Abstract
Synbio milk, containing recombinant bovine β-lactoglobulin (β-LG), produced in the fungus Trichoderma reesei, was deficient nutritionally compared to bovine milk. It also contained both fungal proteins and fungal metabolites never verified safe for human consumption. By three independent methods synbio milk was found to contain predominantly fungal protein, not the 90–99% β-LG, claimed by the product developer. By (1) shotgun proteomics, (2) ELISA and classical protein analysis, and (3) simulated mixing experiments, measuring percent deviation of the amino acid composition of synbio milk from that of bovine milk, fungal protein content was found to be 75.1%, 86.5% and 90–95%, respectively. Additionally, high sensitivity untargeted mass spectrometry revealed substantial levels of 69 nutrients in bovine milk, of which only 7 were present in small amounts in synbio milk. This analysis also revealed 93 compounds in synbio milk, byproducts/waste products of fungal fermentation, whose chemical identities could not be established searching large mass spectral databases, suggesting they are novel compounds. Neither these nor the fungal proteins found in the synbio milk have been tested for safety or allergenicity at exposure levels relevant for synbio milk consumption. Therefore, comprehensive toxicity and allergenicity testing are needed to assess the safety of synbio milk for human consumption.
Data availability
All data used in this research are freely available and can be obtained on reasonable request from the corresponding author. Proteomic mass spectrometry data and search results are available from the Massive data repository (massive.ucsd.edu) and Proteome exchange (www.proteomexchange.org) using the repository numbers MSV000099870 and PXD070657 respectively. For questions, contact ccms@proteomics.ucsd.edu.
References
Brooks, S. M. & Alper, H. S. Applications, challenges, and needs for employing synthetic biology beyond the lab. Nat. Commun. 12, 1390 (2021).
Meige, A. et al. The brave new world of synthetic biology-Major impacts, significant challenges. https://www.adlittle.com/en/insights/report/brave-new-world-synthetic-biology (2024).
Vegconomist. 7 Fermentation Companies Paving the Whey to a Cow-Free Dairy Industry. Cultivated X. https://cultivated-x.com/fermentation/7-companies-fermenting-whey-proteins-cow-free-dairy-industry/ (2024).
Ovadia, Y. Sustainable skim: Making dairy lactose-free, sans cows. CTech-Calcalist. https://www.calcalistech.com/ctech/articles/0,7340,L-3913944,00.html (2021).
Corella, R. Milk without cows? The new era of dairy, synthetic milk and sustainability. Metored. https://www.theweather.com/news/science/milk-without-cows-the-new-era-of-dairy-synthetic-milk-and-sustainability.html (2024).
Kwon, H. C., Jung, H. S., Kothuri, V. & Han, S. G. Current status and challenges for cell-cultured milk technology: A systematic review. J. Anim. Sci. Biotechnol. 15, 81 (2024).
Bojovic, M. Opinion: Synthetic milk is no longer a sci-fi fantasy, it’s here already. Alberta Farmer. https://www.albertafarmexpress.ca/opinion/opinion-synthetic-milk-is-no-longer-a-sci-fi-fantasy-its-here-already/ (2022).
Barnum, C. R., Endelman, B. J. & Shih, P. M. Utilizing plant synthetic biology to improve human health and wellness. Front. Plant Sci. 12, 691462 (2021).
Latham, J. R., Wilson, A. K. & Steinbrecher, R. A. The mutational consequences of plant transformation. Biomed. Res. Int. 2006, 025376 (2006).
Wilson, A. K., Latham, J. R. & Steinbrecher, R. A. Transformation-induced mutations in transgenic plants: Analysis and biosafety implications. Biotechnol. Genet. Eng. Rev. 23, 209–238 (2006).
Schubert, D. A different perspective on GM food. Nat. Biotechnol. 20, 969–969 (2002).
Eckerstorfer, M. F. et al. Biosafety of genome editing applications in plant breeding: Considerations for a focused case-specific risk assessment in the EU. Biotech 10, 10 (2021).
Chu, P. & Agapito-Tenfen, S. Z. Unintended genomic outcomes in current and next generation GM techniques: A systematic review. Plants 11, 2997 (2022).
Koller, F. & Cieslak, M. A perspective from the EU: Unintended genetic changes in plants caused by NGT—their relevance for a comprehensive molecular characterisation and risk assessment. Front. Bioeng. Biotechnol. 11, 1276226 (2023).
Hilbeck, A. et al. No scientific consensus on GMO safety. Environ. Sci. Eur. 27, 4 (2015).
Caradus, J. R. Intended and unintended consequences of genetically modified crops—Myth, fact and/or manageable outcomes?. N. Z. J. Agric. Res. 66, 519–619 (2023).
CONAHCYT. Effects of GM corn on human health, the environment and biodiversity, including the biocultural richness of native corn in Mexico. National Council for Humanities, Science and Technology-Mexico. https://usrtk.org/wp-content/uploads/2024/11/DOSSIER-MAIZ-2024-ENGfinal-5.pdf (2024).
Rodrigues, R. C., Pereira, H. S., Senra, R. L., Ribon, A. D. O. B. & Mendes, T. A. D. O. Understanding the emerging potential of synthetic biology for food science: Achievements, applications and safety considerations. Food Chem. Adv. 3, 100476 (2023).
Zeng, X. et al. Regulation and management of the biosecurity for synthetic biology. Synthetic Syst. Biotechnol. 7, 784–790 (2022).
Rajasekharan, M. & Ahmed, S. Superfood - Unveiling the “Dark Matter” of Food, Diets and Biodiversity. Interpress News Service. https://www.ipsnews.net/2024/06/unveiling-dark-matter-food-diets-biodiversity/ (2024).
Barabási, A.-L., Menichetti, G. & Loscalzo, J. The unmapped chemical complexity of our diet. Nat. Food 1, 33–37 (2019).
Mozaffarian, D. Nutrition’s dark matter of polyphenols and health. Nat. Food 2, 139–140 (2021).
Madureira, A. R., Pereira, C. I., Gomes, A. M. P., Pintado, M. E. & Xavier-Malcata, F. Bovine whey proteins—Overview on their main biological properties. Food Res. Int. 40, 1197–1211 (2007).
Villa, C., Costa, J., Oliveira, M. B. P. P. & Mafra, I. Bovine milk allergens: A comprehensive review. Comp. Rev. Food Sci. Food Safe 17, 137–164 (2018).
Adav, S. S., Chao, L. T. & Sze, S. K. Quantitative secretomic analysis of Trichoderma reesei strains reveals enzymatic composition for lignocellulosic biomass degradation. Mol. Cell. Proteomics 11, 1–15 (2012).
Ngan, S. F. C. & Sze, S. K. Proteomic profiling of the secretome of Trichoderma reesei. In Trichoderma reesei Vol. 2234 (eds Mach-Aigner, A. R. & Martzy, R.) 237–249 (Springer, US, 2021).
Alexander, L. J. et al. Beta-lactoglobulin [Bos taurus]. GenBank. https://www.ncbi.nlm.nih.gov/protein/520 (1989).
Wu, Y., Liu, Z. & Chen, X. Major allergen beta-lactoglobulin [Bos taurus]. GenBank https://www.ncbi.nlm.nih.gov/nuccore/EU883598.1 (2008).
Mohanta, T. K. et al. Virtual 2-D map of the fungal proteome. Sci. Rep. 11, 6676 (2021).
Wiśniewski, J. R., Hein, M. Y., Cox, J. & Mann, M. A “proteomic ruler” for protein copy number and concentration estimation without spike-in standards. Mol. Cell. Proteomics 13, 3497–3506 (2014).
Schwanhäusser, B. et al. Genome-wide parallel quantification of mRNA and protein levels and turnover in mammalian cells. Nature 473, 337–342 (2011).
Krey, J. F. et al. Accurate label-free protein quantitation with high- and low-resolution mass spectrometers. J. Proteome Res. 13, 1034–1044 (2014).
Millán-Oropeza, A., Blein-Nicolas, M., Monnet, V., Zivy, M. & Henry, C. Comparison of different label-free techniques for the semi-absolute quantification of protein abundance. Proteomes 10, 2 (2022).
Wang, H. et al. Tissue-based absolute quantification using large-scale TMT and LFQ experiments. Proteomics 23, 2300188 (2023).
Tyanova, S., Temu, T. & Cox, J. The MaxQuant computational platform for mass spectrometry-based shotgun proteomics. Nat. Protoc. 11, 2301–2319 (2016).
Verma, A. & Ambatipudi, K. Challenges and opportunities of bovine milk analysis by mass spectrometry. Clin. Proteom. 13, 8 (2016).
Bradshaw, C. V. et al. Shotgun proteomics of homogenate milk reveals dynamic changes in protein abundances between colostrum, transitional, and mature milk of swine. J. Anim. Sci. 99, skab240 (2021).
Vincent, D. et al. Milk bottom-up proteomics: Method optimization. Front. Genet. 6, 360 (2016).
Borin, G. P. et al. Comparative secretome analysis of Trichoderma reesei and Aspergillus niger during growth on sugarcane biomass. PLoS ONE 10, e0129275 (2015).
EFSA Panel on Food Enzymes (FEZ) et al. Safety evaluation of the food enzyme 6‐Phytase from the genetically modified Trichoderma reesei strain AR‐766. EFS2 23 (2025).
EFSA Panel on Food Contact Materials, Enzymes and Processing Aids (CEP) et al. Safety evaluation of the food enzyme cellulase from the genetically modified Trichoderma reesei strain AR‐852. EFS2 20 (2022).
EFSA Panel on Food Contact Materials, Triacylglycerol lipases and Processing Aids (EFSA CEP Panel) et al. Safety evaluation of the food enzyme triacylglycerol lipase from Trichoderma reesei (strain RF10625). EFS2 17 (2019).
Motoyama, A. & Yates, J. R. Multidimensional LC separations in shotgun proteomics. Anal. Chem. 80, 7187–7193 (2008).
Morales-Amparano, M. B., Huerta-Ocampo, J. Á., Pastor-Palacios, G. & Teran, L. M. The role of enolases in allergic disease. J. Allergy Clin. Immunol. Pract. 9, 3026–3032 (2021).
Achatz, G. et al. Molecular cloning of major and minor allergens of Alternaria alternata and Cladosporium herbarum. Mol. Immunol. 32, 213–227 (1995).
Glenn, K. C. et al. Biochemical and clinical studies of putative allergens to assess what distinguishes them from other non-allergenic proteins in the same family. Transgenic Res. 31, 507–524 (2022).
Sinitcyn, P. et al. Global detection of human variants and isoforms by deep proteome sequencing. Nat. Biotechnol. 41, 1776–1786 (2023).
Bekker-Jensen, D. B. et al. An optimized shotgun strategy for the rapid generation of comprehensive human proteomes. Cell Syst. 4, 587-599.e4 (2017).
Fonslow, B. R. et al. Improvements in proteomic metrics of low abundance proteins through proteome equalization using ProteoMiner prior to MudPIT. J. Proteome Res. 10, 3690–3700 (2011).
Guzman, U. H. et al. Ultra-fast label-free quantification and comprehensive proteome coverage with narrow-window data-independent acquisition. Nat. Biotechnol. 42, 1855–1866 (2024).
Jiang, X. et al. Improved production of majority cellulases in Trichoderma reesei by integration of cbh1 gene from Chaetomium thermophilum. Front. Microbiol. 11, 1633 (2020).
Thomas, K. GRN32-GRAS Notification for pectin lyase enzyme preparation (pectin transeliminase enzyme preparation) produced with Trichoderma reesei carrying a gene from aspergillus niger. FDA. https://www.hfpappexternal.fda.gov/scripts/fdcc/index.cfm?set=GRASNotices&id=32 (1999).
EFSA Panel on Food Contact Materials, Enzymes and Processing Aids (CEP) et al. Safety evaluation of the food enzyme pectin lyase from the genetically modified Trichoderma reesei strain RF6199. EFS2 20 (2022).
Food and Drug Administration. Food Labeling; Gluten-Free Labeling of Foods. Federal Register. http://data.europa.eu/eli/reg/2015/2283/oj (2013).
European Parliament and the Council. Regulation (EU) 2015/2283 of the European Parliament and of the Council of 25 November 2015 on novel foods, amending Regulation (EU) No 1169/2011 of the European Parliament and of the Council and repealing Regulation (EC) No 258/97 of the European Parliament and of the Council and Commission Regulation (EC) No 1852/2001. http://data.europa.eu/eli/reg/2015/2283/oj.
Canadian Parliament. Regulation and Guidelines for the Safety Assessment of Novel Foods - Canada.ca. Consolidated Regulations of Canada. https://www.canada.ca/en/health-canada/services/food-nutrition/legislation-guidelines/guidance-documents/guidelines-safety-assessment-novel-foods-2006.html (2015).
Campden BRI (Chipping Campden) Ltd. Comparing International Approaches to Food Safety Regulation of GM and Novel Foods. https://www.food.gov.uk/research/research-projects/comparing-international-approaches-to-food-safety-regulation-of-gm-and-novel-foods (2021).
GFSI-India. Novel food regulations around the world. GFSI India Online. https://gfi-india.org/novel-food-regulations-around-the-world/ (2024).
Center for Food Safety. U.S. Tries to Force Potentially Hazardous GM Corn on Mexico. Center for Food Safety. https://www.centerforfoodsafety.org/press-releases/6894/us-tries-to-force-potentially-hazardous-gm-corn-on-mexico (2024).
Zimmerman, S. USDA must do more to regulate genetically modified crops, judge rules _ Agriculture Dive. Agriculture Dive. https://www.agriculturedive.com/news/usda-crop-gene-editing-rule-trump-overturned/734970/ (2024).
GLP. Which genetically engineered crops and animals are approved in the US. Genetic Literacy Project. https://geneticliteracyproject.org/gmo-faq/which-genetically-engineered-crops-and-animals-are-approved-in-the-us/.
EFSA. Genetically modified organisms. EFSA. https://www.efsa.europa.eu/en/topics/genetically-modified-organisms (2025).
European Commission. GMO legislation—Food Safety—European Commission. European Commission. https://food.ec.europa.eu/plants/genetically-modified-organisms/gmo-legislation_en (2025).
EPA, FDA & USDA. Report on Stakeholder Outreach Related to Ambiguities, Gaps, Uncertainties, in Regulation of Biotechnology Under Coordinated Framework. https://www.epa.gov/system/files/documents/2023-11/eo14081-8a-stakeholder-engagement.pdf (2023).
EPA, FDA & USDA. The Coordinated Framework for the Regulation of Biotechnology Plain language information on the Biotechnology Regulatory System. https://usbiotechnologyregulation.mrp.usda.gov/sites/default/files/coordinated-framework-plain-language.pdf (2023).
McHughen, A. A critical assessment of regulatory triggers for products of biotechnology: Product vs. process. GM Crops Food 7, 125–158 (2016).
Wang, D. et al. Nano-enabled pesticides for sustainable agriculture and global food security. Nat. Nanotechnol. 17, 347–360 (2022).
Singh, R. Identifying ambiguities, gaps, inefficiencies, and uncertainties in the coordinated framework for the regulation of biotechnology. Ind. Biotechnol. 19, 79–82 (2023).
Lee-Muramoto, M. R. Reforming the “uncoordinated”. Drake J. Agricult. Law 17(2), 312–366 (2012).
Dooley, C. Regulatory silos: Assessing the United States’ regulation of biotechnology in the age of gene drives. Georgetown Environ. Law Rev. 30, 547–568 (2018).
Peck, A. The failure of federal biotechnology regulation. Valparaiso Univ. Law Rev. 51, 483–517 (2017).
Wunderlich, S. & Gatto, K. A. Consumer perception of genetically modified organisms and sources of information. Adv. Nutr. 6, 842–851 (2015).
McMullen, M. Food transparency translates to trust: what manufacturers need to know. Omnivore ([McMullen, 2024, p. 1] (zotero://select/groups/488871/items/978GKDK3)) https://omnivoreagency.com/2024/07/31/food-transparency-translates-to-trust-what-manufacturers-need-to-know/ (2024).
Zhang, M., Fan, Y., Chen, C., Cao, J. & Pu, H. Consumer perception, mandatory labeling, and traceability of GM soybean oil: Evidence from Chinese urban consumers. GM Crops Food 12, 36–46 (2021).
Food Insight. Survey: Nearly Half of U.S. Consumers Avoid GMO Foods; Large Majority Primarily Concerned About Human Health Impact. https://foodinsight.org/survey-nearly-half-of-u-s-consumers-avoid-gmo-foods-large-majority-primarily-concerned-about-human-health-impact/ (2018).
Todi, S. & Dixson, H. People must be at the heart of GMO policies. Nature India. https://www.nature.com/articles/d44151-024-00149-w (2024).
Bancroft, D. Synbio Perfumery: Making Smells and Flavors with Microbes. Labiotech https://www.labiotech.eu/trends-news/synbio-perfumery-tasty-smells-ingredients-microbes/ (2022).
Friends of the Earth. Synthetic Biology Vanillin: not natural, not sustainable, not likely to be labeled, and coming to an ice-cream cone. Friends of the Earth. http://www.synbiowatch.org/wp-content/uploads/2013/09/synbio_vanillin_fact_sheet.pdf (2013).
Xi, X. et al. Ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry MSE-based untargeted milk metabolomics in dairy cows with subclinical or clinical mastitis. J. Dairy Sci. 100, 4884–4896 (2017).
Kang, M. et al. Analytical strategies based on untargeted and targeted metabolomics for the accurate authentication of organic milk from Jersey and Yak. Food Chem. X 19, 100786 (2023).
Sharma, Y., Velamuri, R., Fagan, J. & Schaefer, J. Full-spectrum analysis of bioactive compounds in rosemary (Rosmarinus officinalis L.) as influenced by different extraction methods. Molecules 25, 4599 (2020).
Sharma, Y., Velamuri, R., Fagan, J. & Schaefer, J. UHPLC-ESI-QTOF-mass spectrometric assessment of the polyphenolic content of salvia officinalis to evaluate the efficiency of traditional herbal extraction procedures. Rev. Bras. Farmacogn. 30, 701–708 (2020).
Acknowledgements
This study was supported by grants from the Organic and Natural Health Association, the Non-GMO Project, Abby Rockefeller, the Foundation for Agricultural Integrity, as well as in kind support from Health Research Institute. We thank Churchtown Dairy and Radiance Dairy for providing milk samples. We thank Dr. Gabriela Grigorean for performing the shotgun proteomic analysis, including sample prep, the proteomic LC-MS/MS analysis and data analysis and draft writeup of the proteomics method, working in the Proteomics Core Facility of the Genome Center, University of California, Davis. The Bruker timsTOF HT LC/MS system was supported by the Howard Hughes Medical Institute, Investigator Award for Dr. Neal Hunter, UC Davis.
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All authors contributed to design of the research. R.V. and T.Y. carried out analytical procedures. R.V. conducted UPLC-QTOF data analysis and compound identification. All authors analyzed results. J.F. wrote the first draft, except for Methods, which were written by R.V. All authors reviewed and revised the manuscript. J.F. conceived of, planned and supervised the research.
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T.Y. declares no competing interests. J.F. is Chief Science Officer and CEO, and K.V. is Laboratory Director at the Health Research Institute, which provides authenticity, nutritional and residue testing to companies within the food, agriculture and nutritional supplements sectors including organic companies. None of the authors hold patents or have financial investments related to the content of this manuscript. JF has collaborated with Rodale Institute on research regarding organic agriculture. Maharishi International University has a department that specializes in regenerative organic agriculture. The funders of this project did not have input into the design, execution or interpretation of the research, nor did they have input into the writing and publication of the article.
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Velamuri, R., Yertha, T. & Fagan, J. Multi-omics profiling finds synbio milk differs nutritionally from bovine milk and contains 93 uncharacterized fungal metabolites and 236 fungal proteins. Sci Rep (2026). https://doi.org/10.1038/s41598-026-38994-7
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DOI: https://doi.org/10.1038/s41598-026-38994-7