Enzymatic hydrolysates from tuna backbone and the subsequent Maillard reaction with different...

Original article

Enzymatic hydrolysates from tuna backbone and the subsequent

Maillard reaction with different ketohexoses

Yan Zeng, Xiaoxi Zhang, Yuping Guan & Yuanxia Sun*

Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China

(Received 2 August 2011; Accepted in revised form 15 January 2012)

Summary Enzymatic hydrolysates from tuna backbone were prepared, and the subsequent Maillard reaction with rare

sugars (d-psicose, d-sorbose and d-tagatose) was investigated. The hydrolysates were found to contain a

large number of short peptides under 1000 Da and were good sources of essential amino acids. In assays of

free radical scavenging activity and reducing power, the Maillard reaction products from rare sugars,

especially d-tagatose, performed better than that from d-fructose. After heating at 55 �C for 48 h, the

scavenging capacity of the hydrolysates for 1,1-diphenyl-2-picryl-hydrazyl radicals improved by 8.9- and

16-fold in the presence of d-fructose and d-tagatose. Hence, hydrolysates with high nutrient contents could

be prepared from fish by-products via proteolysis, and the Maillard reaction of rare sugars may greatly

promote the antioxidant activity of the hydrolysates.

Keywords Antioxidant activity, enzymatic hydrolysates, Maillard reaction, rare sugars, tuna backbone.

Introduction

There has been an increasing recognition of limitedbiological resources in recent years. By-products fromfood processing, which are common environmentalcontaminants, are valuable in many fields. Conse-quently, the demand for the effective utilisation of theseby-products has increased, and some of their relatedapplications have also been reported (Arvanitoyannis &Giakoundis, 2006; Arvanitoyannis et al., 2007; Arvani-toyannis & Ladas, 2008; Arvanitoyannis & Tserkezou,2008). For example, the treated fish by-products havebeen used in animal feed, fertilizers, dietic applications,food packaging and so on (Arvanitoyannis & Kassaveti,2008).Among fish by-products, those from tunas have

drawn particular interest because of the global economicimportance of tunas and their roles in the internationalcanning trade. According to Langley et al. (2009), theglobal tuna catch exceeded four million metric tons in2005, and the total value of the catch exceeded threebillion dollars. Because only the white meat is used intuna canning, the amount of generated solid by-prod-ucts, including viscera, heads, skin, bones and somemuscle tissues, can be as high as 70% of the originalmaterial. Considerable attention has been paid to the

recovery of bioactive proteins and peptides from theseby-products by enzymatic hydrolysis (Klomklao et al.,2007; Ahn et al., 2010). Je et al. (2009) have evaluatedthe antioxidant and antihypertensive activities of proteinhydrolysates prepared from tuna liver under theapplication of Flavourzyme, Alcalase, Protamex andNeutrase. Nguyen et al. (2011) have investigated theproteolysis of tuna head, viscera and tail by the wide-spectrum protease Protamex. Tuna backbone, whichcontains around 30% proteins and is considered as agood nutraceutical, has also been hydrolysed by differ-ent proteases (alcalase, a-chymotrypsin, neutrase, pa-pain, pepsin and trypsin) for antioxidant peptideproduction (Je et al., 2007). Although many proteinhydrolysates with good functional properties have beenreported, these hydrolysates can still be modified toobtain new or enhanced functional properties forproducing more profitable end products (Oliver et al.,2006).The Maillard reaction, also called non-enzymatic

browning, is a set of reactions resulting from the initialcondensation between the amino group of amino acids,peptides, or proteins and the carbonyl group of reducingcarbohydrates (Hodge, 1953). This reaction produces alarge number of so-called Maillard reaction products(MRPs), which play important roles in the quality andacceptability of stored or processed foods. The Maillardreaction has been widely applied to improve thefunctional properties of fish proteins, such as solubility,

*Correspondent: Fax: +86-22-84861961;

e-mail: [email protected]

International Journal of Food Science and Technology 2012, 47, 1293–1301 1293

doi:10.1111/j.1365-2621.2012.02973.x

� 2012 The Tianjin Institute of Industrial Biotechnology. International Journal of Food Science and Technology � 2012 Institute of Food Science and Technology

thermal stability, emulsifying properties, etc. (Sanmartinet al., 2009). However, studies on the effect of theMaillard reaction on the antioxidant activity of fishhydrolysates are limited. Such studies can evaluate thepotential use of fish hydrolysates in the food industry assafe natural antioxidants. Hence, the antioxidantpotential of glycated hydrolysates from tuna backboneis interesting to explore.Rare sugars are defined as ‘monosaccharides and their

derivatives that rarely exist in nature’ by the Interna-tional Society of Rare Sugars. These sugars haveinteresting biological activities, such as the inhibitionof cancer cell proliferation and oxygen radical produc-tion, as well as anti-diabetic and anti-atheroscleroticeffects (Levin, 2002; Matsuo et al., 2002; Sui et al.,2005). Proteins glycated with rare sugars also haveexcellent properties. d-Psicose can more efficientlypromote the foaming property of egg white than glucoseand fructose with increasing whipping time (Sun et al.,2004). a-Lactalbumin modified with d-allose exhibitshigher tetrazolium salt reducibility and higher ABTS+

•

radical scavenging activity than that modified with d-fructose or d-glucose (Sun et al., 2006a). In addition, theMaillard reaction with d-tagatose can be uniquely usedin optimised and cost-efficient ways to manufacturefoods in which caramelized, malt flavour profiles arecharacteristic, such as milk-based crumb chocolates andtraditionally cooked cereals (Skytte, 2006). At present,rare sugars are still difficult to manufacture and theirselling prices are very high. Nevertheless, the greatpotential for the large-scale production of rare sugarsfrom microbial or enzymatic reactions has been dem-onstrated (Izumori, 2002; Granstrom et al., 2004). Thisfinding means that rare sugars may be approved forcommercial use as substitutes of alimentary sugars infoodstuffs. Hence, the effects of rare sugars in theMaillard reaction in relation to functional food need tobe explored.To evaluate the effect of rare sugars on the antioxi-

dant activity of the modified fish hydrolysates, hydro-lysates from tuna backbone were prepared by enzymatichydrolysis, and their Maillard reaction with four keto-hexoses, namely, d-fructose, d-psicose, d-sorbose, andd-tagatose (Fig. 1), was carried out. The antioxidant

activities of the corresponding MRPs at different reac-tion times were also determined.

Materials and methods

Materials

Frozen yellowfin tuna (Thunnus albacores) backboneswere provided by the Zhonglu Oceanic Fishery Co. Ltd.(Shangdong, China) and were stored at )20 �C untiluse. Neutrase, Papain, Protemax and Flavourzyme wereprovided by the Noao Sci & Tech Development Co. Ltd.(Tianjin, China). d-Psicose, d-sorbose and d-tagatosewere donated by the Rare Sugar Research Center,Kagawa University, Japan. 2, 2¢-Azinobis (3-ethyl-benzothiazoline-6-sulphonic acid) diammonium salt(ABTS), 6-hydroxy-2,5,7,8-tetramethylchroman-2-car-boxylic acid (Trolox), 1,1-diphenyl-2-picryl-hydrazyl(DPPH), o-phthaldialdehyde (OPA) and K2S2O4 werepurchased from the Sigma Chemical Co. (St Louis, Mo,USA). Trichloroacetic acid was purchased from the AlfaAesar Chemical Co. (Ward Hill, MA, USA). All otherreagents used were analytical grade.

Methods

Preparation of enzymatic hydrolysatesThawed tuna backbone (750 g) was cut into 1 to 2 cmpieces and autoclaved in deionised water (3000 mL) at121 �C for 1 h. After cooling to room temperature, thesupernatant oil of the mixture was removed. Thepurpose of this step was to crisp the backbone andreduce the emulsification in the subsequent hydrolysis.Then, the pH of the treated mixture was adjusted to 7.5,and Neutrase, Papain, Protemax and Flavourzyme wereadded, at the same enzyme ⁄ substrate concentration of0.25% by wet weight. The enzymatic hydrolysis wasperformed at 45 �C for 12 h, with the re-adjustment ofthe pH to 7.5 at 6 h. The soluble fraction was clarifiedby centrifugation at 4500 · g for 20 min to remove oilyand solid residues. After microfiltration and lyophilisa-tion, enzymatic hydrolysates from the soluble fractionwere obtained as faint yellow powders and stored at)20 �C until use.

Nitrogen recovery (NR) and protein contentThe nitrogen in the substrate (dried tuna backbone) andin the hydrolysates was determined by the Kjeldahlmethod. NR was calculated as follows:

NR ¼ Nitrogen in the hydrolysates

Nitrogen in the substrate

�the product yield� 100%

where the product yield was calculated based on thedried weight of the hydrolysates and the tuna backbone.

Figure 1 Structural formulas (Fischer projection) of the four keto-

hexoses.

Tuna backbone hydrolysates and their glycation Y. Zeng et al.1294

International Journal of Food Science and Technology 2012 � 2012 The Tianjin Institute of Industrial Biotechnology

International Journal of Food Science and Technology � 2012 Institute of Food Science and Technology

The protein content (%) in the hydrolysates wascalculated by multiplying the nitrogen in the hydroly-sates by 6.25.

Determination of the degree of hydrolysisThe degree of hydrolysis (DH) was determined usingOPA according to the method of Nielsen et al. (2001). l-Serine was used as the standard at a concentration of0.1 mg L)1. The sample, standard or deionised water(400 lL) was vigorously mixed with 3 mL of OPA andwas incubated at room temperature for 2 min. Theabsorbance at 340 nm was then measured as Asample,Astandard and Ablank, respectively. DH was calculated asfollows:

Serine-NH2 ¼Asample � Ablank

Astandard � Ablank

� 0:9516meqvL�1 � 0:1� 100

X� P

Serine-NH2 = meq serine NH2 g)1 protein, X = g

sample, P = protein % in the sample, and 0.1 is thesample volume in litre.

DH ¼ h

htot� 100%

where h = (Serine-NH2 ) b) ⁄ a meq g)1 protein. Forfish, htot = 8.6, b = 0.40, and a = 1.00.

Measurement of molecular weight distributionThe molecular weight distribution of the hydrolysateswas determined by high-performance liquid chromatog-raphy (HPLC) ⁄mass spectrometry (MS) using an Agi-lent 1100 series HPLC system coupled with an Agilent6210 ESI-TOF MS (Agilent Technologies, Palo Alto,CA, USA).In the HPLC analysis, a Vydac C18 column (300 A,

2.1 mm · 150 mm; Separations Group, Hesperia, CA,USA) was eluted with two solvents (A: 0.1% formic acidin deionised water, and B: 0.1% formic acid in aceto-nitrile) at a flow rate of 0.2 mL min)1. The gradientelution was 0% to 5% B in 5 min (desalination on line),5% to 50% B in 10 min, 50% to 95% B in 2 min with aholding time of 3 min, then 5% B in 2 min with a post-time of 10 min.

Amino acid analysisThe amino acid composition of the hydrolysates wasmeasured according to the method of Vidotti et al.(2003) with slight modifications. The sample washydrolysed with 6 N hydrochloric acid and 2% phenolat 110 �C for 22 h. The acid hydrolysis products werethen analysed using an automatic amino acid analyzerL-8900 (Hitachi, Tokyo, Japan) with a ninhydrin

reagent and lithium buffer system. Tryptophan contentwas not determined.

Preparation of Maillard reaction products (MRPs)The Maillard reaction of the enzymatic hydrolysateswas performed with four ketohexoses (d-fructose, d-psicose, d-sorbose and d-tagatose). In a preliminaryexperiment, the optimal reactant ratio of the hydro-lysates and d-fructose (or d-psicose) by weight wasfound to be 6:1. Hence, the hydrolysates (6%, w ⁄v)and reducing sugar (1%, w ⁄v) were dissolved incarbonate buffer (10 mm, pH 9.0). The solutions werelyophilised and the dried samples were incubated in aKBF115 climate chamber (Binder, Germany) at 55 �Cand 65% relative humidity for 48 h. At the same time,control experiments without sugar addition wereconducted.

Monitoring the degree of Maillard reactionBrowning was analysed by the absorbances of the MRPs(10 mg mL)1) at 420 nm. The content of free aminogroups was determined using the OPA reagent (Churchet al., 1983) and was expressed as the relative concen-tration (%) compared with the original hydrolysatecontent.

Trolox equivalent antioxidant capacity (TEAC) assayA TEAC assay was performed according to the methoddescribed by Re et al. (1999). The previously preparedABTS+

•solution was diluted to an absorbance of

0.7 ± 0.02 at 734 nm using sodium phosphate buffer(10 mm, pH 7.4). The sample (5 mg mL)1, 50 lL) wasvigorously mixed with the diluted ABTS+

•solution

(3 mL) and was allowed to stand in the dark for 6 minat room temperature. The absorbance at 734 nm wasthen recorded as Asample. Control and blank experimentswere carried out in the same way, except that the MRPswere substituted by the hydrolysates and deionisedwater, respectively. The percentage of ABTS+

•radical

scavenging activity was calculated as follows:

ABTSþ�radical scavenging activity (%)

¼Ablank � Asample � Acontrol

� �

Ablank� 100%

A standard curve of Trolox ranging from 0.12 to1.2 mm was prepared in the same manner. The finalABTS+

•radical scavenging activity of the MRPs was

expressed as lmol Trolox equivalents per gram.

DPPH assayThe DPPH

•radical scavenging activity of the MRPs was

determined according to the method of Yen & Hsieh(1995). The sample (2 mg mL)1, 400 lL) was mixed

Tuna backbone hydrolysates and their glycation Y. Zeng et al. 1295

� 2012 The Tianjin Institute of Industrial Biotechnology International Journal of Food Science and Technology 2012


with 2 mL of 0.25 mm DPPH in methanol with vigor-ously stirring and allowed to stand in the dark at roomtemperature for 30 min. The absorbance of the mixtureat 517 nm was then immediately recorded as Asample.The percentage of DPPH

•radical scavenging activity

was calculated as follows:

DPPH�radical scavenging activity (%)

¼Ablank � Asample � Acontrol

� �

Ablank� 100%

Measurement of reducing powerThe reducing power of the MRPs was determinedaccording to the method of Oyaizu (1986). Onemillilitre of sample (2 mg mL)1) was mixed with1 mL of 0.2 m sodium phosphate buffer (pH 6.6) and1 mL of 1% K3Fe(CN)6. The mixture was incubated at50 �C for 20 min, and 1 mL of trichloroacetic acid(10%) was added. After centrifugation at 750 · g for10 min at room temperature, 1 mL of the mixturesupernatant was collected. Addition of 1 mL of deion-ised water and 200 lL of 0.1% FeCl3 followed. Theabsorbance of the reaction mixture was measured at700 nm and recorded as Asample. The reducing power(A) was calculated by the increase in the absorbance at700 nm, as follows:

A = Asample ) Ablank ) Acontrol

Statistical analysis

All analyses were run in triplicate, and all resultswere expressed as mean ± standard deviation. Thedifferences were calculated by one-way anova usingSPSS 12.0 for windows (SPSS Inc., Chicago, IL, USA).Differences at P < 0.05 were considered significant. ThePearson’s correlation coefficients were also calculatedto quantify the association between the progress ofMaillard reaction and the antioxidant activity of theMRPs.

Results and discussion

Proteolysis

The hydrolysis of tuna backbone was carried out at45 �C for 12 h under the combination of Flavourzyme,Neutrase, Papain and Protemax. To evaluate the effectof enzymatic hydrolysis, NR and DH were measured as86.2% and 22.3%, respectively. The NR and DH resultswere similar with those of the hydrolysis of some tunawastes or backbones from marine fishes, as shown inTable 1 (Slizyte et al., 2009; Hou et al., 2011; Naqash &Nazeer, 2011).The molecular weight distribution may play possible

key roles in the physiological activity of the hydroly-sates. Table 2 showed that the molecular weight distri-bution of the enzymatic hydrolysates ranged from 300 to5000 Da, and most of the peptides were under 2000 Da.About 25.6% and 45.9% of the hydrolysates were

Table 1 Enzymatic hydrolysis of some marine fish by-products

Fish waste Enzyme

Enzyme

dosage (%)

pH Time

(h)

Temperature

(�C) DH (%) NR (%) References

Tuna stomach Alcalase 3 8 5.5 50 23.3 – Guerard et al. (2001)

Tuna stomach Neutrase 1.5 7 4 45 22.5 57 Guerard et al. (2002)

Tuna head 32.3 73.6

Tuna viscera Protamex 0.1 – 12 45 16.8 82.7 Nguyen et al. (2011)

Tuna tail 22.2 85.8

Tuna backbone Alcalase 1 7 50 <60

Papain 1 6 8 37 <60 – Je et al.(2007)

Neutrase 1 8 50 74.5

Alaska pollock frame MEAP 1.2 8 6 45 18.1 76.9 Hou et al. (2011)

Exocoetus volitans

backbone

Trpsin 8.8

Pepsin – 2 4 37 – >60 Naqash & Nazeer (2011)

Papain 6

Gadus morhua

backbone

Protamex 0.1 – 1 55 24.3 – Slizyte et al. (2009)

Tuna backbonea Papain 0.25

Protemax 0.25

Neutrase 0.25 7.5 12 45 22.3 86.2 –

Flavourzyme 0.25

DH, degree of hydrolysis; NR, Nitrogen recovery; MEAP, mixed enzymes for animal proteolysis.aThe hydrolysis of tuna backbone was carried out in this study.




fractions of 300 to 500 Da and 500 to 1000 Da,respectively. This result was also similar with that ofsome enzymatic hydrolysates from marine fishes (Guer-ard et al., 2002; Je et al., 2009).The amino acid composition is a very important index

used to measure the nutritive quality of food. In thepresent study, glycine was the most abundant aminoacid in the enzymatic hydrolysates, with the content of12.4%. The second most abundant amino acid wasglutamic acid at about 10%, followed by proline atabout 7% (Table 3). According to the report of Wuet al. (2001), the high level of glycine and proline in thehydrolysates was related to the great amount of connecttissue protein in tuna backbone. Moreover, the essentialamino acids in the hydrolysates exceeded the humanadult requirements suggested by the Food and Agricul-tural Organization ⁄World Health Organization (FAO ⁄WHO). The hydrolysates contained 4.6% lysine and2.1% methionine, whereas the amounts of lysine andmethionine recommended by FAO ⁄WHO (1989) forhumans are 16 and 17 mg g)1 protein, respectively.Therefore, the hydrolysates could be considered as goodsources of lysine, methionine and other essential aminoacids. In addition, the very slightly bitter taste of theenzymatic hydrolysates may be attributed to the highcontent of hydrophobic amino acids such as leucine andisoleucine (Hou et al., 2011).

Maillard reaction

Proteins modified with rare sugars have several advan-tages, as mentioned in the Introduction. Nevertheless,there is no report on the application of rare sugars in theglycation of fish hydrolysates. In the present study, theMaillard reaction of rare sugars (d-psicose, d-sorboseand d-tagatose) with the enzymatic hydrolysates of tunabackbone at 55 �C and 65% relative humidity wasinvestigated.Browning is the easiest measurable parameter of the

Maillard reaction and is often used as an indicator of thedegree of reaction. The browning intensities of theMRPs from four ketohexoses (d-fructose, d-psicose, d-sorbose and d-tagatose) were investigated by theirabsorbances at 420 nm. As shown in Fig. 2, thebrowning of the four systems significantly increasedwith the reaction time. In contrast, the absorbance ofthe native hydrolysates (the control) at 420 nm did notchange even after heating for 48 h. During the entirereaction, the browning in the reaction systems of raresugars, especially in the system of d-tagatose, was moreobvious than in the system of d-fructose.Apart from browning, the loss of free amino groups

induced by the formation of a Schiff base at the firststage of the Maillard reaction is also commonly used todetect the Maillard reaction extent. In the current study,the free amino group content of the modified hydroly-sates was measured using the OPA reagent andexpressed as a relative value (%) to the free aminogroups of the native hydrolysates. As illustrated inFig. 3, the free amino groups in the four systems sharply

Table 2 Molecular weight distribution of the hydrolysates from tuna

backbone

Molecular weight (Da) Proportion (%)

300–500 25.6

500–1000 45.9

1000–1500 15.9

1500–2000 6.5

2000–3000 4.1

3000–4000 1.5

>4000 0.5

Table 3 Amino acid composition of the hydrolysates from tuna

backbone

Essential

amino acids

The content

(mg g)1)

Nonessential

amino acids

The content

(mg g)1)

Lysine 46 Histidine 23

Methionine 21 Serine 33

Threonine 32 Arginine 61

Isoleucine 17 Cysteine 22

Leucine 41 Tyrosine 16

Phenylalanine 11 Alanine 68

Valine 26 Aspartic acid 60

Glutamic acid 100

Glycine 124

Proline 72

Figure 2 Browning of the hydrolysates during the thermal treatment

with four ketohexoses. The control sample was carried out without the

addition of sugars. Different capital superscripts (A to E) denote the

significant difference (P < 0.05) in the browning of the different

reaction systems at the same heating time.




decreased, especially in the system of d-tagatose, inwhich the content of free amino groups was reduced to48.2% after heating for 48 h. Between the systems of d-psicose and d-sorbose, there were slight differences(P > 0.05) in the loss of free amino groups during thethermal treatment.To detect the radical scavenging ability of the MRPs,

TEAC and DPPH assays were carried out in aqueousand organic media, respectively. Although enzymatichydrolysates from tuna backbone could removeABTS+

•radicals well, their scavenging efficiencies could

still be promoted by the modification with sugars,especially rare sugars. As shown in Fig. 4, the scaveng-ing capacity of the four MRPs for ABTS+

•radicals

increased with heating time. The initial TEAC value ofthe hydrolysates was about 82 lmol equivalents ofTrolox per gram. After incubating with d-fructose andd-tagatose at 55 �C for 48 h, the TEAC value reached102 and 111 lmol equivalents of Trolox per gram,respectively. Moreover, the MRPs from d-psicose andd-sorbose showed a small distinction in the TEACvalue, which was in accordance with the observation ofbrowning and the loss of amino groups.The results of the DPPH assay differed from those of

the scavenging activity for ABTS+•radicals (Fig. 5).

First, the DPPH•radical scavenging activity of the

native hydrolysate was low. The radical scavengingactivity of the enzymatic hydrolysates for DPPH

•was

only 5.2% at 0.33 mg mL)1, whereas their radicalscavenging capacity for ABTS+

•was 52.4% at

0.08 mg mL)1. Second, the scavenging activity forDPPH

•during the Maillard reaction increased more

noticeably than that for ABTS+•. After heating for 48 h,

the antioxidant effect of the hydrolysates on ABTS+•

radicals only improved by about 24% and 35% in thepresence of d-fructose and d-tagatose, whereas that onDPPH

•radicals improved by about 8.9- and 16-fold.

Different radical scavenging capacities for DPPH•and

ABTS+•have also been found in other food by-

Figure 4 ABTS+•radical scavenging activity of the hydrolysates

during the thermal treatment with four ketohexoses (indicated as

equivalents of Trolox per gram). The control sample was carried out

without the addition of sugars. Different capital superscripts (A to E)

denote the significant difference (P < 0.05) in the free amino group

content of the different reaction systems at the same heating time.

Figure 5 DPPH· radical scavenging activity of the hydrolysates during

the thermal treatment with four ketohexoses. The control sample was

carried out without the addition of sugars. Different capital super-

scripts (A to E) denote the significant difference (P < 0.05) in the free

amino group content of the different reaction systems at the same

heating time.

Figure 3 Free amino group content in the hydrolysates during the

thermal treatment with four ketohexoses. The control sample was

carried out without the addition of sugars. Different capital super-

scripts (A to E) denote the significant difference (P < 0.05) in the free

amino group content of the different reaction systems at the same

heating time.




products. Nakkarike & Narayan (2008) have reportedthat fermented shrimp by-products exhibited 40%scavenging activity for DPPH

•at a concentration of

1.0 mg mL)1, compared with 95% activity againstABTS+

•at a concentration of 0.5 mg mL)1. Moreover,

the remarkable change of DPPH•scavenging activity in

the Maillard reaction has also been detected by otherresearchers. After heating at 40 mg mL)1 glucose con-centration for 4 h, residual DPPH

•radicals in the

presence of glycated cod viscera hydrolysate dramati-cally decreased from 95% to almost zero (Guerard &Sumaya-Martinez, 2003). The most probable explana-tion for the observation was that a substantial amountof molecules were generated during the Maillard reac-tion, and these molecules could readily react withDPPH

•in organic media.

Changes in reducing power in the Maillard reactionwere also detected, considering that MRPs could act asreducing agents by electron donation to form morestable products. As shown in Fig. 6, the reducing powerof the tuna hydrolysates continued to increase in theMaillard reaction with the four ketohexoses. The mostobvious growth trend was observed in the system of d-tagatose, in which the absorbance at 700 nm changedfrom 0.1 to more than 1.0 after heating for 48 h.Meanwhile, from beginning to end, d-psicose and d-sorbose exhibited similar effects on the reducing powerof the modified hydrolysates (P > 0.05).The characteristics and mechanisms of the three

assays indicated that the MRPs might efficiently elim-inate free radicals in organic media and act well aselectron donors in aqueous solutions. Furthermore, the

enhanced antioxidant activity was closely related to theprogress of the Maillard reaction. The browning or lossof amino groups, which indicated the Maillard reactionextent, had linear correlations with the improvement inantioxidant activity. In the system of d-tagatose, thecorrelation coefficient between the browning and thechange in ABTS+

•radical scavenging activity was 0.993

(P < 0.01, N = 5). In the system of d-fructose, thecorrelation coefficient between the loss of amino groupsand the improvement in reducing power was )0.968(P < 0.01, N = 5). It is worth noting that the enhancedantioxidant activity in the MRPs may be adjusted underother practical operating conditions because the Mail-lard reaction can be influenced by numerous factors (e.g.reactant concentration, temperature, heating time, ini-tial pH and so on). Sumaya-Martinez et al. (2005) havefound that the increase in DPPH

•radical scavenging of

MRPs from sugar-tuna stomach hydrolysate is not onlyattributed to the temperature but also to the buffer typeand buffer concentration. Similar phenomena have alsobeen found in glycated porcine haemoglobin hydroly-sates (Sun & Luo, 2011) and the silver carp (Hypoph-thalmichthys molitrix) protein hydrolysate-glucosesystem (You et al., 2011).

d-Psicose and d-sorbose, a pair of epimers isomerisedat the C-4 position (Fig. 1), played similar roles in theMaillard reaction of tuna hydrolysates. However, theeffects of d-tagatose and d-fructose, which were alsoisomerised at the C-4 position, were significantly differ-ent. Among the four ketohexoses, the MRPs of d-tagatose showed the best antioxidant activity, whereasthe MRPs of d-fructose had the weakest antioxidantactivity. Sun et al. (2006b) have found that in the non-enzymatic glycation between ovalbumin and seven d-aldohexoses, the configuration of OH groups at C-3 andC-4 in the sugars may be very important for theformation of MRPs and their antioxidant behaviours.The MRPs of d-allose, d-altrose and d-tagatose had thebest antioxidant activities in their corresponding tests.Hence, based on the configurations of these sugars, ahexose may perform better in the Maillard reactionwhen its OH groups at C-3 and C-4 are at the same side.According to Benjakul et al. (2005), the effect of aketohexose in glycation may be related to its relativestructural stability, including its mutarotation, openingof the hemiacetal ring and enolisation.

Conclusions

In the present study, enzymatic hydrolysates wereprepared from the fish by-product tuna backbone. TheMaillard reaction of the hydrolysates with rare sugars(d-psicose, d-sorbose and d-tagatose) was subsequentlyinvestigated. The hydrolysates had a large molecularweight distribution that ranged from 300 to 1000 Daand could be used as good supplements of essential

Figure 6 Reducing power of the hydrolysates during the thermal

treatment with four ketohexoses. The control sample was carried out

without the addition of sugars. Different capital superscripts (A to D)

denote the significant difference (P < 0.05) in the free amino group

content of the different reaction systems at the same heating time.




amino acids. Via the Maillard reaction, rare sugars(especially d-tagatose) induced a more remarkableimprovement than d-fructose in the radical scavengingactivity and oxidation–reduction potential of thehydrolysates. According to the good correlation be-tween the antioxidant activity and the Maillard reaction,the higher antioxidant activities of the MRPs from raresugars were directly related to the more effective role ofrare sugars in the Maillard reaction. In conclusion,proteolysis was confirmed to be an effective andvaluable way to utilise fish by-products. More impor-tantly, the Maillard reaction with rare sugars wasintroduced into the modification of fish raw materialsand demonstrated great potential in food industryapplications.

Acknowledgments

The research was supported by the National HighTechnology and Research Development Program(2009AA02Z201), National Natural Science Foundationof China (20972181) and the Academy-LocalityCooperation Project of Chinese Academy of Sciences(DJBJ-2011-019).

References

Ahn, C.B., Lee, K.H. & Je, J.Y. (2010). Enzymatic production ofbioactive protein hydrolysates from tuna liver: effects of enzymesand molecular weight on bioactivity. International Journal of Foodscience and technology, 45, 562–568.

Arvanitoyannis, I.S. & Giakoundis, A. (2006). Current strategies fordairy waste management: a review. Critical Reviews in Food Scienceand Nutrition, 46, 379–390.

Arvanitoyannis, I.S. & Kassaveti, A. (2008). Fish industry waste:treatments, environmental impacts, current and potential uses.International Journal of Food Science and Technology, 43, 726–745.

Arvanitoyannis, I.S. & Ladas, D. (2008). Meat waste treatmentmethods and potential uses. International Journal of Food Scienceand Technology, 43, 543–559.

Arvanitoyannis, I.S. & Tserkezou, P. (2008). Corn and rice waste: acomparative and critical presentation of methods and current andpotential uses of treated waste. International Journal of Food Scienceand Technology, 43, 958–988.

Arvanitoyannis, I.S., Kassaveti, A. & Stefanatos, S. (2007). Olive oilwaste treatment: a comparative and critical presentation of methods,advantages and disadvantages. Critical Reviews in Food Science andNutrition, 47, 187–229.

Benjakul, S., Visessanguan, V., Phongkanpai, V. & Tanaka, M. (2005).Antioxidative activity of caramelisation products and their pre-ventive effect on lipid oxidation in fish mince. Food Chemistry, 90,231–239.

Church, F.C., Swaisgood, H.E., Porter, D.H. & Catignani, G. (1983).Spectrophotometirc assay using o-phaldialdehyde for determinationof proteolysis in milk and isolated milk proteins. Journal of DairyScience, 66, 1219–1227.

FAO ⁄WHO. (1989). Protein quality evaluation: report of a jointFAO ⁄WHO expert consultation held in Bethesda. MD, USA, Dec4–8.

Granstrom, T.B., Takata, G., Tokuda, M. & Izumori, K. (2004).Izumoring: a novel and complete strategy for bioproduction of raresugars. Journal of Bioscience and Bioengineering, 97, 89–94.

Guerard, F. & Sumaya-Martinez, M.-T. (2003). Antioxidant effects ofprotein hydrolysates in the reaction with glucose. Journal of theAmerican Oil Chemists’ Society, 80, 467–470.

Guerard, F., Dufosse, L., Broise, D.D.L. & Binet, A. (2001).Enzymatic hydrolysis of proteins from yellowfin tuna (Thunnusalbacares) wastes using Alcalase. Journal of Molecular Catalysis B:Enzymatic, 11, 1051–1059.

Guerard, F., Guimas, L. & Binet, A. (2002). Production of tuna wastehydrolysates by a commercial neutral protease preparation. Journalof Molecular Catalysis B: Enzymatic, 19, 489–498.

Hodge, J.E. (1953). Dehydrated foods, chemistry of browning reac-tions in model systems. Journal of Agricultural and Food Chemistry,1, 928–943.

Hou, H., Li, B., Zhao, X., Zhang, Z. & Li, P. (2011). Optimization ofenzymatic hydrolysis of Alaska pollock frame for preparing proteinhydrolysates with low-bitterness. LWT – Food Science and Technol-ogy, 44, 421–428.

Izumori, K. (2002). Bioproduction strategies for rare sugars. Natur-wissenschafte, 89, 120–124.

Je, J.-Y., Qian, Z.-J., Byun, H.-G. & Kim, S.-K. (2007). Purificationand characterization of an antioxidant peptide obtained from tunabackbone protein by enzymatic hydrolysis. Process Biochemistry, 42,840–846.

Je, J.-Y., Lee, K.-H., Lee, M.H. & Ahn, C.-B. (2009). Antioxidantand antihypertensive protein hydrolysates produced from tunaliver by enzymatic hydrolysis. Food Research International, 42,1266–1272.

Klomklao, S., Benjakul, S., Visessanguan, W., Kishimura, H. &Simpson, B.K. (2007). Purification and characterisation of trypsinsfrom the spleen of skipjack tuna (Katsuwonus pelamis). FoodChemistry, 100, 1580–1589.

Langley, A., Wright, A., Hurry, G., Hampton, J., Aqorua, T. &Rodwell, L. (2009). Slow steps towards management of the world’largest tuna fishery. Marine Policy, 33, 271–279.

Levin, G.V. (2002). Tagatose, the new GRAS sweetener and healthproduct. Journal of Medicinal Food, 5, 23–36.

Matsuo, T., Suzuki, H., Hashiquchi, M. & Izumori, K. (2002). d-Psicose is a rare sugar that provides no energy to growing rats.Journal of Nutritional Science and Vitaminology, 48, 77–80.

Nakkarike, M.S. & Narayan, B. (2008). In vitro antioxidant activity ofliquor from fermented shrimp biowaste. Bioresource technology, 99,9013–9016.

Naqash, S.Y. & Nazeer, R.A. (2011). Evaluation of bioactiveproperties of peptide isolated from Exocoetus volitans backbone.International Journal of Food science and Technology, 46, 37–43.

Nguyen, H.T.M., Sylla, K.S.B., Randriamahatody, Z. et al. (2011).Enzymatic hydrolysis of yellowfin tuna (Thunnus albacares) by-products using Protamex protease. Food Technology and Biotech-nology, 49, 48–55.

Nielsen, P.M., Petersen, D. & Dambmann, C. (2001). Improvedmethod for determining food protein degree of hydrolysis. Journal ofFood Science, 66, 642–646.

Oliver, C.M., Melton, L.D. & Stanley, R.A. (2006). Creating proteinswith novel functionality via the Maillard reaction: a review. CriticalReviews in Food Science and Nutrition, 46, 337–350.

Oyaizu, M. (1986). Studies on products browning reaction, antioxida-tive activity of products of browning reaction prepared fromglucosamine. Japanese Journal of Nutrition, 44, 307–315.

Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M. & Rice-Evans, C. (1999). Antioxidant activity applying an improved ABTSradical cation decolorization assay. Free Radical Biology andMedicine, 26, 1231–1237.

Sanmartin, E., Arboleya, J.C., Villamiel, M. & Moreno, F.J. (2009).Recent advances in the recovery and improvement of functionalproteins from fish processing by-products: use of protein glycationas an alternative method. Comprehensive Reviews in Food Scienceand Food Safety, 8, 332–344.




Skytte, U.P. (2006). Tagatose. In: Sweeteners and Sugar Alternatives inFood Technology (edited by H. Mitchell). Pp. 276–280. Oxford, UK:Blackwell.

Slizyte, R., Mozuraityte, R., Martınez-Alvarez, O., Falch, E., Fou-chereau-Peron, M. & Rustad, T. (2009). Functional, bioactive andantioxidative properties of hydrolysates obtained from cod (Gadusmorhua) backbones. Process Biochemistry, 44, 668–677.

Sui, L., Dong, Y.Y., Watanabe, Y. et al. (2005). The inhibitory effectand possible mechanisms of d-allose on cancer cell proliferation.International Journal of Oncology, 27, 907–912.

Sumaya-Martinez, M.T., Thomas, S., Linard, B., Binet, A. & Guerard,F. (2005). Effect of Maillard reaction conditions on browning andantiradical activity of sugar–tuna stomach hydrolysate modelsystem. Food Research International, 38, 1045–1050.

Sun, Q. & Luo, Y. (2011). Effect of Maillard reaction conditions onradical scavenging activity of porcine haemoglobin hydrolysate–sugar model system. International Journal of Food Science &Technology, 46, 358–364.

Sun, Y., Hayakawa, S. & Izumori, K. (2004). Antioxidative activityand gelling rheological properties of dried egg white glycated with arare keto-hexose through the Maillard reaction. Journal of FoodScience, 69, 427–434.

Sun, Y., Hayakawa, S., Puangmanee, S. & Izumoring, K. (2006a).Chemical properties and antioxidative activity of glycated a-lactal-

bumin with a rare sugar, d-allose, by Maillard reaction. FoodChemistry, 95, 509–517.

Sun, Y., Hayakawa, S., Chuamanochan, M., Fujimoto, M., Innun, A.& Izumori, K. (2006b). Antioxidant effects of Maillard reactionproducts obtained from ovalbumin and different d-aldohexoses.Bioscience Biotechnology and Biochemistry, 70, 598–605.

Vidotti, R.M., Viegas, E.M.M. & Carneiro, D.J. (2003). Amino acidcomposition of processed fish silage using different raw materials.Animal Feed Science and Technology., 105, 199–204.

Wu, T.H., Nigg, J.D., Stine, J.J. & Bechtel, P.J. (2001). Nutritionaland chemical composition of by-product fractions produced fromwet reduction of individual red salmon (Oncorhynchus nerka) headsand viscera. Journal of Aquatic Food Product Technology., 20, 183–195.

Yen, G.C. & Hsieh, P.P. (1995). Antioxidative activity and scavengingeffects on active oxygen of xylose-lysine maillard reaction-products.Journal of the Science of Food and Agriculture, 67, 415–420.

You, J., Luo, Y., Shen, H. & Song, Y. (2011). ffect of substrate ratiosand temperatures on development of Maillard reaction and antiox-idant activity of silver carp (Hypophthalmichthys molitrix) proteinhydrolysate-glucose system. International Journal of Food Scienceand Technology, 46, 2467–2474.




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