Enzymes in the production of cultivated meat products

Abstract

Enzymes play a pivotal role in meat processing and maturation, starting with endogenous enzymes that catalyse a partial proteolysis in natural meat that enhances texture and flavor, to exogenous enzymes that may be incorporated for additional processing. Therefore, a question is raised whether endogenous and exogenous proteases could be used to improve the quality of meat products originated from in vitro cell-based technology. This manuscript aims at presenting the main applications of enzymes in meat processing, starting with conventional meat products and expanding to reported and potential applications in cultured meat technologies. Enzymes are applied since the early stages of cell isolation, multiplication and detaching from supports. The use of endogenous enzymes, such as cathepsins and calpains, has been proposed to improve the palatability of cultivated meat, and the application of transglutaminases has already been reported in scientific papers and patent documents, with the aim of promoting crosslinking reactions among proteins. This feature can be used to modulate the texture of cultivated meat products. Also, their use has been directed to the manufacture of scaffolds and microcarriers for animal cell cultivation. As concluded from the scientific and technological literature survey, it is evident that a substantial area of study remains unexplored regarding the utilization of enzymes in cultivated meat products.

Graphical Abstract

Keywords

Protease / Transglutaminase / Animal cell culture / Alternative proteins

References

[1] Good Food Institute. (2021) Building an ecosystem for cultivated meat in India. In: https://gfi-india.org/wp-content/uploads/2022/01/Building-an-ecosystem-for-cultivated-meat-in-India.pdf.

[2] Soccol CR, Karp SG, Letti LAJ, et al. . Is the development of low-cost media one of the greatest challenges to produce cultivated meat on an industrial scale?. Biotechnol Res Innov. 2022, 6: e2022005

[3] Thyden R, Dominko T, Weathers P, et al. . Recycling spent animal cell culture media using the thermally resistant microalga Chlorella sorokiniana. Syst Microbiol Biomanufacturing. 2024

[4] Pang C, Yin X, Zhang G, et al. . Current progress and prospects of enzyme technologies in future foods. Syst Microbiol Biomanufacturing. 2021, 1: 24-32

[5] Ding Z, Wei Q, Liu C, Zhang H, Huang F. The quality changes and proteomic analysis of cattle muscle postmortem during rigor mortis. Foods. 2022;11. https://doi.org/10.3390/foods11020217.

[6] Abril B, Bou R, García-Pérez JV, Benedito J. Role of enzymatic reactions in meat processing and use of emerging technologies for process intensification. Foods. 2023;12:1940. https://doi.org/10.3390/foods12101940.

[7] Ordoñez JA. Tecnologia De Alimentos. 2005, Porto Alegre, Artmed

[8] Kaur L, Hui SX, Morton JD, et al. . Endogenous Proteolytic systems and Meat Tenderness: influence of Post-mortem Storage and Processing. Food Sci Anim Resour. 2021, 41: 589-607

[9] Madhusankha GDMP, Thilakarathna RCN. Meat tenderization mechanism and the impact of plant exogenous proteases: a review. Arab J Chem. 2021, 14: 102967

[10] Mohd Azmi S, Kumar P, Sharma N, et al. . Application of Plant proteases in Meat Tenderization: recent trends and Future prospects. Foods. 2023, 12: 1336

[11] Melloni E, Averna M, Stifanese R, et al. . Association of Calpastatin with Inactive Calpain. J Biol Chem. 2006, 281: 24945-54

[12] Zhang H, Pan J, Wu Z. Investigation of the effects of high pressure processing on the process of rigor in pork. Meat Sci. 2018, 145: 455-60

[13] Bai Y, Hou C, Huang C, et al. . Phosphorylation of calpastatin negatively regulates the activity of Calpain. Life. 2023, 13: 854

[14] Warner RD, Kerr M, Kim YHB, Geesink G. Pre-rigor carcass stretching counteracts the negative effects of high rigor temperature on tenderness and water-holding capacity– using lamb muscles as a model. Anim Prod Sci. 2014, 54: 494

[15] Lyu J, Puolanne E, Ertbjerg P. Relationship between pre-rigor temperature of pork longissimus muscle, myofibril-bound calpain activity and protein degradation. Meat Sci. 2023, 198: 109094

[16] Álvarez C, Morán L, Keenan DF, et al. . Mechanical and biochemical methods for Rigor Measurement: relationship with eating Quality. J Food Qual. 2019, 2019: 1-13

[17] Ramos PM, Wright SA, Delgado EF, et al. . Resistance to pH decline and slower calpain-1 autolysis are associated with higher energy availability early postmortem in Bos taurus indicus cattle. Meat Sci. 2020, 159: 107925

[18] Peng Y, He W, Teng S, Jamali MA. The degradation of intramuscular connective tissue in Vitro with purified cathepsin L from bovine pancreas. Foods. 2023, 12: 3517

[19] Kaur L, Hui SX, Boland M. Changes in cathepsin activity during low-temperature Storage and Sous Vide Processing of Beef Brisket. Food Sci Anim Resour. 2020, 40: 415-25

[20] Morellon-Sterling R, El-Siar H, Tavano OL, et al. . Ficin: A protease extract with relevance in biotechnology and biocatalysis. Int J Biol Macromol. 2020, 162: 394-404

[21] Naveena BM, Mendiratta SK, Anjaneyulu ASR. Tenderization of buffalo meat using plant proteases from Cucumis Trigonus Roxb (Kachri) and Zingiber officinale roscoe (ginger rhizome). Meat Sci. 2004, 68: 363-9

[22] Kwatra B. A review on potential properties and therapeutic applications of bromelain. World J Pharm Pharm Sci. 2019, 8: 488-500

[23] Israelian V, Holembovska N, Slobodianiuk N. Application of the papain enzyme in meat products technology. Anim Sci FOOD Technol. 2021, 12: 60-7

[24] Sullivan GA, Calkins CR. Application of exogenous enzymes to beef muscle of high and low-connective tissue. Meat Sci. 2010, 85: 730-4

[25] Zhang B, Sun Q, Liu H-J, et al. . Characterization of actinidin from Chinese kiwifruit cultivars and its applications in meat tenderization and production of angiotensin I-converting enzyme (ACE) inhibitory peptides. LWT. 2017, 78: 1-7

[26] Naqvi ZB, Campbell MA, Latif S, et al. . Improving tenderness and quality of M. biceps femoris from older cows through concentrate feeding, zingibain protease and sous vide cooking. Meat Sci. 2021, 180: 108563

[27] Kaur L, Mao B, Bailly J, Oladeji O, Blatchford P, McNabb WC. Actinidin in green and SunGold kiwifruit improves digestion of alternative proteins—An in vitro investigation. Foods. 2022;11. https://doi.org/10.3390/foods11182739.

[28] Lanzoni D, Bracco F, Cheli F, et al. . Biotechnological and Technical challenges related to cultured meat production. Appl Sci. 2022, 12: 6771

[29] Yuen JSK, Saad MK, Xiang N, et al. Aggregating in vitro-grown adipocytes to produce macroscale cell-cultured fat tissue with tunable lipid compositions for food applications. Elife. 2023;12. https://doi.org/10.7554/eLife.82120.

[30] Dominguez-Hernandez E, Salaseviciene A, Ertbjerg P. Low-temperature long-time cooking of meat: eating quality and underlying mechanisms. Meat Sci. 2018, 143: 104-13

[31] Lee J-H, Kim T-K, Kang M-C, et al. . Methods to isolate muscle stem cells for cell-based cultured meat production: a review. Animals. 2024, 14: 819

[32] Spinazzola J, Gussoni E. Isolation of primary human skeletal muscle cells. Bio Protoc. 2017;7. https://doi.org/10.21769/BioProtoc.2591.

[33] Feige P, Rudnicki MA. Isolation of satellite cells and transplantation into mice for lineage tracing in muscle. Nat Protoc. 2020, 15: 1082-97

[34] Takahashi H, Yoshida A, Gao B, et al. . Harvest of quality-controlled bovine myogenic cells and biomimetic bovine muscle tissue engineering for sustainable meat production. Biomaterials. 2022, 287: 121649

[35] Mitterer A, Tauer C, Reiter M, Mundt W. Method of isolation and purification of trypsin from pronase protease and use thereof. Patent document US6830917 B2. 2004.

[36] Miersch C, Stange K, Röntgen M. Effects of trypsinization and of a combined trypsin, collagenase, and DNase digestion on liberation and in vitro function of satellite cells isolated from juvenile porcine muscles. Vitro Cell Dev Biol Anim. 2018, 54: 406-12

[37] Huang H-L, Hsing H-W, Lai T-C, et al. . Trypsin-induced proteome alteration during cell subculture in mammalian cells. J Biomed Sci. 2010, 17: 36

[38] Lai T-Y, Cao J, Ou-Yang P, et al. . Different methods of detaching adherent cells and their effects on the cell surface expression of Fas receptor and Fas ligand. Sci Rep. 2022, 12: 5713

[39] Derakhti S, Safiabadi-Tali SH, Amoabediny G, Sheikhpour M. Attachment and detachment strategies in microcarrier-based cell culture technology: a comprehensive review. Mater Sci Engineering: C. 2019, 103: 109782

[40] Kim N-S, Yu H-Y, Chung N-D, et al. . High-level production of recombinant trypsin in transgenic rice cell culture through utilization of an alternative carbon source and recycling system. Enzyme Microb Technol. 2014, 63: 21-7

[41] Zhang Y, Ling Z, Du G, et al. . Improved production of active Streptomyces griseus trypsin with a Novel Auto-Catalyzed Strategy. Sci Rep. 2016, 6: 23158

[42] Matinfar A, Dezfulian M, Haghighipour N, et al. Replacement of trypsin by proteases for medical applications. Iran J Pharm Res. 2022;21. https://doi.org/10.5812/ijpr-126328.

[43] Zhang Y, Zhang Y, Zhang Y, et al. Improving production of Streptomyces griseus trypsin for enzymatic processing of insulin precursor. Microb Cell Fact. 2020;19. https://doi.org/10.1186/s12934-020-01338-9

[44] Biagini G, Senegaglia AC, Pereira T, et al. 3D poly(lactic acid) scaffolds promote different behaviors on endothelial progenitors and adipose-derived stromal cells in comparison with standard 2D cultures. Front Bioeng Biotechnol. 2021;9. https://doi.org/10.3389/fbioe.2021.700862.

[45] Weber C, Pohl S, Pörtner R, et al. . Expansion and harvesting of hMSC-TERT. Open Biomed Eng J. 2007, 1: 38-46

[46] Nienow AW, Rafiq QA, Coopman K, Hewitt CJ. A potentially scalable method for the harvesting of hMSCs from microcarriers. Biochem Eng J. 2014, 85: 79-88

[47] Rodrigues AL, Rodrigues CAV, Gomes AR, Vieira SF, Badenes SM, Diogo MM, et al. Dissolvable microcarriers allow scalable expansion and harvesting of human induced pluripotent stem cells under xeno- free conditions. Biotechnol J. 2019;14. https://doi.org/10.1002/biot.201800461.

[48] Van Beylen K, Papantoniou I, Aerts J-M. Microcarrier screening and evaluation for dynamic expansion of human periosteum-derived progenitor cells in a xenogeneic free medium. Front Bioeng Biotechnol. 2021;9. https://doi.org/10.3389/fbioe.2021.624890

[49] Tzimorotas D, Solberg NT, Andreassen RC, et al. Expansion of bovine skeletal muscle stem cells from spinner flasks to benchtop stirred-tank bioreactors for up to 38 days. Front Nutr. 2023;10. https://doi.org/10.3389/fnut.2023.1192365.

[50] Rafiq QA, Brosnan KM, Coopman K, et al. . Culture of human mesenchymal stem cells on microcarriers in a 5 l stirred-tank bioreactor. Biotechnol Lett. 2013, 35: 1233-45

[51] Hanga MP, de la Raga FA, Moutsatsou P, et al. . Scale-up of an intensified bioprocess for the expansion of bovine adipose‐derived stem cells (bASCs) in stirred tank bioreactors. Biotechnol Bioeng. 2021, 118: 3175-86

[52] Wang Z, Zhang X, Xue L, et al. A controllable gelatin-based microcarriers fabrication system for the whole procedures of MSCs amplification and tissue engineering. Regen Biomater. 2023;10. https://doi.org/10.1093/rb/rbad068.

[53] Fraeye I, Kratka M, Vandenburgh H, Thorrez L. Sensorial and nutritional aspects of cultured meat in comparison to traditional meat: much to be inferred. Front Nutr. 2020;7. https://doi.org/10.3389/fnut.2020.00035.

[54] Broucke K, Van Pamel E, Van Coillie E, Herman L, Van Royen G. Cultured meat and challenges ahead: a review on nutritional, technofunctional and sensorial properties, safety and legislation. Meat Sci. 2023;195:109006. https://doi.org/10.1016/j.meatsci.2022.109006.

[55] Lambert EG, O’Keeffe CJ, Ward AO, Anderson TA, Yip Q, Newman PLH. Enhancing the palatability of cultivated meat. Trends Biotechnol. 2024;2476. https://doi.org/10.1016/j.tibtech.2024.02.014.

[56] Heck T, Faccio G, Richter M, Thöny-Meyer L. Enzyme-catalyzed protein crosslinking. Appl Microbiol Biotechnol. 2013, 97: 461-75

[57] Yang X, Zhang Y. Expression of recombinant transglutaminase gene in Pichia pastoris and its uses in restructured meat products. Food Chem. 2019, 291: 245-52

[58] Abril B, Bou R, García-Pérez JV, Benedito J. Role of enzymatic reactions in meat processing and use of emerging technologies for process intensification. Foods 2023;12(10):1940. https://doi.org/10.3390/foods12101940.

[59] Dong H, Wang P, Yang Z, Xu X. 3D printing based on meat materials: challenges and opportunities. Curr Res Food Sci. 2023;6:100423. https://doi.org/10.1016/j.crfs.2022.100423.

[60] Yokoyama K, Nio N, Kikuchi Y. Properties and applications of microbial transglutaminase. Appl Microbiol Biotechnol. 2004;64:447–54. https://doi.org/10.1007/s00253-003-1539-5.

[61] Yen FC, Glusac J, Levi S, et al. Cultured meat platform developed through the structuring of edible microcarrier-derived microtissues with oleogel-based fat substitute. Nat Commun. 2023;14. https://doi.org/10.1038/s41467-023-38593-4.

[62] Liu Y, Wang R, Ding S, et al. Engineered meatballs via scalable skeletal muscle cell expansion and modular micro-tissue assembly using porous gelatin micro-carriers. Biomaterials. 2022;287. https://doi.org/10.1016/j.biomaterials.2022.121615.

[63] Siddiqui SA, Bahmid NA, Karim I, Mehany T, Gvozdenko AA, Blinov AV, t al. Cultured meat: processing, packaging, shelf life, and consumer acceptance. LWT 2022;172:114192. https://doi.org/10.1016/j.lwt.2022.114192.

[64] Kang DH, Louis F, Liu H, et al. Engineered whole cut meat-like tissue by the assembly of cell fibers using tendon-gel integrated bioprinting. Nat Commun. 2021;12. https://doi.org/10.1038/s41467-021-25236-9.

[65] Kuppusamy P, Kim D, Soundharrajan I, Hwang I, Choi KC. Adipose and muscle cell co-culture system: a novel in vitro tool to mimic the in vivo cellular environment. Biology. 2020;10(1):6. https://doi.org/10.3390/biology.

[66] Rubio NR, Xiang N, Kaplan DL. Plant-based and cell-based approaches to meat production. Nat Commun. 2020;11:6276. https://doi.org/10.1038/s41467-020-20061-y.

[67] Letti LAJ, Karp SG, Molento CFM, et al. . Cultivated meat: recent technological developments, current market and future challenges. Biotechnol Res Innov. 2021, 5: e2021001

[68] Karp SG, Herrmann LW, Biagini G, et al. Patents and innovations in cultivated meat production. In: Soccol CR, Molento CFM, Reis GG, Karp SG, editors. Cultivated meat - technologies, commercialization and challenges. Springer Nature; 2024.

[69] Liu S, Wang X, Zhou J et al. (2020) Improving morphology of artificial meat derived from animal cells used in food engineering, by mixing and stirring animal stem cell cultured meat emulsion, salt, phosphate, water, casein powder and substrate transglutaminase. Patent document CN111513263-A.

[70] Hong W, Wu Z, Tao T et al. (2023) Edible cell cultured meat hydrogel bioscaffold useful in preparing cell culture foods comprises hydrogel system formed by plant protein, transglutaminase and polyphenols. Patent document CN117247886-A.

[71] Hong JK, Choi BK. Scaffold to produce cultured meat, comprises biocompatible enzymatically cross-linked polymer comprising gelatin-containing polypeptide, patterned surface in some areas, and third surface region of planarized surface. Patent document KR2023081978-A. 2023.

[72] Machluf M, Fishman A, Davidovich-Pinhas M et al. (2023) Composition-of-matter used to produce food products comprises edible microparticles portion of edible microparticles including plant protein. Patent document WO2023026291-A1.

[73] Yang F, Li S, Li Y et al. Preparation raw material of the biological ink comprises pectin, transglutaminase, the first protein component and the second protein component. Patent document CN115747197-A/B. 2023.

Funding

Fundação Araucária(19.810.961-4)

Conselho Nacional de Desenvolvimento Científico e Tecnológico(309237/2021-1)

RIGHTS & PERMISSIONS

Jiangnan University