Valorisation of waste animal bones to aminoacids and bonegrafts by using different high-pressure extraction technologies

Valorisation of waste animal bones to aminoacids and bonegrafts by using different high-pressure extraction technologies | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Valorisation of waste animal bones to aminoacids and bonegrafts by using different high-pressure extraction technologies Lenka Matějová, Ivana Troppová, Nikol Krusberská This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9689010/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The extraction of raw beef bones by using a supercritical carbon dioxide without and with a modifier (water), subcritical water and pressurized hot ethanol was investigated in order to valorise them to natural value-added products, namely amonoacids and bonegrafts. It was revealed the utilization of high-pressure extraction technologies represents an ecologic and sustainable approach for valorisation of secondary animal residues to natural value-added products. Individual high-pressure extraction technologies should be select with respect to required yielded products. Biotechnology and Bioengineering secondary animal residues raw beef bones green high-pressure technologies extraction supercritical carbon dioxide extraction subcritical water extraction pressurized hot ethanol extraction aminoacid bonegraft Figures Figure 1 Figure 2 1. Introduction Biogenic waste (namely secondary animal residues – i.e. bones, cartilage, skins, plumage, talons etc.) and mixed animal biomass waste ( e.g. residues from supermarkets) represent the group of large-volume biomass waste, which is commonly sell to rendering plants to be treated. However, it can be further valorised to value-added natural products such as collagen, amino acids, cholesterol, fatty acids and hydroxyapatite-based solid bone residues (bonegrafts) by using the up-to date sustainable and green high-pressure extraction technologies. Supercritical CO 2 extraction (without or with a modifier), subcritical water extraction and pressurized hot solvent extraction belong among these technologies [ 1 , 2 ]. The yielded natural substances can be used for animal nutrition in agriculture sector or for production of animal food and cosmetics. Moreover, developed green extraction technologies may offer a novel alternative way to treat/sterilize and dilapidate even the human bonegrafts, e.g. before being implanted in orthopaedic and dentist surgery practice [ 3 ]. With respect to finding new solutions for both, the waste animal bones valorisation to value-added natural products and the medical practice to yield hydroxyapatite-based human bonegrafts, this study reveals the effect of used high-pressure extraction technology on the quality and quantity of yielded value-added natural substances such as aminoacids and bonegrafts. Namely, supercritical CO 2 extraction (without and with a modifier) (SFE, SFE with water, respectively), pressurized hot ethanol extraction (PFE) and subcritical water extraction (SubWE) were compared. 2. Materials and methods 2.1. Input material and high-pressure extraction processing Waste raw beef bones (ribs) were taken from Tešínské Jatky Holding, s.r.o. (Czech Republic). They were crashed cryogenically (using liquid nitrogen) and sieved to a bone particle-size fraction of ∼5 mm. After crushing, 30 g of bones were extracted using (i.) supercritical CO 2 (without and with water − 30 wt.% ( i.e. SFE, SFE with 30 wt.% water, respectively), (ii.) subcritical water (SubWE) and (iii.) pressurized hot ethanol (PFE) on the commercial SFE 200 ML supercritical CO 2 extractor (EXTRATEX, France) or a laboratory-made high-pressure set-up for subcritical water and pressurized hot solvent extraction [ 4 ]. 2.2. Characterisation techniques High-pressure liquid chromathography with mass-spectroscopy (HPLC-MS) was measured on Shimadzu (Nexera X2) to determine extracted aminoacids. Thermogravitemic analysis (TGA) of extracted bones (bonegrafts) was performed on the thermogravimetric analyser TGA 701 (LECO, USA) according to the ASTM D7582 standard to reach the proximate analysis including determination of moisture, volatile matter, fixed carbon and ash in wt.%. Organic elementary analysis (OEA) of extracted bones (bonegrafts) was realized using CHNS 628 (LECO, USA) according to the standard ASTM D3172-13 and D5373-16 in order to perform the ultimate analysis including determination of elementary C, H, N in wt.%, calculating O in wt.%. Nitrogen physisorption at 77 K on extracted bones (bonegrafts) was measured to determine their textural properties. 3. Results and discussion 3.1. Effect of high-pressure extraction technology on aminoacids yields Since each fluid used for waste raw beef bones extraction shows a different dielectric constant, characterizing its polarity/non-polarity and dissolving power ( e.g. CO 2 – nonpolar vs. water – polar), this affects crucially the solubility of valuable natural substances obtained in waste raw bones in fluids, thus yielding different spectrum of aminoacids in different quantity and residual bonegrafts of different chemical structure and textural properties. Using subcritical water extraction (SubWE), the highest yields of alanine, isoleucine and glycine were achieved (Fig. 1 .). From essential aminoacids, higher yields were achieved for isoleucine, leucine, phenylalanine, valine and threonine. Using supercritical CO 2 extraction (SFE), one order different (lower) yields of aminoacids were yielded. Using supercritical CO 2 extraction with addition of a modifier (water) (SFE with water), the aminoacids yields were increased, yielding mainly isoleucine, leucine, phenylalanine, valine, being all from the group of essential aminoacids. Using pressurized hot ethanol extraction (PFE), the yields of aminoacids were very low, similar to those from SFE. 3.2. Effect of high-pressure extraction technology on bonegraft chemical structure The chemical structure of yielded bonegrafts by using different high-pressure extraction technologies is summarized in Fig. 2 . While subcritical water extraction (SubWE) yields a bonegraft rich on mineral part with almost no carbon part and minimum volatiles, supercritical CO 2 extraction (SFE) yields a bonegraft rich on mineral part as well as volatiles and no fixed carbon. This explains and corresponds to the significantly different yields of aminoacids using both technologies (Fig. 1 ). The addition of water as a modifier to supercritical CO 2 (SFE with water) reduces the volatiles at the expanse of fixed carbon compared to using supercritical CO 2 (SFE) (Fig. 2 ). This feature corresponds to a different spectrum of natural substances extracted by using more polar solvent (supercritical CO 2 with water) than nonpolar (supercritical CO 2 ). Using pressurized hot ethanol extraction (PFE) results in a bonegraft with a similar chemical structure such as using supercritical CO 2 with water (SFE with water), however, with less fixed carbon and more ash. 4. Conclusion This study focused on the revelation of the effect of different used high-pressure extraction technologies on the aminoacids yields and bonegraft chemical structure from raw beef bones. By using subricrtical water extraction (SubWE) the highest yields of aminoacids were obtained, including essential ones, besides a minerals-rich bonegraft wit minimum of volatiles. By using supercritical CO 2 extraction (SFE) the minimum of aminoacids was extracted. The addition of a polar modifier in supercritical CO 2 extraction significantly and positively affects the aminoacids yield and the quality of bonegraft. It can be concluded that the utilization of high-pressure extraction technologies represents an ecologic and sustainable approach for valorisation of secondary animal residues to natural value-added products. Using of individual high-pressure extraction technologies should be select with respect to required products. Declarations Funding: The work was supported by the OP JAK project "INOVO!!!", No. CZ.02.01.01/00/23_021/0008588 supported by the Ministry of Education, Youth and Sports and co-financed by the European Union. The financial support of the European Union under the REFRESH - Research Excellence For REgion Sustainability and High-tech Industries project No. CZ.10.03.01/00/22_003/0000048 via the Operational Programme Just Transition is also gratefully acknowledged. Experimental results were accomplished by using Large Research Infrastructure ENREGAT supported by the Ministry of Education, Youth and Sports of the Czech Republic under project No. LM2023056. Data availability: Open research data will be available on Zenodo in the final version of the manuscript. References Baiano A (2014) Recovery of Biomolecules from Food Wastes — A Review. Molecules 19:14821–14842 Silva JC, Barros AA, Aroso IM, Fassini D, Silva TH, Reis RL, Duarte ARC (2016) Extraction of Collagen/Gelatin from the Marine Demosponge Chondrosia reniformis (Nardo, 1847) Using Water Acidified with Carbon Dioxide – Process Optimization. Ind Eng Chem Res 55:6922–6930 Fages J, Jean E, Frayssinet P, Mathon D, Poirier B, Autefage A, Larzul D (1998) Bone allografts and supercritical processing: effects on osteointegration and viral safety. J Supercrit Fluids 13:351–356 Matějová L, Matěj Z, Fajgar R, Cajthaml T, Šolcová O (2012) TiO 2 Powders Synthesized by Pressurized Fluid Extraction and Supercritical Drying: Effect of Water and Methanol on Structural Properties and Purity . Mater Res Bullet 47:3573–3579 Additional Declarations The authors declare no competing interests. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9689010","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":638838810,"identity":"5cf5cb70-7694-43c5-bff6-1456c383925e","order_by":0,"name":"Lenka Matějová","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9klEQVRIiWNgGAWjYBACPgYGZhg7AYhtGBgkCGhhg2nhgWhJI00LCBwmQgt772GDHwzb5O3ZGx4+5qk5nzh/dgPjxx/4tPCcS07sYbht2MNzINmY59jtxA13DjBL4LOJTSLH+AAPw23GHomENMkZbLeNDSQS2BgM8GmRf2N88A/Dbfse+QdALf/OGcvPAGpJwGsLj3Ey0JbEHgmGNImPbQfkGG4AtRzA65ccY2MZg9vJPWcSkg0+9iXLGdw52CzZgEcLP/sZY8k3Fbdt29vPJD5I+GbHIz+7+SDeEIMAsG95YB5gxGcHCmA/QKzKUTAKRsEoGGEAAGnMR5rEvUnQAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-8816-0070","institution":"Institute of Environmental Technology, Centre for Energy and Environmental Technologies, VSB–Technical University of Ostrava","correspondingAuthor":true,"prefix":"","firstName":"Lenka","middleName":"","lastName":"Matějová","suffix":""},{"id":638843990,"identity":"eb29fa39-1cf6-4749-b1fd-9e04b2a12556","order_by":1,"name":"Ivana Troppová","email":"","orcid":"https://orcid.org/0000-0003-0451-6402","institution":"Institute of Environmental Technology, Centre for Energy and Environmental Technologies, VSB–Technical University of Ostrava","correspondingAuthor":false,"prefix":"","firstName":"Ivana","middleName":"","lastName":"Troppová","suffix":""},{"id":638843991,"identity":"113c846d-dea1-44f0-9807-5992ef063b18","order_by":2,"name":"Nikol Krusberská","email":"","orcid":"","institution":"VSB–Technical University of Ostrava","correspondingAuthor":false,"prefix":"","firstName":"Nikol","middleName":"","lastName":"Krusberská","suffix":""}],"badges":[],"createdAt":"2026-05-12 08:34:49","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-9689010/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9689010/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109274916,"identity":"f2b2c5e5-b6c1-446b-bafe-693d3eeb3cfb","added_by":"auto","created_at":"2026-05-14 14:53:34","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":62599,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 1.\u003c/strong\u003e Comparison of individual aminoacids yields from raw beef bones by using different high-pressure extraction technologies; SFE - supercritical CO\u003csub\u003e2\u003c/sub\u003e extraction, SFE with 30 wt.% water - supercritical CO\u003csub\u003e2\u003c/sub\u003e extraction with a modifier (water), \u0026nbsp;PFE - pressurized hot ethanol extraction, SubWE - subcritical water extraction.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9689010/v1/5aad543d747097b84d44d4fb.jpg"},{"id":109275000,"identity":"f65c0210-11eb-469c-8018-514e04127102","added_by":"auto","created_at":"2026-05-14 14:53:51","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":36301,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 2.\u003c/strong\u003e Comparison of chemical structure of obtained bonegrafts from raw beef bones by using different high-pressure extraction technologies; SFE - supercritical CO\u003csub\u003e2\u003c/sub\u003e extraction, SFE with 30 wt.% water - supercritical CO\u003csub\u003e2\u003c/sub\u003e extraction with a modifier (water), \u0026nbsp;PFE - pressurized hot ethanol extraction, SubWE - subcritical water extraction.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9689010/v1/8bd351fdc6ce4c748d298482.jpg"},{"id":109275280,"identity":"45db91cc-628d-4ec1-b279-c937051f26dc","added_by":"auto","created_at":"2026-05-14 14:54:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":211758,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9689010/v1/af93503d-e7d6-4bb1-88b4-aa1b6d7c672a.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eValorisation of waste animal bones to aminoacids and bonegrafts by using different high-pressure extraction technologies\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eBiogenic waste (namely secondary animal residues \u0026ndash; \u003cem\u003ei.e.\u003c/em\u003e bones, cartilage, skins, plumage, talons etc.) and mixed animal biomass waste (\u003cem\u003ee.g.\u003c/em\u003e residues from supermarkets) represent the group of large-volume biomass waste, which is commonly sell to rendering plants to be treated. However, it can be further valorised to value-added natural products such as collagen, amino acids, cholesterol, fatty acids and hydroxyapatite-based solid bone residues (bonegrafts) by using the up-to date sustainable and green high-pressure extraction technologies. Supercritical CO\u003csub\u003e2\u003c/sub\u003e extraction (without or with a modifier), subcritical water extraction and pressurized hot solvent extraction belong among these technologies [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The yielded natural substances can be used for animal nutrition in agriculture sector or for production of animal food and cosmetics. Moreover, developed green extraction technologies may offer a novel alternative way to treat/sterilize and dilapidate even the human bonegrafts, \u003cem\u003ee.g.\u003c/em\u003e before being implanted in orthopaedic and dentist surgery practice [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWith respect to finding new solutions for both, the waste animal bones valorisation to value-added natural products and the medical practice to yield hydroxyapatite-based human bonegrafts, this study reveals the effect of used high-pressure extraction technology on the quality and quantity of yielded value-added natural substances such as aminoacids and bonegrafts. Namely, supercritical CO\u003csub\u003e2\u003c/sub\u003e extraction (without and with a modifier) (SFE, SFE with water, respectively), pressurized hot ethanol extraction (PFE) and subcritical water extraction (SubWE) were compared.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Input material and high-pressure extraction processing\u003c/h2\u003e \u003cp\u003eWaste raw beef bones (ribs) were taken from Teš\u0026iacute;nsk\u0026eacute; Jatky Holding, s.r.o. (Czech Republic). They were crashed cryogenically (using liquid nitrogen) and sieved to a bone particle-size fraction of \u0026sim;5 mm. After crushing, 30 g of bones were extracted using (i.) supercritical CO\u003csub\u003e2\u003c/sub\u003e (without and with water\u0026thinsp;\u0026minus;\u0026thinsp;30 wt.% (\u003cem\u003ei.e.\u003c/em\u003e SFE, SFE with 30 wt.% water, respectively), (ii.) subcritical water (SubWE) and (iii.) pressurized hot ethanol (PFE) on the commercial SFE 200 ML supercritical CO\u003csub\u003e2\u003c/sub\u003e extractor (EXTRATEX, France) or a laboratory-made high-pressure set-up for subcritical water and pressurized hot solvent extraction [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Characterisation techniques\u003c/h2\u003e \u003cp\u003eHigh-pressure liquid chromathography with mass-spectroscopy (HPLC-MS) was measured on Shimadzu (Nexera X2) to determine extracted aminoacids.\u003c/p\u003e \u003cp\u003eThermogravitemic analysis (TGA) of extracted bones (bonegrafts) was performed on the thermogravimetric analyser TGA 701 (LECO, USA) according to the ASTM D7582 standard to reach the proximate analysis including determination of moisture, volatile matter, fixed carbon and ash in wt.%.\u003c/p\u003e \u003cp\u003eOrganic elementary analysis (OEA) of extracted bones (bonegrafts) was realized using CHNS 628 (LECO, USA) according to the standard ASTM D3172-13 and D5373-16 in order to perform the ultimate analysis including determination of elementary C, H, N in wt.%, calculating O in wt.%.\u003c/p\u003e \u003cp\u003eNitrogen physisorption at 77 K on extracted bones (bonegrafts) was measured to determine their textural properties.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Effect of high-pressure extraction technology on aminoacids yields\u003c/h2\u003e \u003cp\u003eSince each fluid used for waste raw beef bones extraction shows a different dielectric constant, characterizing its polarity/non-polarity and dissolving power (\u003cem\u003ee.g.\u003c/em\u003e CO\u003csub\u003e2\u003c/sub\u003e \u0026ndash; nonpolar \u003cem\u003evs.\u003c/em\u003e water \u0026ndash; polar), this affects crucially the solubility of valuable natural substances obtained in waste raw bones in fluids, thus yielding different spectrum of aminoacids in different quantity and residual bonegrafts of different chemical structure and textural properties.\u003c/p\u003e \u003cp\u003eUsing subcritical water extraction (SubWE), the highest yields of alanine, isoleucine and glycine were achieved (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.). From essential aminoacids, higher yields were achieved for isoleucine, leucine, phenylalanine, valine and threonine. Using supercritical CO\u003csub\u003e2\u003c/sub\u003e extraction (SFE), one order different (lower) yields of aminoacids were yielded. Using supercritical CO\u003csub\u003e2\u003c/sub\u003e extraction with addition of a modifier (water) (SFE with water), the aminoacids yields were increased, yielding mainly isoleucine, leucine, phenylalanine, valine, being all from the group of essential aminoacids. Using pressurized hot ethanol extraction (PFE), the yields of aminoacids were very low, similar to those from SFE.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Effect of high-pressure extraction technology on bonegraft chemical structure\u003c/h2\u003e \u003cp\u003eThe chemical structure of yielded bonegrafts by using different high-pressure extraction technologies is summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. While subcritical water extraction (SubWE) yields a bonegraft rich on mineral part with almost no carbon part and minimum volatiles, supercritical CO\u003csub\u003e2\u003c/sub\u003e extraction (SFE) yields a bonegraft rich on mineral part as well as volatiles and no fixed carbon. This explains and corresponds to the significantly different yields of aminoacids using both technologies (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe addition of water as a modifier to supercritical CO\u003csub\u003e2\u003c/sub\u003e (SFE with water) reduces the volatiles at the expanse of fixed carbon compared to using supercritical CO\u003csub\u003e2\u003c/sub\u003e (SFE) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This feature corresponds to a different spectrum of natural substances extracted by using more polar solvent (supercritical CO\u003csub\u003e2\u003c/sub\u003e with water) than nonpolar (supercritical CO\u003csub\u003e2\u003c/sub\u003e). Using pressurized hot ethanol extraction (PFE) results in a bonegraft with a similar chemical structure such as using supercritical CO\u003csub\u003e2\u003c/sub\u003e with water (SFE with water), however, with less fixed carbon and more ash.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThis study focused on the revelation of the effect of different used high-pressure extraction technologies on the aminoacids yields and bonegraft chemical structure from raw beef bones. By using subricrtical water extraction (SubWE) the highest yields of aminoacids were obtained, including essential ones, besides a minerals-rich bonegraft wit minimum of volatiles. By using supercritical CO\u003csub\u003e2\u003c/sub\u003e extraction (SFE) the minimum of aminoacids was extracted. The addition of a polar modifier in supercritical CO\u003csub\u003e2\u003c/sub\u003e extraction significantly and positively affects the aminoacids yield and the quality of bonegraft. It can be concluded that the utilization of high-pressure extraction technologies represents an ecologic and sustainable approach for valorisation of secondary animal residues to natural value-added products. Using of individual high-pressure extraction technologies should be select with respect to required products.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThe work was supported by the OP JAK project \"INOVO!!!\", No. CZ.02.01.01/00/23_021/0008588 supported by the Ministry of Education, Youth and Sports and co-financed by the European Union. The financial support of the European Union under the REFRESH - Research Excellence For REgion Sustainability and High-tech Industries project No. CZ.10.03.01/00/22_003/0000048 via the Operational Programme Just Transition is also gratefully acknowledged. Experimental results were accomplished by using Large Research Infrastructure ENREGAT supported by the Ministry of Education, Youth and Sports of the Czech Republic under project No. LM2023056.\u003c/p\u003e\u003ch2\u003eData availability:\u003c/h2\u003e \u003cp\u003eOpen research data will be available on Zenodo in the final version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBaiano A (2014) Recovery of Biomolecules from Food Wastes \u0026mdash; A Review. Molecules 19:14821\u0026ndash;14842\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSilva JC, Barros AA, Aroso IM, Fassini D, Silva TH, Reis RL, Duarte ARC (2016) Extraction of Collagen/Gelatin from the Marine Demosponge Chondrosia reniformis (Nardo, 1847) Using Water Acidified with Carbon Dioxide \u0026ndash; Process Optimization. Ind Eng Chem Res 55:6922\u0026ndash;6930\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFages J, Jean E, Frayssinet P, Mathon D, Poirier B, Autefage A, Larzul D (1998) Bone allografts and supercritical processing: effects on osteointegration and viral safety. J Supercrit Fluids 13:351\u0026ndash;356\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMatějov\u0026aacute; L, Matěj Z, Fajgar R, Cajthaml T, Šolcov\u0026aacute; O (2012) \u003cem\u003eTiO\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e \u003cem\u003ePowders Synthesized by Pressurized Fluid Extraction and Supercritical Drying: Effect of Water and Methanol on Structural Properties and Purity\u003c/em\u003e. Mater Res Bullet 47:3573\u0026ndash;3579\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"d809cb53-81d1-46e5-ad63-c02e872736ba","identifier":"10.13039/501100001823","name":"Ministerstvo Školství, Mládeže a Tělovýchovy","awardNumber":"CZ.02.01.01/00/23_021/0008588 ","order_by":0},{"identity":"fb5d6bfa-98f9-4300-944c-54320739c164","identifier":"10.13039/501100000780","name":"European Commission","awardNumber":"CZ.10.03.01/00/22_003/0000048 ","order_by":1},{"identity":"606a7202-6768-4980-8cef-69c8a563e590","identifier":"10.13039/501100001823","name":"Ministerstvo Školství, Mládeže a Tělovýchovy","awardNumber":"LM2023056","order_by":2}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"VSB–Technical University of Ostrava","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"secondary animal residues, raw beef bones, green high-pressure technologies, extraction, supercritical carbon dioxide extraction, subcritical water extraction, pressurized hot ethanol extraction, aminoacid, bonegraft","lastPublishedDoi":"10.21203/rs.3.rs-9689010/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9689010/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe extraction of raw beef bones by using a supercritical carbon dioxide without and with a modifier (water), subcritical water and pressurized hot ethanol was investigated in order to valorise them to natural value-added products, namely amonoacids and bonegrafts. It was revealed the utilization of high-pressure extraction technologies represents an ecologic and sustainable approach for valorisation of secondary animal residues to natural value-added products. Individual high-pressure extraction technologies should be select with respect to required yielded products.\u003c/p\u003e","manuscriptTitle":"Valorisation of waste animal bones to aminoacids and bonegrafts by using different high-pressure extraction technologies","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-14 14:53:15","doi":"10.21203/rs.3.rs-9689010/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d9fec43b-5e86-462f-8548-175eb4eef01f","owner":[],"postedDate":"May 14th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":67994639,"name":"Biotechnology and Bioengineering"}],"tags":[],"updatedAt":"2026-05-14T14:53:15+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-14 14:53:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9689010","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9689010","identity":"rs-9689010","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}