evaluation of catalytic effects of chymotrypsin and for...

Research ArticleEvaluation of Catalytic Effects of Chymotrypsin andCu2+ for Development of UV-Spectroscopic Method forGelatin-Source Differentiation

Anis Hamizah,1 Ademola Monsur Hammed,1,2,3

Tawakalit Tope Asiyanbi-H,2 Mohamed Elwathig Saeed Mirghani,1,3

Irwandi Jaswir,1,3 and Nurrulhidayah binti Ahamad Fadzillah1

1 International Institute for Halal Research and Training (INHART), International Islamic University Malaysia (IIUM),Gombak, Kuala Lumpur, Malaysia2Plant Sciences Department, North Dakota State University, Fargo, ND, USA3Biotechnology Engineering Department, International Islamic University Malaysia (IIUM), Gombak, Kuala Lumpur, Malaysia

Correspondence should be addressed to Ademola Monsur Hammed; [email protected]

Received 24 February 2017; Revised 24 July 2017; Accepted 7 September 2017; Published 8 October 2017

Academic Editor: Salam A. Ibrahim

Copyright © 2017 Anis Hamizah et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The consumers interest in gelatin authentication is high due to allergic reactions and adoption of Halal and Kosher eating cultures.This research investigated browning development due to enzymatic hydrolysis and presence of Cu2+ duringMaillard reaction of fish,porcine, and bovine gelatin.The rate of browning index samples showed twophases—rapid and slow—for all the gelatin samples andchanges in browning index (Δ𝐵index) were increased (>100%) in presence of Cu2+. Δ𝐵index of enzymatic hydrolysates were differentamong the gelatin species. Fish gelatin hydrolyzate displayed > 400% increase in browning in the first six hours compared to gelatinhydrolyzates from porcine (200%) and bovine (140%).The variation inΔ𝐵index of chymotrypsin digested gelatin in presence of Cu2+could be valuable for the development of an efficient UV-spectroscopic method for gelatin differentiation.

1. Introduction

Maillard reaction is a nonenzymatic browning leading toformation of numerous compounds when proteins (such asgelatin) are heated in presence of sugar. Maillard reactionproducts (MRP) have fluorescence and browning capabilitysuitable to measure reaction progress [1]. The degree ofbrowning during Millard reaction depends on the type ofsugar, temperature, pH, reaction time, concentration of reac-tants, presence of inorganic compounds, and most especiallyprotein amino acids profiles [1–3]. The colour intensity ofMRP from basic amino acids was reportedly greater than thatof acidic amino acids, while nonpolar amino acids were ofintermediate colour intensity. Also, browningwas acceleratedby the presence ofmetal ions (Fe2+ and Cu2+) but not affectedby Na+ [3]. Study of Maillard reaction of essential aminoacids with glucose revealed that amino acids were degradeddifferently and exhibited a varying browning degree [2].

Previous studies have shown that the amino acid profilesand film properties of different gelatin sources varied, mostespecially methionine and histidine [4].The content of iminoacids proline and hydroxyproline of fish collagen is generallylower than that of mammalian collagen [5]. Another reportshowed that properties of gelatin film depend on the sourceof the gelatin such that fish gelatin exhibited lowest watervapor permeability while pork gelatin exhibited least watersolubility [6].Therefore, there is a possibility that variation inthe degree of browning inMillard reaction of different gelatinsources could be observed. The degree of browning can bedetected by UV-spectroscopy and the readings can then beused to differentiate gelatin of varying sources.

We presume that distribution of aromatic amino acid ongelatin is species specific. Chymotrypsin cleaves peptide bondby aromatic residues, unlike trypsin that only cleaves peptidebond at lysine and arginine. Therefore, chymotrypsin digestof gelatin will possibly produce peptides and hydrolysates

HindawiInternational Journal of Food ScienceVolume 2017, Article ID 2576394, 5 pageshttps://doi.org/10.1155/2017/2576394

https://doi.org/10.1155/2017/2576394

2 International Journal of Food Science

unique to different gelatin species. Previous studies havereported enzyme hydrolysis of gelatin yield peptides as abiomarker for gelatin-source differentiation [7].Most of thesestudies made use of expensive and difficult procedures suchas GC-MS, PCR, ELISA, and HPLC. This research is basedon two principles: (i) the chymotrypsin hydrolysis of gelatinfrom a different source could produce peptides of varyingproperties that will yield a different degree of browningduringMaillard reaction and (ii) the introduction ofCu2+willexpedite the rate of reaction and reduce the time for browncolour development.

Till date, there is only one study on the use of UV-spectroscopy reading of the degree of browning of MRP ofgelatin for species-specific differentiation purpose. Bovineand porcine gelatin were successfully differentiated with UV-spectroscopy reading after ribose-induced Maillard reaction[8]. A literature search revealed a lack of studies on Maillardreaction of gelatin hydrolyzate and the effect of Cu2+ onthe degree of browning of gelatin. In a preliminary study,Cu2+ has shown to improve browning index during Maillardreaction. Improvement of UV-spectroscopic measurementfor differentiation of gelatin power could be achieved byconsidering effects of chymotrypsin and Cu2+ on the degreeof browning after Maillard reaction process.

Hence, the aim of this study is to investigate the devel-opment of brown colour during Maillard reaction of fish,porcine, and bovine gelatin as affected by chymotrypsinhydrolysis and the presence of Cu2+. It is hoped thatdetail understanding of reaction kinetics will be valuabletowards the development of more efficient and reliableUV-spectroscopy based method for species-specific gelatin-source differentiation.

2. Methodology

2.1. Production of Gelatin Hydrolyzates. Gelatin hydrolysateswere produced using chymotrypsin to digest gelatin fromfish, porcine, and bovine. The digestion was carried out for4 h at 25∘C with the enzyme-gelatin ratio of 1 : 250 (w : w).The reaction was stopped by heating the mixture at 100∘C for10min.The solutions were centrifuged at 3000 rpm for 15minand the supernatants were discounted off and referred to asgelatin hydrolyzates solution.

2.2. Nonenzymatic Browning of Gelatin and GelatinHydrolysate. An equal volume of 0.25% xylose solutionsand 1.0% of gelatin/hydrolyzates were thoroughly mixedto make a final solution containing 0.125 and 0.5% ofxylose and gelatin/hydrolyzates, respectively. In anotherset of experiments, about 2mM of CuCl

2was added to

the mixture. The nonenzymatic browning was carried outby heating the mixture at 95∘C for 6, 12, and 24 h. Themixture was allowed to cool to room temperature before thedetermination of browning index.

2.3. Determination of Browning Index. The browning index(𝐵index) of a cooled mixture of gelatin/hydrolyzate containingxylose with or without Cu2+ was measured at 420 nm using

a microplate spectrophotometer.The change in the browningindex (Δ𝐵index) was used to determine the effect of enzymehydrolysis and presence of Cu2+. 𝐵index and Δ𝐵index weredetermined using

𝐵index of sample = 𝐴x-g − 𝐴g,

Δ𝐵index of xylose-hydrolyzate =(𝐴x-h − 𝐴x-g) × 100𝐴x-g

,

Δ𝐵index of xylose-gelatin in presence of Cu2+

=(𝐴x-gCu − 𝐴x-g) × 100

𝐴x-g,

Δ𝐵index of xylose-hydrolyzate in presence of Cu2+

=(𝐴x-hCu − 𝐴x-g) × 100

𝐴x-g,

(1)

where 𝐴g is the absorbance of gelatin; 𝐴x-g is the absorbanceof xylose-gelatin mixture; 𝐴x-h is the absorbance of xylose-hydrolyzate; 𝐴x-gCu is the absorbance of xylose-gelatin inpresence of Cu2+, and 𝐴x-hCu is the absorbance of xylose-hydrolyzate in presence of Cu2+.

2.4. Data Analysis. All data were collected in triplicateand their average and standard deviations were calculated.Diagrammatic representations were used to present the resultfor easy understanding.

3. Results and Discussion

3.1. Nonenzymatic Browning of Fish, Porcine, and Bovine.Figure 1 shows that the browning index of gelatin fromdifferent samples increases with increase in heating time.There was an initial rise in the browning index for all the3 gelatin samples in the first 6 hours followed by a steadyincrease. This is similar to previous reports that colourformation during Maillard reactions is usually rapid at theearly reaction stage [9].

Several studies have shown that the amino acids composi-tions of gelatin vary according to the species [5]. It is possiblethat different regions/parts of the gelatin responded differ-ently during the nonenzymatic browning. The variation inthe reaction rate might be due to differences in the reactivityof amino acids present in the peptides. A study of Maillardreaction of essential amino acids with glucose revealedthat amino acids were degraded differently and exhibited avarying degree of browning [2]. Compared to acidic aminoacids, the basic amino acids contributed to browning inten-sity, while the nonpolar amino acids exhibited intermediatecolour intensity [3]. Lysine participated in browning reactioninduced by glucose compared to other amino acids and thatthreonine contributed very little towards browning [2].

This pattern can be explained that there are two regionsin the gelatin polymer including the fast responding regionand the slow responding region. The slow responding region

International Journal of Food Science 3

Porcine

Bovine

Fish

0 2010 30

Time (h)

0.000

0.039

0.078

0.117

0.000

0.035

0.070

0.105

0.000

0.035

0.070

0.105

Brow

ning

inde

x (4

20nm

)

Figure 1: Browning index of xylose-inducednonbrowning of gelatinfrom different sources (fish, bovine, and porcine) at 95∘C.

might need to undergo initial reactions as a sequel tobrowning reaction. This surmised that gelatin of differentsources has similar configurations which eventually reflectsimilarities in their Maillard reaction kinetic. However, theproportion of the two (slow and quick) regions might varyamong the gelatin samples.

3.2. Effect of Chymotrypsin Hydrolysis of Gelatin on Degreeof Xylose-Induced Nonenzymatic Browning. Structurally,collagen—the parent material of gelatin—is composed ofnearly one-third of glycine and another 15 to 30% of prolineand 4-hydroxyprolyl. 3-Hydroxyprolyl and 5-hydroxylysylresidues are also present in a smaller amount. Proline andhydroxyproline are responsible for the unique secondarystructure of collagen as they limit rotation of the polypeptidebackbone and thus contribute to the stability of triple helix[10]. Stabilization of collagen structure involves hydrogenbond (between glycine of the N terminal and proline ofthe adjacent chain), hydrophobic interaction, and Van derWalls interaction [11]. The collagen intermolar forces arelikely inherited by gelatin, therefore, affecting the chemicalproperties of gelatin during Maillard reaction. The presenceof intermolecular forces in gelatin is likely responsible fora reduction on the accessibility of amino acids to xyloseduring Maillard reaction. Hydrolysis of gelatin might result

FishBovinePorcine

5 10 15 20 25 300Time (h)

−10

−5

0

5

10

15

20

25

30

35

40

ΔBＣＨ＞？Ｒ

(%)

Figure 2: Change in the browning index due of chymotrypsindigestion on xylose-induced nonenzymatic browning of gelatinfrom different sources. Δ𝐵index: change in browning index.

into peptides that are more reactive and accessible duringMaillard reaction.

In order to determine the effect of chymotrypsin digestof gelatin on contribution to browning index, the percentagedifference in browning index of hydrolysates compared totheir respective gelatin was estimated and referred to asa change in the browning index (Δ𝐵index). According toFigure 2, the effect of chymotrypsin digestion on Δ𝐵indexvaries among the gelatin samples. Δ𝐵index of fish gelatinhydrolysate was the highest among all the samples through-out the heating period. This suggests that digestion of fishgelatin produces peptides that contributed to Δ𝐵index com-pared to other samples.

Previous reports have shown that enzymatic digestion ofgelatin produced different peptides fractions among species.The peptides might contribute differently towards the devel-opment of browning during Maillard reaction process and,hence, caused variation of Δ𝐵index among the samples.

The trends in browning development that follow were in2 reaction phases, namely, increasing and decreasing phases.The increasing phase is early and high in fish hydrolysatecompared to that of porcine and bovine.The increasing phaseof bovine phase occurred only between 6 and 12 h. After6 h reaction time, there was a decrease in browning in fishhydrolysate while the decreasing phase of porcine hydrolysateoccurred after 12 h.

The reduction effect of chymotrypsin hydrolysis onΔ𝐵index observed in all the 3 samples might be explainedby the lack of stability of their Maillard’s reaction products.This reduction in browning occurred during the first sixhours of heating in bovine gelatin hydrolysate, while that offish hydrolysate occurred after the first six hours. Also, therise in Δ𝐵index observed in all samples suggests that initialconfiguration of gelatin structure hindered the progress ofMaillard reaction. Enzymatic hydrolysis did not only increasethe surface area of gelatin and expose the amino acid but also

4 International Journal of Food Science

FishBovinePorcine

6 12 18 24 300Time (h)

0

20

40

60

80

100

120

140

ΔBＣＨ＞？Ｒ

(%)

Figure 3: Change in the browning index due to the catalytic effectof Cu ion on the degree of xylose-induced nonenzymatic browningof gelatin from different sources.Δ𝐵index: change in browning index.

reduce the hindering effect from gelatin configuration duringMaillard reaction. In line with the findings of Su et al. [12],the smaller molecular peptides obtained from peanut proteinexhibited higher reaction degree during Maillard reaction.

As stated earlier, the kinetic of nonenzymatic browning ofgelatin of the three samples is similar and comprises fast andslow responding regions. However, enzymatic hydrolysis ofgelatin resulted in hydrolysates that varied among the species.The rise in Δ𝐵index of fish hydrolysates might be becausechymotrypsin hydrolysed the slow reactive regions of fishgelatin to produce more reactive peptides, compared to othergelatin samples. Another reason might be that chymotrypsinhydrolysed bovine gelatin might have produced peptides thatparticipated in Maillard reaction after 6 h.

3.3. Catalytic Effect of Cu2+ on Degree of Xylose-InducedNonenzymatic Browning of Gelatin from Different Sources.The presence of Cu2+ during the nonenzymatic browningof gelatin samples causes an increase in the rate of morethan 100% of 𝐵index. The effect of Cu2+ on Δ𝐵index of thethree gelatin samples was similar such that Δ𝐵index increaseddrastically until the 12 h of heating and then stabilized forbovine and porcine, while that of fish slightly decreased toabout 100%. It is possible that Cu2+ enhanced formationof MRP and results in loss of slow reaction phase earlierobserved in Figure 1. Also, Cu2+might catalyse the formationof MRP from the majority of amino acids and reducedhindering effect of gelatin configuration during the reactionprocess. This observation agreed with the previous reportthat stated that Cu2+ contributed to the colour intensity ofMillard reaction product [3].The transitionmetals have beenreported to have catalysed Maillard reaction by the oxidativepathway [3]. The presence of transition ions promotes theformation of chromophores and fluorophores during Mail-lard reaction of DNA with d-Fructose 6-Phosphate [13]. The

FishBovinePorcine

5 10 15 20 25 300Time (h)

0

50

100

150

200

250

300

350

400

450

ΔBＣＨ＞？Ｒ

(%)

Figure 4: Change in the browning index due to combined catalyticeffect of Cu2+ and chymotrypsin digestion on the degree of Maillardreaction of gelatin from different sources. Δ𝐵index: change in brown-ing index.

observed slight reduction in Δ𝐵index of fish might be due toloss of stability of MRP after extended heating.

3.4. Catalytic Effect of Cu2+ on Degree of Xylose-InducedNonenzymatic Browning of Hydrolysates of Gelatin from Dif-ferent Sources. The combined effect of enzymatic hydrolysisand Cu2+ on the development of browning in Maillardreaction of the gelatin samples is shown in Figure 4. Δ𝐵indexof fish hydrolysate was highest with a value of 400% followedby that of porcine (200%) and then bovine (140%). In thefirst 6 h, Δ𝐵index increased drastically in all samples anddecreased steadily in porcine and fish while that of bovinekept increasing slightly.

Compared to that of the result in Figure 3, Δ𝐵index wasincreased in all samples.This suggests that Cu2+ catalysed thedevelopment of browning of peptides compared to gelatin.At this final stage, differentiating between gelatin from adifferent source can be achieved by comparing Δ𝐵index atdifferent stages. In the early stage, fish gelatin exhibitedhighest Δ𝐵index followed by that of porcine and then bovine.The sharp reduction inΔ𝐵index observed in fish gelatin can beused to discriminate fish gelatin from mammalian gelatin.

4. Conclusion

Maillard reaction of gelatin from different sources exhib-ited similar reaction phases—the slow and the fast phases.Enzymatic degradation of gelatin prior to Maillard reactioncaused a difference in the production of browning productsamong the species. Fish gelatin hydrolysate displayed mul-tifold increase in browning in the first six hours comparedto gelatin hydrolysates from porcine and bovine. Althoughthe catalytic effect of Cu2+ during Maillard reaction was

International Journal of Food Science 5

relatively similar in all the gelatin samples, Cu2+ affectsgelatin hydrolysates differently. The variation in Δ𝐵index ofchymotrypsin digested gelatin in presence of Cu2+ could bevaluable for the development of an efficient UV-spectroscopymethod for gelatin differentiation. Future works will inves-tigate the effects of other reaction conditions on Δ𝐵index ofenzymatic hydrolysates from gelatin.

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper.

References

[1] A. Etxabide, M. Urdanpilleta, P. Guerrero, and K. De La Caba,“Effects of cross-linking in nanostructure and physicochemicalproperties of fish gelatins for bio-applications,” Reactive andFunctional Polymers, vol. 94, article 3544, pp. 55–62, 2015.

[2] E. H. Ajandouz and A. Puigserver, “Nonenzymatic browningreaction of essential amino acids: effect of pH on caramelizationandMaillard reaction kinetics,” Journal of Agricultural and FoodChemistry, vol. 47, no. 5, pp. 1786–1793, 1999.

[3] E.-J. Kwak and S.-I. Lim, “The effect of sugar, amino acid, metalion, and NaCl on model Maillard reaction under pH control,”Amino Acids, vol. 27, no. 1, pp. 85–90, 2004.

[4] M. C. Gomez-Guillen, M. Perez-Mateos, J. Gomez-Estaca, E.Lopez-Caballero, B. Gimenez, and P. Montero, “Fish gelatin: arenewable material for developing active biodegradable films,”Trends in Food Science and Technology, vol. 20, no. 1, pp. 3–16,2009.

[5] A. A. Karim and R. Bhat, “Gelatin alternatives for the foodindustry: recent developments, challenges and prospects,”Trends in Food Science and Technology, vol. 19, no. 12, pp. 644–656, 2008.

[6] Z. A. Nur Hanani, Y. H. Roos, and J. P. Kerry, “Use of beef,pork and fish gelatin sources in the manufacture of films andassessment of their composition and mechanical properties,”Food Hydrocolloids, vol. 29, no. 1, pp. 144–151, 2012.

[7] A. Rohman and Y. B. Che Man, “Analysis of pig derivatives forhalal authentication studies,” Food Reviews International, vol.28, no. 1, pp. 97–112, 2012.

[8] T.-C. Tan, A. F. M. Alkarkhi, and A.M. Easa, “Assessment of theribose-inducedMaillard reaction as a means of gelatine powderidentification and quality control,” Food Chemistry, vol. 134, no.4, pp. 2430–2436, 2012.

[9] M. Van Boekel, “Kinetic aspects of the Maillard reaction: acritical review,” Molecular Nutrition Food Research, vol. 45, no.3, pp. 150–159, 2001.

[10] R. Schrieber and H. Gareis, GelaTine Handbook: Theory andIndustrial Practice, John Wiley & Sons, Hoboken, New Jersey,NJ, USA, 2007.

[11] T. Nogrady and D. F.Weaver,Medicinal Chemistry: AMolecularand Biochemical Approach, Oxford University Press, Oxford,UK, 2005.

[12] G. Su, L. Zheng, C. Cui, B. Yang, J. Ren, and M. Zhao, “Char-acterization of antioxidant activity and volatile compoundsof Maillard reaction products derived from different peptidefractions of peanut hydrolysate,” Food Research International,vol. 44, no. 10, pp. 3250–3258, 2011.

[13] J. Morita and N. Kashimura, “The Maillard reaction of DNAwith D-Fructose 6-Phosphate,” Agricultural and BiologicalChemistry, vol. 55, no. 5, pp. 1359–1366, 1991.

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