Effects of a Proline Endopeptidase on the ... - ACS Publications

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Effects of a Proline Endopeptidase on the Detection and Quantitation of Gluten by Antibody-Based Methods during the Fermentation of a Model Sorghum Beer Rakhi Panda,*,† Katherine L. Fiedler,‡ Chung Y. Cho,† Raymond Cheng,§ Whitney L. Stutts,‡ Lauren S. Jackson,∥ and Eric A. E. Garber† †

Division of Bioanalytical Chemistry, Office of Regulatory Science, Center for Food Safety and Applied Nutrition (CFSAN), FDA, College Park, Maryland 20740, United States ‡ Division of Analytical Chemistry, Office of Regulatory Science, Center for Food Safety and Applied Nutrition (CFSAN), FDA, College Park, Maryland 20740, United States § Joint Institute for Food Safety and Applied Nutrition (JIFSAN), University of Maryland, College Park, Maryland 20740, United States ∥ Division of Food Processing Science and Technology, Office of Food Safety, CFSAN, FDA, Bedford Park, Illinois 60501, United States S Supporting Information *

ABSTRACT: The effectiveness of a proline endopeptidase (PEP) in hydrolyzing gluten and its putative immunopathogenic sequences was examined using antibody-based methods and mass spectrometry (MS). Based on the results of the antibody-based methods, fermentation of wheat gluten containing sorghum beer resulted in a reduction in the detectable gluten concentration. The addition of PEP further reduced the gluten concentration. Only one sandwich ELISA was able to detect the apparent low levels of gluten present in the beers. A competitive ELISA using a pepsin-trypsin hydrolysate calibrant was unreliable because the peptide profiles of the beers were inconsistent with that of the hydrolysate calibrant. Analysis by MS indicated that PEP enhanced the loss of a fragment of an immunopathogenic 33-mer peptide in the beer. However, Western blot results indicated partial resistance of the high molecular weight (HMW) glutenins to the action of PEP, questioning the ability of PEP in digesting all immunopathogenic sequences present in gluten. KEYWORDS: gluten, proline endopeptidase, beer, fermentation

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process, FDA “recognized that some food matrices, such as fermented or hydrolyzed foods, may lack currently available scientifically valid methods that can be used to accurately determine if these foods contain ≥20 ppm gluten.”6 Several commercial sandwich ELISAs based on R5 monoclonal (RIDASCREEN Gliadin), MIoBS polyclonal (Morinaga Institutes of Biological Sciences, Inc., Wheat Protein/Gliadin), G12 monoclonal (AgraQuant Gluten G12), A1 and G12 monoclonal (GlutenTox Sandwich), and Skerritt monoclonal (ALLER-TEK Gluten) antibodies have been validated and adapted for detection and quantitation of intact gluten in food.7−14 The Skerritt antibody utilized in the ALLER-TEK Gluten ELISA was originally developed against the ω1, 2-gliadin.7 However, studies have shown the specificity of the antibody towards the HMW glutenins.15,16 Competitive ELISAs based on R5 (RIDASCREEN Gliadin Competitive) and G12 (GlutenTox Competitive) monoclonal antibodies are also available for detection and quantitation of gluten in fermented and hydrolyzed foods.11,12,17,18 The RIDASCREEN

INTRODUCTION Celiac disease (CD) is an immune-mediated disorder triggered by the interaction of the prolamin and glutelin fractions of proteins from wheat, barley, and rye with the intestinal mucosa of sensitive individuals.1 In addition to the protein fractions in wheat, barley, and rye, a small fraction (15-fold. Similar results were obtained by the analysis of the 20 mg/L gluten containing beer samples using the MIoBS ELISA, which was able to detect gluten in all samples, from the beginning to the end of the fermentation, both in the presence and absence of PEP (results not shown). As measured with the MIoBS ELISA, the average gluten level of the 0 mg/L gluten containing beer samples was 0.6 mg/L

during the fermentation process. The study allowed a preliminary evaluation of the impact of PEP on known immunopathogenic sequences suspected of causing CD. MS method was used to identify peptides in the beer samples and no attempt was made to quantitate gluten concentration using the MS method. Relative quantitation using isotopically labeled peptides was conducted in order to compare different brew replicates and for MS run-to-run comparison and normalization. Sorghum beer brewed in the presence and absence of PEP, with wheat gluten (0, 20, and 200 mg/L) added prior to the fermentation process, was used as a model system. This model system allowed us to explore directly specific questions that were not easily possible using barley malt and other brewing formats. By using gluten and not whole grains, the amount of gluten exposed to fermentation conditions was known and could be directly varied at levels of concern as defined by governmental regulations chosen in part to reflect consumer concerns. Similarly, the effectiveness of PEP could be more directly ascertained using this model system. This model system also directly focused on Gluten-free beers, such as sorghum beer, for which inadvertent cross-contact is a serious concern. This research allowed us to gain critical information regarding the processes while developing the analytical methodology that can be applied to more complex model systems. The three levels of gluten used in the study were chosen to represent levels of regulatory importance. The results cannot be extrapolated to higher levels of gluten due to comingling, as this more likely will result in a different pool of proteinaceous substrate with a different content of immunopathogenic elements. During the fermentation of sorghum beer, the sugar content of the wort gradually decreased from 13.7% to 7.4% brix irrespective of whether gluten (0, 20, or 200 mg/L) or PEP was present. The alcohol concentration in the beer sample collected at the end of the fermentation ranged from 5.3 to 5.9% ABV (alcohol by volume). The pH of the wort dropped from approximately 5.5 to 4.0 during fermentation. ELISA Analysis. Four sandwich ELISAs and one competitive ELISA were used to analyze the samples collected throughout the brewing process. Figure 1 shows the results of the ELISA analysis of one of the two replicate trials of the 200 mg/L gluten containing beer brewed in the presence and absence of five different levels of PEP. Similar results were obtained from the analysis of the other replicate trial. Only the MIoBS ELISA was able to detect gluten in all the 200 mg/L gluten containing samples, brewed in the presence and absence of PEP, from the beginning to the end of the fermentation. The detectable gluten concentration for the PEP-containing beers collected after 2 weeks of fermentation was below the limit of quantitation (LOQ) of the R5-sandwich, G12-sandwich, and R5-competitive ELISAs. Detection of gluten in the PEPcontaining beers after 2 weeks of fermentation, by the MIoBS ELISA, was possible due to the low limit of quantitation (LOQ) of the ELISA kit (0.25 mg/kg). According to the results of the MIoBS (Figure 1A), R5sandwich (Figure 1B), and G12-sandwich ELISAs (Figure 1C), the detectable gluten concentration in the non-PEP containing samples gradually decreased as fermentation proceeded. Addition of PEP increased the reductions in the gluten concentration. Relative to non-PEP containing samples, increased reduction in gluten concentration was observed after 1 h of fermentation for the beers brewed with the addition of 806, 323, 129, and 64.5 μL per 3.8 L of PEP, and after 24 h 10529

DOI: 10.1021/acs.jafc.5b04205 J. Agric. Food Chem. 2015, 63, 10525−10535

Article

Journal of Agricultural and Food Chemistry

Figure 2. Lateral flow assay analysis of samples collected throughout the brewing process using the RIDA Quick Gliadin (A), Gluten Rapid Kit (B), EZ Gluten (C), AgraStrip Gluten G12 Test Strips (D), Gluten Residue Lateral Flow Test, sandwich format (E), and Gluten Residue Lateral Flow Test, competitive format (F). Plotted are the mean mAbs measurements of triplicate analyses (conducted on different days) of the “Test” line in the presence (green triangle) and absence (red square) of PEP. Error bars represent ± one standard deviation. Inserts depict calibration curves of intact gluten, not subjected to the fermentation process.

(±0.5). This minimal level of gluten detected by the highly sensitive MIoBS ELISA may reflect gluten protein release from the yeast preparation. Lateral Flow Assays. Gluten standards suspended in PBST were analyzed with each LFD for the quantitation purpose (insets of Figure 2). The competitive LFD displayed an enhanced sensitivity to the presence of anything that affected the binding of the labeled antibody to the immobilized gluten (inset of Figure 2F). All LFDs were able to detect the presence of ≥20 mg/L gluten added to sorghum syrup prior to the onset of fermentation. Though the LFDs could detect the presence of 20 mg/L gluten in sorghum syrup, the variances made it impossible to distinguish between slight concentration changes below 20 mg/L or measure the effects of the PEP. All six LFDs

were able to detect and quantitate the changes in 200 mg/L gluten during the fermentation process (Figure 2). Similar to the ELISA analysis, the presence of PEP increased the loss of detectable gluten. The LFDs that relied on a sandwich format displayed a rapid loss in detectable gluten during the first 24 h followed by a gradual loss until the end of the fermentation (Figure 2A−E). In contrast, the LFD that relied on a competitive format displayed a gradual decrease in detectable gluten throughout the fermentation process (Figure 2F).The difference between the sandwich and competitive LFDs observed is consistent with the sandwich-based LFDs requiring the presence of two antibody binding sites (epitopes), while the competitive LFD required only one. Any hydrolysis that eliminates the presence of two epitopes on a single polypeptide 10530


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Figure 3. Western blots of the sorghum syrup wort immediately after addition of yeast and the final beer product (2 weeks) of the PEP and non-PEP containing beers. The detector antibodies A, G12; B, Skerritt; C, R5; and D, MIoBS of sandwich ELISA test kits were used. Lane information: lane 1, intact gluten standard (80 mg/L); lane 2, molecular weight marker (Mr = 250 000−5000); lane 3, intact gluten standard (2.5 mg/L); lane 4, empty; lane 5, non-PEP containing sorghum syrup wort immediately after addition of yeast; lane 6, empty; lane 7, non-PEP containing sorghum beer final product; lane 8, empty; lane 9, 16 μL per 3.8 L PEP containing sorghum syrup wort immediately after addition of yeast; lane 10, empty; lane 11, 16 μL per 3.8 L PEP containing sorghum beer final product; lane 12, empty; lane 13, 806.5 μL per 3.8 L PEP containing sorghum syrup wort immediately after addition of yeast; lane 14, empty; lane 15, 806.5 μL per 3.8 L PEP containing sorghum beer final product.

average a 4-fold reduction in detectable gluten concentration was observed, from beginning to the end of the fermentation (Figure 1). Further similar to the ELISA results, the presence of PEP (16 or 806 μL per 3.8 L of wort) resulted in >15-fold reduction in signal intensity in the final beers (lanes 11 and 15 respectively) compared to their respective initial levels (lanes 9 and 13 respectively), using the MIoBS, R5 and G12 antibodies. These results indicate the susceptibility of gliadin and LMW glutenin proteins to the action of PEP during the brewing process. However, with the Western blots using the Skerritt antibody, only approximately 8 to 9 fold reduction in signal intensity was observed in the final beer (lane 11 and 15), compared to the initial level (lane 9 and 13), by adding PEP. This indicates greater resistance of the HMW glutenins (relative to the gliadins and LMW glutenins) to the action of PEP during the fermentation of beer. The estimated gluten concentration in the beer samples, as determined by the Western blot using the Skerritt antibody, were similar to those indicated by the MIoBS, R5, and G12 antibodies and therefore seem to be more reliable than the values obtained using the Skerritt-sandwich ELISA, where overestimation of gluten concentration was observed. The Western blot did not show differences in signal intensities between the 20 mg/L gluten containing beers brewed in the presence and absence of PEP, which could be explained by the concentrations of gluten in the beers getting below or close to the limit of detection of the Western blot (results not shown). MS Analysis. MS was conducted in order to get information on the peptide profile of the PEP and non-PEP containing beer

chain would result in a loss of detectable gluten by the sandwich LFDs (the rapid decrease observed during the first 24 h using the sandwich LFDs). Only when the epitopes are completely hydrolyzed, a decrease in detectable gluten would be observed by the competitive LFD. Western Blot Analysis. Figure 3 shows the Western blot of the sorghum syrup wort immediately after addition of yeast and the beer containing 200 mg/L gluten brewed in the presence and absence of 16 or 806 μL per 3.8 L of PEP after 2 weeks of fermentation. Using intact gluten standards at 80 and 2.5 mg/L (lane 1 and 3 respectively) in each Western blot, it was possible to estimate the gluten concentration in the beer samples. Gluten suspended in PBST, which was used as a standard for Western blot, has been shown to perform quantitatively identical to the protein standards used in the MIoBS and RIDASCREEN sandwich ELISAs.16 As previously observed16 the MIoBS (Figure 3D), R5 (Figure 3C), and G12 (Figure 3A) antibodies showed preferential binding to protein bands corresponding to the molecular weight of the gliadins and LMW glutenin fractions of wheat gluten (Mr = 27 000− 50 000), whereas the Skerritt antibody (Figure 3B) preferentially bound to protein bands corresponding to the molecular weight of HMW glutenins (Mr = 70 000−100 000). The signal intensity of non-PEP containing beer, collected at the completion of the fermentation process (lane 7), was reduced by approximately 3−5 fold compared to the initial sample at the beginning of the fermentation (lane 5). These findings were similar to the ELISA analysis of the 200 mg/L gluten containing beers brewed in the absence of PEP, where on an 10531


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Table 2. Number of Unique Peptides from the 12 Most Confidently Identified Gluten Proteins along with Their UniProt Accession Numbers, in the Samples Collected throughout the Production Process of the 2nd Replicate Brew of the 200 mg/L Gluten Containing Sorghum Beers Brewed in the Presence (w/PEP) and Absence (No PEP) of PEP No PEP top 12 identified proteins α-gliadin (Aegilops tauschii) γ-gliadin (T aestivum) γ-gliadin (T aestivum) γ-gliadin (T aestivum) LMW glutenin (T aestivum) γ-prolamin (A retrof ractum) γ-gliadin (T macha) α-gliadin (Aegilops tauschii) α-gliadin (T aestivum) ω-gliadin (T aestivum) α/β-gliadin (T aestivum) HMW glutenin (T aestivum)

accession no.

pre‑ boil

after yeast

w/PEP

1 h 4 h 24 h 3 d 7 d 2 wk

E2CSZ8_AEGTA

2

B6DQD5_9POAL B6UKP6_WHEAT B6DQE1_9POAL B2Y2S0_WHEAT H8Y0M7_9POAL B9VRI0_9POAL A5JSA0_AEGTA

pre‑ boil

after yeast

1 h 4 h 24 h

3d

7d

2 wk

3

7

9

8

11

2

6 5

14 5

16 5

18

4

5

12

2

3

6 2

2

2

2

10 4 3 3

2

2

2

2

4

2 2

A5JSA9_WHEAT Q402I5_WHEAT I0IT59_WHEAT

5

GLT0_WHEAT

4

samples collected throughout the brewing process. A preliminary analysis of the identified peptides, for known immunopathogenic epitopes of CD, was also conducted. PEP reduced the time it took to detect gluten peptides from 24 h of fermentation to within 1 h of addition of the yeast, for the second replicate brew (Table 2). For the first replicate brew, peptides started to appear only after 7 days of fermentation for the non-PEP containing beer and PEP reduced the time of detection of peptides to within 1 h of addition of yeast (Table S1). The difference in the appearance of hydrolyzed peptides between the two replicate brews of the non-PEP containing beer can likely be explained by the different batches of yeast (with different fermentation activity) used during the brewing process. Although some of the same peptides were detected in the beer collected after 2 weeks of fermentation for both the brews (Figure S1), due to nature of data acquisition and processing, multiple analyses of the same sample will produce different peptide identifications. However, only peptides that were identified in at least two of the three acquisition replicates were included to mitigate this limitation. Addition of PEP altered the hydrolyzed gluten peptide profile to predominantly include peptides ending in proline residues (Table S2). Out of the four isotopically labeled peptides included in this study, only the LQLQPFPQPQLPY peptide was detected in the samples fractionated using the Mr = 30 000 MWCO filter. The peptide was observed after 7 days of fermentation of non-PEP containing beer, indicating the generation of the peptide toward the end of the fermentation process. After 2 weeks of fermentation, for one of the replicate brews, 7.1 ± 1.6 fmol LQL peptide per μL of beer was detected and for the other replicate, 1.6 ± 0.4 fmol LQL peptide per μL of beer was detected. However, this peptide was not detected in the beer brewed in the presence of PEP (Figure 4). The LQLQPFPQPQLPY peptide is a fragment of a putative toxic 33-mer peptide present in α2-gliadin. The detection of the peptide fragment of the 33-mer in beers brewed in this study is consistent with a previous study, where gliadin 33-mer equivalent peptides were detected in several commercial wheat and barley beers.26 The absence of this peptide from

3 8 3

2 4 4

3 2

2 4

5

9

11

11

Figure 4. Mass spectrometric determination of fmol of LQLQPFPQPQLPY peptide per μL of beer present in the samples collected throughout the production process of one of the replicate brews of the 200 mg/L gluten containing sorghum beers brewed in the presence (blue dashed line) and absence (bule solid line) of PEP.

the PEP-containing samples is probably due to hydrolysis of the peptide at the sites of the four proline residues. Cleavage of this peptide and the 33-mer peptide, by PEP, has been shown in recently published studies45,46 The peptide profile of the HWP was compared with that of the beers (sample collected after 2 weeks of fermentation as shown in Table 2 and Table S1) brewed with or without the addition of PEP (Figure 5). Out of 274 unique gluten peptides detected in HWP, only 4 or 5 peptides were represented in the peptide profile of the 200 mg/L gluten containing beer brewed without PEP and 1 or 2 were represented in that of the PEP containing beer (Figure 5A,B). Peptides containing the epitope recognized by the R5 (QQPFP) and the G12 (QPQLPY) antibodies (antibodies used in gluten ELISAs) were also compared between the beers and the HWP. The QQPFP epitope was only identified in the HWP (19 times in 13 peptides) and was not found in the peptides identified in the beers brewed in the presence and absence of PEP. The 10532


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celiac sequences, HMW glutenin proteins have also been shown to stimulate celiac small intestinal T cells and can induce toxic response in celiac patients.50−52 The partial resistance of HMW glutenins to the proteolytic action of PEP during fermentation poses questions regarding PEP’s ability to digest all potential immunopathogenic elements in gluten during the brewing of beer. Detailed analysis of the identified gluten peptides in the PEP and non-PEP containing beers, for the presence of other potential immunopathogenic T-cell epitopes besides the 33-mer, is currently underway in our laboratory and will provide additional information on the gluten degradation effects of PEP during the fermentation of beer.

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Figure 5. Venn diagram of the total number of unique gluten peptides identified in the 1st (A) and 2nd (B) brew replicates of the PEP and non-PEP containing beers (collected after 2 weeks of fermentation), comparing it to that of the HWP. Each brew was analyzed in triplicate and only peptides that were identified in at least two of the three injections were included.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b04205. Table S1: Number of unique peptides from the 12 most confidently identified gluten proteins, along with their UniProt accession numbers, in the samples collected throughout the production process of the 1st replicate brew of the 200 mg/L gluten containing sorghum beers brewed in the presence (w/PEP) and absence (No PEP) of PEP. Table S2: Representative gluten peptides identified in the sample collected after 2 weeks of fermentation of the 1st replicate brew of the 200 mg/L gluten containing sorghum beers brewed in the presence (w/PEP) and absence (no PEP) of PEP. Figure S1: Comparison of number of peptides detected between the 1st and 2nd brew replicates, for the beer (sample collect after 2 weeks of fermentation), brewed in the presence and absence of PEP. (PDF)

QPQPLY epitope was found one time in one of the peptides identified in one of the replicate beer brewed in the absence of PEP, and two times in two identified peptides in the HWP. The reliability of a calibrant to accurately quantitate gluten depends on the similarity between the gluten protein/peptides present in the calibrant and the test samples.47 The inconsistency in the peptide profile as well as the number of R5 and G12 epitopes in the identified peptides, between the beers and the HWP indicates that the HWP is unsuitable for use as a calibrant to quantitate hydrolyzed gluten in the beers brewed in this study. Beers brewed from different grain sources and different methods of brewing would most likely yield different sets of peptides. Further, when digested with PEP, the rate of digestion as well as generation of peptides, would be different for gluten derived from wheat (used in this study) and barley (most commonly used in brewing), not only due to differences between hordein and wheat gluten, but also due to differences in accessibility to hordein as it is released from the barley. Therefore, the likelihood of similarity between those peptides and the HWP is questionable. In summary, fermentation of beer resulted in a gradual reduction in detectable gluten concentration, and addition of PEP increased the effect. Only the MIoBS ELISA was able to reliably detect gluten in the beers brewed in the presence of PEP. However, accurate quantitation of hydrolyzed gluten, containing single epitopes, is not possible using the sandwich ELISA. The R5-competitive ELISA was not able to accurately quantitate gluten in the beers brewed in the presence of PEP because of the LOQ (10 mg/kg) of the ELISA and the high variance in the data at low concentrations of gluten. In addition, inconsistency in the peptide profile and the number of R5 epitopes between the peptides identified in the HWP and the peptides generated by fermentation, as demonstrated by the MS analysis, makes the R5-competitive ELISA utilizing HWP as the calibrant, unreliable for accurate quantitation of gluten in beer. Using MS, a fragment of a putative immunopathogenic 33-mer peptide,48,49 one of several potential immunopathogenic T-cell epitopes,50 was not detected in the PEP-containing beer, indicating that PEP may affect immunopathogenicity. Western blot results indicated that HMW glutenins are less susceptible to the action of PEP during the fermentation of beer. Although gliadin is believed to be the main protein fraction in wheat gluten responsible for exacerbating celiac disease due to the presence of a number of immunopathogenic

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AUTHOR INFORMATION

Corresponding Author

*Phone: 240-402-1970. E-mail: [email protected]. Funding

This project was supported in part by an appointment to the Research Participation Program at the Center for Food Safety and Applied Nutrition administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S Department of Energy and the U.S. Food and Drug Administration. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to express gratitude to Sylvie Van Zandycke, Ph.D. (DSM Food Specialties) for generously providing the proline endopeptidase and Hans Zoerb, Ph.D. (University of WisconsinMadison) for technical advice on brewing. The authors also acknowledge Cargill for their kind donation of sorghum syrup for this project.

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REFERENCES

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