Influence of Roasting Temperature on the Detectability of Potentially Allergenic Lupin by SDS-PAGE, ELISAs, LC-MS/MS, and Real-Time PCR

Abstract

Seeds of "sweet lupins" have been playing an increasing role in the food industry. Lupin proteins may be used for producing a variety of foods, including pasta, bread, cookies, dairy products, and coffee substitutes. In a small percentage of the population, lupin consumption may elicit allergic reactions, either due to primary sensitization to lupin or due to cross-allergy with other legumes. Thus, lupin has to be declared on commercial food products according to EU food regulations. In this study, we investigated the influence of roasting seeds of the L. angustifolius cultivar "Boregine" on the detectability of lupin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), ELISAs, LC-MS/MS, and real-time PCR. Seeds were roasted by fluidized bed roasting, and samples were drawn at seed surface temperatures ranging from 98 °C to 242 °C. With increasing roasting temperature, the extractability of proteins and DNA decreased. In addition, roasting resulted in lower detectability of lupin proteins by ELISAs and LC-MS/MS and lower detectability of DNA by real-time PCR. Our results suggest reduced allergenicity of roasted lupin seeds used for the production of "lupin coffee"; however, this has to be confirmed in in vivo studies.

Keywords: ELISA; LC-MS; Lupinus angustifolius; SDS-PAGE; cultivar Boregine; detectability; food allergen; lupin; real-time PCR; roasting.

Figure 1

Seeds of lupin (L. angustifolius L., cultivar “Boregine”) before and after roasting in a professional pilot coffee roasting machine. Samples were drawn at surface temperatures of 98 °C, 120 °C, 140 °C, 160 °C, 175 °C, 195 °C, 220 °C, and 242 °C, respectively.

Figure 2

Influence of roasting temperature on protein recovery. (a) Extraction protocol 1: 0.1 M Tris, 0.5 M glycine, pH 8.7; 3 h. Mean and standard deviation of four replicates. (b) Extraction protocol 2: 2 N urea, 0.2 N Tris-HCl, pH 9.2; 30 min, followed by sonication for 15 min. Mean and standard deviation of three replicates. Small letters indicate homogeneous subgroups based on ANOVA (p ≤ 0.05) and post hoc test according to Scheffe.

Figure 3

SDS-PAGE of extracts obtained with extraction protocol 1 from raw and roasted lupin seeds. (a): seeds roasted at 98 °C, 120 °C, 140 °C, 160 °C, 175 °C; (b): seeds roasted at 98 °C, 120 °C, 195 °C, 220 °C, 242 °C. Extracts from raw seeds and seeds roasted at temperatures ≤ 98 °C were diluted to a protein concentration of 8.3 mg/mL; extracts from seeds roasted at higher temperatures were loaded undiluted. OV: ovalbumin (45 kDa), MY: myoglobin (17 kDa), BSA: bovine serum albumin (66 kDa), IgG: human immunoglobulin G (heavy chain: 50 kDa, low chain: 25 kDa). Protein sizes of the standard proteins (in kDa) are indicated on the left and right side of the gels.

Figure 4

Influence of roasting temperature on the detectability by lupin ELISAs. (a) competitive ELISA, (b) sandwich ELISA. Protein extracts from raw and roasted lupin seeds were obtained with extraction protocol 1.

Figure 5

Heatmap showing the influence of roasting temperature on relative intensities based on raw lupine via LFQ by LC-MS/MS. Warmer tones like red and orange indicate higher values, while cooler tones like yellow and green represent lower values. * based on shared peptides, no unique peptides found; n.d. not detected.

Figure 5

Heatmap showing the influence of roasting temperature on relative intensities based on raw lupine via LFQ by LC-MS/MS. Warmer tones like red and orange indicate higher values, while cooler tones like yellow and green represent lower values. * based on shared peptides, no unique peptides found; n.d. not detected.

Figure 6

Influence of roasting temperature on (a) DNA recovery (means of two technical replicates) and (b) amplification by real-time PCR.