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Journal of Cereal Science 56 (2012) 504e509
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Journal of Cereal Science
journal homepage: www.elsevier .com/locate/ jcs
Malt hydrolysates for gluten-free applications: Autolytic and prolineendopeptidase assisted removal of prolamins from wheat, barley and rye
Sanna Luoto a, Zhongqing Jiang a, Outi Brinck a, Tuula Sontag-Strohm a, Päivi Kanerva a,Maaike Bruins b, Luppo Edens b, Hannu Salovaara a, Jussi Loponen a,*
aDepartment of Food and Environmental Sciences, P.O. Box 66, Agnes Sjöbergin katu 2, 00014 University of Helsinki, FinlandbDSM Food Specialties B.V., Alexander Fleminglaan 1, 2613 AX Delft, The Netherlands
a r t i c l e i n f o
Article history:Received 2 March 2012Received in revised form2 June 2012Accepted 4 June 2012
Keywords:Gluten-freeProlaminProline endopeptidase (PEP)Proteolysis
Abbreviations: AN-PEP, Proline-specific endopeptSDS-PAGE, Sodium dodecyl sulphate polyacrylamideexclusion chromatography; FAN, Free amino nitroimmunoassay.* Corresponding author. Present address: Fazer Gr
00941 Helsinki, Finland. Tel.: þ358 40 732 9772.E-mail addresses: [email protected] (S. Luoto
(Z. Jiang), [email protected] (O. Brinck), tu(T. Sontag-Strohm), [email protected] (M. Br(L. Edens), [email protected] (H. Salovaa(J. Loponen).
0733-5210/$ e see front matter � 2012 Elsevier Ltd.http://dx.doi.org/10.1016/j.jcs.2012.06.004
a b s t r a c t
Cereal based products intended for gluten sensitive individuals, particularly to celiac disease patients,tend to have poor organoleptic qualities and they contain low levels of healthy whole grain compounds.Adding whole grain ingredients, such as malt hydrolysates, could compensate these defects providedthat the ingredients are adequately free from toxic prolamin epitopes. Here we demonstrate that thelevel of toxic prolamin epitopes in the malt autolysates (wheat, barley, rye) were substantially lower thanin the native malts but too high to allow “very low in gluten” labelling. To further eliminate the residuallevels of toxic prolamin epitopes, a proline-specific endoprotease from Aspergillus niger was added to themalt autolysates. In the resulting malt hydrolysates (of wheat and rye but not barley), the prolamins wereindeed greatly reduced and were below the very low gluten limit of 100 mg/kg. Malt hydrolysates withadequately low gluten levels may potentially be used as novel ingredients within gluten-free foods.
� 2012 Elsevier Ltd. All rights reserved.
1. Introduction
Malt autolysates present a rich source of small peptides andamino acids, as a source of baking enzymes and a source of healthywhole grain compounds such as minerals, vitamins, and fibres. Viatransformation to aroma compounds, such as 3-methylbutanal and2-phenylacetalaldehyde, free amino acids such as isoleucine,leucine and phenylalanine play a major role in malty flavourformation (Hansen and Schieberle, 2005) so that malt autolysatesused as a baking ingredient can be expected to add flavour. Addi-tionally, enzymes activated during germination like amylases,hemicellulases, lipases and proteases can to some extent also act asimprovers in gluten-free baking while minerals, vitamins and fibrecomponents present in the malt products will add to the level of
idase from Aspergillus niger;gel electrophoresis; SEC, Sizegen; ELISA, Enzyme-linked
oup, P.O. Box 4, Fazerintie 6,
), [email protected]@helsinki.fiuins), [email protected]), [email protected]
All rights reserved.
desirable healthy ingredients. These quality aspects are of partic-ular interest to celiac disease patients who are dependent ongluten-free products that often have poor taste and overall quality.An industrial example of such an approach is the use of barley maltextract flavourings in breakfast cereals like corn flakes and crispedrice.
Reducing the levels of toxic gluten epitopes within cerealproducts is gaining interest as prospective means to diversify thenutrition and sensory profile of gluten-free diets. In this respect it isworthwhile to note that germination-induced enzymes of wheat,barley, and rye can hydrolyse gluten storage proteins into frag-ments that are non-toxic (Hartmann et al., 2006). Previously, weshowed that sourdough fermentation conditions are very favour-able for the germination-induced enzymes to hydrolyse theprolamins. More specifically, we noted that 95% of the wheat and99.5% of the rye prolamins degraded under sourdough conditionswhen enzyme active malts were used as raw materials (Loponenet al., 2007, 2009).
In Triticeae cereals (e.g. wheat, barley and rye) the proline-richprolamin fraction represents a problematic compound. Thehuman digestive proteases (e.g. pepsin, trypsin and chymotrypsin)are unable to adequately cleave these proline-rich structures andconsequently specific prolamin fragments remain intact andimmunoreactive in the gastro-intestinal lumen and thereby exertinflammatory effects in celiac disease patients. During the past
Fig. 1. Process chart of the malt autolysate and malt hydrolysate production.
S. Luoto et al. / Journal of Cereal Science 56 (2012) 504e509 505
decade, several studies have demonstrated the capacity of a specificgroup of microbial proline-specific proteases (EC 3.4.21.26) toeliminate such prolamin-derived toxic epitopes (Hausch et al.,2002; Piper et al., 2004; Shan et al., 2002). Among these proline-specific endopeptidases, the enzyme from Aspergillus niger (AN-PEP; Edens et al., 2005; Lopez and Edens, 2005; Sebela et al., 2009;Stepniak et al., 2006) is of particular interest as it represents theonly industrial food-grade proline-specific enzyme with an acidicpH optimum. No attempts to utilize such proline specific proteasesin gluten-free food applications, for instance in the elimination ofresidual gluten, have this far been made.
In the present study, we investigated whether the malts fromwheat, barley, and rye differ in their auto-proteolytic potentialregarding prolamin hydrolysis and whether addition of a supple-mentary AN-PEP enzyme can result in malt hydrolysates withprolamin contents sufficiently low such that these might be used ingluten-free foods.
2. Materials and methods
2.1. Malts and AN-PEP enzyme
Malted wheat, barley and rye grains were provided by LaihianMallas Oy (Laihia, Finland). The germinated grains were manufac-tured on an industrial scale using conventional malting practiceswith gentle drying to guarantee high enzyme activity. Grains werefinely ground with a Retsch ZM-200 ultracentrifugal mill (Retsch,Haan, Germany) equipped with a 0.5 mm sieve yielding flours thatwere used in the experiments. The A. niger-derived proline-specificendopeptidase (AN-PEP) was provided by DSM Food Specialties(Delft, The Netherlands). The activity of the enzyme preparationwas 14 PPU/mL, where one PPU is defined as the amount of enzymerequired to release 1 mmol of pNA from Z-Gly-Pro-pNA in 1 minunder the defined assay conditions (pH 4.6, T ¼ 37 �C and ata substrate concentration of 0.37 mM Z-Gly-Pro-pNA).
2.2. Preparation of malt autolysates
Autolysates of wheat, barley and rye malt were prepared in50 mL test tubes bymixing 5.0 g of malt flour and 30 mL of 0.2 mol/L sodium acetate buffer, pH 4.0. Such acidic conditions proved to beessential for malt enzyme activity. Malt suspensions (final pH 4.1)were incubated at 40 �C for 24 h and then freeze-dried and kept at5 �C for further studies. The overall procedure for the preparation ofmalt samples is shown in Fig. 1.
2.3. Preparation of AN-PEP malt hydrolysates
Hydrolysates of wheat, barley and rye malt were prepared bysuspending 0.4 g of each autolysate in 4 mL 0.1 mol/L sodiumacetate buffer, pH 4.0. Per 1.0 g of malt autolysate, 0.5 PPU of theAN-PEP preparation was added and the suspension incubated foreither 2 or 22 h with continuous agitation at 40 �C. Two separateincubations were performed. Hydrolysis was terminated byneutralizing the suspensions with sodium hydroxide and freezingthe samples immediately. The resulting hydrolysates were freeze-dried and stored at 5 �C. With rye malt autolysates also AN-PEPdosages of 0.2 PPU and 0.05 PPU were tested in order to find outthe influence of dosage on the extent of prolamin hydrolysis.
2.4. SDS-PAGE and AN-PEP activity on zymogram gels
To find out the resistance of the AN-PEP to the action of maltenzymes, malt enzyme extracts were incubated with AN-PEP. Thepossible degradation of AN-PEP was followed by SDS-PAGE analysis
on NuPAGE Novex� Bis-Tris 12% gels (Invitrogen, Carlsbad, CA,USA), and the residual AN-PEP activity was visualized on zymogramgels. Malt enzyme extracts of wheat, barley, and rye were preparedusing a modified extraction procedure (Jones et al., 2000). Threegrams of each malt flour was suspended in 15 mL of 0.1 mol/Lsodium acetate buffer, pH 4.5. The suspension was incubated ina shaker at 5 �C for 45 min and then centrifuged (5 �C, 10,000�g,20 min) to recover the malt enzymes containing supernatant. Twoseparate extractions were performed.
Per 1 mL of malt enzyme extract, 0.1 PPU of AN-PEP was addedand incubated for either 2 or 22 h with continuous shaking at pH4.0 and 50 �C. Residual AN-PEP activity was qualitatively analysedusing Novex Zymogram 12% Casein gels (Invitrogen, Carlsbad, CA,USA). Casein denaturation during incubation and sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) was pre-vented by keeping the temperature below 40 �C. After SDS-PAGE,the gels were developed in renaturing buffer according to themanufacturer’s instructions. A parallel SDS-PAGE gel was producedand stained conventionally.
2.5. Size exclusion chromatography (SEC) analysis
In order to evaluate the breakdown of proteins and the forma-tion of protein hydrolysis products in malt autolysates and hydro-lysates, SDS extracts of samples were analysed in a tandem-columnSEC system. Freeze-dried samples (40 mg) were extracted 1:10 (w/v) with 1.5% SDS and 0.05 mol/L sodium phosphate, pH 6.9, at roomtemperature (21 �C) for 1 h and centrifuged (21 �C, 10,000�g,10 min). One volume (2.5 mL) of the supernatant was mixed with 1volume of the elution buffer (0.1% SDS, 20% acetonitrile and0.05 mol/L sodium phosphate, pH 6.9), centrifuged again and thesupernatants were subjected to SEC analysis.
To separate the proteins and peptides over selected size ranges,SEC columns Superdex Peptide 10/300 GL (separation range100e7000) and Superdex 200 10/300 GL (separation range10,000e600,000) (GE Healthcare Biosciences AB, Uppsala, Sweden)were coupled in series in a model 1200 HPLC system equipped withmultiple wavelength detector (Agilent Technologies, Waldbronn,Germany). The setup was calibrated by analysing the elution frac-tions of the wheat, barley and rye prolamin isolates (Mw rangew20,000e100,000) with SDS-PAGE, and by using low molecularmass prolamin peptide standards (9-mer and 33-mer, forsequences see Kanerva et al., 2011). The injection volume was100 mL and the flow rate 0.5 mL/min. The analysis was performed atroom temperature for 150 min and proteins were detected at
S. Luoto et al. / Journal of Cereal Science 56 (2012) 504e509506
210 nm. The quantitative results on different SEC fractions werecalculated as area under the curve at selected elution volumes.
2.6. Analysis of free amino nitrogen
Free amino nitrogen (FAN) content of malts, malt autolysates,and AN-PEP malt hydrolysates were determined by extracting40 mg of each individual sample with 0.4 mL of 0.1 mol/L sodiumphosphate, pH 8.0 at room temperature for 1 h. After centrifugation(21 �C, 10,000�g, 10 min), 20 mL of each supernatant was diluted ina ratio of 1:100 (v/v) with the extraction buffer and amino nitrogenconcentrations were analysed with a ninhydrin method (ASBC,1992). The ninhydrin solution contained 10.0 g of Na2HPO4$12H2O, 6.0 g of KH2PO4, 0.3 g of fructose, and 0.5 g of ninhydrin(Merck KGaA, Darmstadt, Germany) in 100 mL of deionized water.One hundred mL of the ninhydrin solutionwasmixed with 200 mL ofeach diluted supernatant and incubated at 100 �C for 16 min. After20 min cooling at room temperature, 0.5 mL of KIO3 solution [2.0 g/L of 40% (v/v) ethanol] was added and the absorbance wasmeasured at 570 nm. Glycine was used as a standard. The data wasobtained from triplicate analyses of two independent incubations.
Fig. 2. SEC-HPLC analysis of native malts, malt autolysates and AN-PEP malt hydro-lysates of wheat (A), barley (B) and rye (C). The results are representative for twoindependent fermentations. An arrow is indicating the peak of intact AN-PEP.
2.7. Prolamin determination and quantification
Prolamin contents of the different malts, malt autolysates, andAN-PEP malt hydrolysates were determined using a commercialcompetitive enzyme-linked immunoassay (ELISA). In the compet-itive assay (Ridascreen Gliadin Competitive, R-Biopharm, Darm-stadt, Germany), samples (125 mg) were extracted with 60% (v/v)ethanol containing 10% fish gelatin (SigmaeAldrich, MO, USA) ina ratio of 1:10 (w/v) at 21 �C for 10 min. After centrifugation (21 �C,10,000�g, 10 min) the prolamin concentration of supernatant wasdetermined and the obtained peptide concentration divided by 250to convert to prolamin concentration as described in the manu-facturer’s instructions. Two different dilutions of malt material(from two independent incubations of malt autolysates and AN-PEPmalt hydrolysates) were each analysed in duplicate.
Prolamin levels present in rye AN-PEP malt hydrolysates werealso investigated using a commercial sandwich ELISA kit(Ridascreen Gliadin, R-Biopharm, Darmstadt, Germany). Of each ryemalt AN-PEP hydrolysate, 125mgwas extractedwith 1.25mL of thekit’s cocktail solution at 50 �C for 40 min. After the mixture wascooled down to room temperature, 3.75 mL of 80% (v/v) ethanolwas added, and the suspension was extracted for 60 min at roomtemperature. The prolamin concentration of each supernatant wasdetermined by following the manufacturer’s instructions with theexception that the result was not multiplied by two, because themajority of the malt prolamins are soluble under the extractionconditions. Two different dilutions of the malt material were eachanalysed in duplicate.
3. Results
3.1. Properties of the malt autolysates
The size distributions of SDS-soluble malt proteins and peptidespresent in wheat, barley and rye malt autolysates were determinedby SEC. From the data obtained (Fig. 2), it is clear that in all autol-ysates an increasing proportion of the prolamins degraded to yieldfree amino acids and peptides of 5e10 amino acid residues long.According to their free amino nitrogen levels, autolysis increasedthe level of free amino groups of the native wheat, barley and ryeproduct by a factor 2e3 (Fig. 3).
3.2. Properties of AN-PEP malt hydrolysates
AN-PEPwith its activity optimum at pH 4.2 represents a proline-specific protease with well-predicted potential to complementprolamin hydrolysis by the endogenous malt proteases. Thestability of AN-PEP towards malt enzymes was first investigated byadding the AN-PEP to the enzyme extracts of the various malts andincubating the mixture for 2 and 22 h at 40 �C. SDS-PAGE analysisverified that AN-PEP remained intact during the whole incubationperiod with wheat malt enzymes (Fig. 4A) and also in the presenceof barley and rye malt enzymes (data not shown). Instead it wasevident that all malt extract derived proteins disappeared duringthe incubation, which indicates that they were hydrolysed by AN-PEP. Accordingly, qualitative enzyme activity analysis withzymography on casein gels confirmed that AN-PEP retained itsactivity in the presence of wheat malt enzymes (Fig. 4) and simi-larly with barley and rye malt enzymes (data not shown).
According to SEC data (area under the curve), the quantities ofshort peptides (Mw < 1.1 kDa) were 1.6-fold, 1.8-fold and 2.1-fold inthe wheat, barley and rye autolysates compared to native malt,respectively. Addition of 0.5 PPU AN-PEP per gram of either wheat,
Fig. 3. Free amino nitrogen levels in soluble fractions of native malts, malt autolysates, and AN-PEP malt hydrolysates incubated for 2 and 22 h as determined by a ninhydrinmethod.
S. Luoto et al. / Journal of Cereal Science 56 (2012) 504e509 507
barley or ryemalt, the quantities of small peptides inwheat, and ryehydrolysates were 3-fold and in the barley hydrolysate 2-foldcompared with the levels present in the native malts. This obser-vation is corroborated by the significantly increased free aminonitrogen levels (Fig. 3).
Thus, a supplementation with AN-PEP effectively com-plemented the autolytic proteolysis of the cereal malts, leading to
Fig. 4. Wheat malt enzyme extract was incubated with AN-PEP for 0, 2, 4 and 22 h, andthe reaction mixtures analysed on SDS-PAGE (panel A) and casein zymogram gel (panelB). The sample codes shown above the gels: MW ¼ molecular weight standard(molecular mass labels on the left of the gels); 1 ¼ AN-PEP (only the enzyme); 2 ¼ AN-PEP incubated with wheat malt enzymes for 0 h; 3 ¼ AN-PEP incubated with wheatmalt enzymes for 2 h; 4¼ AN-PEP incubated with wheat malt enzymes for 4 h, 5 ¼ AN-PEP incubated with wheat malt enzymes for 22 h. AN-PEP (Mw z 66 000) activity isvisible as a clear band against a Coomassie Brilliant Blue stained background where thecasein was degraded (panel B).
a clear and substantial increase in the degradation of the cerealproteins (Fig. 2).
3.3. Prolamin quantification by immunoassays
The standard method for gluten determination endorsed by theCodex Alimentarius is the R5 antibody ELISA; this requirement isfulfilled by the sandwich and competitive ELISA (CodexAlimentarius Commission, 2008). Although the competitive ELISAis not yet tested as extensively as the sandwich R5-ELISA, themethod is more accurate in detecting hydrolysed gluten(Gessendorfer et al., 2009). Residual prolamin concentrations in thevarious autolysates and hydrolysates were quantified by either thesandwich or the competitive R5 ELISA.
In the first instance, the effect of increasing AN-PEP dosages onresidual rye prolamin levels was explored. Although all AN-PEPconcentrations significantly reduced the rye secalin content(p < 0.05), only the highest concentration added (35 mL; 0.5 PPU/g)resulted in a hydrolysate with a prolamin content below 20 mg/kgupon analysis by the competitive ELISA method, and 70 mg/kgupon analysis by the sandwich method (Table 1).
Subsequently, the levels of residual prolamin concentrations ofthe various cereal malts were quantified by ELISA before and afterautolysis, and after hydrolysis with AN-PEP at the highestconcentration. Using the competitive R5 approach, native rye maltwas found to contain less (35 000 mg/kg) prolamins than eitherwheat (40 000 mg/kg) or barley malt (41 000 mg/kg) (Table 2).After autolysis for 22 h (in the absence of AN-PEP), the prolamincontent of rye malt autolysate (400 mg/kg) was significantly lowerthan the prolamin content of either wheat (10 800mg/kg) or barley
Table 1Prolamin concentrations of rye malt AN-PEP hydrolysates determined by thecompetitive and the sandwich R5 ELISA immunoassays. AN-PEP incubations werecarried out for 22 h at pH 4 and 50 �C.
Rye prolamin concentration(mg/kg ¼ ppm, dm)
AN-PEP concentration(PPU per g malt autolysate)
0.5 0.2 0.05
Competitive 018 � 7 50 � 20 80 � 20Sandwich 070 � 10 130 � 28 210 � 23
Table 2Prolamin concentrations of native malt flour, malt autolysates and AN-PEP malthydrolysates as determined by competitive and sandwich(*) R5 ELISA immunoassay.Hydrolysis conditions were pH 4 and 50 �C. 0.05 PPU of AN-PEP was added per gmalt autolysate.
Prolamin concentration (mg/kg ¼ ppm, dm)
Malt flour(native)
Malt autolysate(24 h)
AN-PEP malthydrolysates
Hydrolysis time (h)
2 22 22*
Wheat 40 000 � 10 000 10 800 � 2250 800 � 300 20 � 7 044 � 10Barley 41 000 � 9 5000 21 000 � 4900 7000 � 2000 340 � 92 590 � 60Rye 35 000 � 11 000 0400 � 200 130 � 420 18 � 7 070 � 10
S. Luoto et al. / Journal of Cereal Science 56 (2012) 504e509508
(21 000 mg/kg) malt autolysate. But the efficacy of digestion wasshown to be greatly enhanced by the subsequent incubation withAN-PEP (at 0.5 PPU/g), lowering prolamins by 99.95%, 99.17% and99.95% in the wheat, barley and rye native malt material, respec-tively (p < 0.05; Table 2). AN-PEP reduced residual prolamin levelsto 20 mg/kg or less in wheat and rye but not in barley (340 mg/kg).In relative terms, the barley digestion with AN-PEP was lesssuccessful than for wheat and rye. As shown in Table 2, the residualprolamin levels of wheat, rye, and barley hydrolysates measured bysandwich ELISA were 44, 70, and 590 mg/kg, respectively, signifi-cantly (p < 0.05) higher than by using the competitive assay.Surprisingly the barley hydrolysate showed a significantly higherresidual prolamin level.
4. Discussion
In this study we characterised the efficacy of proteolysis onprolamin degradation in malted wheat, barley and rye by endoge-nous enzymes (in so-called “malt autolysates”) as well as furtherhydrolysis by exogenous AN-PEP enzyme (in so-called “malthydrolysates”). AN-PEP represents a commercially availableproline-specific endopeptidase, derived from A. niger, which hasthe potential to assist in the digestion of proline-rich cerealproteins under acidic digestive conditions.
Using size-exclusion chromatography and free amino nitrogenanalysis, the extent of protein hydrolysis in the various hydrolysisstages was determined. Our findings clearly show that endogenousenzymes were effective in degrading high molecular weightproteins to small peptides. Extended incubation with AN-PEP,however, led to more extensive peptide digestion and, mostimportantly, greatly reduced the levels of prolamins that could bedetected by R5 ELISA. More specifically; while the wheat, barley,and rye autolysates (24 h digestion) were measured to contain10,800, 21,000, and 400 mg/kg of prolamin using the R5 competi-tive assay, AN-PEP (at 0.5 PPU/g and 22 h digestion) furtherhydrolysed prolamin levels to 20, 340, and 18 mg/kg. An explana-tion for the higher resistance of barley to hydrolysis probably is thatbarley hordeins have a complex secondary structure and thuspresent challenging and compact substrate for endoproteases(Simpson, 2001). On the other hand, the rye secalins, which werehydrolysed to the greatest extent, are less-polymeric and moresoluble than wheat and barley prolamins.
The Codex Alimentarius provides the gluten-free standard forEuropean food manufacturers. According to the Codex Alimentar-ius, food products containing 20 mg/kg or less and between 21 and100mg/kg of gluten can be labelled as “gluten-free”, or “very low ingluten”, respectively. In the US, many manufacturers are voluntaryfollowing the same labelling standards as proposed by the Food andDrug Administration (FDA), probably going into effect in 2012. The
residual prolamin levels in the wheat and rye hydrolysates weremarginally higher and for barley substantially higher whenmeasured by sandwich ELISA as compared to competitive ELISA.This is a contradictory result compared to previous findings ofGessendorfer et al. (2009) who reported that the competitive assaywas more sensitive in the detection of pepsin and trypsin/chymo-trypsin digested gluten. Noteworthy, the hydrolytic enzymes weredrastically different between the two studies, which results inprotein hydrolysis products with different structures. This may beone explanation for the contradictory result. Another explanationcan be the more efficient extraction method used in the sandwichELISA, which may have been more effective in dissolving prolaminsfrom freeze-dried sourdough matrix. In general, the competitiveELISA is deemed to be the most suitable method to accuratelyquantify hydrolysed gluten and the sandwich R5 ELISA particularlyoverestimates prolamins from barley (Kanerva et al., 2006;Thompson andMéndez, 2008). In other words, the digests of wheatand rye but not barley, made by a combined action of endogenousenzymes and AN-PEP enzyme, are believed to meet the “gluten-free” labelling requirement and, thus, adding such malt hydroly-sates to gluten-free recipes would keep the prolamin levels of thefinal products below the Codex gluten-free thresholds. Typicaladdition levels to gluten-free product recipes could range between5 and 30% on a flour basis. Moreover, the use as a liquid extract isvery possible. It can be presumed that these kinds of cerealhydrolysates could be used with other ingredients in gluten-freebaking, for instance. This procedure could be applicable in furtherreducing gluten to trace levels that would meet the “gluten-free”standards and would be safe to consume in a gluten-free diet.Therefore, the preparation of cereal digests offers new insights forthe development of novel ingredients; such material would bringa rich source of amino acids and peptides (known to be importantfor the development of flavour in baked products) while retainingthe natural benefits of the micronutrients and fibre components ofthe whole grain. Given that the hydrolytic approach described hereappeared highly effective in reducing the level of the glutenepitopes linked to toxicity, it is suggested that the technologicalevaluation of such hydrolysates within foods, and gluten-free foodsin particular, merits further attention.
Acknowledgements
SL, ZJ, OB, TS-S, HS, and JL acknowledge Academy of Finland,University of Helsinki Funds and the Finnish Coeliac Society forfinancial support.
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