Analysis and entrapment of select antioxidants from chokecherry and Saskatoon berry fruits

The major objectives of this research were to produce a phenolic rich isolate from two locally grown Saskatchewan fruits, chokecherries and saskatoons, develop an encapsulation system for the phenolic isolate, and test this system for the delivery of the phenolic isolate in an animal (rat) model. Natural phenolic compounds present in plants such as fruits have antioxidant and free radical scavenging activities, which have been proposed to have health benefits. The extraction of these compounds from plants is commonly performed using methanol despite being toxic to both humans and animals. As such, ethanol was investigated for its ability to extract phenolics from plants as a food safe alternative to methanol. Phenolic extraction from chokecherries with ethanol:formic acid:water (EFW) resulted in higher concentrations (9.83 mg gallic acid equivalents (GAE)/g fresh weight) than with methanol:formic acid:water (MFW) (7.97 mg GAE/g fresh weight). Results from saskatoons showed similar phenolic levels of 4.26 and 4.21 mg GAE/g fresh weight with MFW and ethanol (EFW), respectively. These results showed that EFW was a suitable substitute for MFW in phenolic compound isolation from chokecherries and saskatoons, and could be used to produce extracts that were safe for use in foods and feeds. High performance liquid chromatography with photodiode array detection (HPLC-PDA) was used to determine the phenolic compound composition of the raw fruits and their phenolic rich isolates. Chlorogenic acid was identified in both chokecherry and saskatoon samples, and rutin was also shown to be present in saskatoons. These identifications were based on the relative retention time and ultra violet-visual spectra comparisons to standards. Solid phase extraction (SPE) using Amberlite XAD-16 was employed to produce phenolic isolates from chokecherries and saskatoons. HPLC-PDA results determined that there was a ~2.7x and ~1.6x increase in peak area for chokecherries and saskatoons, respectively when SPE was employed. The antioxidant activity of the extracts and isolates was determined using in vitro radical scavenging tests including 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2´-azinobis-3-ethylbenzthiazoline-sulphonic acid (ABTS). The EFW chokecherry extract and isolate had the highest overall free radical scavenging activity. Crude fruit extracts exhibited lower free radical scavenging values compared to the isolate samples in both of the assays performed. The fruit phenolic isolates were encapsulated in chitosan (CH) sodium tripolyphosphate (TPP) nanoparticles at a ratio of 4.0:1.0 (CH:TPP). HPLC-PDA was used to determine the entrapment efficiency of phenolic isolates to be 15.9 ± 2.7% and 23.0 ± 7.1% for chokecherries and saskatoons, respectively. Characteristics such as the size, surface potential and phenolic release were determined for the two fruit isolate containing nanoparticles. The size of the nanoparticles were 527.90 ± 74.57 nm and 443.03 ± 15.79 nm for chokecherries and saskatoons, respectively. Both of the nanoparticle systems had positive surface charges at 52.70 ± 2.93 mV and 54.43 ± 1.27 mV for chokecherries and saskatoons, respectively. The release properties of the CH:TPP nanoparticles containing fruit phenolics were examined in enzymatic simulated intestinal fluid and resulted in ~23% and ~28% release of chokecherry and saskatoon phenolics, respectively. Saskatoon phenolic isolates and isolates encapsulated in CH:TPP were gavage fed to rats (six animals in each of the two groups) at a dosage rate of 276.36 ± 9.74 mg/kg body weight. The saskatoon isolate contained 12.44 ± 0.44 mg/kg body weight anthocyanins (~3.30 mg anthocyanin per rat). These animals were sacrificed after 1 h and all stomach tissue samples in each of the treatment groups contained detectable levels of anthocyanins. In the small intestine tissues all six of the saskatoon isolate and three of the encapsulated isolate groups had detectable amounts of anthocyanins, while in the large intestine tissue, only one sample from the isolate group showed detectable amounts of anthocyanins. Although other tissues were tested (brain, heart, kidney and liver), anthocyanins were not detected. Therefore anthocyanins were detected in the gastrointestinal tract of both of the treatment groups. The research performed therefore illustrated that phenolic compounds can be extracted from fruit sources using EFW and can be successfully encapsulated in chitosan tripolyphosphate capsules allowing for targeted delivery in an animal model

Hiding in plain sight: discovery and phylogeography of a cryptic species of Trichinella (Nematoda: Trichinellidae) in wolverine (Gulo gulo)

Understanding parasite diversity and distribution is essential in managing the potential impact of para- sitic diseases in animals and people. Imperfect diagnostic methods, however, may conceal cryptic species. Here, we report the discovery and phylogeography of a previously unrecognized species of Trichinella in wolverine (Gulo gulo) from northwestern Canada that was indistinguishable from T. nativa using the stan- dard multiplex PCR assay based on the expansion segment 5 (ESV) of ribosomal DNA. The novel genotype, designated as T13, was discovered when sequencing the mitochondrial genome. Phylogenetic analyses of the mitochondrial genome and of 15 concatenated single-copy orthologs of nuclear DNA indicated a com- mon ancestor for the encapsulated clade is shared by a subclade containing Trichinella spiralis and Trichinella nelsoni, and a subclade containing T13 and remaining taxa: T12 + (T2 + T6) + [(T5 + T9) + (T 3 + T8)]. Of 95 individual hosts from 12 species of mammalian carnivores from northwestern Canada from which larvae were identified as T. nativa on multiplex PCR, only wolverines were infected with T13 (14 of 42 individuals). These infections were single or mixed with T. nativa and/or T6. Visual examination and motility testing confirmed that T13 is encapsulated and likely freeze-tolerant. We developed a new Polymerase Chain Reaction-Restriction Fragment Length Polymorphism which unequivocally distinguishes between T13 and T. nativa. We propose Trichinella chanchalensis n. sp. for T13, based on significant genetic divergence from other species of Trichinella and broad-based sampling of the Trichinella genome. Exploration of Alaskan and Siberian isolates may contribute to further resolution of a phylogeo- graphically complex history for species of Trichinella across Beringia, including Trichinella chanchalensis n. sp. (T13

Identification of Trichinella taxa by ITS-1 amplicon next-generation sequencing with an improved resolution for detecting underrepresented genotypes in mixed natural infections

Abstract Background Amplicon-based next-generation sequencing (NGS) has rapidly gained popularity as a powerful method for delineating taxa in complex communities, including helminths. Here, we applied this approach to identify species and genotypes of zoonotic nematodes of the Trichinella genus. A known limitation of the current multiplex PCR (mPCR) assay recommended by the International Commission on Trichinellosis is that it does not differentiate Trichinella nativa from T. chanchalensis. Methods The new assay entails deep sequencing of an amplified variable fragment of the ribosomal cistron's (rDNA) internal transcribed spacer 1 using the Illumina platform. The assay was evaluated using first-stage larvae (L1) of select laboratory strains of various Trichinella taxa mixed in known proportions and then validated using archived L1 from 109 wildlife hosts. The species/genotypes of these L1 isolates from wildlife were previously determined using mPCR. Results NGS data analysis for Trichinella laboratory strains selected as representative of North American fauna revealed a sequence representation bias. Trichinella pseudospiralis, a non-encapsulated species, was the most underrepresented when mixed with T. spiralis, T. murrelli, T. nativa and Trichinella T6 in equal quantities. However, five L1 of T. pseudospiralis were readily revealed by NGS in a mix with 2000 L1 of T. nativa (1:400 ratio). From naturally infected wildlife, all Trichinella taxa revealed by mPCR were also identified by NGS in 103 of 107 (96.3%) samples amplified on both assays. NGS identified additional taxa in 11 (10.3%) samples, whereas additional taxa were revealed by mPCR in only four (3.7%) samples. Most isolates comprised single or mixed infections of T. nativa and Trichinella T6. On NGS, T. chanchalensis (T13) was detected in combination with Trichinella T6 in a wolverine (Gulo gulo) and in combination with T. nativa and Trichinella T6 in a marten (Martes americana) from the Northwest Territories, Canada. Conclusions This new NGS assay demonstrates strong potential as a single assay for identifying all recognised Trichinella taxa as well as improved sensitivity for detecting under-represented and novel genotypes in mixed infections. In addition, we report a new host record for T. chanchalensis in American marten. Graphical Abstrac

Hiding in plain sight: discovery and phylogeography of a cryptic species of Trichinella (Nematoda: Trichinellidae) in wolverine (Gulo gulo)

Understanding parasite diversity and distribution is essential in managing the potential impact of para- sitic diseases in animals and people. Imperfect diagnostic methods, however, may conceal cryptic species. Here, we report the discovery and phylogeography of a previously unrecognized species of Trichinella in wolverine (Gulo gulo) from northwestern Canada that was indistinguishable from T. nativa using the stan- dard multiplex PCR assay based on the expansion segment 5 (ESV) of ribosomal DNA. The novel genotype, designated as T13, was discovered when sequencing the mitochondrial genome. Phylogenetic analyses of the mitochondrial genome and of 15 concatenated single-copy orthologs of nuclear DNA indicated a com- mon ancestor for the encapsulated clade is shared by a subclade containing Trichinella spiralis and Trichinella nelsoni, and a subclade containing T13 and remaining taxa: T12 + (T2 + T6) + [(T5 + T9) + (T 3 + T8)]. Of 95 individual hosts from 12 species of mammalian carnivores from northwestern Canada from which larvae were identified as T. nativa on multiplex PCR, only wolverines were infected with T13 (14 of 42 individuals). These infections were single or mixed with T. nativa and/or T6. Visual examination and motility testing confirmed that T13 is encapsulated and likely freeze-tolerant. We developed a new Polymerase Chain Reaction-Restriction Fragment Length Polymorphism which unequivocally distinguishes between T13 and T. nativa. We propose Trichinella chanchalensis n. sp. for T13, based on significant genetic divergence from other species of Trichinella and broad-based sampling of the Trichinella genome. Exploration of Alaskan and Siberian isolates may contribute to further resolution of a phylogeo- graphically complex history for species of Trichinella across Beringia, including Trichinella chanchalensis n. sp. (T13