{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,24]],"date-time":"2026-04-24T13:49:49Z","timestamp":1777038589913,"version":"3.51.4"},"reference-count":30,"publisher":"Public Library of Science (PLoS)","issue":"5","license":[{"start":{"date-parts":[[2022,5,19]],"date-time":"2022-05-19T00:00:00Z","timestamp":1652918400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001871","name":"Funda\u00e7\u00e3o para a Ci\u00eancia e a Tecnologia","doi-asserted-by":"publisher","award":["UIDB\/00276\/2020 to CIISA"],"award-info":[{"award-number":["UIDB\/00276\/2020 to CIISA"]}],"id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001871","name":"Funda\u00e7\u00e3o para a Ci\u00eancia e a Tecnologia","doi-asserted-by":"publisher","award":["DL57\/2016\/CP1438\/CT0007"],"award-info":[{"award-number":["DL57\/2016\/CP1438\/CT0007"]}],"id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001871","name":"Funda\u00e7\u00e3o para a Ci\u00eancia e a Tecnologia","doi-asserted-by":"publisher","award":["SFRH\/BD\/126198\/2016"],"award-info":[{"award-number":["SFRH\/BD\/126198\/2016"]}],"id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:p>Over the last decades, microalgae have gained popularity due to demand for novel environmental green solutions and development of innovative mass-production sources for multiple processes, including animal feed and human diet, turning microalgae into an exquisite candidate for several ecofriendly technologies. Notwithstanding, there is a catch. Most species of microalgae, as the case of common <jats:italic>Chlorella vulgaris<\/jats:italic> (<jats:italic>C<\/jats:italic>. <jats:italic>vulgaris<\/jats:italic>) display a recalcitrant cell wall, characterized by a complex matrix of polysaccharides and glycoproteins, which constitutes a major barrier for monogastric species digestibility and extraction of inner valuable nutritional compounds. To overcome this limitation, the development of feed enzymes, in particular Carbohydrate-Active enZymes (CAZymes) with capacity to disrupt <jats:italic>C<\/jats:italic>. <jats:italic>vulgaris<\/jats:italic> cell wall may contribute to improve the bioavailability of these microalgae compounds in monogastric diets, namely at high levels of incorporation. In order to disclosure novel combination of feed enzymes to disrupt <jats:italic>C<\/jats:italic>. <jats:italic>vulgaris<\/jats:italic> cell wall, a lab protocol was implemented by our research team containing the following key steps: after microalgae cultivation and having available a repertoire of two hundred pre-selected CAZymes produced by high-throughput technology, the step 1 is the individual screening of the most functional enzymes on disrupting <jats:italic>C<\/jats:italic>. <jats:italic>vulgaris<\/jats:italic> cell wall (versus a control, defined as the microalgae suspension incubated with PBS) and the determination of reducing sugars released by the 3,5-dinitrosalicylic acid (DNSA) method; step 2 concerns on finding the best CAZymes cocktail, testing the synergistic effect of enzymes, to disrupt <jats:italic>C<\/jats:italic>. <jats:italic>vulgaris<\/jats:italic> cell wall (in parallel with running the control) along with characterization of each enzyme thermostability and resistance to proteolytic attack, to which feed enzymes are subjected in the animal gastrointestinal tract; step 3 is the assessment of <jats:italic>C<\/jats:italic>. <jats:italic>vulgaris<\/jats:italic> cell wall degradation degree by measuring the amount of reducing sugars released by the DNSA method, fatty acid analysis by gas chromatography (GC) with flame ionization detector (FID), oligosaccharides quantification by high performance liquid chromatography (HPLC) equipped with an electrochemical detector (ECD), protein content by the Kjeldahl method, and various pigments (chlorophylls a and b, and total carotenoids) in the supernatant. In the correspondent residue, we also assessed cellular counting using a Neubauer chamber by direct observation on a bright-field microscope and fluorescence intensity, after staining with Calcofluor White for both control and CAZymes cocktail treatments, on a fluorescence microscope. Beyond animal feed industry with impact on human nutrition, our lab protocol may increase the yield in obtaining valued constituents from <jats:italic>C<\/jats:italic>. <jats:italic>vulgaris<\/jats:italic> microalga for other biotechnological industries.<\/jats:p>","DOI":"10.1371\/journal.pone.0268565","type":"journal-article","created":{"date-parts":[[2022,5,19]],"date-time":"2022-05-19T17:46:03Z","timestamp":1652982363000},"page":"e0268565","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":9,"title":["Testimony on a successful lab protocol to disrupt Chlorella vulgaris microalga cell wall"],"prefix":"10.1371","volume":"17","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6755-4572","authenticated-orcid":true,"given":"Paula A.","family":"Lopes","sequence":"first","affiliation":[]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6151-8186","authenticated-orcid":true,"given":"Diogo","family":"Coelho","sequence":"additional","affiliation":[]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1032-5987","authenticated-orcid":true,"given":"Jos\u00e9 A. 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