 target samples and 30 non-target samples. All target samples successfully amplified, while no amplification was observed from any non-target samples including other B. longum strains. The identification method was evaluated for sensitivity using three DNA dilution series and the limit of detection was 2 pg. of DNA. Coupled with a viability dye, the method was further validated for quantitative use to enumerate viable cells of UABl-14. The viability dye treatment (PMAxx) was optimized, and a final concentration of 50 µM was found as an effective concentration to inactivate DNA in dead cells from reacting in PCR. The reaction efficiency, linear dynamic range, repeatability, and reproducibility were also evaluated. The reaction efficiency was determined to be 97.2, 95.2, and 95.0% with R2 values of 99%, in three replicates. The linear dynamic range was 1.3 × 102 to 1.3 × 105 genomes. The relative standard deviation (RSD%) for repeatability ranged from 0.03 to 2.80, and for reproducibility ranged from 0.04 to 2.18. The ability of the validated enumeration method to monitor cell counts during shelf life was evaluated by determining the viable counts and total counts of strain UABl-14 in 18 multi-strain finished products. The viable counts were lower than label claims in seven products tested post-expiration and were higher than label claims in products tested pre-expiration, with a slight decrease in viable counts below label claim in three samples that were tested 2-3 months pre-expiration. Interestingly, the total counts of strain UABl-14 were consistently higher than label claims in all 18 products. Thus, the method enables strain-specific stability monitoring in finished products during shelf life, which can be difficult or impossible to achieve using the standard plate count method. The validated methods allow for simultaneous and cost-effective identification and enumeration of strain UABl-14 and represent an advancement in the quality control and quality assurance of probiotics.

The power of DNA based methods in probiotic authentication.

Introduction: The global probiotic market is growing rapidly, and strict quality control measures are required to ensure probiotic product efficacy and safety. Quality assurance of probiotic products involve confirming the presence of specific probiotic strains, determining the viable cell counts, and confirming the absence of contaminant strains. Third-party evaluation of probiotic quality and label accuracy is recommended for probiotic manufacturers. Following this recommendation, multiple batches of a top selling multi-strain probiotic product were evaluated for label accuracy. Methods: A total of 55 samples (five multi-strain finished products and 50 single-strain raw ingredients) containing a total of 100 probiotic strains were evaluated using a combination of molecular methods including targeted PCR, non-targeted amplicon-based High Throughput Sequencing (HTS), and non-targeted Shotgun Metagenomic Sequencing (SMS). Results: Targeted testing using species-specific or strain-specific PCR methods confirmed the identity of all strains/species. While 40 strains were identified to strain level, 60 strains were identified to species level only due to lack of strain-specific identification methods. In amplicon based HTS, two variable regions of 16S rRNA gene were targeted. Based on V5-V8 region data, ~99% of total reads per sample corresponded to target species, and no undeclared species were detected. Based on V3-V4 region data, ~95%-97% of total reads per sample corresponded to target species, while ~2%-3% of reads matched undeclared species (Proteus species), however, attempts to culture Proteus confirmed that all batches were free from viable Proteus species. Reads from SMS assembled to the genomes of all 10 target strains in all five batches of the finished product. Discussion: While targeted methods enable quick and accurate identification of target taxa in probiotic products, non-targeted methods enable the identification of all species in a product including undeclared species, with the caveats of complexity, high cost, and long time to result.

Probiotic and postbiotic analytical methods: a perspective of available enumeration techniques.

Probiotics are the largest non-herbal/traditional dietary supplements category worldwide. To be effective, a probiotic strain must be delivered viable at an adequate dose proven to deliver a health benefit. The objective of this article is to provide an overview of the various technologies available for probiotic enumeration, including a general description of each technology, their advantages and limitations, and their potential for the future of the probiotics industry. The current "gold standard" for analytical quantification of probiotics in the probiotic industry is the Plate Count method (PC). PC measures the bacterial cell's ability to proliferate into detectable colonies, thus PC relies on cultivability as a measure of viability. Although viability has widely been measured by cultivability, there has been agreement that the definition of viability is not limited to cultivability. For example, bacterial cells may exist in a state known as viable but not culturable (VBNC) where the cells lose cultivability but can maintain some of the characteristics of viable cells as well as probiotic properties. This led to questioning the association between viability and cultivability and the accuracy of PC in enumerating all the viable cells in probiotic products. PC has always been an estimate of the number of viable cells and not a true cell count. Additionally, newer probiotic categories such as Next Generation Probiotics (NGPs) are difficult to culture in routine laboratories as NGPs are often strict anaerobes with extreme sensitivity to atmospheric oxygen. Thus, accurate quantification using culture-based techniques will be complicated. Another emerging category of biotics is postbiotics, which are inanimate microorganisms, also often referred to as tyndallized or heat-killed bacteria. Obviously, culture dependent methods are not suitable for these products, and alternative methods are needed for their quantification. Different methodologies provide a more complete picture of a heterogeneous bacterial population versus PC focusing exclusively on the eventual multiplication of the cells. Alternative culture-independent techniques including real-time PCR, digital PCR and flow cytometry are discussed. These methods can measure viability beyond cultivability (i.e., by measuring cellular enzymatic activity, membrane integrity or membrane potential), and depending on how they are designed they can achieve strain-specific enumeration.

Ameliorative effect of Lactobacillus rhamnosus GG on acetaminophen-induced hepatotoxicity via PKC/Nrf2/PGC-1α pathway.

BACKGROUND: Acetaminophen (APAP) overdose is a common cause of hepatotoxicity. Antioxidants like N-acetyl cysteine are recommended as a therapeutic option; nevertheless, it has limitations. The search for efficient alternatives is ongoing. Probiotics are live microorganisms that maintain a healthy gut microecology. Lactobacillus rhamnosus GG (LGG) is one of the widely used probiotics. Our study aimed to assess the protective and therapeutic effects of probiotic LGG on APAP-induced hepatotoxicity and evaluate the molecular pathways behind this effect. METHODS: Wistar Albino male rats were randomly distributed into the following experimental groups: group 1, non-treated rats (vehicle); group 2, rats received oral gavage of suspension of probiotic LGG (5 × 1010 CFU GG/0.5 ml in PBS) daily for 2 weeks (probiotic control); group 3, rats received APAP dose of 2 g/kg body weight (positive control); group 4, rats received oral gavage of suspension of probiotic LGG for 2 weeks followed by a single dose of APAP injection (prophylactic); and group 5, rats received a single dose of APAP and then 24 h later treated with oral gavage of probiotic LGG daily for 2 weeks (treatment). RESULTS: Our study revealed that administration of probiotic LGG (either as prophylactic or treatment) exhibited a remarkable reduction in APAP-induced liver injury as resembled by the decrease in liver enzymes (ALT and AST) and the histopathological features of liver sections. Moreover, the significant reduction in the oxidative marker malondialdehyde, along with the enhancement in glutathione reductase, and the significant reduction in inflammatory markers (nitric oxide and tumor necrosis factor-α) were all indicators of the efficiency of LGG in ameliorating the alterations accompanied with APAP-induced hepatotoxicity. Our findings also demonstrate that LGG administration boosted the expression of Nrf2 and PGC-1 while decreasing the expression of protein kinase C (PKC). As a result, the nuclear abundance of Nrf2 is increased, and the expression of various antioxidants is eventually upregulated. CONCLUSION: Our study shows that probiotic LGG supplementation exerts a prophylactic and therapeutic effect against APAP-induced hepatotoxicity through modulating the expression of PKC and the Nrf2/PGC-1α signaling pathway and eventually suppressing oxidative damage from APAP overdose.

Evaluation of serum levels of interleukins 1-beta, 10 and 12 in patients with acne vulgaris.

INTRODUCTION: One of the most prevalent skin disorders is acne vulgaris. It is a distressing problem among young adults and adolescents. The pathophysiology of AV is significantly influenced by certain immune response cells. Acne's localized tissue inflammatory reactions are brought on by the bacteria Cutibacterium acnes, which also causes the release of several inflammatory mediators. AIM OF THE WORK: To measure the serum levels of IL-1 and IL-12, two pro-inflammatory cytokines, and IL-10, an anti-inflammatory cytokine, in AV patients and link those levels with the severity of the condition. RESULTS: This study included 19 males and 23 females with AV(n = 42), with mean age of 21.79 ± 3.5. The control group included 19 males and 23 female (n = 42) with mean age of 22.05 ± 3.3 (p = 0.729). Serum levels of IL-1ß was significantly higher in patients with AV as than controls (p < 0.001). Similarly, the serum levels of IL-12 were significantly higher in AV cases than controls (p < 0.001). On the contrary, the median IL-10 was significantly lower in AV cases than controls (p = 0.015). The correlation between serum levels of IL-1ß and IL-12 in AV patients and disease severity was insignificant (r = -0.04, p = 0.404) and (r = -0.19, p = 0.118, respectively). (r = -0.19, p = 0.118) On the contrary, there was a significant negative correlation between serum levels of IL-10 in AV patients and disease severity (r = -0.43, p = 0.003). CONCLUSION: Our results indicate significant alterations in the monocyte-derived cytokine (MDCs) profile in patients with AV, IL-1 and IL-12 serum levels rose while IL-10 levels fell, reflecting an increase in pro-inflammatory cytokines and a decrease in anti-inflammatory cytokines. These highlight the important role played by monocytes in the pathogenesis of AV.

Real-time polymerase chain reaction methods for strain specific identification and enumeration of strain Lacticaseibacillus paracasei 8700:2.

Introduction: Reliable and accurate methods for probiotic identification and enumeration, at the strain level plays a major role in confirming product efficacy since probiotic health benefits are strain-specific and dose-dependent. In this study, real-time PCR methods were developed for strain specific identification and enumeration of L. paracasei 8700:2, a probiotic strain that plays a role in fighting the common cold. Methods: The assay was designed to target a unique region in L. paracasei 8700:2 genome sequence to achieve strain level specificity. The identification assay was evaluated for specificity and sensitivity. The enumeration viability real-time PCR (v-qPCR) method was first optimized for the viability treatment, then the method was evaluated for efficiency, limit of quantification, precision, and its performance was compared to plate count (PC) and viability droplet digital PCR (v-ddPCR) methods. Results: The identification method proved to be strain specific and highly sensitive with a limit of detection of 0.5 pg of DNA. The optimal viability dye (PMAxx) concentration was 50 µM. The method was efficient (> 90% with R 2 values > 0.99), with a linear dynamic range between 6*102 and 6*105 copies. The method was highly precise with a relative standard deviation below 5%. The Pearson correlation coefficient (r) was 0.707 for PC and v-qPCR methods, and 0.922 for v-qPCR and v-ddPCR. Bland-Altman method comparison showed that v-qPCR always gave higher values compared to PC method (relative difference ranging from 119% to 184%) and showed no consistent trend (relative difference ranging from -20% to 22%) when comparing v-qPCR and v-ddPCR methods. Discussion: The difference between PC and v-PCR methods can potentially be attributed to the proportion of cells that exist in a viable but non culturable (VBNC) state, which can be count by v-PCR but not with PC. The developed v-qPCR method was confirmed to be strain specific, sensitive, efficient, with low variance, able to count VBNC cells, and has shorter time to results compared to plate count methods. Thus, the identification and enumeration methods developed for L. paracasei 8700:2 will be of great importance to achieve high quality and efficacious probiotic products.

Exosomal expression of RAB27A and its related lncRNA Lnc-RNA-RP11-510M2 in lung cancer.

OBJECTIVES: Examine the diagnostic role of serum exosomal RAB27A mRNA in lung cancer and evaluate the relation of LncRNAs to lung cancer in association to RAB27A mRNA in Egyptian population. METHODS: Exosomal RNA-based biomarkers RAB27A mRNA and Lnc-RNA-RP11-510M2.10 were selected based on bioinformatic methods, followed by RT-qPCR validation of their expression in serum of 20 patients with lung cancer, 10 patients with COPD and 10 healthy volunteers. we examined their expression in 10 bronchoalveolar lavage samples and assessed correlation with the serum levels. RESULTS: There was an inverse relationship between expression of serum exosomal RAB27A mRNA and Lnc-RNA-RP11-510M2.10 (r = -0.62, p = .00). Both serum exosomal RAB27A mRNA and Lnc-RNA-RP11-510M2.10 showed a significant positive and negative association with lung cancer patients respectively in comparison to patients with COPD and healthy persons (p < .001). CONCLUSION: RAB27A mRNA and Lnc-RNA-RP11-510M2.10 could be used as diagnostic and prognostic biomarker tools for lung cancer.

Enumeration of Probiotic Strain Lacticaseibacillus rhamnosus GG (ATCC 53103) Using Viability Real-time PCR.

Probiotic health benefits are strain specific and are dose dependent. Hence, administering the correct strains, at the recommended doses is essential to achieve probiotic health benefits. Reliable methods are needed to facilitate probiotic strain identification and enumeration. Plate count methods are the most commonly used methods for probiotic enumeration. However, these methods are time-consuming, laborious, highly variable, and non-specific. Here, we developed a real-time PCR method for enumeration of a commonly used strain, Lacticaseibacillus rhamnosus GG. The method utilizes PMAxx as a viability dye to enumerate viable cells only. Optimization of viability treatment showed that PMAxx at a final concentration of 50 µM was effective in inactivating DNA from dead cells, and that bead beating for 5 min at 3000 rpm was effective in liberating DNA. The assay demonstrated high efficiency between 93 and 102%, with R2 values > 0.99. The assay showed high precision with relative standard deviation (RSD%) below 2.3%. Assay performance was compared to a plate count method in which there was a strong correlation between both methods (Pearson r = 0.8443). This method offers a 10 × shorter time for results and a higher precision compared to plate count methods. Furthermore, this method enables specific enumeration of L. rhamnosus GG in multi-strain products, which is not possible to achieve using plate count methods. This novel method facilitates faster and more accurate enumeration of L. rhamnosus GG as a raw ingredient as well as in finished products which enables better quality assurance and efficacy of probiotics for consumers.

Niche specificity and functional diversity of the bacterial communities associated with Ginkgo biloba and Panax quinquefolius.

Plant-associated bacteria can establish mutualistic relationships with plants to support plant health. Plant tissues represent heterogeneous niches with distinct characteristics and may thus host distinct microbial populations. The objectives of this study are to investigate the bacterial communities associated with two medicinally and commercially important plant species; Ginkgo biloba and Panax quinquefolius using high Throughput Sequencing (HTS) of 16S rRNA gene, and to evaluate the extent of heterogeneity in bacterial communities associated with different plant niches. Alpha diversity showed that number of operational taxonomic units (OTUs) varied significantly by tissue type. Beta diversity revealed that the composition of bacterial communities varied between tissue types. In Ginkgo biloba and Panax quinquefolius, 13% and 49% of OTUs, respectively, were ubiquitous in leaf, stem and root. Proteobacteria, Bacteroidetes, Actinobacteria and Acidobacteria were the most abundant phyla in Ginkgo biloba while Proteobacteria, Bacteroidetes, Actinobacteria, Plantomycetes and Acidobacteria were the most abundant phyla in Panax quinquefolius. Functional prediction of these bacterial communities using MicrobiomeAnalyst revealed 5843 and 6251 KEGG orthologs in Ginkgo biloba and Panax quinquefolius, respectively. A number of these KEGG pathways were predicted at significantly different levels between tissues. These findings demonstrate the heterogeneity, niche specificity and functional diversity of plant-associated bacteria.

Locked Nucleic Acid Hydrolysis Probes for the Specific Identification of Probiotic Strains Bifidobacterium animalis subsp. lactis DSM 15954 and Bi-07™.

Probiotic health benefits are now well-recognized to be strain specific. Probiotic strain characterization and identification is thus important in clinical research and in the probiotic industry. This is becoming especially important with reports of probiotic products failing to meet the declared strain content, potentially compromising their efficacy. Availability of reliable identification methods is essential for strain authentication during discovery, evaluation and commercialization of a probiotic strain. This study aims to develop identification methods for strains Bifidobacterium animalis subsp. lactis DSM 15954 and Bi-07 (Bi-07™) based on real-time PCR, targeting single nucleotide polymorphisms (SNPs). The SNPs were targeted by PCR assays with locked nucleic acid (LNA) probes, which is a novel application in probiotic identification. The assays were then validated following the guidelines for validating qualitative real-time PCR assays. Each assay was evaluated for specificity against 22 non-target strains including closely related Bifidobacterium animalis subsp. lactis strains and were found to achieve 100% true positive and 0% false positive rates. To determine reaction sensitivity and efficiency, three standard curves were established for each strain. Reaction efficiency values were 86, 91, and 90% (R square values > 0.99), and 87, 84, and 86% (R square values > 0.98) for B. animalis subsp. lactis DSM 15954 and Bi-07 assays, respectively. The limit of detection (LOD) was 5.0 picograms and 0.5 picograms of DNA for DSM 15954 and Bi-07 assays, respectively. Each assay was evaluated for accuracy using five samples tested at three different DNA concentrations and both assays proved to be highly repeatable and reproducible. Standard deviation of Cq values between two replicates was always below 1.38 and below 1.68 for DSM 15954 and Bi-07 assays, respectively. The assays proved to be applicable to mono-strain and multi-strain samples as well as for samples in various matrices of foods or dietary supplement ingredients. Overall, the methods demonstrated high specificity, sensitivity, efficiency and precision and broad applicability to sample, matrix and machine types. These methods facilitate strain level identification of the highly monophyletic strains B. animalis subsp. lactis DSM 15954 and Bi-07 to ensure probiotic efficacy and provide a strategy to identify other closely related probiotics organisms.

Real-Time PCR Assays for the Specific Identification of Probiotic Strains Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC (NCIMB 30242).

The broad spectrum of health benefits attributed to probiotics has contributed to a rapid increase in the value of the probiotic market. Probiotic health benefits can be strain specific. Thus, strain-level identification of probiotic strains is of paramount importance to ensure probiotic efficacy. Both Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC (NCIMB 30242) strains have clinically proven health benefits; however, no assays were developed to enable strain-level identification of either of these strains. The objective of this study is to develop strain-specific PCR-based methods for Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC strains, and to validate these assays according to the guidelines for validating qualitative real-time PCR assays. Using RAST (Rapid Annotation using Subsystem Technology), unique sequence regions were identified in the genome sequences of both strains. Probe-based assays were designed and validated for specificity, sensitivity, efficiency, repeatability, and reproducibility. Both assays were specific to target strain with 100% true positive and 0% false positive rates. Reaction efficiency for both assays was in the range of 90 to 108% with R square values > 0.99. Repeatability and reproducibility were evaluated using five samples at three DNA concentrations each and relative standard deviation was < 4% for repeatability and < 8% for reproducibility. Both of the assays developed and validated in this study for the specific identification of Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC strains are specific, sensitive, and precise. These assays can be applied to evaluate and ensure compliance in probiotic products.

Draft Genome Sequences of Lactobacillus delbrueckii subsp. bulgaricus Strains CBC-LB69 and CBC-LB8, Isolated from Homemade Dairy Foods in Bulgaria.

Here, we report the draft genome sequences of Lactobacillus delbrueckii subsp. bulgaricus strains CBC-LB69 and CBC-LB8. The strains were isolated from naturally processed, homemade dairy foods in Bulgaria. The two genome assemblies each resulted in 39 contigs with total lengths of 1,752,493 and 1,759,908 bp and GC contents of 49.80% and 49.90%, respectively.

Characterization of the microbiota of commercially traded finfish fillets.

The profile of human gut microbiota is known to be affected by diet and is linked to human health. Seafood is a highly consumed food and it accounts for a large proportion of food-borne illness. The objective of this study is to characterise the microbiota of fish fillets of various species sold in the Canadian market. We test 19 fish fillet samples from nine species in five fish families, ten of which were previously determined to be mislabeled as different species. The microbiota profiles were characterized using 16S rRNA gene high-throughput sequencing. Despite the complexities of the supply chain to produce these fillets, the major microbial groups were fairly consistent across samples. Significant differences in microbial taxa were observed between species, families, and based on labelling accuracy. Several putative spoilage and putative pathogenic taxa were identified. Studying food-associated microbiota can provide comprehensive information on food safety, authenticity, and traceability.

A Validated Real-Time PCR Method for the Specific Identification of Probiotic Strain Lactobacillus rhamnosus GG (ATCC 53103).

BACKGROUND: Strain Lactobacillus rhamnosus GG is one of the best-studied and most widely used probiotic strains, with various health benefits. Because probiotic health benefits and safety are strain specific, the availability of a reliable assay for specific identification of Lactobacillus rhamnosus GG is vital to ensure probiotic efficacy. OBJECTIVE: To design and validate a probe-based real-time PCR assay for specific identification of strain Lactobacillus rhamnosus GG. METHOD: Rapid Annotation using Subsystem Technology (RAST) was used to find a unique sequence region in the genome of Lactobacillus rhamnosus GG. A probe-based assay was designed and evaluated for specificity, sensitivity, efficiency, repeatability, and reproducibility. RESULTS: RAST identified a unique gene coding for a hypothetical protein in the genome of Lactobacillus rhamnosus GG. The assay successfully amplified all 22 target samples and did not amplify any of the 28 non-target strains, achieving 100% true positive and 0% false positive results. The Limit of Detection (LOD) was determined to be 0.001 ng. Reaction efficiency values, from three dilution series, were 96.4%, 93.3%, and 96.8% with R square values of 0.9974, 0.9981, and 0.9998, respectively. Relative standard deviation (RSD, %) of repeatability was below 1% and RSD of reproducibility was below 4%. CONCLUSIONS: This Lactobacillus rhamnosus GG specific assay proved to be specific, sensitive, efficient, and reproducible. Since the assay was evaluated on two real-time PCR platforms, including a portable one, the assay can be used for onsite testing throughout the supply chain. HIGHLIGHTS: The availability of validated and reliable assays for strain-specific identification plays a vital role in achieving compliance in probiotic products.

Draft Genome Sequence of Lactobacillus rhamnosus Strain CBC-LR1, Isolated from Homemade Dairy Foods in Bulgaria.

Here, we report the draft genome sequence of Lactobacillus rhamnosus strain CBC-LR1, which was isolated from naturally processed, homemade dairy foods in Bulgaria. The genome was assembled in 29 contigs with a total length of 2,892,155 bp and a GC content of 46.7%. Genome annotation predicted 2,638 coding genes and 49 tRNA genes.

Draft Genome Sequence of Streptococcus thermophilus Strain CBC-S77, Isolated from Homemade Dairy Foods in Bulgaria.

Here, we report the draft genome sequence of Streptococcus thermophilus strain CBC-S77. The strain was originally isolated from naturally processed, homemade dairy foods in West Rhode Mountain, Bulgaria. The genome was assembled in 148 contigs with a total length of 1,707,130 bp, with 1,563 coding genes and a GC content of 39.11%.

Combined Targeted and Non-targeted PCR Based Methods Reveal High Levels of Compliance in Probiotic Products Sold as Dietary Supplements in United States and Canada.

Probiotics are defined as "live microorganisms that, when administered in adequate amounts, confer a health benefit on the host." The diverse health benefits have contributed to rapid increase in probiotic consumption and in the value of probiotic market, valued at USD 46 billion in 2019. For probiotics to be effective, the correct species/strains should be delivered viable in an adequate dose. The most commonly used methods for species/strain identification are DNA based methods including targeted and non-targeted methods (e.g., high-throughput sequencing, HTS). Using different DNA based methods, previous studies reported several cases of non-compliance in probiotic products. The objectives of this study are to evaluate levels of compliance in probiotic products (presence of all declared species/strains, absence of any contaminants or undeclared species, and meeting the declared minimum viable cell count) and to compare the performance of targeted and non-targeted methods in probiotic authentication. To the best of our knowledge, this is the largest study of its kind, testing 182 probiotic products, containing a total of 520 strains, collected from United States and Canada. Using species-specific assays, 11 species could not be detected in ten products. Missing species were Lactobacillus casei in seven products, Bifidobacterium longum and Bifidobacterium bifidum in one product, B. longum in one product while B. longum subsp. longum was mislabeled as B. longum subsp. infantis in another. Additionally, undeclared Bifidobacterium animalis subsp. lactis was detected in one product. Viable count was determined for 72 samples and was found to be lower than declared in five samples, including one product showing no viable cells. Overall, non-compliance was observed in 15 out of 182 products (8%). Additionally, undeclared species at relative abundance of ∼1-2% were found in 14 products using HTS, however, their presence could not be confirmed using species-specific assays. The results show that targeted PCR based methods enable species and strain level identification. The results also highlight the need to continue to develop strain-specific assays appropriate for use with multi-strain products. True strain-specific assays will enable strain authentication in both single-strain products and multi-strain products to ensure probiotic products meet the label claims and ensure probiotic efficacy.

Investigating appropriate molecular and chemical methods for ingredient identity testing of plant-based protein powder dietary supplements.

Plant-based protein powders are rapidly growing in popularity, and outdated quality assurance tools expose vulnerabilities to adulteration via different methods of "protein spiking". Adequate diagnostic tools are urgently needed to be able to authenticate protein source ingredients and screen for potential adulterants. We explored the application of three diagnostic tools for ingredient identification: targeted PCR with Sanger sequencing, NGS, and LC-MS/MS. We collected 33 samples of common commercial products from the plant-based protein powder market and sought to identify botanical components using the three technologies. We found success in detection with all approaches, with at least one main protein source being identified by at least one approach in all samples. The investigation uncovered challenges to data collection or result interpretation with each technology including but not limited to amplification biases with PCR technologies, potential influence of DNA degradation, and issues with protein solubility during isolation. Ultimately, each platform demonstrated utility along with certain caveats, which epitomized the importance of orthogonality of testing.

Re-visiting the occurrence of undeclared species in sausage products sold in Canada.

Meat and poultry are major protein sources for humans worldwide. Undeclared ingredients in processed meat products, like sausage, continue to be identified in retail products all over the world. In collaboration with the Canadian Food Inspection Agency, a previous study of products purchased in Canada showed 20% mislabelling rate in sausage meats when tested for beef, pork, chicken, turkey and horse using DNA barcoding and digital PCR. In a follow-up to this study, an additional 100 "single species" sausage products were collected from Canadian retail markets, one year after our earlier study, to determine the prevalence of undeclared meat species in sausage. A new hierarchy of complementary molecular methods was applied in this study, including the testing of new target species (sheep and goat), in addition to beef, pork, chicken, turkey and horse. First, all samples were tested using DNA barcoding using universal primers, which revealed that 97% of the samples contained the declared species, presumably as the predominant species. Second, all samples were tested using ddPCR assays specifically targeting beef, pork, chicken, and turkey, which revealed that five beef samples, three chicken samples and two turkey samples contained undeclared species. Additionally, ddPCR revealed the presence of undeclared sheep in five samples. Overall, using complementary molecular methods, 14% of the samples contained additional undeclared species. It was encouraging to find a reduced rate of mislabelling compared to the previous study, though it remains clear that meat mislabelling is still an issue affecting Canadian consumers. The results from this study can be used to support decision-making processes for future inspection and monitoring activities in order to control species substitution or adulteration to protect consumers.