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VHHs and other biologics in dry spirulina powder are shelf stable without refrigeration, facilitating distribution, especially into regions lacking high-quality infrastructure. Relative percentage of monomer and dimer peak areas observed under reducing and non-reducing conditions is shown. The concentration of VHH in the spirulina extracts was determined by CEIA. Trends Biotechnol. VHH samples were prepared by dilution of purified protein or spirulina extracts with PBS-TM, and samples in a dilution series were serially diluted with PBS-TM. Untreated water was provided for 1day before C. jejuni challenge. Three-week-old C57BL/6 female mice were housed, five per cage, under standardized conditions (202C, 558% relative humidity, 12/12-h light/dark cycle). CAS Therefore, we developed modular, indoor, 1602,000-l, vertical, flat-panel photobioreactors that were pH controlled and air-mixed. This allowed development of a markerless engineering strategy by replacement of the KmR gene with an exogenous streptomycin resistance gene (aadA), followed by replacement of the exogenous aadA gene with a tandem gene cassette comprising the KmR gene and the gene of interest. Samples were kept on ice throughout. Extended Data Fig. and JavaScript. Our Spirulina Chia supports and promotes the healing of: Roughly 30% of horseswon't eatthe Spirulina Biscuit, so we recommend starting with the loose variety. b, Diagram of primer pairs for PCR genotyping. He would have blow ups constantly making horse shows a difficult time for everyone with trying to manage his allergies. The denatured sample was diluted with 60l of PBS and then digested with 12l of 1mgml1 trypsin and chymotrypsin overnight at 37C. Engineered single-domain antibodies with high protease resistance and thermal stability. To maintain sterility, the following steps were performed in a laminar-flow hood. Cells were washed once with SOT medium at room temperature then resuspended in 2ml of SOT. Data analysis was performed using the Protein Simple Compass software. 3, e201210015 (2012). The vector was transformed into BL-21(DE3) cells and grown overnight at 37C on agar plates with kanamycin, and a single colony was used to inoculate a culture of LB medium containing kanamycin. Its binding site was mapped to the D3 domain of FlaA by phage display of peptides tiled across the FlaA protein (Fig. e, Inhibition of C. jejuni motility by aa682. Keywords: Food and water were withdrawn 30min before treatment, then provided ad libitum. Article Bacterial shedding in stool (CFU10mg1 feces) was measured 7days after challenge. Spirulina is cultivated in open ponds at commercial scale, but uncontrolled exposure to environmental contaminants makes this unsuitable for the manufacture of biopharmaceuticals under FDA cGMP. We will definitely be loyal customers. Natl Acad. Isolation of seed, where plant recombinant proteins are usually expressed, increases apparent potencies but these still fall short of potencies achievable in whole, unfractionated spirulina biomass. adroll_adv_id = "DJ55DRNEPNDMPK2FU4LVTY"; Spirulina was collected at the indicated time points, and full transgene products (MP5+MP6) were amplified from genomic DNA by PCR. J. Mol. Plates were washed three times with 300l of PBS supplemented with 0.05% Tween-20 (PBS-T). Therapeutic cocktails may be transformative for treatment of complex diseases with multiple underlying pathological processes, and for treatment of pathogens commonly exhibiting therapy-evading genetic variation. Extended Data Fig. A 100-l sample of supernatant was carefully pipetted and transferred to either LB or mixed LB/SOT agar plates. Biotinylated MBPFlaA was loaded onto streptavidin biosensors at a loading concentration of 100nM and loading time of 4min. We further report the development of an edible, antibody-based therapeutic targeting gastrointestinal infection by Campylobacter jejuni to illustrate application of the spirulina platform to an important unmet medical need, including validation in animal models of campylobacteriosis and demonstration of its safety and pharmacokinetics in a human phase1 clinical trial. 6e). 24, 11911193 (2006). It has a chemical structure of 14 carbon atoms with no double bonds. Markers for antibiotic resistance in spirulina were cloned between the two homology arms of the plasmid. Glycerol and pigment are subtracted from EE to get TFAee. Uniform expression of GFP in a population of spirulina transformed with a transgene (SP699). Engineering dual-glycan responsive expression systems for tunable production of heterologous proteins in Bacteroides thetaiotaomicron, In situ reprogramming of gut bacteria by oral delivery, Process intensification for the continuous production of an antimicrobial peptide in stably-transformed Sf-9 insect cells, A platform for distributed production of synthetic nitrated proteins in live bacteria, Programmable protein delivery with a bacterial contractile injection system, Targeted and high-throughput gene knockdown in diverse bacteria using synthetic sRNAs, Integrating plant molecular farming and materials research for next-generation vaccines, An anaerobic bacterium host system for heterologous expression of natural product biosynthetic gene clusters, Bacteria as genetically programmable producers of bioactive natural products, https://doi.org/10.1038/s41587-022-01323-0. I cant wait to see the continued benefits this will give my horse. On a weekly basis, cell densities reached ~4gl1 and the biomass was harvested by passage over a series of stainless steel screens. 14, e1007083 (2018). J. Clin. and 37C, back diluted to OD600=0.05 and grown at 37C until cells reached mid-log phase (OD600=0.40.6). Described here are spirulina strains with constitutive VHH expression levels of 0.7, 3.4, 4.0, 4.3, 9.3 and 29% of soluble protein (Fig. 5, 555569 (2007). On day0, mice were given an inoculum of 106live C. jejuni cells (strain 81176, resuspended in PBS) by oral gavage. Product quality specifications, including bioburden and elemental impurities, conformed to industry standards for oral solid-dose therapeutics including United States Pharmacopeia (USP)<1111> and USP<232>. Moreover, the safety of spirulina as an expression host further simplifies the drug manufacturing process. Low productivity and potency in plants not only amplify production costs but may also necessitate expensive downstream processing, which is reported to be a weakness of plant-based production systems11. It is calculated by subtracting plant pigments, esters, and aldehydes from crude fat. 51, 593604 (2011). Lee, E. H., Park, J.-E., Choi, Y.-J., Huh, K.-B. R.T. contributed foundational research. Biotinylated MBPFlaA protein was prepared using an EZ-Link NGS-PEG4-Biotin kit (Thermo Scientific) following the manufacturers guidelines. The study was performed in accordance with ICH guidelines and in compliance with all local and international requirements. Two independent mouse models were used to test the efficacy of spirulina-expressed VHHs in the treatment of C. jejuni infection. Mice were given five doses of a spirulina resuspension before and after challenge. a, Cartoons of multimeric scaffolds with sample expression data for VHHs in spirulina. 10, 720 (2019). Spirulina is a blue green algae derived from freshwater that supports proper immune function. Nature Biotechnology thanks Anurag Rathore and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Not only does Spirulina have incredible immune modulating properties it also supports gastric health through supporting the beneficial bacteria and new research suggests Spirulina has the potential to ameliorate gastric ulcers. Sci. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Spirulina Chia from Stable Feed is a powdered supplement for horses. Wrapp, D. et al. Recombinant aa682 was detected with an anti-His-tag antibody, and data are representative of four independent experiments. High-binding, 96-well plates (Greiner Bio-one or NUNC MaxiSorp) were coated with antigen by the addition of 100l of 15gml1 recombinant protein (FlaA or RBD antigen) in carbonate-bicarbonate buffer (Sigma) to each well and incubation overnight at 4C. What Is Spirulina? Anti-rotavirus protein reduces stool output in infants with diarrhea: a randomized placebo-controlled trial. People were commenting on how beautiful they looked and asked us what we were doing (of course we would share the Stablefeed secret lol). The process was translated to a larger-scale (5kgh1) spray-dryer equipped with a centrifugal atomization system. All proteins contained a C-terminal His-tag. ISSN 1087-0156 (print). Food Sci. performed mass spectrometry experiments. Spirulina cultures were grown for 3days in Innova to optical density (OD700ml1) 0.51.0. Plates were developed using either a SeraCare KPL TMB Microwell Peroxidase Substrate System (Sera Care Life Sciences) or 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Scientific) following the manufacturers recommendations. Functional Composition, Nutritional Properties, and Biological Signup today to be sentnutrition tips, new product alerts, and special discounts! Mice were sacrificed and the cecum was removed, opened longitudinally, carefully separated from cecal content and washed twice with ice-cold PBS. Lyngbya aestuarii BL J was included as an outgroup. After about 8 months, June Bug's sarcoids were eradicated and she is now "healthy as a horse." It has a chemical structure of 16 carbon atoms with no double bonds. Purified aa682 was used to generate a standard curve for binding activity by linear regression using Excel (Microsoft software). Post collection, quality control of powder lots included determination of concentration of the 6x-His-tagged protein using CEIA performed on a Jess system (ProteinSimple). Zimmermann, I., Egloff, P., Hutter, C. & Elife, F. A. Results represent average positional frequency observed in two independent panning experiments. 3). Monomeric (MBP and thioredoxin (TRX)), dimeric (5HVZ), trimeric (cTRP) and heptameric (4B0F) scaffolding proteins were used to multimerize VHHs expressed in spirulina. For more information on Spirulina Chia, clickhere. I started my gelding on the loose spirulina chia this summer. Results represents a single measurement for each strain. StableFeed - "To say my horse has been doing and looking - Facebook 12, 639953 (2021). Modern biotechnology relies on the domestication of cells as biological factories through genetic engineering1,2,3. Drug Discov. 6). Article Yang, W. C., Minkler, D. F., Kshirsagar, R., Ryll, T. & Huang, Y.-M. Google Scholar. United States pharmacopeia safety evaluation of spirulina. PubMed Central The stability and activity of biomass-encapsulated aa682 after exposure to low-pH, simulated gastric buffer was assessed by CEIA and ELISA binding assay. Photosynthetic spirulina is the only microorganism that is commercially farmed worldwide as a food. Consistent with these observations, the 15genes associated with the pathway for natural competence in cyanobacteria are present as complete open reading frames in most available Arthrospira genomes (Extended Data Fig. Savarino, S. J. et al. Nat. It has 18 carbon atoms and a double bond in trans configuartion (between carbons 9 and 10). The polypeptide also contains an N-terminal solubility enhancer MBP fusion and C-terminal 6X-his affinity tag. Spirulina powder as a feed supplement to enhance abalone growth promoting an anti-histamine response to a variety of environmental irritants. Google Scholar. EXTREMELY EFFECTIVE and palatable to most horses. The toxicology profile has been positively reviewed by the FDA, and clinical trials have established its safety in both adult45,46 and pediatric populations47. A single 400-l dose of spray-dried spirulina resuspended in PBS was administered by oral gavage to mice 1.5h before infection with C. jejuni 81176 (108CFU200l1 PBS). Dried spirulina biomass of SP1182 was resuspended in simulated gastric buffer (pH3.0, no pepsin) and the presence of aa682 in buffer was analyzed (A). b. Ribbon representation of a VHH (orange) with a dimerization motif (blue; PDB ID: 5HVZ)56 and the solubility enhancer, MBP (green). L.G., J.D., N.S., D.F., A.M., B.K. Methods Med. Yet, to date little is known about the dynamic changes of Spirulina under real production in this . 60, 6370 (2010). Only available in the loose format. Treatment with SP526 reduced campylobacter fecal shedding by three to four orders of magnitude and significantly reduced two biomarkers of intestinal inflammation, lipocalin-2 (LCN-2) and myeloperoxidase (MPO) (Fig. Mice were challenged on day0 with 106colony-forming units (CFU) of C. jejuni 81176 by oral gavage. A measure of all other fatty acids not mentioned above. 46, 862868 (2011). J.X. Spirulina resuspension was prepared at a concentration of 2% (w/v) in PBS. Denatured aa682 was digested with trypsin and chymotrypsin, with the resulting peptide fragments characterized by LC-MS. Trypsin and chymotrypsin peptide fragments mapping to aa682 are indicated below the sequence of aa682 in blue and red respectively. Moreover, Spirulina shows promising antioxidant and anti-inflammatory properties supporting general equine health as well as increased muscle recovery. The binding activity of aa682 extracted under duodenal conditions was minimally affected by previous incubation of the biomass under the gastric environment (Fig. 1e). CAS c. Ribbon representation of a trimeric VHH (orange). Once homozygous, PCR and DNA sequencing showed that the transgene was genetically stable. They were thermally stable up to 20C. Vaccine 35, 67836789 (2017). For antibiotic selection, medium was supplemented with 70100gml1 kanamycin or 2.55.0gml1 streptomycin. *We use +16 UMF New Zealand Manuka Honey to ensure a beneficial level of MGO for both immune and gastrointestinal support. Wells of an ELISA plate were coated overnight with 100l of 1gml1 FlagV6 VHH in carbonate-bicarbonate buffer, washed with PBS-T and blocked with PBS-TM. How to improve R&D productivity: the pharmaceutical industrys grand challenge. Immunother. 4 Model representations of heterologous proteins designed for expression in spirulina. Weight measurements were made daily for 7days. DNA fragments were gel extracted and characterized by Sanger sequencing. The phagemid library was cleaned up with QiaPrep Spin Minikit columns and transformed into electrocompetent TG1 cells (Lucigen). For support, especially during the critical formative phases of this work, we thank M. McCormick, K. Wolfe, T. Todaro, E. Schlect and D. Baty. Diarrhea was observed in all mice in the control groups, but in none of the mice receiving SP526. Tokuhara, D. et al. PLoS Pathog. were employees of Lumen at the time of data generation. Alfred Haase | Historica Wiki | Fandom The spirulina slurry was rinsed with a trehalose solution and then spray-dried. Cells were pelleted from 5-ml cultures by centrifugation for 10min at 1,600RCF. Weight measurements were made daily for 7days. 1a,b). Google Scholar. His temperament had so improved and he was loving and affectionate. Protein was detected with an anti-His primary antibody followed by an anti-mouse secondary NIR antibody. This blue-green algae's been studied for blood sugar control.and more By Regina C. Windsor, MPH, RDN Updated on October 02, 2022 Medically reviewed by Melissa Nieves, LND, RD Table of Contents View All Uses of Spirulina Side Effects Precautions Dosage Toxicity Nutrients 11, 730 (2019). *We use +16 UMF New Zealand Manuka Honey to ensure a beneficial level of MGO for both immune. Inset CEIA blots for each scaffold demonstrate spirulina expression of a SARS-CoV-2 RBD-binding VHH49 fused to the indicated scaffolding protein. Samples were pelleted by centrifugation at 17,000g for 5min. Identify gaps in your horse's nutrition program to optimize their well-being. 6 CEIA analysis of the oxidation state of disulfides in dimeric proteins expressed in spirulina strain SP1313. and D.C. designed and performed experiments and analyzed data. J. Infect. 3e). At no point were these fragments observed in spirulina, suggesting that there is no integration of the vector outside of the homology arms. The expected abundance ratio for the endogenous gene, promoter and exogenous gene was 1, 2 and 1 respectively. Alfred Haase (29 June 1919-23 February 2009) was a German Wehrmacht sergeant during World War II. Culture volumes ranged from 3 to 100ml. Sci. Benny's irritability, sensitivity, and tenderness resolved. Aliquots of biomass were prepared in duplicate and stored at the indicated temperature with desiccant. Food and water were provided ad libitum throughout. Air-Way EQ (MVP) Additive, Commercial, Minerals, Vitamins, MVP, Pelleted, Respiratory Supplement, United States. Google Scholar. In the meantime, to ensure continued support, we are displaying the site without styles The VHH FlagV6 binds to flagellin (FlaA), a subunit of C. jejuni flagella26. A single homology arm was ineffective, indicating that integration occurs by double crossover, as is typical for natural transformation20. 196, 14711483 (2014). Google Scholar. Prog. Further information on research design is available in the Nature Research Reporting Summary linked to this article. Marles, R. J. et al. Cells were grown to a density of 0.51.0 OD750ml1 in an Innova incubator with appropriate antibiotics. Research findings have associated Spirulina to improvements in animal growth, fertility, aesthetic and nutritional product quality.Spirulina intake has also been linked to an improvement in animal health and welfare. He was holding his weight beautifully. Purified aa682 binding to recombinant FlaA was used to generate a standard curve by linear regression. Spirulina UTEX LB1926 (UTEX culture collection) was not axenic, but microisolation of single filaments was used to prepare axenic strains. Following targeted integration of exogenous genes into the spirulina chromosome (chr), encoded protein biopharmaceuticals can represent as much as 15% of total biomass, require no purification before oral delivery and are stable without refrigeration and protected during gastric transit when encapsulated within dry spirulina. Taxes and shipping calculated at checkout. Treuchtlingen - Wikipedia Koromyslova, A. D. & Hansman, G. S. Nanobodies targeting norovirus capsid reveal functional epitopes and potential mechanisms of neutralization. PubMed PLoS ONE 6, e28218 (2011). To obtain Binding of spirulina-expressed VHHs to a pure culture of C. jejuni was measured by flow cytometry. A large parameter space was evaluated for efficiency of drying, moisture content and retention of antibody activity. Spirulina strain SP526 expressed a monovalent fusion of FlagV6 VHH and MBP, which bound to recombinant FlaA with a KD of 53nM and was constitutively expressed in the cell cytoplasm at 3% of dry biomass (equivalent to 8% of soluble protein) (Fig. Natl Acad. Pigments are plant compounds such as chlorophyll, flavonoids and carotenes that are soluble in ether. Yang, Z. et al. Then, the microbiological analysis . d, ELISA-based, antigen-binding analysis of spirulina lysates prepared as in c assayed for binding to recombinant FlaA. On day0, mice were given an inoculum of 106live C. jejuni cells (strain 81176, resuspended in PBS) by oral gavage. StableFeed | Kasson MN - Facebook Water was replaced with untreated, antibiotic-free water for 1day before C. jejuni challenge. A 1:10,000 dilution of either HRP-conjugated goat anti-mouse antibody or HRP-conjugated donkey anti-rabbit antibody was used as the secondary antibody. Unbound phages were washed from wells with 6,300l of PBS-T. This horse arrived at my training facility with a serious case of scratches on three legs: from the heel bulbs to the bottom of the pastern joint and all the way around the coronet bands. To ease cloning of transgenes into spirulina, a standardized vector was built for markerless integration. Especially for oral delivery, the simplification of both equipment and upstream and downstream processes can more than offset the benefit apparent from a comparison of volumetric productivities. He also had a growing sarcoid on the inside of his hind leg and the most sensitive skin. Pentavalent single-domain antibodies reduce Campylobacter jejuni motility and colonization in chickens. FEBS J. Plant Sci. also supports proper immune responses but is best known for its antimicrobial and antibacterial properties. Food and water were provided ad libitum throughout. A 100-l sample of spirulina-conditioned medium was transferred to either LB agar or mixed LB/SOT agar plates, which were then incubated at 2530C on a light rack (6070Ei) for 57days. 9 VHH stability during spray drying. On day11, levels of LCN-2 and MPO were measured in stool and cecal contents by ELISA (DuoSet ELISA Mouse Lipocalin-2/NGAL, R&D Systems). Provided by the Springer Nature SharedIt content-sharing initiative, Nature Biotechnology (Nat Biotechnol) Bound phages were eluted at low pH by the addtion of 100l of 100mM glycine pH2.0 and incubation for 5min with shaking. (PDF) Spirulina paltensis: Food and Function - ResearchGate Itakura, K. et al. Spirulina . Single colonies were streaked on fresh plates for between five and ten rounds. Campylobacter jejuni is among the most common causes of bacterial gastroenteritis and is a leading cause of infant mortality in the developing world23,24,25. Dried spirulina biomass was resuspended in PBS containing Pierce Protease Inhibitor minitablets and 1mM PMSF at a biomass concentration of 1040mgml1 in 1.7-ml Eppendorf tubes. Spray-dried SP1182 biomass was resuspended in either 50mM bicarbonate buffer or citrate-phosphate buffer pH3.0 with 1mM PMSF. L.P., D.T.B., F.G. and R.G. SP1182, a markerless strain expressing aa682, was constructed for human clinical testing. Highly conserved and degenerate 16S and 23S ribosomal DNA PCR primers (Supplementary Table 1) were used to amplify genomic DNA, following published protocols50,51 from samples derived from both LB and LB/SOT plates. The mass of purified aa682 was analyzed with a 6230 TOF (Agilent) using an ACQUITY UPLC Protein BEH C4 VanGuard pre-column (Waters Corp.). Targeted delivery of a therapeutic to its site of action is a key consideration for efficacy. Aqueous extracts of SP526 were incubated with live C. jejuni 81176 and then stained with anti-His-tag antibody. Using the standard curve, soluble recombinant aa682 was measured at ~3% of total dried biomass. Riazi, A. et al. Syndr. As controls, the parental spirulina strain and a second transgenic strain lacking template for the transgene-specific probes were tested. A synthetic g-block containing the three target loci plus flanking sequences was purchased from IDT as a calibrator. Stability AI, the company behind artificial intelligence (AI) image generator Stable Diffusion, has released a new tool called Uncrop which allows users to "outpaint" images. CAS Protease activity was neutralized by the addition of 50mM NaOH and 1mM PMSF. Bioeng. A randomized study to establish the effects of spirulina in type 2 diabetes mellitus patients. To assess the copy number of an integrated transgene, three sets of primer/taqman probe pairs were designed to target three regions: an endogenous spirulina gene present at a single locus (cpcB), a promoter region present at both an endogenous and transgenic locus (that is, two chromosomal copies) and an exogenous region unique to the transgene. Nat. Peptides derived from the D2/D3/D4 region of FlaA (amino acids 177482) were panned by phage display. Engineering and functionalization of large circular tandem repeat protein nanoparticles. He no longer was experiencing sneezing, snorting, and runny eyes. Spirulina Chia from Stable Feed is a powdered supplement for horses. Spirulina is best known for its ability to modulate the immune system by promoting an anti-histamine response to a variety of environmental irritants. Spirulina as Animal Feed: Opportunities and Challenges - MDPI Transfer of purified herpes virus thymidine kinase gene to cultured mouse cells. Pract. Elaidic acid (C18:1T) is an unsaturated trans fatty acid. After culturing abalones supplemented with Spirulina powder, body weight and shell size were found to increase. The gene for cpcB was used as an endogenous reference gene. To measure intestinal protease resistance, SP1182 lysates were digested with trypsin and chymotrypsin, and VHH binding activity was assessed by ELISA. The promoterterminator pair consisted of a constitutively active, native A. platensis promoter (600bp upstream of the cpcB gene, named Pcpc600) and the terminator of the E. coli ribosomal RNA gene rrnB (named TrrnB). No terrorist motive is suspected. 2a and Extended Data Fig. J. It is necessary for liver health, DNA methylation, neurotransmitter synthesis, and lipid membrane integrity. Spirulina samples were collected 22 and 28days after transformation. This work was supported in part by funding from The Bill and Melinda Gates Foundation (nos. Kusnadi, A. R., Nikolov, Z. L. & Howard, J. Morooka, T., Umeda, A. Article Benny was often tender and sensitive and frequently irritable. Nochi, T. et al. Plasmid DNA was purified from E. coli by the QIAprep Spin Miniprep Kit (Qiagen) and transformed into spirulina strain SP205. Once collected from the hydrocyclone, the powder was sealed and stored in airtight, opaque mylar bags to prevent exposure to moisture or light. Within three days of beginning to feed Pure Form Equines Spirulina Chia product the skin at the edge of the scratches turned a healthy pink.

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