Food Chemistry 128 (2011) 114–122

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Food Chemistry

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Dry heat stress stability evaluation of casein peptide mixture

Matthias D’Hondt a, Wim Demaré a, Sylvia Van Dorpe a, Evelien Wynendaele a, Christian Burvenich b,Kathelijne Peremans b, Bart De Spiegeleer a,⇑a Drug Quality and Registration (DruQuaR) Group, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgiumb Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium

a r t i c l e i n f o

Article history:Received 12 August 2010Received in revised form 15 December 2010Accepted 1 March 2011Available online 5 March 2011

Keywords:Peptide stabilityCasein hydrolysateDry stateStress testingTemperatureOxidative reductive light degradation

0308-8146/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.foodchem.2011.03.004

⇑ Corresponding author. Tel.: +32 9 264 81 00; fax:E-mail address: Bart.DeSpiegeleer@UGent.be (B. D

a b s t r a c t

During food processing, peptide-containing products often experience thermal stress, which can be vol-untary heat treatments to prolong expiration date, or unwanted side-effects, e.g. local heating duringpowder compaction or milling. No information is currently available on the primary structure stabilityof peptides when heated in the dry state. Therefore, the short-term dry heat stress stability of caseinhydrolysate was evaluated by exposure to temperatures of 100, 140 and 180 �C during 1, 3 and 5 mintime intervals. Moreover, the impact of oxidising and reducing agents, as well as photolytic stability wereassessed. Contrary to the general belief that peptides are heat-labile, based on degradation results in solu-tion, all peptides remained stable up to 3 min at 180 �C. The influence of a reducing environment wasfound to be minimal, while the impact of an oxidising environment was significant. Our findings openperspectives for thermal peptide processing techniques.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Bovine milk contains a variety of nutrients, such as proteins, lip-ids, sugars and minerals. The predominant protein in milk is casein,which consists of four different protein chains, i.e. aS1-casein, aS2-casein, b-casein and j-casein (FitzGerald, 1998; Su, He, & Qi, 2007).The biological activities of casein-derived peptides has recentlyattracted much interest: evidence of antihypertensive (Korhonen,2009; Lopez-Fandino, Otte, & van Camp, 2006), antioxidative(Korhonen & Pihlanto, 2006; Pihlanto, 2006), opioid (Meisel &FitzGerald, 2000; Silva & Malcata, 2005), immunomodulatory(Meisel, 2005; Silva & Malcata, 2005), antimicrobial (Exposito &Recio, 2006; Tidona et al., 2009), anticariogenic (Andrews et al.,2006) and mineral binding (Meisel & FitzGerald, 2003) propertiesof casein-derived peptides has become available. Currently, caseinhydrolysates are mainly used as functional food supplements.

During food processing, peptide- or protein-containing foodproducts often experience thermal stress conditions during shorttime intervals, which can be voluntary or unwanted. Thermal pro-cessing of protein-containing food products is performed for a widevariety of reasons, e.g. to prolong the expiration date or to improveprotein digestibility. Rehman and Shah (2005) observed a maxi-mum improvement in digestibility when cooking different vegeta-bles in an autoclave at 121 �C for 10 min. Amnuaycheewa andGonzalez de Mejia (2010) described the heating of soy to 80 �C for

ll rights reserved.

+32 9 264 81 93.e Spiegeleer).

5 min, at pH 2, in order to reduce the amount of profilin, a soyallergen. O’Regan and Mulvihill (2009) reported the glycosylationof sodium caseinate by dry heat treatment of mixtures of sodiumcaseinate and maltodextrine at 60 �C, 79% relative humidity for4 days, hereby improving the emulsifying properties of aforemen-tioned protein. Kiokias, Dimakou, and Oreopoulou (2007) investi-gated the influence of thermally-induced denaturation on theoxidative stability of whey protein emulsions; denaturationexperiments were conducted by heating a whey solution at varioustemperatures between 40 and 85 �C for 15 min. Hidalgo, Brandolini,and Gazza (2008) determined the influence of steam sterilization ofwholemeal flour on its chemical and technological characteristics.While these studies evaluated the solution stability by mimickingvoluntary heat processes, unwanted short-term thermal exposureof the dry protein-mixture can also occur. Typical processes aregrinding, milling, mixing, compression and compaction of powders.When dry powders are mixed or compressed into tablets, local tem-perature increases can occur due to friction between the individualpowders and friction between the powders and the walls of themixing and compression apparatus (Picker-Freyer & Schmidt,2004). Tablet coating, performed in both food and pharmaceuticalindustry, improves visual appearance or masks bad taste of the tab-let. However, this technique requires extensive curing or dryingsteps, e.g. aqueous film coating requires drying at temperatures of100 �C, to remove water from the coating (Porter, 2005).

The majority of thermal processed food products are in solution.Various studies have focused on the heat stability of food proteinsor peptides in solution, mainly focusing on protein denaturation

M. D’Hondt et al. / Food Chemistry 128 (2011) 114–122 115

and peptide activity (Ryan et al., 2008; Sheih, Fang, & Wu, 2009).However, to the best of our knowledge, the heat stability of pro-teins or peptides in dry form has not yet been studied or reported.As conclusions regarding the heat stability of peptides in the dryform are derived from the results obtained with peptides in solu-tion, certain production or processing methods are, potentiallywrongfully, considered as inapplicable for dry peptide or proteinproducts. Therefore, the goal of this research was to perform anevaluation of the short-term dry heat stability of a casein hydroly-sate powder. The dry peptide mixture, i.e. casein hydrolysate pow-der, was exposed to several heat stress conditions, by varyingtemperature, incubation time and additional stress factors, i.e.presence of oxidising and reducing agents. The peptide degrada-tion, resulting from dry heat exposure, was analytically deter-mined by HPLC–UV/MS using a fused-core stationary phase.

2. Materials and methods

2.1. Materials

Acetonitrile (LC–MS grade) was purchased from Romil (Merel-beke, Belgium). Water was purified using an Arium 611 purificationsystem (Sartorius, Gottingen, Germany) yielding P18.2 MX cmquality water. LC–MS grade formic acid was obtained from Fluka(Bornem, Belgium). Potassium permanganate and zinc (granular,30 mesh) powder were bought from Flandria (Ghent, Belgium)and Janssen Chimica (Beerse, Belgium), respectively. The enzymatic(pancreatic) hydrolysed casein digest was a kind gift from DMVInternational (Veghel, the Netherlands). According to informationprovided by DMV International, the casein hydrolysate is preparedby dissolution of the casein proteins and subsequent addition of apancreatic enzyme mixture, containing trypsin, chymotrypsin,aminopeptidase and carboxypeptidase. The digestion process isterminated by heat inactivation of the enzymes, followed by a fil-tration step. Finally, the peptide mixture is concentrated, driedand packaged.

2.2. Liquid chromatography

The HPLC apparatus, implemented for casein pancreatic digestquantification, consisted of a Waters Alliance 2695 separationsmodule and a Waters 2487 dual wavelength absorbance UV detec-tor equipped with an Empower 2 software for data acquisition (allWaters, Milford, MA, USA). The LC–MS apparatus, used for identifi-cation of casein-related peptides, consisted of a Thermo SpectraSystem SN4000 interface, a Spectra System SCM 1000 degasser, aSpectra System P1000XR Pump, a Spectra System AS3000 auto-sampler set at 20 �C and 10 ll injection volume and a FinniganLCQ Classic ion trap mass spectrometer in a positive ion mode(all Thermo, San José, CA, USA) equipped with a Waters 2487 dualwavelength absorbance UV detector (Waters, Milford, MA, USA) setat 195 nm, and Xcalibur 2.0 software (Thermo, San José, CA, USA)for data acquisition. ESI was conducted using a needle voltage of4.5 kV. Nitrogen was used as the sheath and auxiliary gas withthe heated capillary set at 250 �C. Positive mode mass spectra wereobtained in the range of m/z 300 to 2000. A HALO Peptide ES-C18

(75 � 3 mm I.D., 2.7 lm particle size, 160 Å) column (Achrom,Machelen, Belgium), thermostated in an oven set at 30 �C, wasused. The flow rate was set at 0.6 ml/min and the linear gradientused was as follows (where A = 0.1% (w/V) MS grade formic acidin water and B = 0.1% (w/V) MS grade formic acid in MS grade ace-tonitrile (De Spiegeleer, Vergote, Pezeshki, Peremans, & Burvenich,2008): t = 0 min, A:B (95:5, V/V); t = 0–40 min, A:B (71:29, V/V);t = 40–43 min, A:B (10:90, V/V); t = 43–50 min, A:B (10:90, V/V/);t = 50–53 min, A:B (95:5, V/V/); t = 53–60 min, A:B (95:5, V/V/).

2.3. Solutions

The reference solutions were prepared by adding 10.0 ml ofpeptide solvent (i.e. aqueous solution containing 5% V/V acetoni-trile) stored at room temperature (20 ± 5 �C), to 10.0 mg of accu-rately weighed homogenised casein pancreatic digest, resultingin a concentration of 1 mg/ml. The reference solutions were soni-cated until all casein pancreatic digest was dissolved (±5 min).During this sonication process, no significant temperature increasein the reference solutions was seen.

The heat-treated solutions were prepared by adding peptidesolvent to homogenised casein pancreatic digest, subjected to dif-ferent stress conditions as presented in Sections 2.5–2.8. The heat-treated solutions were sonicated until all casein pancreatic digestwas dissolved.

2.4. Non-heat-treated casein pancreatic digest characterisation

First, the individual peptides, present in the casein pancreaticdigest, were identified. For this purpose, a reference solution wasanalysed using the HPLC–MS method as described in Section 2.2.The mass spectra obtained from the different HPLC-peaks werecorrelated with a database containing the amino acid sequencesof the four different casein protein, i.e. aS1-, aS2-, b-, and j-casein,using a software package (Bioworks Browser 3.3.1), thus resultingin tentative identified amino acid sequences. The amino acid se-quences were further confirmed by comparing experimentally ob-tained MS2 spectra with theoretical MS2 spectra obtained fromaforementioned tentative peptide amino acid sequences.

Next, the identified peptides were quantified. In order to assessinter-sample variability, six separate reference solutions were pre-pared and analysed using the HPLC–UV method as described inSection 2.2. The variability between the 6 peak areas, obtainedfrom the 6 separate reference solutions, of each identified peptidewas characterised by calculating the 95% confidence intervalsaround the average peak areas, set as 100%. The limit of quantifica-tion (LoQ) was defined as the concentration equivalent to a signalto noise value of 10.

2.5. Dry heat stress test

Homogenised casein pancreatic digest was accurately weighedinto a 50 ml glass conical flask (Schott Duran�, Mainz, Germany),which were incubated in castor oil, heated at 100, 140 and180 �C (±3 �C) using a Memmert universal oven (Memmert, Schwa-bach, Germany), for 1, 3 and 5 min. Each stress condition was per-formed in duplicate. After incubation, the heat-treated sampleswere immediately stored in ice, in order to limit further degrada-tion. The stress samples were dissolved as described in Section 2.3.

The results were compared to an unstressed reference solutionof equal concentration. The degradation of the identified peptideswas calculated relative to the unstressed reference solution. Therelative peptide amounts after dry heat stress were compared totheir relative 95% confidence intervals of the unstressed samples(see Section 3.1) as well as to the 90–110% interval, which is consid-ered as the specification acceptance limits (Larimore & O’Mathuna,2003; Vergote, Burvenich, Van de Wiele, & De Spiegeleer, 2009;Article 5, EC Directive 2002/46/EC, 2002). The overall total error be-tween both repeats, throughout the different stress conditions, wascalculated.

2.6. Oxidative dry heat stress

During oxidative dry heat stress, 10 parts of homogenised case-in pancreatic digest were mixed with 1 part of potassium perman-ganate using a mortar and pestle. This powder blend was subjected

Table 1Overview of identified peptides.

No. Retention time (min) Observed mass Theoretical mass Amino acidsequence

95% confidenceinterval (%)

LoQ ± SEM (%) Relativeamount (%)a

1 10.22 604.15 604.30 YPVEP 89.34–110.16 8.81 ± 2.78 14.852 12.94 652.15 652.40 VLPVPQ 93.70–106.30 3.60 ± 0.59 19.393b 15.52 659.10 659.34 VAPFPE 95.27–104.73 7.59 ± 1.46 15.58

727.15 727.40 NVPGEIV4b 15.75 684.14 684.39 LEIVPN 95.27–104.73 7.59 ± 1.46 15.58

576.10 576.35 HLPLP5 17.70 755.18 755.39 SDIPNPI 87.48–112.52 46.93 ± 7.26 1.666 19.21 686.34 686.40 RGPFPI 89.98–110.02 10.41 ± 0.91 8.897b 20.90 838.27 838.45 PFPGPIPN 93.38–106.62 14.66 ± 2.58 3.39

659.18 659.34 APFPEV8 24.83 1001.28 1001.51 YPFPGPIPN 90.91–109.09 29.89 ± 2.77 3.849 26.21 1025.33 1025.57 VVPPFLQPE 96.64–103.36 14.43 ± 1.15 8.74

10 27.05 1100.36 1100.58 VYPFPGPIPN 92.66–107.34 11.75 ± 0.94 11.0611 28.77 1373.33 1373.66 TDAPSFSDIPNPI 94.90–105.10 8.93 ± 1.37 8.0612 29.87 889.25 889.48 VYPFPGPI 95.27–104.73 27.36 ± 2.41 4.55

a Calculated in unstressed sample as: average peptide peak/P

(average peptide peaks).b Different [MH+] values, corresponding to two different amino acid sequences.

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to the same heat stress conditions and analysed as described inSection 2.5.

2.7. Reductive dry heat stress

During reductive dry heat stress, 10 parts of homogenised case-in pancreatic digest were mixed with 1 part of zinc powder using amortar and pestle. This powder blend was subjected to the sameheat stress conditions and analysed as described in Section 2.5.

2.8. Dry photolytic stress

Photolytic stress stability tests are performed in a Weiss Tech-nick 500 l light stability chamber set at 25 �C. Four quartz cellswere filled with approximately 80 mg casein pancreatic digest.Two cells, which were packed in aluminium foil, functioned as ref-

Fig. 1. Typical, baseline-corrected, HPLC–UV chromatogram (1

erence samples. One quartz cell and one reference cell were incu-bated under UV and visual light respectively, providing an overallillumination of 1,200,000 lux hours and exposure to an integratednear UV energy of 200 W h/m2. After light incubation, 10.0 mg ofstressed and reference sample were accurately weighed (in dupli-cate) and dissolved by adding 10.0 ml of peptide solvent (seeSection 2.3). The HPLC–UV analysis and data processing of the fourstress samples and four reference samples was performed asdescribed in Section 2.5.

3. Results and discussion

3.1. Non-heat-treated casein pancreatic digest characterisation

The individual peptides present in the casein pancreatic digestwere identified using LC–MS/MS. A detailed overview of the

95 nm) obtained from unstressed casein pancreatic digest.

Fig. 2. Dry heat stress: relative peptide amounts. Relative peptide amounts outside the 95% confidence intervals are indicated in red. (For interpretation of the references incolour in this figure legend, the reader is referred to the web version of this article.)

M. D’Hondt et al. / Food Chemistry 128 (2011) 114–122 117

individually identified peptides is given in Table 1. Only peptidesfree of post-translational modification (PTM), e.g. phosphorylationof serine residues, were selected. The observed monoisotopic molec-ular weight of the peptides, originating either from b- or aS1-casein,ranges between 576 (HLPLP) and 1373 (TDAPSFSDIPNPI) g/mol.Twelve separate peaks, to which the amino acid sequences couldbe attributed, were considered in this stability study. An overviewof previously identified functional peptides, structurally related toour selected peptides, is provided in Supplementary Table S-1.

The identified peptides were then quantified using the HPLC–UV method. A typical baseline-corrected HPLC–UV chromatogram,

obtained from unstressed casein pancreatic digest, is depicted inFig. 1. Given the strong overlap between peptide peak nos. 3(VAPFPE/NVPGEIV) and 4 (LEIVPN/HLPLP), these peaks were inte-grated together. The calculated 95% confidence intervals of thepeptide peak areas, together with the relative peptide amounts,are given in Table 1. The confidence intervals of only three peptidesi.e. YPVEP, SDIPNPI and RGPFPI, are marginally wider than the90–100% interval, which is considered as the specification accep-tance limit. Further investigations revealed normal distributionsof the 6 individual peptide peak areas (Shapiro–Wilk’s test for nor-mality) and the absence of outliers within the sets of integration

Fig. 3. Average temperature (left) and time (right) effect on the dry peptide stability.

118 M. D’Hondt et al. / Food Chemistry 128 (2011) 114–122

results (Grubbs test). The limits of quantification of the identifiedpeptides are presented in Table 1. The peptide represented bythe smallest peak, i.e. SDIPNPI, has a LoQ value of 46.93%. Thus,in order to accurately quantify the SDIPNPI peptide in a stress sam-ple, not more than 53.07% of aforementioned peptide may be de-graded, relative to the same peptide peak area in a referencesample (see Section 2.5).

3.2. Dry heat stress tests

The relative peptide amounts of identified peptides, after dryheat stress stability tests under different conditions, and theirrespective 95% confidence intervals are presented in Fig. 2. In gen-eral, the peptides remained stable up to 3 min incubation at180 �C. However, both specification-significant (i.e. below 90%)and statistically significant degradation, ranging from 10% to 40%,of all identified peptides was seen in the 5 min, 180 �C samples.These samples were also characterised by a yellow brownish discol-oration, as opposed to the native white colour, and a strong odour,indicative for the presence of Maillard reaction products. Based onthe lactose content of the casein pancreatic digest powder (0.8%),it is possible that a part of the peptide degradation can be attributedto the Maillard reaction. In total, 8 of the 12 identified peptide peakscontain amino acid residues sensitive to Maillard reaction i.e. argi-nine (R), lysine (K), asparagine (N) and glutamine (Q). Two majornew peptide peaks were found in the 3 and 5 min 180 �C samples.The degradation peaks shouldered the RGPFPI (peak no. 6, RT:19.19 min) and VPFPGPI (peak no. 12, RT: 29.83 min) peaks, andwere characterised by [MH]+ values of 730.25 and 1315.40 g/mol,respectively. A typical example of a baseline-corrected HPLC–UVchromatogram, obtained from casein pancreatic digest incubatedat 180 �C for 5 min, is depicted in the Supplementary Fig. S-1. Theaforementioned degradation peaks are indicated by an asterix.

The temperature and time effect on the stability of an ‘average’dry peptide, defined as the mean of the relative amounts of the 12identified peptide peaks, in relationship to the specification-signif-icant 90 – 110% interval is presented in Fig. 3. This figure clearlydemonstrates the preservation of the average peptide stability upto 3 min incubation at 180 �C. The overall total error between theexperimental repeats was calculated to be 7.31%.

A degradation model (95% confidence intervals between brack-ets) for the average peptide was constructed (SPSS 17, linearregression):

Average relative peptide amount ð%Þ¼ 109:314ð�4:723Þ þ 4:175ð�1:383Þ � time� 0:036ð�0:033Þ� temperature� 0:045ð�0:010Þ � time� temperature

The model was characterised by a R2 value of 0.610, thusexplaining 61% of the observed variability.

3.3. Oxidative dry heat stress

The relative peptide amounts, after the oxidative dry heat stressstability tests, under the different conditions, and their respective95% confidence intervals, are presented in Fig. 4. An example of abaseline-corrected HPLC–UV chromatogram, obtained from caseinpancreatic digest incubated at 180 �C for 5 min in the presence of10% (w/w) KMnO4, is presented in the Supplementary Fig. S-2.

In general, substantial decreases in the relative peptideamounts were seen throughout the various oxidative stress condi-tions when compared to the respective dry heat stress conditions.Given the information obtained from the previous dry heat stressexperiments, where no degradation was found until 5 min of incu-bation at 180 �C, this degradation was attributed to the presence ofoxidising potassium permanganate. The results can be divided intotwo distinct groups of peptide behaviour. The first group consistedof the following peptides: YPVEP, VLPVPQ, VAPFPE/NVPGEIV, LEI-VPN/HLPLP, SDIPNPI, RGPFPI, PFPGPIPN/APFPEV, VVPPFLQPE andTDAPSFSDIPNPI. These peptides were characterised by more or lessconstant, specification-significant degradation, which was seenthroughout the oxidative stress conditions, with a steep degrada-tion increase in the 5 min, 180 �C stress samples. Three peptideswere placed in the second group, i.e. YPFPGPIPN, VYPFPGPIPN,VYPFPGPI. The structural similarity between these peptides withingroup 2 is striking. In general, massive oxidative peptide degrada-tion was seen throughout all stress conditions for these group 2peptides. The individual peptide profiles were characterised bydegradation ranging from 70% to 90% in the 1 min, 100 �C, incuba-tion sample. An increase in the relative peptide amounts was seenin the 3 min, 140 �C, incubation sample. This increase is more orless constant up until the 1 min, 180 �C, stress sample. Startingfrom the 3 min, 180 �C, stress sample, the relative peptide amountsonce again decreased, but remained elevated when compared tothe relative amounts observed in the oxidative samples incubatedat 100 �C. Further investigations, by re-analysis of the aforemen-tioned oxidative stress samples using the LC–MS method (see Sec-tion 2.2), confirmed our quantitative findings; i.e. that the UVpeptide peaks, used to calculate the relative peptide amounts, aredirectly related to the presence or absence of the identified pep-tides. Hence, the peptides in group 2 are more stable during a5 min incubation period at 140 �C in the presence of approximately10% w/w KMnO4 when compared to a 1 min incubation period at100 �C, under identical oxidative conditions. A hypothesis, stipulat-ing a temperature dependent degradation of potassium permanga-nate resulting in manganese species characterised by loweroxidation potentials, is provided in the Supplementary data.

The average temperature and time effects, calculated as de-scribed in Section 3.2, on the dry peptide stability in the presenceof potassium permanganate, are presented in the SupplementaryFig. S-3. The overall total error between the experimental repeatswas calculated to be 9.31%.

Fig. 4. Oxidative dry heat stress: relative peptide amounts. Relative peptide amounts situated outside the 95% confidence interval or below the limit of quantification areindicated in red or black, respectively. (For interpretation of the references in colour in this figure legend, the reader is referred to the web version of this article.)

M. D’Hondt et al. / Food Chemistry 128 (2011) 114–122 119

3.4. Reductive dry heat stress

The relative peptide amounts, after the reductive dry heat stressstability tests under different conditions, and their respective 95%confidence intervals, are presented in Fig. 5. An example of a base-line-corrected HPLC–UV chromatogram, obtained from a caseinpancreatic digest incubated at 180 �C for 5 min in the presence of10% (w/w) zinc powder, is presented in the SupplementaryFig. S-4.

In general, the relative peptide amounts remained constant upto 1 min of incubation at 180 �C. Incubation for 3 min at 180 �C re-sulted in the peptide degradation, ranging from 10% to 25%, of allidentified peptides. When analysing the results of the 5 min,180 �C samples, additional degradation ranging from 3% (e.g.VLPVPQ, peak no. 2) to 17% (e.g. YPFPGPIPN, peak no. 8) was found.The degradation profile of all peptides was similar to the respectiveprofiles found in the dry heat stress tests. Furthermore, the relativepeptide amounts at the most severe stress condition (5 min,

Fig. 5. Reductive dry heat stress: relative peptide amounts. Relative peptide amounts outside the 95% confidence intervals are indicated in red. (For interpretation of thereferences in colour in this figure legend, the reader is referred to the web version of this article.)

120 M. D’Hondt et al. / Food Chemistry 128 (2011) 114–122

180 �C) are within the same range (i.e. ±5%) as those observed inthe dry heat stress tests. The observation of two additional peptidedegradation peaks, in the 5 min, 140 �C, and in the 3 and 5 min180 �C stress samples, which were also reported in the dry heatstress test (see Section 3.2), further underline the similarity be-tween the dry and reductive dry heat stress tests. The average tem-perature and time effects, calculated as described in Section 3.2, onthe dry peptide stability in the presence of zinc, are presented inSupplementary Fig. S-5. Summarising, these data show only asmall additional degradation effect of the zinc powder, leading toan earlier onset of the degradation of the selected peptides, i.e.starting from 3 min incubation at 180 �C, when compared to dry

heat stress tests. The overall total error between the experimentalrepeats was calculated to be 7.03%.

3.5. Dry photolytic stress

Examples of baseline-corrected HPLC–UV chromatograms, ob-tained from casein pancreatic digest incubated at 25 �C under UVand visual light, are presented in the Supplementary Figs. S-6and S-7, respectively. In general, no significant peptide degradationwas observed. Furthermore, the relative peptide amounts, with theexceptions of peptides VVPPFLQPE (peak no. 9) and TDAPSFSDIPN-PI (peak no. 11), are enclosed in their respective 95% confidence

M. D’Hondt et al. / Food Chemistry 128 (2011) 114–122 121

intervals. A more detailed overview of the dry photolytic stabilityof each individual identified peptide is given in the SupplementaryFig. S-8, while the average photolytic effect is depicted in Supple-mentary Fig. S-9.

3.6. Discussion

In this study, the main focus was given to the primary stabilityof casein-derived peptides in their basic form, i.e. without specificPTMs. Previous stability studies using casein hydrolysates focussedon phosphorylated peptides. Meisel, Andersson, Buhl, Erbersdo-bler, and Schlimme (1991) investigated the impact of dry heattreatment of casein (170 �C for 45, 60 and 75 min) on phosphoserylstability. A time dependant decrease of caseinophosphopeptideswas observed, accompanied by a dephosphorylation and decompo-sition of seryl residues. Furthermore, b-elimination of alanine,yielding dehydroalanine, resulted in the formation of lysinoala-nine. The authors reported the hydrolysis of phosphoseryl to serylresidues as the prevailing degradation step. Zhu and FitzGerald(2010) studied the effects of pH, i.e. 6.0, 7.0 and 8.0, and heat treat-ment at 75 �C on the stability of a tryptic casein hydrolysate insolution. Significant decreases in the peptide sequences were seenin the samples heated at pH 6.0 and 8.0, compared to the sampleheated at pH 7.0. Furthermore, phosphoseryl stability in the pH7.0 samples was higher when compared to the pH 6.0 and 8.0samples.

Besides the aforementioned dephosphorylation and dehydro-alanine b-elimination, other degradation mechanisms can also oc-cur. Chemical instability of peptides can involve one or more of avariety of chemical reactions (Florence & Attwood, 2006). Peptidescan be subjected to transamidation (Asp or Glu) or deamidation(Asn, Gln), as well as isomerization (Pro) of specific amino acid res-idues. Some amino acid residues, such as His, Met, Cys, Trp and Tyr,are vulnerable oxidation sites. Lanthionine-peptides as well asdisulphides, sulphenic, sulphinic and sulphonic acids can thus beformed from cystine residues, with similar oxidative products formethionine. Furthermore, oxidative degradation can yield imineintermediates (Strecker degradation). Finally, the presence ofnon-peptide ‘‘impurities’’ like reducing sugars, e.g. lactose, in thecasein hydrolysate can lead to the formation of Maillard reactionproducts.

4. Conclusions

For the first time, the peptide stability in dry form of a modelpeptide set, i.e. casein pancreatic digest, was evaluated. The stressconditions used consisted of exposure to dry temperatures of 100,140 and 180 �C for 1, 3 and 5 min. Furthermore, the effect of oxidis-ing and reducing substances on the dry peptide stability wasevaluated by adding approximately 10% (w/w) potassium perman-ganate and zinc powder, respectively, to the peptide set. Also,photolytic degradation, using UV and visual light, at 25 �C wasevaluated.

Contrary to general belief, no evidence of massive peptide deg-radation was seen up to 3 min incubation at 180 �C. However, pro-longed exposure to 180 �C did result into significant peptidedegradation. In the oxidative stress samples, high amount of degra-dation was found, with some degradation profiles being time andtemperature dependant. The degradation profiles derived fromthe reductive stress samples were similar to those obtained fromthe dry heat stress samples, with an additional reductive effect. Fi-nally, exposure to UV and visual light stress did not cause any sig-nificant peptide degradation. In conclusion, the short-term heatstress stability evaluation of the peptides in dry form provides sur-prising but promising results. In general, the peptides studied re-

mained stable after a short time exposure to temperatures ashigh as 180 �C and revealed the negative impact of oxidising agentson peptide stability. These results could open new perspectives tothe development of new, more effective peptide processingtechniques.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.foodchem.2011.03.004.

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