Nuclear Magnetic Resonance Fingerprints and Mini DNA Markers for the Authentication of Cinnamon Species Ingredients Used in Food and Natural Health Products.

Cinnamomum verum (syn C. zeylanicum) is considered 'true' cinnamon. However, it is reported that less expensive sources of cinnamon from C. cassia (syn C. aromaticum), C. loureiroi, and C. burmannii (toxic coumarin) may be used in the place of C. verum. We lack the quality assurance tools that are required to differentiate C. verum from other cinnamon species when verifying that the correct species is sourced from ingredient suppliers. The current research on cinnamon species authentication using DNA tools is limited to a few species and the use of high-quality DNA extracted from raw leaf materials. The cinnamon bark traded in the supply chain contains much less DNA and poorer-quality DNA than leaves. Our research advances DNA methods to authenticate cinnamon, as we utilized full-length chloroplast genomes via a genome skimming approach for C. burmannii and C. cassia to facilitate the design of optimal mini DNA markers. Furthermore, we developed and validated the use of NMR fingerprints for several commercial cinnamon species, including the quantification of 16 molecules. NMR fingerprints provided additional data that were useful for quality assessment in cinnamon extract powders and product consistency. Both the new mini DNA markers and NMR fingerprints were tested on commercial cinnamon products.

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.

Identification of circular RNAs of Cannabis sativa L. potentially involved in the biosynthesis of cannabinoids.

MAIN CONCLUSION: We identified circRNAs in the Cannabis sativa L. genome and examined their association with 28 cannabinoids in three tissues of C. sativa. Nine circRNAs are potentially involved in the biosynthesis of six cannabinoids. Cannabis sativa L. has been widely used in the production of medicine, textiles, and food for over 2500 years. The main bioactive compounds in C. sativa are cannabinoids, which have multiple important pharmacological actions. Circular RNAs (circRNAs) play essential roles in growth and development, stress resistance, and the biosynthesis of secondary metabolites. However, the circRNAs in C. sativa remain unknown. In this study, to explore the role of circRNAs in cannabinoid biosynthesis, we performed RNA-Seq and metabolomics analysis on the leaves, roots, and stems of C. sativa. We identified 741 overlapping circRNAs by three tools, of which 717, 16, and 8 circRNAs were derived from exonic, intronic, and intergenic, respectively. Functional enrichment analysis indicated that the parental genes (PGs) of circRNAs were enriched in many processes related to biological stress responses. We found that most of the circRNAs showed tissue-specific expression and 65 circRNAs were significantly correlated with their PGs (P < 0.05, |r|≥ 0.5). We also determined 28 cannabinoids by High-performance liquid chromatography-ESI-triple quadrupole-linear ion trap mass spectrometry. Ten circRNAs, including ciR0159, ciR0212, ciR0153, ciR0149, ciR0016, ciR0044, ciR0022, ciR0381, ciR0006, and ciR0025 were found to be associated with six cannabinoids by weighted gene co-expression network analysis. Twenty-nine of 53 candidate circRNAs, including 9 cannabinoids related were validated successfully using PCR amplification and Sanger sequencing. Taken together, all these results would help to enhance our acknowledge of the regulation of circRNAs, and lay the foundation for breeding new C. sativa cultivars with high cannabinoids through manipulating circRNAs.

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.

Development of Hydrolysis Probe-Based qPCR Assays for Panax ginseng and Panax quinquefolius for Detection of Adulteration in Ginseng Herbal Products.

Authentication of Panax ginseng and Panax quinquefolius products is important to be able to mitigate instances of adulteration and substitution that exist within the international supply chain of ginseng. To address this issue, species-specific hydrolysis probe qPCR assays were developed and validated for both P. ginseng and P. quinquefolius herbal dietary supplements. Performance of the probe-based assays was evaluated using analytical validation criteria, which included evaluation of: (1) specificity, in selectively identifying the target species; (2) sensitivity, in detecting the lowest amount of the target material; and (3) repeatability and reproducibility of the method in detecting the target species in raw materials on a real-time PCR platform (reliability). The species-specific probes were developed and successfully passed the validation criteria with 100% specificity, 80-120% efficiency and 100% reliability. The methods developed in this study are fit for purpose, rapid, and easy to implement in quality assurance programs; authentication of ginseng herbal supplements is possible, even with extracts where DNA is fragmented and of low quality and quantity.

Detection of SAR-CoV-2 on surfaces in food retailers in Ontario.

The COVID-19 pandemic has generated increased interest in potential transmission routes. In food retail settings, transmission from infected customers and workers and customers through surfaces has been deemed plausible. However, limited information exists on the presence and survival of SARS-CoV-2 on surfaces, particularly outside laboratory settings. Therefore, the purpose of this project was to assess the presence of the virus at commonly found surfaces at food retail stores and the potential role that these spaces play in virus transmission. Samples (n=957) were collected twice a week for a month in food-retail stores within Ontario, Canada. High-touch surfaces were identified and surveyed in 4 zones within the store (payment stations, deli counters, refrigerated food section and carts and baskets). The samples were analyzed using a molecular method, i.e., reverse transcriptase quantitative Polymerase Chain Reaction (RT-qPCR). Regardless of the store's location, the sampling day or time, the location of the surface within the store or the surface material, all samples tested negative for SARS-CoV-2. These results suggest that the risk of exposure from contaminated high-touch surfaces within a food retailer store is low if preventive measures and recommended sanitizing routines are maintained.

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.

Validation of a Triplex Quantitative Polymerase Chain Reaction Assay for Detection and Quantification of Traditional Protein Sources, Pisum sativum L. and Glycine max (L.) Merr., in Protein Powder Mixtures.

Several botanicals have been traditionally used as protein sources, including the leguminous Pisum sativum L. and Glycine max (L.) Merr. While a rich history exists of cultivating these plants for their whole, protein-rich grain, modern use as powdered supplements present a new challenge in material authentication. The absence of clear morphological identifiers of an intact plant and the existence of long, complex supply chains behoove industry to create quick, reliable analytical tools to identify the botanical source of a protein product (many of which contain multiple sources). The utility of molecular tools for plant-based protein powder authentication is gaining traction, but few validated tools exist. Multiplex quantitative polymerase chain reaction (qPCR) can provide an economical means by which sources can be identified and relative proportions quantified. We followed established guidelines for the design, optimization, and validation of qPCR assay, and developed a triplex qPCR assay that can amplify and quantify pea and soy DNA targets, normalized by a calibrator. The assay was evaluated for analytical specificity, analytical sensitivity, efficiency, precision, dynamic range, repeatability, and reproducibility. We tested the quantitative ability of the assay using pea and soy DNA mixtures, finding exceptional quantitative linearity for both targets - 0.9983 (p < 0.0001) for soy and 0.9915 (p < 0.0001) for pea. Ratios based on mass of protein powder were also tested, resulting in non-linear patterns in data that suggested the requirement of further sample preparation optimization or algorithmic correction. Variation in fragment size within different lots of commercial protein powder samples was also analyzed, revealing low SD among lots. Ultimately, this study demonstrated the utility of qPCR in the context of protein powder mixtures and highlighted key considerations to take into account for commercial implementation.

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.

Quantification of Actaea racemosa L. (black cohosh) from some of its potential adulterants using qPCR and dPCR methods.

The demand for popular natural health products (NHPs) such as Black Cohosh is increasing considerably, which in turn challenges quality assurance (QA) throughout the supply chain. To detect and quantify the target species present in a given NHP, DNA-based molecular techniques such as Real-time quantitative PCR (qPCR) and digital PCR (dPCR) are standard tools in the food and pathogen testing industries. There is a gap in the literature concerning validated quantitative PCR methods for botanicals that can be utilized for QA and good manufacturing practices. The objective of this study is to develop an efficient quantification method using qPCR and dPCR techniques for the detection and quantification of Actaea racemosa (Black cohosh) NHPs from its potential adulterants. These developed methods are validated for applicability on commercial NHPs. Species-specific hydrolysis probe assays were designed to analyze the black cohosh NHPs using qPCR and dPCR techniques. The results confirmed that the developed qPCR and dPCR methods are highly precise for identifying and quantifying black cohosh NHPs, indicating their potential applicability in future routine industrial and laboratory testing. This enables a single qPCR test to determine not only the presence of a specific botanical, but also the amount when mixed with an adulterant.

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.

Validation and Optimization of qPCR Method for Identification of Actaea racemosa (Black Cohosh) NHPs.

BACKGROUND: Actaea racemosa (black cohosh) herbal dietary supplements are commonly used to treat menopausal symptoms in women. However, there is a considerable risk of contamination of A. racemosa herbal products in the natural health product (NHP) industry, impacting potential efficacy. Authentication of A. racemosa products is challenging because of the standard, multi-part analytical chemistry methods that may be too costly and not appropriate for both raw and finished products. OBJECTIVE: In this paper, we discuss developing and validating quick alternative biotechnology methods to authenticate A. racemosa herbal dietary supplements, based on the use of a species-specific hydrolysis PCR probe assay. METHODS: A qPCR-based species-specific hydrolysis probe assay was designed, validated, and optimized for precisely identifying the species of interest using the following analytical validation criteria: (1) specificity (accuracy) in determining the target species ingredient, while not identifying other non-target species; (2) sensitivity in detecting the smallest amount of the target material; and (3) reliability (repeatability and reproducibility) in detecting the target species in raw materials on a real-time PCR platform. RESULTS: The results show that the species-specific hydrolysis probe assay was successfully developed for the raw materials and powders of A. racemosa. The specificity of the test was 100% to the target species. The efficiency of the assay was observed to be 99%, and the reliability of the assay was 100% for the raw/starting and powder materials. CONCLUSION: The method developed in this study can be used to authenticate and perform qualitative analysis of A. racemosa supplements.

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.

Genome skimming and NMR chemical fingerprinting provide quality assurance biotechnology to validate Sarsaparilla identity and purity.

Sarsaparilla is a popular natural health product (NHP) that has been reported to be one of the most adulterated botanicals in the marketplace. Several plausible explanations are documented including economically motivated product substitution, unintentional errors due to ambiguous trade name associated with several different taxa, and wild harvesting of incorrect non-commercial plants. Unfortunately, this includes the case of an adulterant species Decalepis hamiltonii, a Red listed medicinal plant species by the International Union for Conservation of Nature (IUCN) and declared as a species with high conservation concern by the National Biodiversity Authority of India (NBA). This study provides validated genomic (genome skimming & DNA probes) and metabolomic (NMR chemical fingerprints) biotechnology solutions to prevent adulteration on both raw materials and finished products. This is also the first use of Oxford Nanopore on herbal products enabling the use of genome skimming as a tool for quality assurance within the supply chain of botanical ingredients. The validation of both genomics and metabolomics approach provided quality assurance perspective for both product identity and purity. This research enables manufactures and retailers to verify their supply chain is authentic and that consumers can enjoy safe, healthy products.

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.