Smart waste-derived materials for feed application: chestnut shells and vine pruning biochar

Abstract

Carrier-assisted delivery is a key step for the successful targeted oral delivery of bioactive molecules in functional diets in livestock. The aim is to protect the biomolecule during gastric transit, and ensure its efficient release in the intestine. Biochar is the by-product of the thermochemical conversion of residual biomass in an oxygen-limited environment and has suitable physico-chemical and morphological properties to be a carrier. Two types of biochar were tested as carriers of egg white lysozyme (LY), selected as a representative of bioactive molecules both in terms of molecular size (MW 14.3 kDa) and antibacterial activity, for application in weaned pig feed. One biochar was derived from chestnut shells (CB) and the other from vine pruning (VB). An efficient and environmentally-friendly procedure for LY adsorption was developed, based on a solid/liquid process in mild conditions. The effects of the operating conditions, such as initial LY content, reaction time, and pH were also studied. The optimal conditions were found to be a maximum LY loading of 21–23 mg_LY g_Carrier⁻¹. Both pristine and hybrid materials were extensively characterized by combining morphological and physico-chemical techniques to obtain information on LY allocation and interactions with the carriers. Preliminary experiments on lysozyme release were performed at pH=3 and pH=7, simulating the pH conditions of the stomach and intestine of the weaned pigs, respectively. The results showed a higher releasing capacity when the pH was increased from 3 to 7. Specifically, the release showed a slight increase from 0.8% to 1.2% as the pH shifted from 3 to 7 for CB, and from 1.5% to 2.3% for VB. These results confirmed that biochar can protect LY from the low pH, during the gastric transit, and that LY could be released in the gut. These two benefits are likely related to the homogeneous distribution of LY molecules at the carrier surface, which is facilitated by the interaction of charges of opposite signs.

Keywords

Biochar / Characterization / Animal feed / Lysozyme / Muramidase

References

[1] Albernaz-Gonçalves R, Olmos Antillón G, Hötzel MJ. Linking animal welfare and antibiotic use in pig farming—a review. Animals, 2022, 12: 216

[2] Alsawy T, Rashad E, El-Qelish M, Mohammed RH. A comprehensive review on the chemical regeneration of biochar adsorbent for sustainable wastewater treatment. NPJ Clean Water, 2022, 5: 29

[3] Ange KD, Eisemann JH, Argenzio RA, Almond GW, Blikslager AT. Effects of feed physical form and buffering solutes on water disappearance and proximal stomach pH in swine. J Anim Sci, 2000, 78: 2344

[4] Barth A. Infrared spectroscopy of proteins. Biochim Biophys Acta (BBA) Bioenerg, 2007, 1767: 1073-1101

[5] Batista EMCC, Shultz J, Matos TTS, Fornari MR, Ferreira TM, Szpoganicz B, De Freitas RA, Mangrich AS. Effect of surface and porosity of biochar on water holding capacity aiming indirectly at preservation of the Amazon biome. Sci Rep, 2018, 8 10677

[6] Borchard N, Schirrmann M, Cayuela ML, Kammann C, Wrage-Mönnig N, Estavillo JM, Fuertes-Mendizábal T, Sigua G, Spokas K, Ippolito JA, Novak J. Biochar, soil and land-use interactions that reduce nitrate leaching and N2O emissions: a meta-analysis. Sci Total Environ, 2019, 651: 2354-2364

[7] Brewer CE, Chuang VJ, Masiello CA, Gonnermann H, Gao X, Dugan B, Driver LE, Panzacchi P, Zygourakis K, Davies CA. New approaches to measuring biochar density and porosity. Biomass Bioenergy, 2014, 66: 176-185

[8] Brown RC. The role of pyrolysis and gasification in a carbon negative economy. Processes, 2021, 9: 882

[9] Caporale AG, Pigna M, Sommella A, Conte P. Effect of pruning-derived biochar on heavy metals removal and water dynamics. Biol Fertil Soils, 2014, 50: 1211-1222

[10] Castañeda-Barba S, Top EM, Stalder T. Plasmids, a molecular cornerstone of antimicrobial resistance in the One Health era. Nat Rev Microbiol, 2024, 22: 18-32

[11] Cederlund H, Börjesson E, Stenström J. Effects of a wood-based biochar on the leaching of pesticides chlorpyrifos, diuron, glyphosate and MCPA. J Environ Manage, 2017, 191: 28-34

[12] Cegielska-Radziejewska R, Leśnierowski G, Kijowski J. Properties and application of egg white lysozyme and its modified preparations—a review. Pol J Food Nutr Sci, 2008, 58(1): 5-10

[13] Cha JS, Park SH, Jung S-C, Ryu C, Jeon J-K, Shin M-C, Park Y-K. Production and utilization of biochar: a review. J Ind Eng Chem, 2016, 40: 1-15

[14] 5220 Chemical Oxygen Demand (COD). Standard methods for the examination of water and wastewater, 2017, Washington, DC, American Public Health Association

[15] Chen W, Meng J, Han X, Lan Y, Zhang W. Past, present, and future of biochar. Biochar, 2019, 1: 75-87

[16] Chu GM, Kim JH, Kang SN, Song YM. Effects of dietary bamboo charcoal on the carcass characteristics and meat quality of fattening pigs. Korean J Food Sci Anim Resour, 2013, 33: 348-355

[17] Chu GM, Kim JH, Kim HY, Ha JH, Jung MS, Song Y, Cho JH, Lee SJ, Ibrahim RIH, Lee SS, Song YM. Effects of bamboo charcoal on the growth performance, blood characteristics and noxious gas emission in fattening pigs. J Appl Anim Res, 2013, 41: 48-55

[18] Cristiani C, Finocchio E, Rossi L, Giromini C, Dell’Anno M, Panseri S, Bellotto M. Natural clays as potential amino acids carriers for animal nutrition application. Appl Sci, 2021, 11: 5669

[19] Cromwell GL. Why and how antibiotics are used in swine production. Anim Biotechnol, 2002, 13: 7-27

[20] De S, Medeiros BG, Souza MP, Pinheiro AC, Bourbon AI, Cerqueira MA, Vicente AA, Carneiro-da-Cunha MG. Physical characterisation of an alginate/lysozyme nano-laminate coating and its evaluation on ‘Coalho’ cheese shelf life. Food Bioprocess Technol, 2014, 7: 1088-1098

[21] Dehkhoda AM, West AH, Ellis N. Biochar based solid acid catalyst for biodiesel production. Appl Catal Gen, 2010, 382: 197-204

[22] Dell’Anno M, Hejna M, Sotira S, Caprarulo V, Reggi S, Pilu R, Miragoli F, Callegari ML, Panseri S, Rossi L. Evaluation of leonardite as a feed additive on lipid metabolism and growth of weaned piglets. Anim Feed Sci Technol, 2020, 266 114519

[23] Dell’Anno M, Scaglia E, Reggi S, Grossi S, Sgoifo Rossi CA, Frazzini S, Caprarulo V, Rossi L. Evaluation of tributyrin supplementation in milk replacer on diarrhoea occurrence in preweaning Holstein calves. Anim, 2023, 17 100791

[24] Dragoni I, Balzaretti C, Rossini S, Rossi L, Dell’Orto V, Baldi A. Detection of hen lysozyme on proteic profiles of Grana Padano cheese through SELDI-TOF MS high-throughput technology during the ripening process. Food Anal Methods, 2011, 4: 233-239

[25] Elkordy AA, Forbes RT, Barry BW. Integrity of crystalline lysozyme exceeds that of a spray-dried form. Int J Pharm, 2002, 247: 79-90

[26] Ferraboschi P, Ciceri S, Grisenti P. Applications of lysozyme, an innate immune defense factor, as an alternative antibiotic. Antibiotics, 2021, 10: 1534

[27] Gerlach A. Oral application of charcoal and humic acids to dairy cows influences clostridium botulinum blood serum antibody level and glyphosate excretion in urine. J Clin Toxicol, 2014

[28] González ME, Cea M, Sangaletti N, González A, Toro C, Diez MC, Moreno N, Querol X, Navia R. Biochar derived from agricultural and forestry residual biomass: characterization and potential application for enzymes immobilization. J Biobased Mater Bioenergy, 2013, 7: 724-732

[29] Guagliano M, Cristiani C, Dell’Anno M, Dotelli G, Finocchio E, Lacalamita M, Mesto E, Reggi S, Rossi L, Schingaro E. A commercial clay-based material as a carrier for targeted lysozyme delivery in animal feed. Nanomaterials, 2023, 13: 2965

[30] Guagliano M, Dell’Anno M, Dotelli G, Finocchio E, Lacalamita M, Mesto E, Reggi S, Rossi L, Schingaro E, Staltari E, Cristiani C. Lysozyme-mineral clay systems: comparison of interaction for controlled release in feed application. Minerals, 2023, 13: 660

[31] Guo X, Liu H, Zhang J. The role of biochar in organic waste composting and soil improvement: a review. Waste Manag, 2020, 102: 884-899

[32] Henze LJ, Koehl NJ, Bennett-Lenane H, Holm R, Grimm M, Schneider F, Weitschies W, Koziolek M, Griffin BT. Characterization of gastrointestinal transit and luminal conditions in pigs using a telemetric motility capsule. Eur J Pharm Sci, 2021, 156 105627

[33] Hong M, Zhang L, Tan Z, Huang Q. Effect mechanism of biochar’s zeta potential on farmland soil’s cadmium immobilization. Environ Sci Pollut Res, 2019, 26: 19738-19748

[34] Hu YJ, Cowling BJ. Reducing antibiotic use in livestock, China. Bull World Health Organ, 2020, 98: 360-361

[35] Ibrahim H, Aoki T, Pellegrini A. Strategies for new antimicrobial proteins and peptides: lysozyme and aprotinin as model molecules. Curr Pharm des, 2002, 8: 671-693

[36] Jaiswal AK, Frenkel O, Tsechansky L, Elad Y, Graber ER. Immobilization and deactivation of pathogenic enzymes and toxic metabolites by biochar: a possible mechanism involved in soilborne disease suppression. Soil Biol Biochem, 2018, 121: 59-66

[37] Jeffery S, Verheijen FGA, Van Der Velde M, Bastos AC. A quantitative review of the effects of biochar application to soils on crop productivity using meta-analysis. Agric Ecosyst Environ, 2011, 144: 175-187

[38] Jones S, Bardos RP, Kidd PS, Mench M, De Leij F, Hutchings T, Cundy A, Joyce C, Soja G, Friesl-Hanl W, Herzig R, Menger P. Biochar and compost amendments enhance copper immobilisation and support plant growth in contaminated soils. J Environ Manage, 2016, 171: 101-112

[39] Kalus K, Koziel J, Opaliński S. A review of biochar properties and their utilization in crop agriculture and livestock production. Appl Sci, 2019, 9 3494

[40] Kammann C, Ippolito J, Hagemann N, Borchard N, Cayuela ML, Estavillo JM, Fuertes-Mendizabal T, Jeffery S, Kern J, Novak J, Rasse D, Saarnio S, Schmidt H-P, Spokas K, Wrage-Mönnig N. Biochar as a tool to reduce the agricultural greenhouse-gas burden—knowns, unknowns and future research needs. J Environ Eng Landsc Manag, 2017, 25: 114-139

[41] Kan T, Strezov V, Evans TJ. Lignocellulosic biomass pyrolysis: a review of product properties and effects of pyrolysis parameters. Renew Sustain Energy Rev, 2016, 57: 1126-1140

[42] Kasapidou E, Sossidou E, Mitlianga P. Fruit and vegetable co-products as functional feed ingredients in farm animal nutrition for improved product quality. Agriculture, 2015, 5: 1020-1034

[43] Kim Y-C, Myerson AS. Diffusivity of lysozyme in undersaturated, saturated and supersaturated solutions. J Cryst Growth, 1994, 143: 79-85

[44] Klank, D.; Goverde, T.; Blum, C. Particle World; technical papers of quantachrome; quantachrome: Klundert, The Netherlands, 2009. www.quantachrome.com. Accessed 17 Mar 2023

[45] Kocherbitov V, Latynis J, Misiu̅nas A, Barauskas J, Niaura G. Hydration of lysozyme studied by Raman spectroscopy. J Phys Chem B, 2013, 117: 4981-4992

[46] Kummer N, Huguenin-Elie L, Zeller A, Chandorkar Y, Schoeller J, Zuber F, Ren Q, Sinha A, De France K, Fischer P, Campioni S, Nyström G. 2D foam film coating of antimicrobial lysozyme amyloid fibrils onto cellulose nanopapers. Nanoscale Adv, 2023, 5: 5276-5285

[47] Leistikow KR, Beattie RE, Hristova KR. Probiotics beyond the farm: benefits, costs, and considerations of using antibiotic alternatives in livestock. Front Antibiot, 2022, 1: 1003912

[48] Leng L, Xiong Q, Yang L, Li H, Zhou Y, Zhang W, Jiang S, Li H, Huang H. An overview on engineering the surface area and porosity of biochar. Sci Total Environ, 2021, 763 144204

[49] Li L, Yao Z, You S, Wang C-H, Chong C, Wang X. Optimal design of negative emission hybrid renewable energy systems with biochar production. Appl Energy, 2019, 243: 233-249

[50] Liu Y, Zhao X, Li J, Ma D, Han R. Characterization of bio-char from pyrolysis of wheat straw and its evaluation on methylene blue adsorption. Desalination Water Treat, 2012, 46: 115-123

[51] Liu Q, Zhang Y, Liu B, Amonette JE, Lin Z, Liu G, Ambus P, Xie Z. How does biochar influence soil N cycle? A meta-analysis. Plant Soil, 2018, 426: 211-225

[52] Liu Y, Espinosa CD, Abelilla JJ, Casas GA, Lagos LV, Lee SA, Kwon WB, Mathai JK, Navarro DMDL, Jaworski NW, Stein HH. Non-antibiotic feed additives in diets for pigs: a review. Anim Nutr, 2018, 4: 113-125

[53] Lunney JK, Van Goor A, Walker KE, Hailstock T, Franklin J, Dai C. Importance of the pig as a human biomedical model. Sci Transl Med, 2021, 13 eabd5758

[54] Ma X, Zhou B, Budai A, Jeng A, Hao X, Wei D, Zhang Y, Rasse D. Study of biochar properties by scanning electron microscope – energy dispersive X-ray spectroscopy (SEM-EDX). Commun Soil Sci Plant Anal, 2016, 47: 593-601

[55] Mahfuz S, Shang Q, Piao X. Phenolic compounds as natural feed additives in poultry and swine diets: a review. J Anim Sci Biotechnol, 2021, 12: 48

[56] Mandal A, Singh N, Purakayastha TJ. Characterization of pesticide sorption behaviour of slow pyrolysis biochars as low cost adsorbent for atrazine and imidacloprid removal. Sci Total Environ, 2017, 577: 376-385

[57] Masschalck B, Van Houdt R, Van Haver EGR, Michiels CW. Inactivation of Gram-Negative Bacteria by lysozyme, denatured lysozyme, and lysozyme-derived peptides under high hydrostatic pressure. Appl Environ Microbiol, 2001, 67: 339-344

[58] Mößeler A, Köttendorf S, Große Liesner V, Kamphues J. Impact of diets’ physical form (particle size; meal/pelleted) on the stomach content (dry matter content, pH, chloride concentration) of pigs. Livest Sci, 2010, 134: 146-148

[59] Noritomi H. Abrol V, Sharma P. Application of biochar to enzyme carrier for stress tolerance of enzymes. Biochar—an imperative amendment for soil and the environment, 2019, London, IntechOpen

[60] Nunes LJR, Rodrigues AM, Matias JCO, Ferraz AI, Rodrigues AC. Production of biochar from vine pruning: waste recovery in the wine industry. Agriculture, 2021, 11: 489

[61] O’Dell JW. The determination of chemical oxygen demand by semi-automated colorimetry. Methods for the determination of metals in environmental samples, 1996, Amsterdam, Elsevier509-521

[62] Oliver WT, Wells JE. Lysozyme as an alternative to growth promoting antibiotics in swine production. J Anim Sci Biotechnol, 2015, 6: 35

[63] Oliver WT, Wells JE, Maxwell CV. Lysozyme as an alternative to antibiotics improves performance in nursery pigs during an indirect immune challenge1,2. J Anim Sci, 2014, 92: 4927-4934

[64] Ortiz Sanjuán JM, Manzanilla EG, Cabrera-Rubio R, Crispie F, Cotter PD, Garrido JJ, Ekhlas D, O’Neill L, Argüello H. Fine-tuning of post-weaning pig microbiome structure and functionality by in-feed zinc oxide and antibiotics use. Front Cell Infect Microbiol, 2024, 14: 1354449

[65] Pallarés J, González-Cencerrado A, Arauzo I. Production and characterization of activated carbon from barley straw by physical activation with carbon dioxide and steam. Biomass Bioenergy, 2018, 115: 64-73

[66] Pandey D, Daverey A, Dutta K, Arunachalam K. Bioremoval of toxic malachite green from water through simultaneous decolorization and degradation using laccase immobilized biochar. Chemosphere, 2022, 297 134126

[67] Pawar VK, Meher JG, Singh Y, Chaurasia M, Surendar Reddy B, Chourasia MK. Targeting of gastrointestinal tract for amended delivery of protein/peptide therapeutics: strategies and industrial perspectives. J Controlled Release, 2014, 196: 168-183

[68] Picchi G, Silvestri S, Cristoforetti A. Vineyard residues as a fuel for domestic boilers in Trento Province (Italy): comparison to wood chips and means of polluting emissions control. Fuel, 2013, 113: 43-49

[69] Prestinaci F, Pezzotti P, Pantosti A. Antimicrobial resistance: a global multifaceted phenomenon. Pathog Glob Health, 2015, 109: 309-318

[70] Ramirez BC, Xin H, Halbur PG, Beermann DH, Hansen SL, Linhares DCL, Peschel JM, Rademacher CJ, Reecy JM, Ross JW, Shepherd TA, Koltes JE. At the intersection of industry, academia, and government: how do we facilitate productive precision livestock farming in practice?. Animals, 2019, 9: 635

[71] Reggi S, Frazzini S, Torresani MC, Guagliano M, Cristiani C, Pilu SR, Ghidoli M, Rossi L. Metabolomic profiling and functional characterization of biochar from vine pruning residues for applications in animal feed. Animals, 2024, 14: 3440

[72] Reggi S, Frazzini S, Pedrazzi S, Ghidoli M, Torresani MC, Puglia M, Morselli N, Guagliano M, Cristiani C, Pilu SR, Onelli E, Moscatelli A, Rossi L. Metabolomic insights into the potential of chestnut biochar as a functional feed ingredient. Appl Sci, 2025, 15 1084

[73] Robinson TP, Bu DP, Carrique-Mas J, Fèvre EM, Gilbert M, Grace D, Hay SI, Jiwakanon J, Kakkar M, Kariuki S, Laxminarayan R, Lubroth J, Magnusson U, Thi Ngoc P, Van Boeckel TP, Woolhouse MEJ. Antibiotic resistance: mitigation opportunities in livestock sector development. Animal, 2017, 11: 1-3

[74] Rossi L, Dell’Anno M, Turin L, Reggi S, Lombardi A, Alborali GL, Filipe J, Riva F, Riccaboni P, Scanziani E, Dall’Ara P, Demartini E, Baldi A. Tobacco seed-based oral vaccination against Verocytotoxic O138 Escherichia coli as alternative approach to antibiotics in weaned piglets. Antibiotics, 2023, 12 715

[75] Ruiz B, Ferrera-Lorenzo N, Fuente E. Valorisation of lignocellulosic wastes from the candied chestnut industry. Sustainable activated carbons for environmental applications. J Environ Chem Eng, 2017, 5: 1504-1515

[76] Safaei Khorram M, Zhang Q, Lin D, Zheng Y, Fang H, Yu Y. Biochar: a review of its impact on pesticide behavior in soil environments and its potential applications. J Environ Sci, 2016, 44: 269-279

[77] Sakhiya AK, Anand A, Kaushal P. Production, activation, and applications of biochar in recent times. Biochar, 2020, 2: 253-285

[78] Schmidt H-P, Hagemann N, Draper K, Kammann C. The use of biochar in animal feeding. PeerJ, 2019, 7 e7373

[79] Sha Z, Li Q, Lv T, Misselbrook T, Liu X. Response of ammonia volatilization to biochar addition: a meta-analysis. Sci Total Environ, 2019, 655: 1387-1396

[80] Silvetti T, Morandi S, Hintersteiner M, Brasca M. Silvetti T, Morandi S, Hintersteiner M, Brasca M. Use of hen egg white lysozyme in the food industry. Egg innovations and strategies for improvements, 2017, Cambridge, Academic Press233242

[81] Squillaci G, Apone F, Michele Sena L, Carola A, Tito A, Bimonte M, De Lucia A, Colucci G, La Cara F, Morana A (2018) Chestnut (Castanea sativa Mill.) industrial wastes as a valued bioresource for the production of active ingredients. Process Biochem 64:228-236. https://doi.org/10.1016/j.procbio.2017.09.017. https://www.sciencedirect.com/science/article/pii/S1359511317310073

[82] Waters CL, Janupala RR, Mallinson RG, Lobban LL. Staged thermal fractionation for segregation of lignin and cellulose pyrolysis products: an experimental study of residence time and temperature effects. J Anal Appl Pyrolysis, 2017, 126: 380-389

[83] WHO Regional Office for Europe and European Centre for Disease Prevention and Control (2025). Surveillance of antimicrobial resistance in Europe, 2024 data: executive summary. Copenhagen: WHO Regional Office for Europe

[84] World Health Organization. (2019). Monitoring and evaluation of the global action plan on antimicrobial resistance: framework and recommended indicators.

[85] Wu T, Jiang Q, Wu D, Hu Y, Chen S, Ding T, Ye X, Liu D, Chen J. What is new in lysozyme research and its application in food industry? A review. Food Chem, 2019, 274: 698-709

[86] www.mur.gov.it

[87] www.research-and-innovation.ec.europa.eu

[88] Yan Y, Manickam S, Lester E, Wu T, Pang CH. Synthesis of graphene oxide and graphene quantum dots from miscanthus via ultrasound-assisted mechano-chemical cracking method. Ultrason Sonochem, 2021, 73 105519

[89] Yang H, Yan R, Chen H, Lee DH, Zheng C. Characteristics of hemicellulose, cellulose and lignin pyrolysis. Fuel, 2007, 86: 1781-1788

[90] Yi Q, Qi F, Cheng G, Zhang Y, Xiao B, Hu Z, Liu S, Cai H, Xu S. Thermogravimetric analysis of co-combustion of biomass and biochar. J Therm Anal Calorim, 2013, 112: 1475-1479

[91] Yu S, Wang L, Li Q, Zhang Y, Zhou H. Sustainable carbon materials from the pyrolysis of lignocellulosic biomass. Mater Today Sustain, 2022, 19 100209

[92] Yuan J-H, Xu R-K, Zhang H. The forms of alkalis in the biochar produced from crop residues at different temperatures. Bioresour Technol, 2011, 102: 3488-3497

[93] Zambon I, Colantoni A, Cecchini M, Mosconi E. Rethinking sustainability within the Viticulture Realities Integrating Economy, Landscape and Energy. Sustainability, 2018, 10 320

[94] Zhang X, Wang H, He L, Lu K, Sarmah A, Li J, Bolan NS, Pei J, Huang H. Using biochar for remediation of soils contaminated with heavy metals and organic pollutants. Environ Sci Pollut Res, 2013, 20: 8472-8483

[95] Zhang B, Tao H, Niu X, Li S, Chen H-Q. Lysozyme distribution, structural identification, and in vitro release of starch-based microgel-lysozyme complexes. Food Chem, 2017, 227: 137-141

[96] Zhou P, Adeel M, Guo M, Ge L, Shakoor N, Li M, Li Y, Wang G, Rui Y. Characterisation of biochar produced from two types of chestnut shells for use in remediation of cadmium- and lead-contaminated soil. Crop Pasture Sci, 2022, 74: 147-156

[97] Zou L, Xiong X, Liu H, Zhou J, Liu Y, Yin Y. Effects of dietary lysozyme levels on growth performance, intestinal morphology, immunity response and microbiota community of growing pigs: dietary lysozyme levels in growing pigs. J Sci Food Agric, 2019, 99: 1643-1650

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