译为中文，
 
 
Article	 
https://doi.org/10.1038/s41467-023-42486-x
Food amyloidf ibrils are safe nutrition ingredients based on in-vitro and in-vivo assessment
1234567890():,;	1234567890():,;	Received: 15 February 2023	Dan Xu1,2,8 , Jiangtao Zhou	2,8	, Wei Long Soon2,3, Ines Kutzli2, Adrian Molière4,
		Accepted: 12 October 2023	Sabine Diedrich2 , Milad Radiom	2,5, Stephan Handschin6, Bing Li1, Lin Li1,
			Shana J. Sturla2, Collin Y. Ewald4 & Raffaele Mezzenga	2,7
		Check for updates	Food protein amyloidf ibrils have superior technological, nutritional, sensorial,
			and physical properties compared to native monomers, but there is as yet
			insuff icient understanding of their digestive fate and safety for wide con-
			sumption. By combining SDS-PAGE, ELISA,f luorescence, AFM, MALDI-MS, CD,
microf luidics, and SAXS techniques for the characterization ofβ-lactoglobulin and lysozyme amyloidf ibrils subjected to in-vitro gastrointestinal digestion, here we show that either no noticeable conformational differences exist between amyloid aggregates and their monomer counterparts after the gas-trointestinal digestion process (as inβ-lactoglobulin), or that amyloidf ibrils are digested signif icantly better than monomers (as in lysozyme). Moreover, in-vitro exposure of human cell lines and in-vivo studies withC. elegansand mouse models, indicate that the digestedf ibrils present no observable cyto-toxicity, physiological abnormalities in health-span, nor accumulation off ibril-induced plaques in brain nor other organs. These extensive in-vitro and in-vivo studies together suggest that the digested food amyloids are at least equally as safe as those obtained from the digestion of corresponding native monomers, pointing to food amyloidf ibrils as potential ingredients for human nutrition.
 
Homo sapienshas been consuming dairy and poultry proteins since at least the Neolithic age, and no concerns exist about possible toxicity of these proteins in their native form, besides occasional allergenicity cases in a very restricted part of the human population. Yet, due to safety concerns regarding their consumption in a denatured form, specif ically aggregated into amyloidf ibrils, amyloid dairy and poultry proteins are not widely used. In fact, safety concerns associated with the amyloid status of aggregated proteins in general prevent use of
 

food amyloidf ibrils in foods, independent of the original food source from which the native protein precursor is extracted. From a structural perspective, amyloid f ibrils are f ibrillar aggregates of proteins char-acterized by a robust amyloid core of a highly-ordered cross-β architecture1. Protein amyloidf ibrils were discovered f irst as patholo-gical entities, associated with a variety of human disorders, including certain devastating neurodegenerative diseases, such as Alzheimer’s, Parkinson’s and Huntington’s diseases, as well as many other systemic
 
1School of Food Science and Engineering, Guangdong Province Key Laboratory for Green Processing of Natural Products and Product Safety, Engineering Research Center of Starch and Plant Protein Deep Processing, Ministry of Education, South China University of Technology, 381 Wushan RoadTianhe District Guangzhou 510640, China.2Institute of Food, Nutrition and Health (IFNH), Department of Health Sciences and Technology (HEST), ETH Zurich, Zürich 8092, Switzerland.3Center for Sustainable Materials (SusMat), School of Materials Science and Engineering, Nanyang Technological University, Singapore 639798, Singapore.4Institute of Translational Medicine, Department of Health Sciences and Technology (HEST), ETH Zurich, Schwerzenbach, Switzerland.
5Laboratory of Food Immunology, Institute of Food, Nutrition and Health, ETH Zürich, Zürich, Switzerland.6Scientif ic Center for Optical and Electron Microscopy (ScopeM), ETH Zurich, Otto-Stern-Weg 3, 8093 Zurich, Switzerland.7Department of Materials, ETH Zurich, Zürich 8092, Switzerland.8These
authors contributed equally: Dan Xu, Jiangtao Zhou.	 	e-mail:<PRESIDIO_ANONYMIZED_EMAIL_ADDRESS>;<PRESIDIO_ANONYMIZED_EMAIL_ADDRESS>
1
Nature Communications|        (2023) 14:6806 			

 

Article 	https://doi.org/10.1038/s41467-023-42486-x
 
amyloidoses2,3. However, an increasingly thorough understanding of amyloid aggregation suggests that these proteinaceous nanof ibrils are not limited to pathologies, and the f ibrillization propensity could constitute a generic property of many, if not all, existing proteins4.
In recent years, numerous examples of non-pathological amyloids have been found in various organisms, including humans2,5. The non-disease-related functional amyloids arise from many different pro-teins, including spider silk6, E. coli Curli f ibrils7 and human receptor-interacting protein kinase8. These amyloids play important physiolo-gical roles in living organisms, and in some cases even in humans, such as endocrine hormone peptides, which are stored in secretory gran-ules in the form of amyloids9. More recently, artif icial amyloids from natural proteins or synthetic polypeptides were intensively studied, and employed in diverse advanced nanotechnologies10–12. Among them, food proteins are an ideal source for manufacturing artif icial amyloid materials in-vitro because of the non-toxic nature of the protein precursor, their natural abundance, and low cost4,13.
Due to their remarkable physiochemical and nanomechanical properties, food protein amyloids have been used as biomaterials with a wide variety of superior functionalities, ranging from biomedicine
and	environmental	science	to	material	science	and
nanotechnology12,14. These food protein amyloids, which include milk β-lactoglobulin15, egg-white lysozyme16, ovalbumin17, oat18 and other proteins19–21 could offer substantial potential for food and pharma-ceutical industries4,22. Indeed, due to their rich functional groups and high surface-to-volume ratio, amyloid f ibrils are capable of carrying nutrients for delivery23, and are utilized for encapsulation of drug-like molecules in microgels or gel shells24. Additionally, biocompatible hybrid biomaterials based on amyloid f ibrils could be promising in antitumor therapy25, anti-obesity treatments26, and other biomedical applications27. However, in stark contrast to their remarkable success in many technologies and applications, the implementation of food protein amyloids into the human diet has long been hampered, mostly due to the extrapolation to the pathological amyloid class4. Since no systemic or pathological amyloidosis associated to food protein amyloids is reported to date, current concerns about their use in human nutrition remain associated with either the general nanotoxi-city effects, or more specif ically, the possibility that associated aggregates reach the bloodstream and nucleate the formation of pathological amyloids by cross-seeding4,13. Therefore, the most critical current question concerns whether amyloid nuclei resist gastro-intestinal digestion, whether the digested amyloids are cytotoxic, and whether the digested remaining counterparts cross epithelium into the bloodstream and induce cross-seeded pathological aggregates.
Here, we report a comprehensive investigation of the biological fate of amyloidf ibrils fromβ-lactoglobulin (β-lg) through a simulated in-vitro gastrointestinal digestion, in comparison with the native β-lg protein (Fig.1a). This milk-derived β-lg is one of the most intensively studied food proteins for amyloid formation, and it was therefore chosen as our main focus in this digestion study; we also expand these key results on in-vitro gastrointestinal digestion to hen egg white lysozyme (Lys) amyloids to evaluate the generality of ourf indings. Our results show that β-lg amyloids signif icantly degrade after gastric digestion and can be broken down in the following intestinal digestion, resulting in unfolded oligopeptides, indistinguishable from the pro-ducts of in-vitro digested β-lg monomer. Generalization to lysozyme amyloids shows not only that the residual digested peptides are unfolded oligopeptides but even that the amyloid digestion is more extensive than the corresponding monomers. These results strongly support that physiologically, β-lg and lysozyme amyloid f ibrils would degrade into unfolded amino acids polypeptides, which are, therefore, at least equally safe (β-lg) if not safer (lysozyme), than those obtained from digested native monomers. We further consolidate these results by evaluating the impact of the digestates on the viability of human intestinal epithelium cells and on in-vivo studies carried out on
 

C. elegans and mice. The results further support that the digested amyloids present no detectable cytotoxicity in human cells, nor adverse effects on movements during aging onC. elegans, nor any sign of cross-seeding to pathological amyloids in the investigated animal models upon long-term administration. In brief, these in-vitro and in-vivo results together suggest food amyloid f ibrils as a possible safe nutritional ingredient in human health applications.
Results
SDS-PAGE and ELISA assay 
Wef irst followed the dynamic proteolysis digestions ofβ-lg monomers and amyloid f ibrils with SDS-PAGE. The monomeric β-lg mainly remained intact with a molecular weight of 18 kDa during gastric digestion (Fig. 1b) with only a small proportion of digested β-lg frag-mented into two segments. This indicates the protease resistance ofβ-lg monomer while exposed to pepsin at the acidic condition, and this observed resistance may be due to the hydrophobic globular structure of β-lg monomer at low pH28,29. The indigestibility of β-lg monomers was also ref lected in its high allergenicity, as indicated by ELISA assay (Fig. 1d). This is a technique for detecting and quantifying target macromolecules such as peptides, proteins or antibodies, as antigens via specif ic immobilization with an antibody linked to a reporter enzyme. Here, we applied ELISA to detect the several motifs identif ied to be IgE-binding epitopes associated more frequently with milk allergy30,31 and have been suggested to contain pepsin cleavage sites. These motifs, which are reported to be part of the hydrophobic cluster in the core ofβ-lg under acidic pH32,33, render themselves inaccessible to pepsin hydrolysis and could potentially be the trigger of allergenic responses upon entering the intestinal tract. This resistance to diges-tion was also observed in the appearance of only a small proportion of short polypeptides after gastric digestion, in the case of β-lg mono-mers. Interestingly, we found a different digestion behavior on β-lg amyloid f ibrils (Fig. 1c), observed to be digested into polypeptides of less than 10 kDa within thef irst few minutes after pepsin introduction, during which their allergenicity dropped signif icantly (Fig.1e), hinting that the allergenic epitopes could have been more easily accessible to pepsin. Similarities of the peptide sequences fromβ-lg amyloidf ibrils afterf ibrillization34 with some of the IgE-binding epitopes from earlier studies highly support that these allergenic motifs are more suscep-tible to pepsin hydrolysis in the amyloid form due to surface presentation30,31,33. With the introduction of pancreatin in the intestinal phase, both the remaining undigestedβ-lg monomer and polypeptides from amyloid f ibrils from the gastric phase began to hydrolyze into shorter polypeptides of less than 5 kDa (Fig.1c). An SDS-PAGE gel with a higher resolution at lower molecular weights is shown in Supple-mentary Fig. 1. Their allergenicity was greatly reduced, as cleavage of allergenic motifs resulted from the opening of the compact structure of β-lg monomer due to the increase in pH35, while remaining motifs left on amyloid f ibrils uncleaved by pepsin were further degraded by pancreatin. In essence, this shows that the previously undigestibleβ-lg monomer became much more digestible after f ibrilization starting from the gastric tract. This observation was further supported and strengthened by the study of the other system considered, lysozyme, where lysozyme amyloidf ibrils were also observed to have improved digestibility and low allergenicity compared to its monomeric form, not only after the gastric tract, but even after the complete gastro-intestinal digestion (Supplementary Figs. 2 and 3).
Florescence assay and CD spectroscopy 
To investigate the possibility that amyloid core fragments withstand gastrointestinal digestion, thiazole orange (TO)f luorescence assay was applied to track the enzymatic degradation kinetics ofβ-sheet content in the monomeric and amyloid solution during digestion (Fig.1f–g). As seen in Fig. 1f, TO intensity of β-lg monomer exhibited a relatively slight decrease of 20% in the gastric phase as expected from their
 
Nature Communications|        (2023) 14:6806 	2
 

Article	a	b	β-lg monomer	Amylase	c	https://doi.org/10.1038/s41467-023-42486-x
						β-lgfibril
			Gastricphase	Intestinalphase			Gastricphase	Intestinalphase
		kDa				kDa		
		2 5 0				2 5 0		
			Pepsin			Pepsin	Amylase
		1 5 0			1 5 0		
		1 0 0
75			1 0 0
75		
		50			50		
		37			37		
	Gastric	2 5
2 0	Time(min)	Chymotrypsin 	&Trypsin	2 5
2 0	Chymotrypsin 	&Trypsin
		1 5
1 0				1 5
1 0		
							Time(min)
	digestion						
		d	�-lgmonomer			e	�-lg fibril
	Intestinal 		Gastric phase	Intestinal phase				Gastric phase	Intestinal phase
digestion
f	3	β-lg monomer	g	Absorbance	× 106	Formula
Milk 3	Absorbance	dmol−1)	Formula
Milk 3
						Milk 2
Milk 1		Milk 2
Milk 1
				T im e (m in )		i		T im e (m in )
				β-lgfibril
GastricphaseIntestinalphase	h
dmol−1)				
NormalizedTO signal 			NormalizedTO signal 	3 0
2 0
1 0				β-lg monomer		× 106
		Gastricphase IntestinalphaseI Gastric phase 	Intestinal phase								
										β-lg monomer
								Gastricdigestion		Gastricdigestion
								Intestinaldigestion		Intestinaldigestion
	2									
		Enzymatic signal Enzym Enzymaticsignal y atic sig gnal			Enzymatic signal Enzym Enzymaticsignal y atic sign nal	Molarellipticity(deg	-1	2 2 0	2 4 0	2 6 0	Molarellipticity(deg	-1
	1											
							-2					-2
	0											
							2 0 0					2 0 0	2 2 0	2 4 0	2 6 0
		Time(min)			Time(min)		Wavelength (nm)		Wavelength (nm)

 
Fig. 1 | Molecular and structural investigation ofβ-lg monomers and amyloid f ibrils during in-vitro gastrointestinal digestion. aSimulated gastrointestinal digestion phases of interest. Thisf igure was partly generated using Servier Medical Art provided by Servier licensed under a Creative Commons Attribution 3.0 unported license [https://smart.servier.com/how-to-cite-servier-medical-art/]. b,cSDS-PAGE ofβ-lg monomer (b) and amyloidf ibrils (c). The red arrows indicate theβ-lg monomer (b) and amyloidf ibrils (c).d,eELISA antigenicity assay ofβ-lg
resistance to pepsin hydrolysis, followed by a steep decay in thef irst 20 min of intestinal digestion to the background level of enzymatic solution. In contrast, the f luorescence intensity of β-lg amyloid f ibril decreased exponentially by 70% in the gastric phase, reaching as low as the background enzymatic signal in the intestinal phase (Fig.1g). These results were further conf irmed by Thiof lavin T (ThT) f luorescence assay in Supplementary Fig. 4. A similar trend was also observed for the digestion of lysozyme amyloid f ibrils, during which the addition of pepsin in the gastric phase resulted in a nearly 4-fold reduction in signal, pointing at the facile digestion of lysozyme amyloid f ibrils (Supplementary Fig. 5). A slower digestion in the intestinal phase might be due to the change of the degraded polypeptide charge states upon the gastric to intestinal pH transition with possible aggregation of these polypeptides.
Bulk technique CD spectroscopy was performed to detect the
presence of	β-sheet	content	before	and	after	gastrointestinal
Nature Communications|        (2023) 14:6806 
 

monomer (d) and amyloidf ibrils (e). The boxes cover the range of 25–75 percen-tage, the whisker lines and center lines refer to SD and median value respectively and square symbols indicate the mean value.n= 3 independent experiments. f,gTOf luorescence assay ofβ-lg monomer (f) and amyloidf ibrils (g). The enzy-matic signal refers to background contribution from digestive enzymes.n= 3 independent experiments.h,iCD spectra ofβ-lg monomer (h) and amyloidf ibrils (i) before and after digestion.
digestion.β-lg monomer consisted of a mixture ofβ-sheet andα-helix structures as shown by a broad peak ranging from 210 to 220 nm36
(Fig. 1h), which shifted slightly towards a lower wavelength in the gastric phase due to partial degradation by pepsin and further shifting towards the typical random coil structure in the intestinal phase. β-lg amyloid f ibrils showed a spectrum with a large single minimum at 218 nm (Fig. 1i), indicating the predominant β-sheet structure. This absorption dip shifted signif icantly to 200 nm after gastric digestion and further shifted to 198 nm after intestinal digestion, exhibiting a dominating random coil structures37. Thesef indings provide evidence that both β-lg amyloid f ibril and β-lg monomer can be decomposed into random coil polypeptides after gastrointestinal enzymatic reac-tion, with no detectableβ-sheet residues in the protein solution, sup-porting the observations in Fig.1f, g and suggesting the absence ofβ-sheet amyloid cores after digestion. The gastrointestinal digestion of lysozyme amyloid f ibrils was also shown to be much more eff icient
3
 

Article	Intensity (a.u.)	x 1 04	Before digestion	x 1 03
6	2798	Gastric-digested	https://doi.org/10.1038/s41467-023-42486-x
a							x 1 03
1 5	Intestinal-digested	1508
β-lg monomer		2	9185			x 1 04	1910	3134		x 1 03	1245	
				4		9184	1	879	4415	1 0	5	1059	1313
												1130	
										930
		1	18366	2		0	0	600	9 0 0 1 2 0 0 1 5 0 0
						0	2 0 0 0	4 0 0 0			
			6123			18360			
6122
b	0	2665	2308	1 0 0 0 0	2 0 0 0 0	0	4112	1 0 0 0 0	2 0 0 0 0	0	1 0 0 0 0	2 0 0 0 0
	0					0				0		
				m /z m /z				m /z m /z			m /z	
	x 1 03					x 1 04				x 1 03		
	1 2			9262 12161	2 0 0 0 0	6		x 1 03	878	1988 2802 3992	1 2	x 1 03	1059 1130
						904		1 0				
β-lg fibril	Intensity (a.u.)	8
4									8	1	
													930	1313
							4		5					
										2 0 0 0	4 0 0 0		0		
									0	0					9 0 0 1 2 0 0 1 5 0 0
							2						4		
														6 0 0	
							0						0		
	0							1 0 0 0 0	2 0 0 0 0		1 0 0 0 0	2 0 0 0 0
				1 0 0 0 0								
	0					0				0		
				m /z				m /z			m /z	
Fig. 2 | MALDI-MS spectra ofβ-lg monomer and amyloid f ibrils digested products. a,bβ-lg monomer (a) and amyloidf ibril (b) before digestion, after gastric, and
intestinal digestion. The a.u. refers to arbitrary units. Full range m/z was acquired in linear mode and insets are in ref lector mode.
 
than that of its monomeric form, exhibiting an obvious transition from the β-sheet peak towards random coil structures while the monomer was observed not to have been as well-digested (Supplemen-tary Fig. 6).
MALDI-mass spectrometry analysis 
To further determine the composition of digestedβ-lg monomers and amyloids, matrix-assisted laser desorption/ionization mass spectro-metry (MALDI-MS) f irst was carried out under linear mode detection (Fig. 2). The spectrum of native β-lg monomer showed three main
dominant	peaks,	corresponding	to	singly	(Mavg	≈	18366 Da)
[M + H]+ = (18366 + 1)/1 = 18367,	doubly	[M + 2H]2+ = (18366 + 2)/
2 = 9184, and triply charged [M + 3H]3+ = (18366 + 3)/3 = 6123 full-length β-lg (Fig. 2a). After gastric digestion, we again observed these three peaks of β-lg monomer remaining in addition to partially digested polypeptides below 5 kDa. The inserted plot obtained in ref lector mode provides additional details about the spectrum. After intestinal digestion, along with the disappearance of the main β-lg monomer peaks, the distribution of molecular weight shifted towards species of less than 5 kDa, leaving mostly digested oligopeptides of 1–2 kDa corresponding to ~10–20 amino acids. Amyloids after gastric digestion exhibited four main peaks of less than 4 kDa. Specif ically, some oli-gopeptides were digested below 1 kDa, other larger oligopeptides down to 3 kDa or more. Subsequent intestinal digestion of amyloid produced a large number of polypeptides with a molecular weight lower than 5 kDa centered around 1–2 kDa similar to those of β-lg monomer. Similar spectra were obtained after f iltering the digested amyloid solution with 10 kDa f ilters (Supplementary Fig. 7). Interest-ingly, in the ref lector mode spectrum after intestinal digestion we found a total of 100 peaks in theβ-lg monomers and only 55 peaks in theβ-lg amyloids (see Supplementary Table 1); out of the 55 detected peaks found in digested β-lg amyloid f ibrils, 38 were also found on digested β-lg monomers, 15, did not belong to the β-lg sequence (attributed to enzyme autolysis) and only two peaks (at 815.45 Da and
 

1354.71 Da) were found in the digested f ibrils but not the digested monomers. These two peaks can be sequenced into either VRTPEVD, or NENKVLV or VEELKPT for the 815.45 Da peak and YSLAMAASDISLL or QKVAGTWYSLAM for the 1354.71 Da peak. In contrast, in the case of the intestinally digestedβ-lg monomer we found as many as 22 peaks present only in the monomer sequence (but not in the amyloid sequence) with 40 peaks not belonging to the β-lg sequence (attrib-uted again to enzyme autolysis). This implies that only the two peaks at 815.45 Da and 1354.71 Da are unique to the digestedβ-lgf ibrils, while as many as 37% (22:60) of the fragments are unique to the digestedβ-lg monomers. However and most importantly, we dof ind all the possible sequence candidates for these two peaks at 815.45 Da and 1354.71 Da also occurring in larger fragments within the digestedβ-lg monomers, simply indicating that the digestion of theβ-lg amyloids has proceeded to a signif icantly larger extent than in the case of the β-lg monomers (see Supplementary Table 1), and thus, that residual oligopeptides from digested β-lg f ibrils cannot be more toxic than those from the digested homolog β-lg monomers. This conclusion is also reinforced by the digestion behavior of lysozyme—lysozyme monomers were barely digested and remained even after intestinal digestion, whereas lysozyme amyloid f ibrils were digested mostly into polypeptides of less than 1 kDa (Supplementary Fig. 8). We should note here that the MALDI-MS spectrometry is carried out at low enzyme content to minimize autolysis of the enzyme and that a more detailed sequencing of the digested peptides is carried out in the in-vivo part where the enzyme is present at physiological conditions (see below for details).
In-situ microf luidics screening system under polarized light microscopy 
To further understand the kinetics of amyloid f ibril digestion, a cir-culatory microf luidic system was set up for monitoring the dynamic in-situ digestion process of amyloid f ibrils under cross-polarized light microscope (Fig.3a). Amyloidf ibril solution was incubated in a water bath at 37 °C with constant shaking. Amyloidf ibrils in the solution were
 
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Article	Polarizer	Polarizer	CCD	b	1 0-2	Amyloidfibril	0 .1	0 .2	1 0-2	https://doi.org/10.1038/s41467-023-42486-x
a										c	e
										1 0 0
										Gastric digestion	Intestinal digestion	Amyloid fibril
				d		Gastric-digestion				le xip	80
										per	60
										ytis
						Intestinal-digestion				
										ntegratedniten	4 0
2 0
						MQ water					
										I	0
										0	3600	7 2 0 0	1 0 8 0 0	1 4 4 0 0
										Time (s)	Gastric digestion
				(a.u).		Before digestion				Gastric-digested	1 0-2	In te s tin a l- d ig e s te d
					1 0-3	 MQ water			1 0-3	MQ water	1 0-3	 MQ water
				I								
										 Gastric buffer	 Intestinal buffer
					1 0-4	0 .0 2			1 0-4	0 .0 2	0 .1	0 .2	1 0-4	0 .0 2	0 .1	0 .2
						q(1/Å)				q(1/Å)	q(1/Å)

 
Fig. 3 | In-situ screening of theβ-lg amyloidf ibril degradation behavior during digestion. aSchematic of microf luidic polarized light microscopy setup.
bPolarized light microscopy images of amyloidf ibrils during digestion. The result is verif ied by two independent tests. Scale bar is 1 mm.cIntensity per pixel over time. Error bars represent the standard deviation of 60 images. Data points are
aligned parallel to the shear f low in the cylindrical capillary38. This allows imaging of the aligned f ibrils with cross-polarized light, result-ing in a bright birefringence pattern, as observed in the polarized light images in Fig. 3b. In-vitro digestion was initiated by introducing enzymatic solution to amyloid solution, and the subsequent digestion process was screened by recording microscopy images every 0.5 s. After gastric and intestinal digestion, we observed a signif icant decrease of the birefringence represented by the normalized intensity on the polarized light images. The f inal intensity was comparable to that of deionized water (Fig.3b). Analyzing the integrated intensity of polarized light microscopy images revealed the kinetics of amyloid f ibrils degradation (Fig. 3c). Specif ically, upon the addition of pepsin, birefringence intensity immediately dropped, followed by an expo-nential decay in the f irst 30 min, but remained unstable with large variation in the rest of the gastric phase, which could be ascribed to the remaining shortf ibrils. A further decay of this signal was found in the beginning of the intestinal phase and was quickly stabilized to the range of the signal of deionized water, indicating the total absence of f ibrils in the solution.
SAXS andf luorescence imaging 
The conformational diversity of β-lg monomer and amyloid f ibril during different phases of digestion was investigated also by small-angle X-ray scattering (SAXS). In Fig. 3d, β-lg monomer, β-lg amyloid f ibril, and deionized water showed different prof iles before digestion. The β-lg amyloid f ibril exhibited a higher scattering intensity in the low-q region over the monomer and water, indicating larger aggre-gates in the solution39. After gastric digestion, the prof iles of β-lg monomer and digestion buffer overlapped, but showed differences from those of amyloids and deionized water. Remarkably, SAXS pro-f iles ofβ-lg amyloidf ibril,β-lg monomer and buffer were identical after intestinal digestion, which is conf irmed by the background subtracted prof iles (Supplementary Fig. 9), showing undistinguishable difference from that of deionized water. This indicates, in line with all the other experiments, that there is no detectable conformational diversity between the β-lg monomer and amyloid f ibril solution after gastro-intestinal digestion. This result was verif ied by STORM f luorescence
 

presented as mean value with error bar as SD.dSAXS measurement ofβ-lg monomer and amyloidf ibrils before digestion, after gastric, and intestinal diges-tion.e STORMf luorescence images ofβ-lg amyloidf ibrils before and after gastric digestion. The result is verif ied by two independent tests. Scale bars are 2μm.
images (Fig. 3e), in which amyloid f ibril solution exhibited a large quantity of f ibrillar f luorescent structure that disappeared during gastric digestion, showing a homogenous solution with no f ibrillar aggregates.
Single-Molecule Analysis of in-vitro Digested Amyloids 
While all in-vitro bulk ensemble experimental methods used above indicate full digestion of the amyloidf ibrils, it certainly is possible that at the very low enzyme concentration used (trypsin activity of 0.3 U/mL i.e. >500 times lower than that required by the INFOGEST protocol40), some f ibrils undetectable to bulk methods may not be entirely digested within the digestion time considered. The ratio of enzymevs. digested protein in the in-vitro evaluation was intentionally set as low as possible to minimize autolysis/cross-digestion of the enzyme, thus enabling meaningful MALDI-MS analysis, while allowing comprehensive digestion. However, this is of no concern for two main reasons: f irst, even eventual residual f ibrils found at these low enzy-matic concentrations will be digested at larger enzyme concentrations expected in-vivo; secondly, eventual undigested remnants, will be excreted in-vivo via feces, as shown in the in-vivo sections.
We therefore performed additional experiments to clarify the minimal concentration of enzyme required to digestf ibrils according to both bulk and single molecule methods. We applied AFM large-f ield-scan and random-scan imaging of in-vitro digested amyloids at dif-ferent enzyme concentrations, from a minimum of 0.3 U/mL trypsin to progressively higher concentrations of the enzymes until no f ibril
fragments	were	detected	in	any	samples	(see	Supplementary
Figs. 10–11 for more information). We found that the minimum amount of trypsin enzyme required to completely digest thef ibrils is 20 U/mL to 50 U/mL, which is 5 to 2 times lower than the amount of enzyme recommended for full substrate digestion by the in-vitro INFOGEST protocol. We also realized that the digestion protocol for a given enzyme concentration could be largely improved by stirring instead of shaking, requiring ca. 5–10 times less enzymes for a complete diges-tion (Supplementary Figs. 12–13). These results conf irm both by bulk and single molecule methods that the digestion of the amyloids is completed in-vitro within the boundaries of the INFOGEST method.
 
Nature Communications|        (2023) 14:6806 	5
 

Article	a	(a.u).	 	β-lg fibril	C aco2	1 2 5 0	Flourescence intensity(a.u.)	b	(a.u).	https://doi.org/10.1038/s41467-023-42486-x
										L y s f ib ril
				β-lg monomer						Lys monomer
	Caco2									
		ytilib aiv	1							ytilib aiv	1	
		viteclle							viteclle	
				50	2 5 0	500						50	2 5 0	500	1 2 5 0
		ale									ale				
		R									R				
		0									0				
	c	(a.u).		Concentration (		1 2 5 0		d	(a.u).	Concentration (	 
				β-lg fibril						Lys fibril
				β-lg monomer						Lys monomer
	HCEC									
		ytilib aiv	1							ytilib aiv	1	
		viteclle							viteclle	
				50	2 5 0	500						50	2 5 0	500	1 2 5 0
		ale									ale				
		R									R				
		0									0				
	e	4 0 0 0 0		Concentration (	 		4 0 0 0 0		f	1 0 0 0 0	C oncentration (	 
											4 0 0 0 0
											C aco2
	(a.u).	3 0 0 0 0		 	 	 	H C E C	3 0 0 0 0			(a.u).	8 0 0 0	H C E C
													3 0 0 0 0
2 0 0 0 0
1 0 0 0 0	Flourescence intensity(a.u.)
	ytis													
											ytis	6 0 0 0		
	olurescenceniten	2 0 0 0 0						2 0 0 0 0						
											olurescenceniten	4 0 0 0
2 0 0 0		
		1 0 0 0 0						1 0 0 0 0						
	F	0						0						
											F		

 
Fig. 4 | Effect of in-vitro digested protein monomers and amyloids on cell viability.The ratio of Caco2 cell viability treated withβ-lg (a) and lysozyme (b) f ibrils and monomers respectively, vs. digestion matrix at the treatment con-centration of 50, 250, 500 and 1250µg/mL. The ratio of HCEC cell viability treated withβ-lg (c) and lysozyme (d)f ibrils and monomers respectively, vs. digestion
Impact on viability in human cell lines 
To evaluate the biocompatibility of the in-vitro digested amyloids, we f irst characterized their effect on the viability of two types of human intestinal cell lines (cancer coli-2, Caco2; human colon epithelial cells, HCEC). Caco2 cells are widely used to study intestinal transport, but have phenotypic characteristics of cancer cells, whereas HCEC cells are complementary in being immortalized cells with no pathological fea-tures, and therefore an attractive model for testing potential impact on healthy cells41–43. We characterized cellular responses of exposure to the complex buffer used for the in-vitro digestion buffers (matrix), as well as the effects of digested monomer or digested amyloid from both β-lg and lysozyme (Supplementary Fig. 15). These assays were per-formed for f ibril and monomer concentrations ranging from 50 to 1,250μg/mL, as well as corresponding dilutions of the digestion matrix. When we compared the viability of both cell types following exposures to digested amyloids vs. digestion matrix, or digested
 

matrix at the treatment concentration of 50, 250, 500 and 1250µg/mL. Reactive oxidative species experiments for (e)β-lg and (f), lysozyme digestedf ibrils and their monomer controls. TBHP is the positive control. The a.u. refers to arbitrary units.N= 3 biological replicates.The plots are showed as mean values ± standard deviation.
monomers vs. digestion matrix (Fig.3a), it was apparent that there was no loss of cell viability due to the presence of the digestion products. In fact, in both cell lines there was growth promotion upon exposure to digestedβ-lg and lysozymef ibrils or monomers relative to exposures to the digestion matrix alone (Fig. 4), consistent with an increase in bioavailable nutritive protein components.
No signif icant differences were found when comparing the via-bility of cells upon exposure to digested monomervs. digestedf ibril, in both Caco2 and HCEC cell lines at all concentrations. The highest concentration of digestion matrix alone caused a strong reduction in cell viability (Supplementary Fig. 15), however the relative inf luence of the digestedf ibrils or monomers at any of the concentrations was not associated with any additional negative impacts (Supplementary Fig. 15). To address whether digestion is even capable to suppress toxicity of pathological amyloids, we additionally tested Aβ42 f ibrils as pathological amyloid model. Interestingly, we found that even
 
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Article 	https://doi.org/10.1038/s41467-023-42486-x
 
digested Aβ42 f ibrils did not reduce cell viability (Supplementary Fig. 16). Thus, the observations regarding overall cell viability are fully consistent with predictions from the digestion behavior, supporting that on af irst-tier cytotoxicity assessment, digestedf ibrils are as safe as their digested monomers as there is no detectable cytotoxic effects, and furthermore, that they are well broken down to biocompatible and nutritive peptides, even in the case of Aβ42 f ibrils.
These results were further supported by reactive oxidative spe-cies (ROS) experiments, as shown in Fig.4e, f, where for bothβ-lg and lysozymef ibrils and in both Caco2 and HCEC cells,f luorescence indi-cating the generated stress had comparable values for digestedf ibrils and their monomer controls.
C. elegans model 
To examine any toxic effects of feeding amyloids to C. elegans, we assessed the mobility ofC. elegans(i.e., swimming rates) during aging. We did not observe any detrimental effects of either monomers,f ibrils, or digestedf ibrils on their physical activity while on the contrary,β-lg f ibrils and lysozyme f ibrils prolonged health span during C. elegans aging (Fig. 5a, Supplementary Table 2). Since the same amount of protein (0.1667 mg/ml) among monomers,f ibrils, and digestedf ibrils were fed, we conclude that the f ibrils inf luenced the metabolism of C. elegansto promote health span. We observed higher relative moti-lities and improved health whenC. elegansis fed byβ-lg and lysozyme f ibrils (Fig.5a, Supplementary Fig. 17, Supplementary Table 2).
Next, we asked whether f ibrils could promote aggregation of endogenously expressed disease-related aggregation-prone proteins. Previously, feeding 0.1 mg/ml of human α-synuclein f ibrils but not monomers to C. elegans promoted the endogenous aggregation of muscle-expressed human α-synuclein39. We chose the polyglutamine-expansion protein aggregation model that expresses human Hun-tington’s disease polyglutamine with 35 repeats (polyQ35) tagged with YFP in the muscles ofC. elegans, which does not aggregate on day 1 of adulthood (Fig. 5b), partially aggregated on day 5 of adulthood (Fig. 5c), and fully aggregated at day 10 of adulthood44. We found no change in polyQ aggregation at day 5 of adulthood when 0.25 or 1.5 mg/ml ofβ-lgf ibrils were fed, and a mild increase of the number of polyQ aggregates in muscles when lysozymef ibrils were fed at a higher concentration (1.5 mg/ml) but not at the lower concentration of 0.25 mg/ml lysozyme f ibrils (Fig. 5d, Supplementary Table 3). These results suggest only integrant, undigested lysozymef ibrils at very high concentrations may slightly affect polyQ aggregation in C. elegans mutants via cross-seeding, while all other cases including the digested f ibrils, do not. This implies that, even for amyloid f ibril cross-seeding events at extreme concentrations in polyQ C. elegans mutants with simple epithelia, the in-vitro human-digested amyloid f ibrils are safe and trigger no cross-seeding, proving the protective mechanism of Craniatagastrointestinal system.
Mouse Model 
Having assessed the effect of digested amyloidf ibrils on theC. elegans model, we turned our attention to mouse models. LC-MS/MS analysis on the fate of β-lg/lysozyme amyloids f ibrils digested in-vivo by mice was applied to verify our in-vitro results. To identify the digested products ofβ-lg/lysozymef ibrils, contents in the intestine and colon of mice after 4 h oral administration were measured by LC-MS/MS. The heat map illustrates the identif ied digested oligopeptides of β-lg/ lysozyme f ibrils and monomers in the small intestine after digestion (Fig.6a and Supplementary Fig. 18). A large number of low molecular weight fragments were observed due to proteolysis by intestinal enzymes, and we generally found that digested peptides of lower (Fig. 6a) or comparable molecular weights (Supplementary Fig. 18) were detected for the amyloids compared to the monomer controls, conf irming in-vivo the superior digestibility of amyloids vs monomers. Most of the oligopeptide sequences from the digestion of β-lg f ibrils
 

were also identical to those of their monomers, with the exception of LNENKV and NGECAQK, both non-amyloidogenic sequences. In the case of lysozyme digestion, all peptides observed for amyloids were also found as monomers (Supplementary Fig. 18).
Given current concerns of the possibility that amyloid cores could remain after digestion and traverse the epithelium into the blood-stream, we then sought to determine if the digested oligopeptides from β-lg/lysozyme f ibrils could be detected in serum. Most impor-tantly, after 1 h of oral administration ofβ-lg/lysozyme monomers and f ibrils, no oligopeptides from eitherβ-lg/lysozyme monomer norf ibrils were detected in the serum of mice (see Supplementary Table 4 for details). Unabsorbed oligopeptides proceeded to the colon, extracted and subjected to further MS analysis (Supplementary Fig. 19).
The monitoring of the distribution of β-lg f ibril and monomer during digestion was performed using 5-(4,6-dichlorotriazinyl) ami-nof luorescein (5-DTAF) labeled-β-lg f ibrils and monomer. Mice fed with labeled f ibrils were sacrif iced at different time points and their respective organs and blood were imaged. The migration of labeled-β-lg f ibril and monomer through the intestinal tract could be clearly tracked by the high intensity regions through the 6-h time frame. Unabsorbed digests exited the intestinal tract and f inally were excre-ted as feces (Fig. 6b, c). Further monitoring over 24 h demonstrated the absence off luorescence in mice blood serum, and in the same vein (Fig. 6d, e), no f luorescence was also detected in the organ tissues of the mice (Supplementary Fig. 20), which corroborated no absorption of labeled peptides across epithelium into the bloodstream.
To rule out absorption of degraded f ibrils through the gastro-intestinal epithelia and possible targeting of other organs, we addressed the possibility of the formation in mouse tissues of amyloid plaques induced by long-term administration of β-lg/lysozyme amy-loid f ibrils. As a positive control, amyloid plaques were observed in Congo Red (CR)-stained brain tissue sections from the Alzheimer’s disease (AD) FAD4T mouse group. However, no plaques were observed in the brain tissues or other main organs of mice after 30 days (Sup-plementary Figs. 21–22) nor after 60 days (Fig.6f and Supplementary Fig. 23) β-lg-FL, β-lg-FH, Lys-FL and Lys-FH (where F stands for f ibril, L and H stand for high and low dosage, refer to methods) adminis-
tration.	Thus,	histological	analysis	appeared	undistinguishable
between to those from the non-AD control group fed with a normal diet and those fed dietaryβ-lg-F or Lys-F.
Discussion
A putative overview of the biological fate ofβ-lg monomer and amyloid f ibrils, after entering the gastrointestinal tract, emerging from this work is shown in Supplementary Fig. 24. Native β-lg monomers exhi-biting strong pepsin resistance during gastric digestion, mainly main-tain their monomeric state and partially cleave into polypeptides and oligopeptides. Undigestedβ-lg monomers are then broken down into oligopeptides and amino acids by intestinal enzymes. In stark contrast, β-lg amyloid f ibrils are rapidly disassembled by pepsin hydrolysis, disassembling into polypeptides and oligopeptides with residual short amyloidf ibrils at the end of the gastric phase. These remnants are then entirely degraded into unfolded oligopeptides and amino acids, in the following intestinal phase. The fate of these oligopeptides from there on can therefore only be postulated to be identical, that is, undergo further degradation into di-/tri-peptides or amino acids by the enzy-matic activities of enterocyte membranes, before entering blood cir-culation either via portal vein or lymph system. A generalization of these results is inferred from additional experiments on hen egg white lysozyme amyloidf ibrils digestion compared to that of native homolog monomers (found in supplementary information), where results even indicate an improved digestibility of the amyloids versus the mono-mers. In support of this picture, we found that β-lg/lysozyme f ibrils digested in-vitro as above and then exposed to two distinct types of human epithelium cell lines, behaved essentially as their digested
 
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Article	a	1.5	Control	https://doi.org/10.1038/s41467-023-42486-x
Lysozyme monomer
Relativemotility	1.0	Lysozyme monomer digested
	0.5	Lysozyme fibril	P < 0.0001
		Lysozyme fibril digested
		Digest buffer control
	0.0	
-0.5
6	12	18	24	30
Days of adulthood
1.5	C ontrol
ß -lg m o n o m e r
Relativemotility	1.0	β-lg ß -lg m o n o m e r d ig e s te d
	0.5	β-lg ß-lg fibril	P < 0.0001
		β-lg ß-lgfibril digested
		Digest buffer control
	0.0	
-0.5
6	12	18	24	30
Days of adulthood
b	aggregates/worm	25	d	P = 0.9997	aggregates/worm	20	P = 0.9992
							P = 0 .1 2 8 0
				P = 0 .0 0 3 8			
							P = 0 .3 6 2 1
				P = 0.9999			
							P = 0.8669
							P = 0 .9 9 6 1
c		20		P = 0.9959		15	
		15					
						10	
		10					
		0				0	

 
Fig. 5 | In-vivo effects of amyloid ingestion onC. elegans. aNo mobility impair-ment in any condition after ingestion of either digested or undigested lysozyme andβ-lg monomers andf ibrils. By contrast,β-lgf ibrils and lysozyme f ibrils pro-longed healthspan (active movement) ofC. elegans. Data was analysed using a one-way ANOVA, followed by Dunnett’s multiple comparisons test of the area under the curve. Proteins were supplemented at day 1 of adulthood. Data are presented as mean ± SD. The p value of the control vs.β-lg samples areβ-lg monomer (0,9546), β-lg mono digested (0,9918),β-lgf ibril (<0,0001) andβ-lgf ibril digested (0,9996). The p value of the control vs. lysozyme samples are lysozyme monomer (0,9842),
 

lysozyme mono digested (>0,9999), lysozymef ibril (<0,0001), lysozyme digested (0,3958) and digest buffer control (0,9996).b,cRepresentative transgenic AM140 C. elegansshowing of non-aggregated (day 1) (b) and aggregated (day 5) (c) Q35-YFP signal, respectively.N= 3 biological replicates.dSupplementation of protein at day 1 of adulthood leads to increased number of polyglutamine (Q35) aggregates in transgenic AM140C. elegansexpressing Q35-YFP in muscles ingesting a high con-centration of lysozymef ibrils, but no other condition. Shown is one out of three independent biological trials. Data was analysed using a one-way ANOVA, followed by Dunnett’s multiple comparisons test. Data are presented as mean ± SD.
 
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Article	
1 8 .2 4		5.06		0 .3 3				2 8 .0 5		27.55	
1 2 .7 6	4 2 . 4 3	71.97	17.85	49.95	1 5 .4 1					1 2 .1 3	89.36
16.77	1 2 .6			9.59							
	0 .7 3		1 2 .1	8.49		1 4 .4 2		1 1 .5 8	1 0 .7	1 0 .3 4	
			2 .7 5	1 .5 4			2 4 .6 9				
1 .3 9											
	5.97	2 .8 1	3.63	3.58		53.86	60.38				
4 .1 8	8.85		4.9		2 6 .0 1	15.72			3 2 .6 3	45.17	
1 5 .2 7			25.88	1 7 .7 3							
		4.68	6.14		8.26		1 4 .9 2	37.43	1 6 .8 1	4.81	1 0 .6 4
						1 6			15.63		
5.93	4.9	0 .6 2	5.46		1 2 .8 4						
								2 2 .9 4	2 4 .2 3		
15.68	1 7 .0 8	1 4 .8 5	1 6 .0 6	6.5	3 2 .5 8						
4.8	3 .3 7		5.23								
1 .8 2	1 .0 4										
3 .1 6	3 .0 5			2 .2 9	4.9						
	https://doi.org/10.1038/s41467-023-42486-x
viteabundance(%)		 
ale		
R		

 
Fig. 6 | In-vivo digestion and effects of amyloid ingestion on mice. aIdentif ied digested oligopeptides ofβ-lg monomer andf ibrils in small intestine of mice after 4 h oral administration.b,cFluorescence images of mice small intestines during different digestion time and feces after administration of 5-DTAF-labeledβ-lgf ibril (b) and monomer (c).d,e Fluorescence images of mice blood during different
monomeric counterparts, that is they featured no detectable cyto-toxicity and produced increased viability of the cells compared to the same culture in absence of proteins, indicating that they were broken down to biocompatible and nutritive peptides.
The in-vivo results collaborate and reinforce the in-vitro evidence: no signs of cytotoxicity of digestedf ibrils were observed inC. elegans or mouse models, nor cross-seeding events from digestedf ibrils could be detected in the two animal models. Our mice experimentalf indings demonstrated that β-lg and lysozyme f ibrils can be digested into the same oligopeptides as their monomers, and that any unabsorbed digested oligopeptides of f ibrils do not enter the bloodstream and instead are further metabolized and excreted out of the body together with feces. Accordingly, no peptides from amyloids f ibrils could be detected in the plasma of mice. In addition, no health impact con-cerning the induction of amyloid plaques formation by the long-term ingestion ofβ-lg/lysozymef ibrils were found in mice, ruling out cross-seeding mechanisms.
In summary, we applied multiple methodologies to elucidate the fate of β-lg and lysozyme amyloid f ibrils f irst through in-vitro gastro-intestinal digestion followed by the assessment of potential in-vivo toxicity using two animal models, in both cases withβ-lg and lysozyme monomers as a control. SDS-PAGE and ELISA measurements showed that β-lg amyloids were signif icantly degraded after gastric digestion and completely degraded following intestinal digestion. TO assay and CD spectroscopy indicated neither amyloid cores nor β-sheet con-formation after gastrointestinal digestion. AFM images illustrated that amyloid f ibrils could be entirely digested at enzyme:amyloid ratio within the range of the INFOGEST method specif ications. MALDI-MS
 

digestion time after administration of 5-DTAF-labeledβ-lgf ibril (d) and monomer (e). The results in panels b-e are verif ied by three independent experiments. fRepresentative micrographs of CR-stained mouse brain sections after 60 days of administeringβ-lg/lysozymef ibrils. The result was repeated with six independent experiments. Scale bars represent 200μm.
spectra revealed that these digested polypeptides had molecular weights of approximately 1–2 kDa, and homolog polypeptides series were observed in the in-vitro digestion of both β-lg monomers and f ibrils. Polarized light microscopy study of the kinetics of amyloid digestion showed that the birefringence signal of amyloids completely disappeared after intestinal digestion, and SAXS prof iles indicated that amyloids were digested in the intestinal phase with no conformational diversity, yielding a scattering prof ile in each case identical to the prof ile of deionized water. Parallel experiments on lysozyme amyloid f ibrils further reinforce these conclusions.
In-vivo results showed that no amyloid toxicity or health impact of β-lg/lysozymef ibrils degradation products were found, no crossing to the blood stream in mice and no events of cross-seeding in both polyQ35C. elegansmutants and mice. Based on the above the concern of: (i) cross-seeding between remaining food protein amyloid core and pathological amyloid and (ii) cytotoxicity arising from presence of nanoparticles shall no longer hold.
This work compares comprehensively the digestibility of amy-loids with their monomeric counterparts by molecular, antigenic, spectroscopic, microscopic and scattering techniques, providing the most compelling evidence that digestion of amyloids proceeds to an extent identical—if not greater—to that of the homolog monomers. The fact that food protein amyloid cannot induce toxic polypeptides after entering the human gastrointestinal tract, and further validation of these conclusions on intestinal human cell lines, C. elegans and mice animal models, suggest a possible classif ication of these ingredients—at least—as Generally Regarded as Safe (GRAS) for human consump-tion, although the f inal conclusion can only be left to the relevant
 
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Article 	https://doi.org/10.1038/s41467-023-42486-x
 
regulatory institutions. We nonetheless believe that these conclusions may open the door for promising and unexplored food and nutritional applications for food protein amyloid f ibrils, introducing them as possible ingredients for human diet.
Methods
All animal procedures complied with all relevant ethical regulations and were approved by the Laboratory Animal Committee (LAC) of South China University of Technology (AEC-2019071).
Materials 
β-lactoglobulin (β-lg, purity ≥90%) was extracted and purif ied from whey protein isolated according to a previously developed protocol45, and brief ly described as following. BioPURE-betalactoglobulin (lot JE 003-6-922, from 23-05-2005) from Davisco Foods International, Inc. (Le Sueur, MN) was dissolved in MilliQ water at 10 wt% concentration and adjusted to pH 4.6. Then, it was centrifuged at 15,000 rpm for 15 min at 20 °C, and the supernatant was recovered and adjusted to pH 2, before f iltering through a 0.22μm Millipore f ilter, followed by dialyzingf irst against pH 2 MilliQ water and against MilliQ water, using a Membrane (MWCO of 6−8k Da) at 4 °C for at least 4 h. Final solution was freeze-dried and the powder was stored at 4 °C. Lysozyme from hen egg white (L-6876), pepsin from porcine (P6887), porcine pan-creatin (P7545), thiof lavin T (T3516), Trichloroacetic acid (T6399), p-Toluene-sulfonyl-L-arginine methyl ester (T4626) and the rest of the reagents used were purchased from Sigma-Aldrich (Switzerland).
Amyloidf ibril preparation 
β-lg and lysozyme amyloidf ibrils were obtained by heating a monomer solution (2 wt%) in an oil bath at 90 °C for 6 and 24 h, respectively, with continuous stirring with a magnetic stirrer. The amyloidf ibril solutions were then dialyzed (MWCO 100 kDa) against pH 2 deionized water at 4 °C for 3 days.
Simulated gastrointestinal digestion 
The INFOGEST standard protocol40 for food digestion was applied to simulate the physiological human gastrointestinal digestion process. Various concentrations of enzymes were considered to detect the minimum required concentration of enzyme for complete digestion of the amyloids. The prepared stock solutions of simulated gastric f luid (SGF) and simulated intestinal f luid (SIF) were prepared and equili-brated to 37 °C before use. Prior to the digestion, the pepsin and trypsin activity in the pepsin and pancreatin were investigated according to the INFOGEST protocol. To begin the gastric digestion, SGF stock solution was f irst adjusted to pH 3 by 5 M HCl and then added to the food amyloid solution (1 mg/ml) with a ratio of 1:1 (v/v). Freshly prepared porcine pepsin solution in water was added to the mixture to a f inal pepsin activity of 280 U/mL in the digestion mix-ture. CaCl2(H2O)2 was added to the mixed solution to achieve af inal concentration of 0.15 mM prior to digestion,. The digestion proce-dure was carried out in a water bath (VWR 462-0493) at 37 °C with shaking condition for 120 min. An aliquot was sampled at each time point for the multiple in-vitro analysis, to which NaOH was added to inactivate the pepsin. Intestinal digestion was proceeded immedi-ately after gastric digestion. Pancreatin was dissolved in SIF con-taining 0.6 mM CaCl2 and added to the gastric digests with a ratio of 1:1 (v/v). The lowest used activity of pancreatin in the digestion mixture was adjusted to the low amount of protein used in our study and was 0.3 U/mg of protein. Digestion was likewise carried out for 120 min at 37 °C under shaking with periodic sampling. It should be noted that upon addition of SIF, gastric-digested lysozyme samples displayed turbidity which suggested some aggregation compared to theβ-lg, possibly due to the very different isoelectric point of the two proteins. Samples were frozen to prevent further digestion at each time point before simultaneous analysis of all samples with SDS-
 

PAGE and ELISA, while other analyses were performed immediately on fresh samples.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) 
The dynamic proteolysis ofβ-lg and lysozyme monomer and amyloid f ibrils during digestion were analyzed by using 4–20% stain-free pre-cast gels (Criterion TGX Stain-Free Protein Gel, Biorad Laboratories). The digested samples were diluted with a premade Laemmli sample buffer system including 0.065 M Tris-HCl with pH 6.8, 20 mg/ml SDS, 330 mg/ml glycerol, 0.01% (w/v) bromophenol blue, after which 50μl β-mercaptoethanol was added and heated at 90 °C for 10 min. 15μl of diluted sample was loaded into each well, and a voltage of 200 V was applied with the running buffer solution including 0.025 M Tris-HCl, 0.192 M glycine, and 0.1% (w/v) SDS. The images were obtained by scanning the gels with a molecular imager (ChemiDoc MP Imaging System, Bio-Rad Laboratories).
Circular dichroism (CD) 
The freshly digested protein samples were diluted to 0.02 wt% using deionized water and loaded into a high-quality quartz cuvette with 1 mm optical path length. CD spectra were collected using a Jasco J-815 CD spectrometer with a wavelength ranging from 190–280 nm, bandwidth 2 nm, and scan speed 100 nm/min.
Fluorescence assay 
Thiazole orange (TO) f luorescence assay with high sensitivity and reliability was used for monitoring the presence of β-sheet from amyloidf ibrils over the course of gastrointestinal digestion46. TO stock solution of 2 mM was prepared in dimethyl sulfoxide and stored at 4 °C before use. Gastric-digested samples were diluted with SIF to the same concentration as intestinal digested samples. 200μl of each sample was added followed by 1μl TO solution, and thef luorescence intensity was measured with a microplate reader (Inf inite M200PRO, Tecan) using an excitation wavelength and emission wavelength of 421 nm and 455 nm, respectively. The same digestion process was likewise performed for 4 h only with digestive enzymes without monomer and f ibril to determine the contribution of background f luorescence intensity. Concurrently, Thiof lavin T (ThT) f luorescence was also per-formed to track the digestive behavior of amyloidf ibrils. 2μl of 2 mM ThT stock solution prepared in deionized water was added to each gastric-digested sample diluted with SIF. The f luorescence intensity was measured with an excitation wavelength and emission wavelength of 440 nm and 480 nm, respectively.
Atomic force microscopy (AFM) 
The digested protein solutions were collected and diluted to a f inal concentration of 0.01 wt.%. 10μl of diluted sample solution was deposited on a freshly cleaved mica for 10 min, followed by rinsing with deionized water and drying by a gentle f low of compressed air-f low. AFM measurements were carried out with a Bruker multimode 8 scanning probe microscope (Bruker, U.S.A.) with an acoustic hood to minimize vibrational noise. AFM imaging was operated in soft tapping mode under ambient condition with a commercial silicon nitride cantilever at a vibration frequency of 150 kHz and relatively soft tip-sample interaction. AFM images were simply f lattened using Nano-scope 8.1 software.
Determination of antigenicity 
The antigenicity of all samples was evaluated using with ELISA assay using polyclonal antibodies quantif ication kits (AgraQuant Beta-Lactoglobulin and AgraQuant Lysozyme) purchased from Romer Labs Deutschland GmBH, Germany. All samples were diluted with deionized water to a concentration of 0.5μg/ml for further use. To each well, 100μl of ready-to-use standard solutions and samples were added and
 
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incubated at room temperature for 20 min.β-lg monomer and amyloid f ibrils were detected as the positive control samples, while wells with only deionized water were measured as negative controls. Wells were then emptied and rinsedf ive times with wash buffer, followed by 100μl of the enzyme-conjugate solution. After 20 min of incubation followed by rinsing, 100μl of substrate solution was added and incubated for another 20 min in the dark. The reaction was stopped by introducing 100μl of stop solution into each microwell, and the absorbance of each sample was detected with a microplate reader (Inf inite 200 PRO) at 450 nm. The milk and formula used in the control experiment were purchased from a local retail supermarket (Migros Group, Switzerland). The antigenicity of each sample was assessed according to the adsorption of a standard curve in the experiment.
Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) 
To prepare the samples for mass determination, samples which were prior desalted using C18 Zip Tips (Millipore, USA) were spotted onto a MALDI target (MTP 384 target polished steel TF, Bruker Daltonics, Bremen, Germany) along with 2μl ofα-cyano-4-hydroxycinnamic acid (α-CHCA) solution (10 mg/ml) in acetonitile/water/trif luoroacetic acid (50:50:0.1). We note that the singly charged protein species is not the dominant signal, and we believe this is due to the use of CHCA matrix. MALDI measurements were performed on an ultraf leXtreme MALDI-TOF/TOF mass spectrometer equipped with a smartbeam laser (Bruker Daltonics). FlexControl (Version 3.4) was used with the following programmed measurement parameters: laser frequency of 1,000 Hz in the positive-ion linear mode with acquisition ranging from 500 to 30,000 Da as well as in positive-ion ref lector mode with acquisition ranging from 400 to 6,000 Da. Final spectra consisted of 1,000-3,000 shots per analysis.
Polarized light microscopy of simulated digestion 
A microf luidic system consisting of a peristaltic pump circulating the protein amyloid solution through a water bath maintained at 37 °C was set up to monitor the dynamic digestion process of amyloid f ibrils through a 2 mm diameter capillary under cross-polarized light micro-scope (Zeiss AxioImager Z2). The gastric digestion was started by injecting pepsin into the amyloid solution with an incubation for 120 min, followed by another injection of pancreatin with an incuba-tion for 120 min. Images were recorded using microscope software every 0.5 s. The images were subsequently analyzed using ImageJ. They were converted to 32-bit grayscale before measuring the integrated density per area, expressed as the intensity within a def ined polygon in the middle of the sample jet. The intensity of a pure water jet was subtracted as background from these values.
Fluorescence imaging 
β-lg amyloid f ibril before and after gastric digestion were dyed with ThT and imaged using a Nikon N-STORM microscope (Nikon, UK Ltd.) using an SR Apochromat TIRF 100 ×1.49 N. A. oil immersion objective lens. Fluorescence was detected with an EM-CCD Camera iXon DU897 (Andor). The f ield of view imaged typically covered 512 × 512 camera pixels corresponding to an area of ~80 × 80μm2 on the sample. An in-built focus-lock system was used to prevent axial drift of the sample during data acquisition. The laser excitation was 20% at 405 nm, with a maximum intensity measured at the tip of the opticalf iber of 20 mW and an exposure time of 50 ms. The emission passed through a multi-edge dichroic f ilter with windows at 415–490 nm, 502–560 nm, and 660–800 nm. Images were acquired using the NIS Elements Nikon software.
Small-angle X-ray scattering (SAXS) 
SAXS experiments with Rigaku (Cu Kαradiation,λ= 1.54 Å, and a two-dimensional argon-f illed Triton detector) were performed using the
 

following conditions: the scattering vector was calibrated and
centered	using	silver	behenate,	and	an	active	range
0.014 Å−1 < q < 0.200 Å−1 was probed. Quartz capillaries with a dia-meter of 1.5 mm were f illed with the samples and placed onto a stainless-steel holder. The samples were then measured for 2 h.
Cell culture and cellular response assays 
Caco2 cells were obtained from the Food Biotechnology group at ETH Zürich. Caco2 cells are epithelial cells isolated from colon tissue derived from a patient with colorectal cadenocarcinoma. They are cultured in DMEM (cat. Nr: 31966 021, ThermoFisher Scientifc, Mas-sachusetts, USA) with 10% FBS (cat. Nr: 10270106, ThermoFisher Sci-entifc, Massachusetts, USA) 100 U/ml penicillin and 100µg/ml streptomycin (cat. r.: 15,140,122, ThermoFisher Scientifc, Massachu-setts, USA). HCEC-1CT cells were obtained from Jerry Shay from the UT Suthwestern in August 2011. HCEC-1CT cells are a colon epitelial cell line. They were cultured in ready mixed full medium (cat. Nr: MHT-039, Evercyte, Vienna, Austria). Caco2 cells were seeded at a density of 5000/cells/well in 100μl medium in 96-well plates (cat Nr: 400096, Bioswisstec, Schhaussen, Switzerland), HCEC-1CT cells were seeded at a density of 2500 cells/well in 100μl medium in 96-well plates (cat. Nr: 734-0079, Corning, New York, USA). ATP was quantif ied, as an indi-cator of metabolically active cells (cat. Nr:G7571, Promega, Dübendorf, Schweiz). Caco2 and HCEC cells were exposed to the digested Lyso-zyme and ß-lg monomers andf ibrils at def ined concentrations for 4 h. The digested f ibrils/monomers samples were freeze dried and recon-stituted in deionized water. After INFOGEST digestion, the digested protein samples or digestion matrix were subject f irst to heat-shock (95-degree, 10 min) to inactivate the enzymes and were then freeze-dried. Afterwards the samples were diluted in cell culture media to reach f inal concentrations. A portion (100μl) of each sample was added per well to the cells. Triton X-100 (0.01 %) was used as a positive control. At the end of the 4 h treatment, media containing the digested substances was removed and replaced with 45 ul of fresh media. Afterwards 45 ul of CellTiter-Glo reagent was added to each well. The plate was shacked 2 min on an orbital shaker and incubated 10 min in the dark. Afterwards 75 ul were transferred to a white half area 96 well plate. Luminescence with 1000 ms integration time was measured using a plate reader (Inf inite M200 Pro, Tecan group, Männerdorf, Switzerland).
C. elegans Strains 
TJ1060spe-9(hc88)I;rrf-3(b26)II. AM140rmIs132[unc-54p::Q35::YFP]
Mobility assay 
Mobility assay was adapted from Statzer et al.,47. In brief, gravid TJ1060 C. elegansadults were synchronized by bleaching and L1 arrested over night. L1 worms at day 1 of adulthood were then transferred into U-shaped 96-well plates in S-complete buffer and heat-inactivated bacteria. At day 1 of adulthood the protein samples or controls were added to the wells for af inal concentration of 0.1667 mg/mL. From day 3 of adulthood onwards theC. eleganswere scored for mobility using the MicroTracker (MTK100). For statistical analysis, the area under the curve was measured, and the mean for each run was calculated. Sta-tistical analysis was performed by using a 1-way ANOVA.
Q35 aggregate quantif ication 
Poly-Q35:YFP aggregates were imaged with an upright bright f ield f luorescence microscope (Tritech Research, model: BX-51-F) using the triple band f ilter sets consist of the 69000 ET-DAPI/FITC/TRITC (69000x, 69000 m, 69000bs, EX/EM 25 mm, ringed, DC 25.5 × 36 x 1 mm) (Chroma Technology, catalog number: 69000)48. Transgenic AM140 C. elegans were fed with protein samples from day 1 of adult-hood onwards and imaged at a time-point when aggregation started but the worms were not yet fully aggregated, usually day 4 of
 
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Article 	https://doi.org/10.1038/s41467-023-42486-x
 
adulthood. Images were then analyzed using ImageJ. An intensity threshold was set manually based on the control worms and used for all conditions. Then aggregates were counted using the ‘Analyze par-ticle’ tool. To minimize noise, a minimum value for circularity (0.02–0.03) and size (2–3 pixel) was set.
Mouse strains 
Kunming mice were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. Male mice (5 weeks old) were housed in enclosures on sawdust bedding at 25 °C and 40% relative humidity including a normal light cycle (12 h dark /12 h light cycle). Deionized water and fodder were provided. Mice were acclimatized for one week before in-vivo experiments. All animal procedures complied with all relevant ethical regulations and were approved by the Laboratory Animal Committee (LAC) of South China University of Technology (AEC-2019071).
LC-MS/MS analysis on In-vivo mice gastrointestinal digests The in-vivo digested products of β-lg/lysozyme amyloid f ibrils and monomer and their peptides identif ication in small intestinal tracts, colon and serum were studied by LC-MS/MS analysis49. Six-week-old Kunming male mice (n= 6 per group) were starved for 24 h, after which they were orally gavaged with 200μl 1 mg/ml ofβ-lg/lysozyme amyloid f ibrils and monomer solutions, separately. The contents of each small intestinal tracts and colon were collected after 4 h gastrointestinal digestion for digests identif ication. For serum β-peptides identif ica-tion, mice were orally gavaged with 200μl 1 mg/ml β-lg/lysozyme amyloidf ibrils and monomer solution, serum samples were collected after 1 h digestion.
LC-MS/MS measurements were performed on a Q ExactiveTM mass spectrometer coupled to Easy nLC (Thermo Fisher Scientif ic). Speci-f ically, 15μL of each sample was injected from the autosampler to Zorbax 300SB-C18 peptide traps (Agilent Technologies, Wilmington, DE), and then separated by liquid chromatography column (15 cm long, 75μm I.D., 5μm resin, C18-reversed phase column) which was equilibrated by buffer A (0.1% formic acid solution) and then separated with a linear gradient of buffer B (0.1% formic acid in 84% acetonitrile) at af low rate of 250 nL/min controlled by IntelliFlow technology over 120 min. MS data was acquired using a data-dependent top10 method which dynamically chose the most abundant precursor ions from the survey scan (300–1800 m/z) for high-energy collisional dissociation (HCD) fragmentation. The target value was determined based on predictive automatic gain control (AGC) and the dynamic exclusion duration was 20 s. Survey scans were acquired at a resolution of 70,000 at m/z 200 and resolution for HCD spectra was set to 17,500 at m/z 200. Survey scans were acquired at a resolution of 70,000 at m/z 200 and HCD spectra resolution was set to 17,500. Normalized colli-sion energy of 27 eV was used while the underf ill ratio, which specif ies the minimum percentage of target value likely to be reached at max-imumf ill time, was set at 0.1%. peptide recognition mode was enabled throughout the run. MS data were processed using the MaxQuant software (version 1.5.5.1) by UniProt database without specifying enzyme cleavage rules for the processed MGF f iles. The following search parameters were used: ±20 ppm for peptide mass tolerance, 0.1 Da for MS/MS tolerance. Variable modif ication: Oxidation (M). Label-free peptide quantif ication (LFQ) based on extracted ion chro-matograms and validation were performed in the MaxQuant software. The cutoff value of global false discovery rate (FDR) for peptide identif ication was set to 0.01.
In-vivo distribution off ibrils during digestion 
Six-week-old Kunming male mice (n= 3 per group) were orally gavaged with 200μl 1 mg/ml β-lg monomer and amyloid f ibrils solution, respectively, which labeled using the dye 5-(4,6-dichlorotriazinyl) aminof luorescein (5-DTAF). Small intestine, blood, brain, heart, liver,
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spleen, lung, kidney and feces of mice from each time points were collected, and then imaged using an in-vivo imaging system (IVIS, Lumina XRMS III, PerkinElmer).
Long-term intake off ibrils 
Six-week-old Kunming male mice were randomly separated into f ive groups (n= 8 per group) namely with the control group,β-lg-FL group, lys-FL group,β-lg-FH group and lys-FH group (F stands forf ibril; L for low dosage; H for high dosage). The control group had no further treatment, the β-lg-FL group was daily orally gavaged with 200μL β-lg-f ibrils of 1 mg/ml, the lys-FL group with 200μl lysozymef ibrils of 1 mg/ml, β-lg-FH group of 5 mg/ml and lys-FH group of 5 mg/ml. The mice were sacrif iced after 30 and 60 days, respectively, of adminis-tration and sections of the mainly tissues were processed for amyloi-dosis examination. The major organs including brain, heart, liver, spleen, kidneys and lungs were excised and f ixed in 10% formalin. Congo red (CR) staining method was used for identif ication of amy-loids in tissue sections, brain sections of Alzheimer’s disease (AD) mice were used as positive control.
Reporting summary 
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Data availability
The data used to reproduce the results and to support thef indings are available within the article, Supplementary Information f ile and the Source Dataf i le. The Source Data are provided with this paper [https:// doi.org/10.6084/m9.figshare.24201405]. The mass spectrometry pro-teomics data have been deposited to the ProteomeXchange Con-sortium via the PRIDE partner repository with the dataset identif ier PXD045698 	[https://www.ebi.ac.uk/pride/archive/projects/ PXD045698]. Extra data are available from the corresponding authors upon request. Source data are provided with this paper.
References
1. 	Toyama, B. H. & Weissman, J. S. Amyloid structure: conformational 	diversity and consequences.Annu. Rev. Biochem.80, 557–85 	(2011).
2. 	Chiti, F. & Dobson, C. M. Protein misfolding, Amyloid formation, and 	human disease: a summary of progress over the last decade.Annu. 	Rev. Biochem.86, 27–68 (2017).
3. 	Benson, M. D. et al. Amyloid nomenclature 2018: recommendations 	by the International Society of Amyloidosis (ISA) nomenclature 	committee.Amyloid25, 215–219 (2018).
4. 	Cao, Y. & Mezzenga, R. Food protein amyloidf ibrils: origin, struc-	ture, formation, characterization, applications and health implica-	tions.Adv. Colloid Inter.Sci.269, 334–356 (2019).
5. 	Hammer, N. D., Wang, X., McGuff ie, B. A. & Chapman, M. R. Amy-	loids: friend or foe?J. Alzheimer’s Dis.13, 407–419 (2008).
6. 	Kenney, J. M., Knight, D., Wise, M. J. & Vollrath, F. Amyloidogenic 	nature of spider silk.Eur. J. Biochem.269, 4159–4163 (2002).
7. 	Dueholm, M. S., Albertsen, M., Otzen, D. & Nielsen, P. H. Curli 	functional Amyloid systems are phylogenetically widespread and 	display large diversity in operon and protein structure.PLoS One7, 	e51274 (2012).
8. 	Li, J. et al. The RIP1/RIP3 necrosome forms a functional Amyloid 	signaling complex required for programmed Necrosis.Cell150, 	339–350 (2012).
9. 	Maji, S. K. et al. Functional Amyloids as natural storage of peptide 	hormones in pituitary secretory granules.Science325, 
	328–332 (2009).
10. 	Knowles, T. P. J. & Mezzenga, R. Amyloidf ibrils as building blocks 	for natural and artif icial functional materials.Adv. Mater.28, 	6546–6561 (2016).
12
 

Article 	https://doi.org/10.1038/s41467-023-42486-x
11. 	Ke, P. C. et al. Half a century of amyloids: past, present and future. 	34. Bateman, L., Ye, A. & Singh, H. In vitro digestion ofβ-Lactoglobulin
 
Chem. Soc. Rev.49, 5473–5509 (2020).
12. 	Wei, G. et al. Self-assembling peptide and protein amyloids: from 	structure to tailored function in nanotechnology.Chem. Soc. Rev. 	46, 4661–4708 (2017).
13. 	Jansens, K. J. A. et al. Rational design of Amyloid-likef ibrillary 	structures for tailoring food protein techno-functionality and their 	potential health implications.Compr. Rev. Food Sci. Food Saf.18, 	84–105 (2019).
14. 	Hauser, C. A. E., Maurer-Stroh, S. & Martins, I. C. Amyloid-based 	nanosensors and nanodevices.Chem. Soc. Rev.43, 
	5326–5345 (2014).
15. 	Hamada, D. et al. Competition between folding, native-state 	dimerisation and Amyloid aggregation inβ-Lactoglobulin.J. Mol. 	Biol.386, 878–890 (2009).
16. 	Hill, S. E., Robinson, J., Matthews, G. & Muschol, M. Amyloid pro-	tof ibrils of lysozyme nucleate and grow via oligomer fusion.Bio-	phys. J.96, 3781–3790 (2009).
17. 	Tanaka, N. et al. The mechanism off ibril formation of a non-	inhibitory Serpin ovalbumin revealed by the identif ication of 	Amyloidogenic core regions*.J. Biol. Chem.286, 5884–5894 	(2011).
18. 	Zhou, J. et al. Oat plant Amyloids for sustainable functional mate-	rials.Adv. Sci.9, 2104445 (2022).
19. 	Soon, W. L., Peydayesh, M., Mezzenga, R. & Miserez, A. Plant-based 	amyloids from food waste for removal of heavy metals from con-	taminated water.Chem. Eng. J.445, 136513 (2022).
20. Tang, C.-H. & Wang, C.-S. Formation and characterization of Amyloid-likef ibrils from Soyβ-Conglycinin and Glycinin.J. Agric. Food Chem.58, 11058–11066 (2010).
21. 	Li, T. et al. Plant protein Amyloid Fibrils for multifunctional sus-	tainable materials.Adv. Sustain. Syst.7, 2200414 (2023).
22. Cao, Y. & Mezzenga, R. Design principles of food gels.Nat. Food1, 	106–118 (2020).
23. Shen, Y. et al. Amyloidf ibril systems reduce, stabilize and deliver 	bioavailable nanosized iron.Nat. Nanotech.12, 642–647 (2017).
24. Shimanovich, U. et al. Protein microgels from Amyloid Fibril Net-	works.ACS Nano.9, 43–51 (2015).
25. Mei, E. et al. Self-assembling Collagen/Alginate hybrid hydrogels for combinatorial photothermal and immuno tumor therapy.Col-loids Surf. A Physicochem. Eng. Asp.577, 570–575 (2019).
26. Hu, B. et al. Flavonoid-amyloidf ibril hybrid hydrogels for obesity control via construction of Gut Microbiota.Biomater. Sci.https:// doi.org/10.1039/D2BM00366J(2022).
27. 	Zhao, J. & Yang, P. Amyloid-mediated fabrication of 
	organic–inorganic hybrid materials and their biomedicalapplica-	tions.Adv. Mater. Inter.7, 2001060 (2020).
28. Kontopidis, G., Holt, C. & Sawyer, L. Invited review:β-Lactoglobulin: binding properties, structure, and function.J. Dairy Sci.87, 
785–796 (2004).
29. Sakurai, K., Oobatake, M. & Goto, Y. Salt-dependent 
	monomer–dimer equilibrium of bovineβ-lactoglobulin at pH 3.
Protein Sci.10, 2325–2335 (2001).
30. Miller, K., Meredith, C., Selo, I. & Wal, J.-M. Allergy to bovineβ-lactoglobulin: specif icity of immunoglobulin E generated in the Brown Norway rat to tryptic and synthetic peptides.Clin. Exp.
Allergy29, 1696–1704 (1999).
31. 	Matsuo, H., Yokooji, T. & Taogoshi, T. Common food allergens and 	their IgE-binding epitopes.Allergol. Int.64, 332–343 (2015).
32. Ragona, L., Pusterla, F., Zetta, L., Monaco, H. L. & Molinari, H.
Identif ication of a conserved hydrophobic cluster in partially folded bovineβ-lactoglobulin at pH 2.Fold. Des.2, 281–290 (1997).
33. Niemi, M. et al. Molecular interactions between a recombinant IgE 	antibody and theβ-Lactoglobulin allergen.Structure15,
 

f ibrils formed by heat treatment at low pH.J. Agric. Food Chem.58, 9800–9808 (2010).
35. Blanch, E. W., Hecht, L. & Barron, L. D. New insight into the pH-dependent conformational changes in bovineβ-lactoglobulin from Raman optical activity.Protein Sci.8, 1362–1367 (1999).
36. Tavel, L., Moreau, C., Bouhallab, S., Li-Chan, E. C. Y. & Guichard, E.
Interactions between aroma compounds andβ-lactoglobulin in the heat-induced molten globule state.Food Chem.119, 1550–1556 (2010).
37. Dodero, V. I. Biomolecular studies by circular dichroism.Front 	Biosci.16, 61 (2011).
38. Khadem, S. A., Bagnani, M., Mezzenga, R. & Rey, A. D. Relaxation dynamics in bio-colloidal cholesteric liquid crystals conf ined to cylindrical geometry.Nat. Commun.11, 4616 (2020).
39. Chen, M. et al. Heparan sulfate proteoglycans mediate prion-likeα-synuclein toxicity in Parkinson’s in vivo models.Life Sci. Alliance5, e202201366 (2022).
40. Brodkorb, A. et al. INFOGEST static in vitro simulation of gastro-	intestinal food digestion.Nat. Protoc.14, 991–1014 (2019).
41. 	Roig, A. I. et al. Immortalized epithelial cells derived from human 	colon biopsies express stem cell markers and differentiate in vitro. 	Gastroenterol.138, 1012–1021.e5 (2010).
42. Erzinger, M. M. et al. Sulforaphane preconditioning sensitizes human colon cancer cells towards the bioreductive anticancer prodrug PR-104A.PLoS One11, e0150219 (2016).
43. Ramirez Garcia, A. et al. Pathogenic and commensal gut bacteria harboring Glycerol/Diol dehydratase metabolize glycerol and pro-duce DNA-reactive Acrolein.Chem. Res. Toxicol.35, 
1840–1850 (2022).
44. Morley, J. F., Brignull, H. R., Weyers, J. J. & Morimoto, R. I. The threshold for polyglutamine-expansion protein aggregation and cellular toxicity is dynamic and inf luenced by aging in Cae-norhabditis elegans.Proc. Natl Acad. Sci.99, 10417–10422 (2002).
45. Jung, J.-M., Savin, G., Pouzot, M., Schmitt, C. & Mezzenga, R.
Structure of heat-inducedβ-Lactoglobulin aggregates and their complexes with Sodium-dodecyl sulfate.Biomacromol.9, 2477–2486 (2008).
46. Gao, G.-Q. & Xu, A.-W. A newf luorescent probe for monitoring amyloidf ibrillation with high sensitivity and reliability.RSC Adv.3, 21092–21098 (2013).
47. Statzer, C., Reichert, P., Dual, J. & Ewald, C. Y. Longevity interven-tions temporally scale healthspan in Caenorhabditis elegans.
iScience25, 103983 (2022).
48. Teuscher, A. C. & Ewald, C. Y. Overcoming autof luorescence to assess GFP expression during normal physiology and aging in Caenorhabditis elegans.Bio. Protoc.8, e2940 (2018).
49. Wang, S. et al. Large-scale discovery of non-conventional peptides in Maize and Arabidopsis through an integrated peptidogenomic pipeline.Mol. Plant13, 1078–1093 (2020).
Acknowledgements
The authors gratefully acknowledge Dr. Serge Chesnov from the Func-tional Genomics Center Zurich and Pinotsi Dorothea from the Scientif ic Center for Optical and Electron Microscopy (ScopeM), ETH Zurich. D.X.
also acknowledges the Guangzhou Elite Project forf inancial support (GEP No: JY202032). Chia-Wei Lin is kindly acknowledged for assistance with the MALDI analysis. R.M. wishes to posthumously acknowledge Sir. Christopher Dobson for inspiring past discussions on the digestion of amyloidf ibrils.
Author contributions
R.M. conceptualized the project; R.M., J.Z. and S.J.S. supervised the research and designed studies; D.X, J.Z., W.L.S., I.K., S.D., A.M. and S.H.
 
1413–1421 (2007). 	conducted the experiments. D.X., J.Z., W.L.S., I.K., A.M., S.H. B.L., L.L.,
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Article 	https://doi.org/10.1038/s41467-023-42486-x
 
C.Y.E. and M.R. contributed to experimental and data analyses. J.Z., D.X. and R.M. wrote the manuscript and all authors contributed to evaluating data and writing thef inal version.
Competing interests
The authors declare no competing interests.
Additional information
Supplementary informationThe online version contains supplementary material available at 
https://doi.org/10.1038/s41467-023-42486-x.
Correspondenceand requests for materials should be addressed to Jiangtao Zhou or Raffaele Mezzenga.
Peer review informationNature Communicationsthanks <PRESIDIO_ANONYMIZED_PERSON> and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer reviewf ile is available.
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