Autoclaved Peanuts Exhibit Reduced Immunoglobulin E Binding and Improved Oral Tolerability

ABSTRACT

Background

Major peanut allergens are stable and resistant to denaturation under standard cooking conditions, contributing to allergenicity and low rates of developing natural tolerance in allergic individuals. We evaluated the effects of heat and pressure autoclaving on peanut proteins, IgE binding, and oral tolerability.

Methods

Raw, roasted, and autoclaved peanut protein extracts were evaluated by Bradford assay, ELISA, and mass spectrometry‐proteomics to compare relative amounts of protein, specific IgE binding, and allergen fragmentation. To assess changes in clinical reactivity, we performed skin prick testing (SPT) in 41 subjects using standard and autoclaved peanut extracts. We also performed double‐blind oral food challenges (OFC) in 10 peanut‐allergic subjects with standard and autoclaved peanuts.

Results

Autoclaving at 130°C, 2.4 atm, for 30 min significantly degraded allergens Ara h 1 and 2, and completely degraded Ara h 8. Mass spectrometry‐proteomics analysis of size‐filtered extracts (< 10 kDa) showed greater numbers and diversity of peptides from peanut proteins and allergens in autoclaved extracts. Autoclaving fragmented proteins into shorter peptides, against which sera from highly allergic patients exhibited a 74% reduction in IgE binding compared to raw peanuts. SPT demonstrated significant decreases in wheal diameters using autoclaved peanut extract (median [IQR] = 5 mm [2, 9]) compared to commercial extract (10 mm [6, 15]; p < 0.001). All OFC subjects tolerated the maximum cumulative autoclaved peanut dose (444 mg) versus standard peanut (median: 9 mg, range: [1, 44]).

Conclusions

Autoclaving peanuts induces important chemical changes including fragmentation, leading to decreased peanut allergenicity and consequently increased tolerability. This has the potential for novel immunotherapeutic approaches with more favorable side effect profiles.

• This study evaluates the effects of heat and pressure autoclaving on peanut proteins, IgE binding, and oral tolerability.

• Heat and pressure autoclaving breaks down peanut proteins and allergens, contributing to reduced peanut‐specific IgE binding.

• Autoclaved peanuts are associated with decreased skin test reactivity and greater oral tolerability in allergic subjects, suggesting a potentially safer substrate for immunotherapy.

Abbreviations: IgE immunoglobulin E; ELISA enzyme‐linked immunosorbent assay.

1. Introduction

The common peanut ( Arachis hypogaea ) is an important legume crop worldwide, serving as an affordable source of protein, unsaturated fatty acids, vitamins, and minerals [1, 2]. However, for an increasing proportion of the population in Western societies, allergy to peanuts prevents their consumption and requires constant vigilance to avoid accidental exposure. Peanut allergy is a leading cause of fatal anaphylaxis affecting over 1.5% of children and adults in North America, Australia, and the UK [3, 4, 5, 6]. The allergic reaction to peanut is characterized by specific immunoglobulin E (sIgE) antibodies binding to peanut protein allergens, Ara h 1 through 18, which triggers a cascade of symptoms such as hives, swelling, difficulty breathing, and in some cases, anaphylaxis [6].

Raw peanuts are prepared for consumption in different ways around the world [7, 8]. In North America and Western Europe, peanuts are commonly dry roasted, such as in the preparation of peanut butter, while in Asia it is common to boil peanuts in briny water. Unlike other foods like cow's milk or hen's egg, major peanut allergens are resistant to denaturation following traditional processing methods. This may contribute to their high allergenicity and difficulties associated with desensitization. The mechanisms by which processing can affect peanut allergenicity remain a subject of active study.

Other non‐traditional processing methods have also been assessed for their ability to affect peanut allergenicity. For example, enzymatic hydrolysis, high‐moisture extrusion, and autoclaving have shown promise in degrading peanut allergens and reducing peanut‐sIgE binding [9, 10, 11]. Autoclaving at high temperature, in contrast with other non‐traditional processing methods, has the advantage of keeping the seed intact and requiring minimal manipulation. Preliminary studies suggested that autoclaving may reduce sIgE binding and allergenicity to peanuts [11, 12, 13]. Despite these findings, the mechanism for autoclave‐mediated decreased sIgE binding remains unclear and autoclaved peanuts have never directly been compared to standard peanuts in oral food challenges (OFC).

In this study, we aimed to evaluate the effects of two thermal processing methods—roasting and autoclaving—on peanut proteins, allergens, and binding to sIgE from highly allergic individuals. Our findings show that autoclaving induces changes in both solubility and protein fragmentation, reducing sIgE binding and clinical reactivity as measured by SPT and improved oral tolerability.

2. Methods

2.1. Preparation of Peanuts and Protein Extracts

Commercially available red‐skinned, shelled raw peanuts (Jumbo Shandong Luhua, China) were purchased in Montreal (Marché Victoria Oriental). Raw peanuts were either roasted in a convection oven at 150°C for 30 min or autoclaved (T‐Edge 10S, Tuttnauer; VWR AS12 AccuSterilizer) at 100°C–130°C (1–2.4 atm) for 30 min or at 130°C (2.4 atm) for 5–60 min. Twelve peanuts of each processing condition were ground into a paste, suspended in n‐hexane, and vacuum filtered to obtain defatted peanut flour.

For soluble protein extracts, 50 mg of flour was dissolved in 1.5 mL of 20 mM Tris buffer (pH 8.5), vortexed, and rotated overnight at 4°C. After centrifugation (12,600 g, 5 min), the supernatant was collected. Protein concentrations were determined by Bradford assay using a bovine serum albumin (BSA; Sigma‐Aldrich) standard curve. Analyses were performed in triplicate in comparison with raw, unprocessed peanuts.

2.2. Soluble and Insoluble Fraction Analyses

Peanuts (raw, roasted, autoclaved, blanched, blanched‐roasted, blanched‐autoclaved) were processed into defatted flour and proteins extracted in three steps (Figure 1A): an initial Tris buffer extraction yielding the soluble fraction, a second UTC buffer (7 M urea, 2 M thiourea, 2% CHAPS, 30 mM Tris–HCl, pH 8.5) extraction of the pellet, and a final filtration through a 3 kDa cutoff to obtain the insoluble fraction. Protein concentrations were measured by Bradford assay, and ELISA plates coated with 1 μg/mL protein were probed with pooled sera from five peanut‐allergic participants (sIgE Ara h 1: 884kU/L, Ara h 2: 398kU/L, Ara h 6: 675kU/L) to compare IgE reactivity between soluble and insoluble fractions. Additionally, equal masses of defatted raw, roasted, and autoclaved peanut flours were extracted, fractionated into soluble (supernatant) and insoluble (pellet) components, lyophilized, and weighed to compare mass recovery across processing conditions. See Supporting Information for more detail.

FIGURE 1.

(A) Workflow for sequential extraction of soluble and insoluble protein fractions from defatted peanut flour. Samples were first extracted in 20 mM Tris buffer (pH 8.5) to obtain the soluble fraction, followed by re‐extraction of pellets in UTC buffer (7 M urea, 2 M Thiourea, CHAPS 2%, 30 mM Tris–HCl, pH 8.5) and buffer exchange back into 20 mM Tris buffer (pH 8.5) through a 3 kDa filter to obtain the insoluble fraction. (B) Coomassie stain of protein gels following SDS‐PAGE comparing soluble and insoluble fractions of raw, raw‐roasted, raw‐autoclaved, blanched, blanched‐roasted, and blanched‐autoclaved conditions. (C) Protein concentrations of soluble and insoluble fractions measured by Bradford assay. (D) Total peanut‐specific IgE ELISA using pooled sera from five peanut‐allergic subjects evaluating IgE binding to soluble and insoluble fractions. Plates were coated with extracts normalized by protein concentration (1 μg/mL). For both (C) and (D), data represent mean ± SEM (n = 3). Statistical comparisons were performed using paired t‐tests; ELISA data were log‐transformed prior to analysis. *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant.

2.3. SDS‐PAGE Analysis

Whole protein extracts were created by dissolving equal masses of defatted peanut flour (50 mg) for each condition in equal volumes of extraction buffer (1.5 mL). Extracts were diluted 1 in 30 to achieve 1 mg/mL for raw peanut extract and separated by SDS‐PAGE under reducing conditions (2.5% β‐mercaptoethanol). Protein gels were stained with 0.1% Coomassie Brilliant blue to visualize protein bands. See Supporting Information for more detail.

2.4. Determination of sIgE Responses and Relative Quantification of Allergens

ELISAs were used to measure sIgE binding to peanut extracts, compare the relative detection of peanut allergens, and quantify peanut‐sIgE and sIgG4 concentrations in subject sera. Microplates were coated overnight at 4°C with peanut extracts or with recombinant peanut allergens Ara h 1, 2, or 8 at concentrations of 0.0001, 0.01, 1, and 100 μg/mL. To assess antibody binding to serial dilutions of peanut extracts, coated plates were incubated with pooled serum from 4 allergic individuals with high levels of peanut‐sIgE (1:250; median sIgE: 474 kU/L, median age: 15, 75% male), or with polyclonal antibodies against Ara h 1, 2, or 8. For inhibition ELISAs, pooled serum from 8 highly peanut‐allergic individuals (median sIgE: 1254 kU/L, median age: 14, 75% male) was instead preincubated with PBS (control), or raw, roasted, or autoclaved peanut extracts as inhibitors and then added to the plate coated with raw extracts. The percentage decrease in sIgE binding (i.e., inhibition) was calculated using the formula [(1–(AI/AN))] x 100%, where AI represents the OD value obtained from sera preincubated with each peanut sample, and AN represents the OD value of uninhibited sera. See Supporting Information for more detail.

2.5. Mass Spectrometry (MS) and Proteomics Analyses

Extracts generated from equal flour masses of raw, roasted, and autoclaved peanuts were passed through 2 mL Amicon Ultra‐2 centrifugal filters with a 10 kDa cut‐off (UFC200324, Millipore Sigma, Canada). Protein filtrates were mixed with proteomics‐grade trypsin (12 ng/μL; Promega Gold) and digested overnight at room temperature. Peptide analysis was performed via high‐performance liquid chromatography (HPLC) followed by MS/MS, and peptides were identified by mapping to the Uniprot Peanut database. See Supporting Information for more detail.

2.6. Skin Prick Test (SPT) Study

Forty‐one subjects with peanut allergy aged 6 to 53 were recruited to the Montreal Children's Hospital for SPT (REB approval: 2021–7628). Subjects were considered allergic based on previous history of peanut allergy and at least one of: positive SPT to peanut (wheal diameter ≥ 3 mm), serum peanut‐sIgE (≥ 0.35 kU/L), or a previous positive OFC to peanut. Autoclaved peanut (130°C, 30 min) protein extracts were diluted to a protein concentration of 0.5 mg/mL (50% glycerol), matching the protein concentration in commercial standard peanut extract (ALK‐Abello, Allergy Canada Limited) measured by Bradford Assay. SPTs were conducted by placing a drop of autoclaved or commercial extract on the forearm followed by a scratch using a solid bore needle; saline and histamine (1 mg/mL, ALK‐Abello) were used as negative and positive controls, respectively. After 10 min, wheal boundaries were outlined with pen and translucent medical tape was used to transfer imprints to a paper and kept as a permanent SPT record. Wheal diameters were then measured in millimeters. Patients were instructed to abstain from using antihistamine‐containing medications for at least 5 days before the procedure.

2.7. OFC Study

We conducted a randomized, double‐blinded OFC with 10 participants aged 5 to 45 with a confirmed peanut allergy to compare responses to blanched and autoclaved peanuts (REB approval: 2024–9682). Blanched peanuts were used for the OFC as it is the substrate used in our center's OIT studies. They are commercially processed with high consistency, with skins removed for homogeneity after grinding and encapsulation. Participants required a positive peanut SPT (≥ 3 mm) and/or peanut‐sIgE levels ≥ 0.35 kU/L at screening for eligibility. Exclusion criteria included unstable respiratory conditions, non‐IgE‐mediated or non‐immunological adverse reactions to peanuts, immunosuppressor therapy or beta‐blockers, cardiovascular disease, severe hypertension, and eosinophilic gastrointestinal disorder.

Blanched Runner peanuts (Yupik, Montreal, Canada) were autoclaved (130°C, 2.4 atm, 30 min), then ground into a coarse powder. Unprocessed blanched or blanched‐autoclaved peanuts were then weighed into capsules (4, 12, 40, 120, 400, or 1200 mg) by a licensed pharmacy (Uniprix Pharmacy, Montreal, Canada) and stored at 4°C.

Participants underwent two OFCs on separate days, 3 to 6 weeks apart, randomly receiving autoclaved peanut on 1 day and blanched peanut on the other. Prior to the OFC, participants abstained from antihistamines and other medications that could affect the outcomes for at least 5 days. Increasing peanut doses from capsules were mixed into a food vehicle (pudding or fruit puree) and administered every 30 min, starting with 4 mg of peanut (1 mg of peanut protein; 25% of total mass) and escalating to a maximum of 1200 mg (300 mg protein; Table S1). Participants were closely monitored throughout the OFC for signs of an allergic reaction, at which point the challenge was stopped and treatment was provided. Allergic reactions were categorized using a published classification system [14] which distinguishes two main categories: non‐anaphylactic allergic reactions (AR) and anaphylactic allergic reactions (AAR), each further subclassified into mild, moderate, and severe reactions. See Supporting Information for more detail. Participants remained under observation for at least 2 h post‐challenge until clinical stability was attained.

2.8. Statistical Analyses

Statistical and data analyses were performed in R (version 4.2.2). Data are expressed as mean ± standard error of the mean (SEM) of 3 replicates. All analyses were performed in comparison to raw peanut unless otherwise specified. Statistical tests used are specified in figure captions; a p < 0.05 was considered statistically significant in all cases.

3. Results

3.1. Autoclaving Reduces Peanut Protein Detection and Solubility

We first sought to characterize the impact of roasting and autoclaving (130°C, 30 min) on commercially available raw and blanched peanuts. SDS‐PAGE and Coomassie staining revealed substantial differences in the distribution of protein bands between treatments (Figure 1B). Roasting raw or blanched peanuts revealed multiple protein bands of similar molecular weight, while in contrast, autoclaving peanuts exhibited protein degradation with less intense, diffuse bands at lower molecular weights. There were minimal differences in proteins detected in raw peanuts compared to blanched. While roasting led to a significant decrease in the amount of protein detected by Bradford assay compared to raw or blanched peanuts, autoclaving led to a significantly greater decrease in protein detected (Figure 1C).

We determined the conditions for autoclaving that led to the greatest decrease in protein detection by evaluating extracts from raw peanuts autoclaved at a range of times (5–60 min, fixed 130°C) and temperatures (100°C–130°C, fixed 30 min). At 130°C, protein and allergen detection dropped in as little as 5 min, with further decreases observed following longer processing times (Figures S1 and S2). We established that 130°C (2.4 atm) at 30 min was the optimal parameters for autoclaving and was used for further experiments.

To understand the discrepancy between amounts of protein introduced and that which was detected by the Bradford assay, we assessed the impact of autoclaving on soluble and insoluble extract fractions using stronger extraction buffers and via freeze‐drying experiments. Autoclaving substantially reduced protein solubility, demonstrated by the increased pellet mass (Table S2), decreased protein detection in the soluble extract, and increased protein detection in the insoluble extract (Figure 1C). To compare the potential allergenicity following roasting or autoclaving in the soluble and insoluble fractions, we then performed a total peanut‐specific IgE ELISA coating the plate with equal protein concentrations determined by Bradford and using a pooled serum from allergic patients. Autoclaving resulted in a significant decrease in sIgE binding in both the soluble and insoluble fractions when compared to raw or roasted (Figure 1D). These results suggest autoclaving is—in addition to reducing protein solubility—inducing other structural changes to proteins in both fractions that impair IgE binding. Independent of solubility changes, we further observed a substantial presence of mass in the soluble fraction of autoclaved peanuts not detected by Bradford (Table S2, % Protein of extract).

3.2. Autoclaving Induces Peanut Protein and Allergen Fragmentation

We hypothesized that autoclaved extracts contained substantial amounts of peptides that would not be detected by Bradford [15]. Protein extracts created from raw, roasted, and autoclaved peanuts were passed through size‐exclusion centrifugal filters (10 kDa) and then analyzed via Bradford assay and mass spectrometry (MS)‐proteomics. Bradford assay did not detect protein in any filtrate. In contrast, MS‐proteomics detected a wide spectrum of peptides following autoclaving that were not present in either raw or roasted filtrates.

Using MS, we detected higher total and unique spectral counts in autoclaved peanut compared to raw or roasted (Figure 2A–2D and Appendix S1). Moreover, upon evaluating specific peptide sequences of major allergens Ara h 1, Ara h 2, and Ara h 3, we found far greater numbers and diversity of peanut allergen peptides in the autoclaved samples (Figure 2E–2G). When analyzing filtrates with a 10‐kDa cut‐off, we detected a wide range of allergen‐derived peptides originating from proteins larger than 10 kDa in the autoclaved peanut sample, and not in raw or roasted, indicating that autoclaving induces extensive peanut protein fragmentation.

FIGURE 2.

Mass spectrometry followed by proteomics analyses on raw, roasted, and autoclaved (130°C, 30 min) peanut protein extract filtrates (10 kDa cut‐off). (A, B) Total spectral counts and (C, D) total unique spectral counts corresponding to peptides mapped to proteins in the peanut proteome. Percentages represent the proportion of proteins above and below the y = x line. Error bars represent standard errors (n = 3). (E–G) Peptide sequence coverage maps illustrating detected fragments spanning from the N‐ to C‐terminus of (E) Ara h 1 (accession: B3IXL2), (F) Ara h 2 (accession: A8VT44), and (G) Ara h 3 (accession: A0A445CPR7), demonstrating widespread fragmentation across each allergen sequence under autoclaving conditions, and not raw nor roasted. P values were determined by ANOVA & Tukey's HSD. *p < 0.05, ***p < 0.001. Error bars represent standard errors (n = 3).

3.3. Peanut‐Specific IgE Binding Is Reduced to Autoclaved Peanut Extracts

We performed ELISA experiments to determine if IgE binding was affected by autoclaving. We coated plates with serial dilutions of raw, roasted, and autoclaved peanut extracts (normalized to flour mass) and compared antibody binding using either commercial antibodies against allergens Ara h 1, Ara h 2, and Ara h 8 or pooled sera from subjects with high levels of peanut‐sIgE antibodies. Autoclaving significantly reduced the detection of major allergens Ara h 1 and Ara h 2 (Figure 3A,B), and minor allergen Ara h 8 was no longer detectable (Figure 3C). Similarly, autoclaving—but not roasting—significantly decreased sIgE binding to peanut extracts (74% reduction, 95% CI: [63%, 85%]) when compared to raw peanuts (Figure 3D). We then performed a competitive ELISA with peanut‐allergic pooled sera to further evaluate IgE binding in solution. Plates were coated with either raw peanut extracts or recombinant Ara h 2 exclusively and raw, roasted, or autoclaved extracts were used as inhibitors of IgE binding. In solution, autoclaved extracts inhibited IgE binding to both total peanut and Ara h 2 significantly less than raw and roasted extracts (Figure 3E,F).

FIGURE 3.

(A–C) Relative quantification of peanut protein allergens (A) Ara h 1, (B) Ara h 2, and (C) Ara h 8 via ELISA. (D) Total peanut‐specific IgE ELISA using a pooled serum of highly peanut‐allergic subjects evaluating IgE binding. Plates were coated with peanut extracts of each condition at a range of concentrations. (E–F) Inhibition ELISA using plates coated with (E) total raw peanut or (F) recombinant Ara h 2, pre‐incubating pooled serum with serial dilutions of raw, roasted, or autoclaved peanut extracts as inhibitors of IgE binding to the plate. In all assays, protein concentrations were normalized by peanut flour mass; x‐axis values correspond to raw extract concentrations. P values were determined by ANOVA & Tukey's HSD at each concentration; statistical annotations denote comparisons to raw extracts for 1 μg/mL. *p < 0.05, **p < 0.01, ***p < 0.001. Error bars represent standard errors (n = 3).

3.4. Autoclaved Peanut Extracts Induce Smaller Skin Test Responses Compared to Standard Extracts

To evaluate the impact of autoclaving on skin testing, we performed SPTs on 41 peanut‐allergic individuals comparing standard commercial peanut extracts and soluble extracts created from autoclaved peanuts (130°C, 30 min). Participant median age was 20 years, and 17 subjects were female (Table 1). Wheal diameters were recorded, and serum samples were obtained to compare peanut‐sIgE levels via ELISA. There was a significant decrease in SPT wheal diameter in peanut‐allergic subjects using autoclaved peanut extract (median [IQR] = 5 mm [2, 9]) compared to commercial extracts (10 mm [6, 15]; p < 0.001, Figure 4A,B).

TABLE 1.

Study subject demographics and clinical characteristics of peanut‐allergic patients participating in SPT study using commercial standard and autoclaved peanut protein extracts. Total peanut‐ and component‐specific IgE levels were determined via ELISA. Subjects were further stratified into 2 groups based on SPT wheal diameter difference between standard and autoclaved peanut extracts (≥ 3 mm).

Characteristics	Median (interquartile range)
	Total	SPT wheal decrease (≥ 3 mm)	No SPT wheal decrease
Number of patients (N)	41	30	11
Age (y)	20 (15, 28)	24 (14, 28)	18 (15, 25)
Sex (male)	24 (59%)	18 (60%)	6 (55%)
SPT standard wheal diameter (mm)	10 (6, 15)	11 (7, 15)	6 (4, 12)
SPT autoclaved wheal diameter (mm)	5 (2, 9)	3 (2, 6)	8 (5, 15)
ImmunoCAP (kU/L)	10.9 (1.43, 100) (N = 28)	2.21 (1.14, 49.2) (N = 19)	100 (9.16, 100) (N = 9)
sIgE total peanut, raw (kU/L)	197 (28.6, 2922)	147 (14.5, 2001)	5342 (1140, 8047)
sIgE total peanut, autoclaved (kU/L)	61.8 (1.78, 727)	48.3 (1.49, 641)	421 (133, 1768)
sIgE Ara h 1 (kU/L)	42.1 (0.76, 656)	25.2 (0.35, 186)	1328 (36.7, 2037)
sIgE Ara h 2 (kU/L)	45.4 (4.95, 529)	39.7 (2.51, 140)	529 (149, 1270)
sIgE Ara h 8 (kU/L)	0.0 (0.0, 2.09) [max = 33.3]	0.0 (0.0, 0.0) [max = 24.3]	2.17 (0.0, 7.53) [max = 33.3]

Abbreviations: ELISA, enzyme‐linked immunosorbent assay; sIgE, specific immunoglobulin E; SPT, skin prick test.

FIGURE 4.

(A) Peanut‐specific IgE levels to raw and autoclaved (130°C, 30 min) peanut and (B) Skin prick test (SPT) wheal diameters using standard and autoclaved peanut extracts in 41 allergic subjects. Subjects with a significant autoclaved wheal decrease compared to standard (≥ 3 mm decrease; SPT decrease group) are highlighted. Horizontal dashed line represents the threshold of SPT positivity (3 mm). (C) Total raw and autoclaved peanut‐ and (D) component‐specific IgE levels quantified by ELISA. Subjects were stratified into two groups based on SPT wheal diameter difference between standard and autoclaved peanut extracts, as described in (B). p values were determined by Wilcoxon signed‐rank test (A, B, and C; #: p < 0.001) and Mann–Whitney U test (C and D; *p < 0.05, **p < 0.01).

We detected variability in the responses to autoclaved peanut extracts, with a subset having similar wheal sizes to both extracts. We explored possible contributors by stratifying participants into two groups by differences in wheal size: those with substantially smaller autoclaved wheal sizes (≥ 3 mm; N = 30) and < 3 mm decrease (N = 11). Subjects with a substantial decrease in autoclaved wheal sizes had significantly lower sIgE levels to total peanut (p < 0.05) and allergen components Ara h 1 (p < 0.05), Ara h 2 (p < 0.01), and Ara h 8 (p < 0.05; Figure 4C,D). This suggests testing to autoclaved peanut extract may help stratify peanut‐allergic subgroups. Additionally, basophil activation testing on 3 allergic patients showed reduced activation (%CD63⁺) in response to autoclaved peanut soluble extracts when compared to raw (Figure S3).

3.5. Peanut‐Allergic Subjects Tolerate Significantly Higher Doses of Autoclaved Peanuts on OFC

While SPT may act as a proxy for predicting the potential for food allergy, OFC is the gold standard for diagnosis and assessing severity. As such, we then set out to compare if peanut‐allergic individuals would react to a challenge using doses of autoclaved peanuts up to 300 mg, compared to similar amounts of standard peanut (Table S1). We recruited 13 peanut‐allergic subjects, of which 10 eligible subjects completed two double‐blinded OFCs with each peanut on different days (Figure 5A, Table S3). Equal masses of blanched peanuts, either unprocessed or autoclaved, were provided to subjects to evaluate their tolerability.

FIGURE 5.

(A) CONSORT flow diagram of oral food challenge (OFC) study. Ten (10) peanut‐allergic subjects completed two double‐blind, randomized OFCs: One to blanched and the other to autoclaved (130°C, 30 min) peanut. (B) Skin prick test (SPT) wheal diameters using blanched and autoclaved peanut extracts in 10 allergic subjects. Horizontal dashed line represents the threshold of SPT positivity (3 mm). (C) Cumulative tolerated dose of blanched and autoclaved peanut by study subjects; all 10 subjects tolerated the maximum dose of autoclaved peanut. (D) Heatmap summarizing adverse events following OFC with blanched and autoclaved peanuts based on the classification system by De Schryver et al. (2019) [14]. AR: Allergic reaction; AAR: Anaphylactic allergic reaction. Subjects with AARs required at least one dose of epinephrine. P values were determined by Wilcoxon signed‐rank test (**p < 0.01).

The 10 subjects had reduced wheal diameters when skin tested with autoclaved peanuts compared to standard extract (Figure 5B). All 10 subjects tolerated the maximum cumulative dose (444 mg) of autoclaved peanut. In contrast, all subjects ingesting blanched peanut had symptoms requiring treatment including epinephrine, with variable cumulative doses (median: 9 mg, range: [1, 44]; Figure 5C). Reactions to autoclaved peanuts were limited to mild and moderate non‐anaphylactic allergic reactions (AR), such as mild throat itchiness or abdominal discomfort (Figure 5D, Table S4). Blanched peanuts, in contrast, induced more severe symptoms such as abdominal pain, nausea, throat tightness and itchiness, facial edema, and in one case, hypotension.

4. Discussion

Peanut allergy is among the highest causes of food‐induced anaphylaxis, and a common indication for oral immunotherapy (OIT). This can be addressed by determining safer substrates to use in the treatment of peanut allergy. Our study is the first to directly demonstrate both reduced peanut‐sIgE binding because of heat and pressure autoclaving and potential clinical impact due to increased oral tolerability. In vitro, autoclaved peanut extracts exhibited decreased amounts of intact allergens Ara h 1, 2, and 8, and reduced sIgE binding to sera from allergic patients, in part due to protein fragmentation. Autoclaving peanuts at 130°C (2.4 atm) for 30 min significantly decreased SPT wheal diameters in peanut‐allergic subjects and increased the tolerated dose of peanut by OFC.

The presence of high moisture and vapor pressure distinguishes autoclaving from roasting and may drive hydrolysis—in addition to other chemical reactions—that result in protein fragmentation and reduced allergenicity. Cabanillas et al. (2012 & 2015) showed that autoclaving reduced peanut‐sIgE binding and the detection of major allergens Ara h 1, 2, and 6 and is accompanied by a decrease of α‐helix content and an increase in random coils and loops in the soluble fraction as a function of pressure and duration [11, 13]. Additionally, previous reports have shown that autoclaving reduces the solubility of other food proteins like soy [16, 17], buckwheat [18], and egg [19]. Likewise, our study suggests that autoclaving chemically changes the peanut, inducing protein fragmentation and decreasing solubility, which may be due to processes such as denaturation, aggregation, or crosslinking. For our OFCs, we used whole peanuts to eliminate bias from any post‐autoclaving processing including both soluble and insoluble fractions.

Prior studies using mass spectrometry on selected protein bands (> 15 kDa) from processed peanuts did not directly confirm autoclaving‐induced fragmentation [12, 20, 21, 22]. Building on our previous NMR profiling of soaked and intact peanuts [23], which revealed extensive protein hydrolysis but no alteration in small molecules such as glucose, sucrose, or triglycerides, the present study provides direct molecular confirmation of autoclave‐induced fragmentation. Analysis of peanut extract filtrates enriched for fragments < 10 kDa demonstrated a marked increase in peptide frequency and diversity in autoclaved samples compared with raw and roasted controls, consistent with widespread hydrolytic cleavage of peptide bonds. Most peptides mapped to allergens with intact sizes > 10 kDa, indicating fragmentation across multiple sites rather than selective degradation typical of biological processing. While a few common peptides—such as those near the N‐terminus of Ara h 1—were also detected in raw and roasted filtrates, their abundance was three‐ to five‐fold higher after autoclaving, confirming that heat and pressure promote extensive allergen fragmentation consistent with prior reports [21, 24, 25].

The capacity to evaluate protein fragmentation is influenced by differences in relative protein size and abundance. More unique peptides mapped to Ara h 3 in autoclaved filtrates than Ara h 1, 2, and 6, likely due to its greater abundance and large size affording more possible fragmentation sites [26]. In contrast, peptides from Ara h 8—a small, very low abundance protein—were not detected. Differences in relative solubility, fragmentation above the 10 kDa threshold, and degradation beyond the point of detection are also possible contributors. Further peptide analysis at different sizes may help understand how peanut allergens may be differentially affected by autoclaving. Our study also highlights the importance of carefully considering the assays used to measure protein, as the Bradford assay failed to detect a substantial fraction of the soluble autoclaved peanut extract due to fragmentation below the size threshold for detection.

Studies in other foods—including cashews [27], pistachios [27], and walnuts [28]—also showed reduced allergenicity after autoclaving, evidenced by diminished sIgE binding via ELISA, reduced SPT wheal sizes, and reduced basophil activation. Previous studies with smaller cohorts reported decreased wheal diameters using autoclaved peanut extracts [11, 12] but used subjects with low to moderate levels of peanut‐sIgE (mean ± SD: 2.5 ± 2.3 kU/L [11] or 9.4 ± 3.3 kU/L [12]). In this study, we employed a large cohort with subjects who had very high levels of peanut‐sIgE (38.9 ± 42.9 kU/L). While we did have subjects who did not show significant reductions in wheal size to autoclaved peanuts, they were more likely to be those with the highest peanut‐sIgE levels, a metric negatively associated with success in other OIT trials such as with cow's milk [29]. Importantly, all peanut‐allergic subjects who received an OFC tolerated all doses of autoclaved peanut with minimal allergic symptoms compared with blanched peanuts, regardless of their SPT wheal sizes or peanut‐sIgE. To our knowledge, this is the first study to perform OFC with autoclaved food on allergic subjects and directly demonstrate reduced peanut allergenicity in a clinical setting.

Autoclaving may alter peanut epitopes, disrupting some through conformational changes and hydrolysis while producing more linear epitopes. Other cooked products like milk and eggs have a decreased reactivity in allergic individuals, and baked egg and milk are used as a desensitization strategy for some children [30, 31, 32]. In the case of peanuts with highly stable allergens like Ara h 2, and little change in these proteins with dry roasting, autoclaving may be a method capable of introducing advantageous changes that traditional cooking methods such as roasting cannot. Our ELISA data indicate that detectable allergens like Ara h 1 and 2 remain; however, SPT and OFC results demonstrate reduced reactivity in allergic subjects, suggesting that autoclaved peanuts could be valuable for OIT. Unlike existing OIT trials using boiled peanut [33, 34], which dilutes peanuts by leaching small molecular weight allergens into the water [35], autoclaving reduces intact allergens and creates novel fragments and epitopes, providing a potentially more efficacious substrate for desensitization. Other novel approaches to improving oral tolerance include using peanut seeds harvested early; immature peanuts may be lower in allergenic proteins such as Ara h 2 [36, 37]. Further research is required to understand what impact this modified, heavily degraded peanut can have on peanut sensitization or desensitization potential compared to current treatments such as Palforzia, a lyophilized standardized peanut available in the USA.

These findings demonstrate that autoclaving peanuts induces chemical changes that significantly reduce allergenicity and risk of severe allergic reaction upon consumption. This modified peanut may be a useful substrate for future peanut OIT formulations.

Author Contributions

Bruce D. Mazer and Bertrand J. Jean‐Claude conceptualized and supervised the study. Casey G. Cohen, Diana Toscano‐Rivero, Eisha A. Ahmed, Wei Zhao, Carmen H. Li, and Kurt Dejgaard contributed to laboratory data collection. Casey G. Cohen, Diana Toscano‐Rivero, Adnan Al Ali, Danbing Ke, Duncan Lejtenyi, Liane Beaudette, Hana Chazbey, and Abdulaziz S. Alrafiaah contributed to clinical data collection and administration. Casey G. Cohen, Diana Toscano‐Rivero, Eisha A. Ahmed, Albert M. Berghuis, Bertrand J. Jean‐Claude, Christine McCusker, Thomas Eiwegger, Moshe Ben‐Shoshan, and Bruce D. Mazer contributed to formal data analysis. Casey G. Cohen and Diana Toscano‐Rivero curated the data. All authors contributed to the interpretation of the data. Casey G. Cohen, Diana Toscano‐Rivero, and Eisha A. Ahmed drafted the original draft. All authors were involved in writing, review and editing. All authors approved the manuscript before submission. All authors had final responsibility for the decision to submit for publication.

Funding

This work was supported by Fondation de l'Hôpital de Montréal pour enfants. McGill University Health Centre. AllerGEN NCE. Canadian Institutes of Health Research.

Conflicts of Interest

Dr. Moshe Ben‐Shoshan is a principal investigator on studies by ALK evaluating sublingual immunotherapy with peanut. Dr. Thomas Eiwegger reports to act/recently acted as local P.I. for company‐sponsored trials by D.B.V. Therapeutics, Greer Stallergens, and sub‐investigator for Regeneron and ALK‐Abello. He is Co‐Investigator or scientific lead in three investigator‐initiated oral immunotherapy trials supported by the SickKids Food Allergy and Anaphylaxis Program and serves as an associate editor for Allergy. His lab received unconditional/in‐kind contributions from Macro Array Diagnostics and an unrestricted grant from ALK‐Abello. He holds advisory board roles for ALK‐Abello, Nutricia/Danone, Hipp, Greer Stallergens, Allergy Therapeutics/Bencard, and Sanofi. He reports lecture fees from Novartis, Thermo Fisher, Schwabe, Nutricia/Danone, Aimmune, ALK‐Abello, Sanofi. All other authors declare that they have no conflicts of interest.

Supporting information

Figure S1: (A–B) Coomassie stain of protein gels following SDS‐PAGE analysis comparing raw, roasted, autoclaved peanut samples at a range of (A) temperatures for a constant time of 30 min and (B) times at a constant temperature of 130°C. (C–D) Concentration of protein extracts derived from raw, roasted, and autoclaved peanuts measured by Bradford assay. Peanuts were autoclaved at a range of (C) temperatures or (D) times. #: p < 0.001 (compared to raw), ANOVA & Tukey's HSD. (E–F) Total peanut‐specific IgE ELISA using a pooled serum of highly peanut‐allergic subjects evaluating IgE binding to autoclaved peanuts at a range of (E) temperatures and (F) times. Plates were coated with peanut extracts of each condition at a range of concentrations and were normalized by peanut flour mass; x‐axis values correspond to raw extract concentrations. P values were determined by ANOVA & Tukey's HSD at each concentration; statistical annotations denote comparisons to raw extracts for 1 μg/mL. *p < 0.05, **p < 0.01. Error bars represent standard errors (n = 3).

Figure S2: Effect of (A–C) time and (D–F) temperature of autoclaving on peanut protein allergens (A, D) Ara h 1, (B, E) Ara h 2, and (C, F) Ara h 8. Relative quantification of each allergen was done via ELISA. Plates were coated with peanut extracts of each condition at a range of concentrations normalized by peanut flour mass; x‐axis values correspond to raw extract concentrations. Error bars represent standard errors (n = 3).

Figure S3: Percent CD63+ basophils following activation testing in response to raw and autoclaved (130°C, 30 min) peanut extracts. Tests were performed using 3 extract replicates to compute the mean activation for each subject at 3 concentrations: 1, 10, 100 ng/mL. Serum used for BAT came from 3 individual patients, noted to have ImmunoCAP levels between 22 and > 100 kUL for peanut. Presented data summarizes results of 3 subjects; error bars represent standard errors (n = 3).

Table S1: Oral food challenge (OFC) dosing protocol. Double‐blind, randomized OFC up to a cumulative dose of 444 mg of blanched or autoclaved peanut protein.

Table S2: Relative extracted masses from defatted peanut flours dissolved in extraction buffer (20 mM Tris, pH 8.5) following lyophilization experiments.

Table S3: Demographics and clinical characteristics of ten subjects that completed two double‐blind OFC.

Table S4: Reaction severity grading of each study subject following OFC with either blanched or autoclaved peanut using the De Schryver, CoFAR, and WAO grading schemes.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.