at SciVerse ScienceDirect

Food Hydrocolloids 28 (2012) 267e274

Contents lists available

Food Hydrocolloids

journal homepage: www.elsevier .com/locate/ foodhyd

Functional properties of isolated porcine blood proteins modifiedby Maillard’s reaction

Carlos Álvarez, Vanessa García, Manuel Rendueles, Mario Díaz*

Department of Chemical Engineering and Environmental Technology, University of Oviedo, C/Julián de Clavería n� 8, 33006 Oviedo, Spain

a r t i c l e i n f o

Article history:Received 1 August 2011Accepted 3 January 2012

Keywords:Maillard’s reactionProteinedextran conjugatesFunctional propertiesPlasma proteinsHaemoglobin

* Corresponding author. Fax: þ34 985103434.E-mail address: mariodiaz@uniovi.es (M. Díaz).

0268-005X/$ e see front matter � 2012 Elsevier Ltd.doi:10.1016/j.foodhyd.2012.01.001

a b s t r a c t

Plasma proteins (albumin and immunoglobulins) and haemoglobin from porcine blood can be recoveredfrom slaughterhouse waste. These proteins are employed as ingredients in food products on account oftheir functional properties. There are different methods to improve these properties, Maillard’s reactionprobably being the most promising technique for food purposes. In the present study, 10 kDa dextranwasemployed to produce the conjugates. Three reaction temperatures were assayed, 80 �C for 60 min beingfound to be the most suitable to produce conjugates with enhanced functional properties. Both thermalstability and emulsification capacity were improved; gelling temperature was increased 15 �C; and gelstrength was lowered 50% compared to native proteins. However, solubility decreased slightly. It hasbeen demonstrated that the functional properties of blood proteins are enhanced through conjugation,showing a improvement in the application of the blood proteins in food products.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Research into applications for blood proteins originating fromslaughterhouse waste gives rise to several possibilities for theirrecovery and use. The use of proteins as a food component has beenwidely reported due in part to their high nutritional value, as wellas to their functional properties. The development of new tech-niques and tools to improve functional properties (Oliver, Melton, &Stanley, 2006) has resulted in new production methods such asacetylation, deamidation, succinylation and different types ofchemical modification that lead to changes in the structure of theprotein and hence in its chemical and physical properties. It hasbeenwidely reported and established that the functional propertiesof proteins can be improved by covalent binding of severalcompounds, the most suitable system in the food industry beingthe use of polysaccharides or monosaccharides (Akhtar &Dickinson, 2003; Aoki, Hiidome, Sugimoto, Ibrahim, & Kato, 2001;Campbell, Raikos, & Euston, 2003; Sun, Hayakawa, & Izumori,2004). Hence, the use of the Maillard reaction is one of the mostpromising techniques for foodstuff applications seeing as themethod does not require the addition of chemical products that areincompatible with food industry requirements.

In the meat industry, functional properties such as emulsifica-tion and gelification are desirable. The former property is employed

All rights reserved.

in the elaboration of restructured products such as sausages andmortadella. Gelling properties are used in products that requiremajor retention of water, e.g. minced meat, surimi and bacon. Goodthermal stability enables the use of processes involving the appli-cation of high temperatures (pasteurization or cooking) withoutthe loss of other functional properties.

The early stages of Maillard’s reaction involve condensationbetween the carbonyl group of a reducing carbohydrate with anavailable amino group, mainly the amino group of the lysine resi-dues in proteins. With aldoses, such as glucose, this leads to a Schiffbase with a release of water. The Schiff base subsequently cyclizesto the corresponding N-glycosylamine, which then undergoes anirreversible Amadori rearrangement to produce the Amadoricompound. The next stage involves the degradation of the Amadoricompound via divergent pathways to afford a large array of poorlycharacterized compounds. The last stage takes place when highlycoloured insoluble nitrogen-containing polymeric compounds areformed. For food applications, this last stage must be avoided.Therefore, the Maillard reaction must be carried out undercontrolled conditions.

Conjugation of carbohydrates and several proteins such as wheyproteins, egg proteins, lysozyme, soy proteins or bovine serumalbumin (BSA) has been reported previously (Bautista et al., 2000;Kato, Murata, & Kobayashi, 1993; Rich & Foegeding, 2000).However, protein fractions from porcine plasma have not yet beenreported.

In a previous study, the authors reported the functional prop-erties of fractions from plasmatic proteins and haemoglobin from

C. Álvarez et al. / Food Hydrocolloids 28 (2012) 267e274268

porcine species (Alvarez, Bances, Rendueles, & Díaz, 2009). The fourfractions from blood plasma are composed of fibrinogen, gammaglobulins, alpha and beta globulins and albumin. The use of eachprotein in the food industry has been determined in accordancewith these properties. In the present study, the improvement ofthese functional properties was carried out via the Maillard reac-tion by linking the proteins with dextran. 10 kDa dextran wasemployed on the basis of previous research (Jimenez-Castaño,Villamiel, & López-Fandiño, 2007) which confirmed that thisdextran size blocked the higher amounts of available lysines inindividual whey proteins.

The aim of this study was to produce and characterize conju-gates obtained via the Maillard reaction between dextran and themain protein fractions and haemoglobin of porcine blood. Once theconjugates had been produced in suitable conditions, the subse-quent goal was to evaluate their functional properties so as toascertain the feasibility of these compounds as additives in food-stuffs, especially in meat products.

2. Materials and methods

2.1. Plasma fractionation

Blood from porcine species was obtained from Junquera BobesCo. slaughterhouse (Noreña, Asturias, Spain). This was collecteddirectly from the animal in plastic bottles using sodium citrate as ananticoagulant at a concentration of 1.5% w/v of the total samplevolume. The blood was then centrifuged at 3000� g for 15 min at6e8 �C to separate the plasma (60%) from the cells (40%) ina Kubota 6700 centrifuge using 250 mL test tubes. The plasma thusobtained must be kept at 4 �C to avoid bacterial proliferation.

Plasma was fractionated following a modification of the CohnMethod (Cohn et al., 1946) based on differential solubility usingethanol as the fractionation agent. The plasma was diluted to 50%with distilled water to obtain a final protein concentration ofbetween 30 and 35 mg/mL. A volume of diluted plasma (200 mL)was then taken and ethanol was added progressively until reachinga desired concentration. In our case, four fractionation steps wereperformed using this method: the first with an ethanol concen-tration of 8% v/v and 7.2 pH to precipitate fibrinogen (Fraction I orFI); the second using 19% ethanol and the same pH to obtain g-globulins (Fraction II or FII); the third (40% ethanol and 5.5 pH) toobtain a- and b-globulin (Fraction III or FIII); and, finally, albumin(Fraction IV or FIV) was precipitated with an ethanol concentrationof 40% v/v, decreasing the pH to 4.5. Each precipitation step wascarried out for 3 h, maintaining these conditions for 30 min to reachequilibrium. All temperature steps were close to 0 �C to minimizeprotein denaturation due to ethanol. On completion of eachprecipitation, the phases were separated by centrifugation at10,000� g for 10 min and the precipitated proteins were re-dissolved in a phosphate buffer at pH 7.5. Finally, the fractionswere freeze dried (Telstar Cryodos) and kept at �20 �C until use. Itwas determined by means of the Kjeldahl method that the powderwas composed of �95% protein.

The haemoglobin was extracted from red cells by osmotic shockby adding distilled water (1:1 v/v) and gently mixing the solutionfor 5 min. Chloroform was then added (1:4, v/v chloroform/aqueous red cell solution) and the solution was gently stirred for5 min and centrifuged (5000� g, 10 min) to remove plasmamembranes from the media. The supernatant is composed of anaqueous phase containing the haemoglobin, which is recoveredand freeze dried (Telstar Cryodos) and stored at �20 �C until use.Haemoglobin purity was determined at �98% by the Kjeldahlmethod.

2.2. Preparation of proteinedextran conjugates

Dextran, with an average molecular size of 10 kDa produced byLeuconostoc sp., was supplied by SIGMA. Lyophilized proteins weredissolved in a 0.1 phosphate buffer (pH 7.6), subsequently addingdextran to the solution in a weight proportion of 1:3 protein/dextran. This proportion was chosen on the basis of bibliographicdata (Jung, Choi, Kim, & Moon, 2006), having been shown to be themost suitable to afford a good yield in the conjugation of BSA anddextran. The protein/dextran mixture was freeze dried to obtaina homogeneous powder composed of protein and carbohydrates.Four grams of powder was placed in hermetic vessels and heated to70, 75 and 80 �C to determine the influence of temperature on theconjugation process. This range of temperatures was chosen on thebasis of previous research, which confirmed that 24 h at 60 �C wererequired to obtain a high conjugation yield (Jimenez-Castaño et al.,2007), while only 2 h were required at 80 �C (Akhtar & Dickinson,2003). Samples were taken at different times to monitor the reac-tion. The process was carried out twice in order to obtain dupli-cates. A blank employing only protein was likewise run.

2.3. SDS-polyacrylamide gel electrophoresis and glycoprotein stain

SDS-PAGE was performed using a 12% acrylamide separationgel. Conjugates (15 mL, 4 mg/mL) were prepared in a TriseHCl 0.1 Mbuffer at 6.8 pH. Electrophoresis was carried out for 60 min at 200 Vin a TriseGlycine buffer. The gels weremade in duplicate, one beingstained with Coomassie Blue and the other using the GelCode�

Glycoprotein staining kit supplied by Pierce Biotechnology.

2.4. Determination of free amino groups

Free amino groups were determined so as to ascertain thedegree of conjugation of the proteins. The trinitrobenzene acidmethod developed by Fields (1971), was employed for this purpose,mixing 500 mL of sample with 0.5 mL of potassium borate buffer0.11 M (pH 9.1) and 20 mL of TNBS 1.8 M. The mixture was kept at25 �C for 5 min. 2 mL of buffer was added (0.1 sodium phosphatemonobasic, 1.5 mM sodium sulphite) to stop the reaction and theabsorbance was measured at 420 nm on a spectrophotometer.Leucine standards were prepared to calibrate the method, in the0.21e2.1 mM range.

2.5. Solubility

The solubility of conjugates was studied in aqueous media.Protein solubility was determined by the method reported by DeVouno, Penteado, Lajolo, and Pereira dos Santos (1975). Thisconsists in dissolving 0.5 g of the native or conjugated proteinsample in 10 mL of distilled water. The solution was centrifuged at2400� g for 30 min in a Kubota Model 6700 centrifuge. pH wasvaried to test its effect on solubility. Different pH solutions wereprepared by adding HCl or NaOH 1 N to the solution to reach thedesired pH, the tested pH ranging between 3 and 8. The amount ofsoluble protein before and after centrifugation was determined bythe Lowry method. All the experiments were carried out in tripli-cate. Solubility was calculated as follows:

%S ¼ PdPt

� 100 (1)

where %S is the percentage of solubility, Pd the amount of solubleprotein (g) and Pt the amount of total protein (g) used in the assay.

C. Álvarez et al. / Food Hydrocolloids 28 (2012) 267e274 269

2.6. Thermal stability

In order to determine the thermal stability (Jimenez-Castañoet al., 2007) of the conjugates, 1 mg/mL solutions of the conju-gates and native protein were prepared. The fact that the conju-gates are formed by dextran and protein in 1:3 w/w proportionwastaken into account when calculating the results. pH was adjusted to4 or 7 using HCl or NaOH 1 M, respectively. The samples were thenheated to 85 �C for 15 min, cooled at room temperature and finallycentrifuged for 5 min at 3500� g. The conjugated concentration inthe supernatant obtained was measured by Lowry’s method. Thepercentage of stability was calculated as:

% Thermal estability ¼ CpoCpt

� 100 (2)

where Cpo is the conjugated concentration in the supernatant andCpt is the initial conjugated concentration.

2.7. Emulsifying capacity

Emulsifying capacity was determined by the Inklaar and Fortuin(1969) method. Freeze-dried native proteins and conjugates wereused in concentrations of 4 and 10 mg/mL in a final volume of10 mL. pH was not adjusted, as previous experiments showed thatthis value was situated between 6.5 and 7.5, depending on theprotein employed. The solvent used to prepare the samples wasdistilled water with NaCl 0.075% w/v. This salt increases theemulsifying capacity of the protein because protein folding isinduced. Subsequently, 13 mL of cotton oil was added to eachsample. After stirring for 15 min at 1000 rpm in a magnetic stirrer,the sample was centrifuged at 1200� g for 10 min (Kubota 6700centrifuge), the non-emulsified oil being decanted andmeasured ina suitable measuring cylinder. The percentage of emulsificationwascalculated as:

%E ¼ VeVa

� 100 (3)

where %E is the percentage of emulsification, Ve the volume of theemulsified oil (mL) and Va the volume of the added oil (mL).

2.8. Gelling properties

The gelling properties of the conjugates were measured in threeways analyzing: a) the lowest gelation concentration (LGC)(Coffman & García, 1977); b) the temperature of gelification (Tgel);and c) the strength of the gel (G0 measured in pascals). A range ofconcentrations needs to be tested to determine the LGC of eachconjugate. In this study, the tested range was 1e16% w/v aqueoussolutions of freeze-dried conjugate. pH was adjusted to 6 usingNaOH or HCl and the solutions were heated in a thermostatic bathto 85 �C for 30 min. After heat-induced gelation, samples werecooled and stored at 4 �C for 24 h. The LGC is the lowest concen-tration of protein in the tested range in which the test tube isinverted and the gel does not slide; i.e. it is consistent.

The Tgel measurements were carried out using a rheometer(Haake MARS II) fitted with a plateeplate system and a 1 mm gapbetween plates. Freeze-dried conjugates and native proteins weredissolved in water to achieve a final concentration of 10% w/w andthe pHwas adjusted to 6, as in the LGC tests (Dávila, Parés, Cuvelier,& Relkin, 2006). Temperature sweeps at 2 �C/min were recordedfrom 20 �C to 90 �C and samples were covered with mineral oil toprevent evaporation. The deformation (g) was adjusted to 1% andthe oscillation frequency was 1 rad/s. The storage modulus (G0) andelastic modulus (G00) were recorded. The Tgel is determined when

a crossover between the plots of G0 and G00 takes place (Lamsal, Jung,& Johnson, 2007). When the temperature reaches 90 �C, the G0

modulus is recorded to determine the strength of the gel formed.This value is called G0

max.

2.9. Differential scanning calorimetry (DSC)

Differential scanning calorimetry tests were carried out inhermetically sealed aluminium pans. A Mettler-Toledo DSC 822eapparatus was used, implementing temperature ramps from 25 �Cto 120 �C at a heating rate of 0.6 �C/min in a nitrogen atmosphere.Denaturation temperatures were determined using the equipmentsoftware. DSC measurements were performed for 12% w/v solu-tions of conjugates and native proteins (Laca, Paredes, & Díaz,2010).

2.10. Isothermal microcalorimetry (IMC)

An isothermal calorimetric experiment was conducted tomeasure the variation in energy produced in the conjugation ofhaemoglobin and dextran. A mixture of freeze-dried haemoglobinand dextran (1:3 w/w) was employed. This protein was chosen asthe model because it is the only fraction formed by one singleprotein. The instrument used was a Mettler-Toledo CSC 4400apparatus. The temperature was fixed at 60 �C. The sampleemployed (800 mg) was deposited in a 40 mL volume glass cell.

3. Results and discussion

3.1. Effect of temperature on conjugation

Temperature is one of the most important factors affectingMaillard’s reaction. In order to determine the appropriatetemperature to obtain a conjugate in a short time, conjugation wascarried out at different temperatures: 70, 75 and 80 �C. Theevolution of the reaction was monitored analyzing the free aminogroups detected in each sample over the experimental period. Fig. 1shows the decrease in the amounts of free amino groups (mM,equivalent to the calibration curve of leucine).

It can be seen that the decrease in free amino groups at thelower temperatures (70 and 75 �C) is more marked than at thehighest temperature. This fact is appreciable in all the cases, exceptin Fraction IV, where the values are practically the same. Thedecrease is more evident in Fractions II and IV, reaching values forthe conjugated free amino group of 60% and 70%, respectively. Ascan be observed, an increase in 5 �C produces an increase in thekinetic reaction that reduces the time employed by half, except inthe case of Fraction III, where the temperature does not seem tohave a clear-cut effect. In the experiment carried out at 70 �C, atleast 4 h were needed to obtain a stable minimum value of freeamino groups. The assays carried out at 75 and 80 �C reach thisvalue of free amino groups after 2e4 h and 1.5e2 h, respectively,depending on the fraction studied. Table 1 shows the difference infree amino groups between native protein and conjugated proteinonce a stable value of amino groups is reached.

The different value obtained in each fraction is due to the aminoacid composition of each protein. Conjugation takes place prefer-entially between the sugar and lysine and secondarily with histi-dine, tryptophan and arginine (Aoki et al., 2001). If the amino acid islocated inside the protein structure, there are spatial impedimentsto the conjugation and hence not all the free amino groups can beconjugated with dextran.

The haemoglobin sequence contains 44 residues of lysine anda total of 110 free amino groups suitable for conjugation withcarbohydrates. The data in Table 1 show how all the amino groups

Fig. 1. Evolution of free amino groups with the time for the fractions proteins time in the assayed temperatures. 70 �C: ; 75 �C: and 80 �C: .

C. Álvarez et al. / Food Hydrocolloids 28 (2012) 267e274270

in the haemoglobin are free at the outset. Finally, over 20 residuesconjugated with dextran at 70 and 75 �C, though only 10 of thesereacted with dextran at 80 �C.

These results suggest that a greater degree of conjugation can bereached at low temperatures, but the time employed to achieve thisis longer compared to that required at 80 �C.

3.2. SDS-PAGE electrophoresis of proteinedextran conjugates

To confirm that the decrease in free amino groups is related tothe formation of glycoproteins, electrophoresis was performed inan acrylamide gel. The gel was stained in two different ways: totalprotein, and specifically for glycoproteins. These gels are shown inFigs. 2 and 3.

Fig. 2A shows in the first line the proteins of the fraction II, andtheir conjugates throughout the reaction time, composed mainlyfor alpha and beta globulins. Fig. 2B was composed by samesamples than 2A, but stained following a protocol that only revealsglycoproteins. It can be seen how the glycoproteins are onlyrevealed after 2 h of heating, their concentration and sizeincreasing with reaction time. No glycoproteins are detected in

Table 1Free amino groups (mM) detected in native and conjugated protein. In brackets thetime at which maximum blockage is reached, giving in minutes. Column b for Hbindicates the ratio between the number of molecules of free amino groups and thenumber of molecules of haemoglobin.

Temp., �C FII FIII FIV Hb

a b

Initial 70 0.22 0.21 0.25 0.44 11275 0.23 0.21 0.24 0.45 11580 0.21 0.28 0.24 0.44 112

Final 70 0.08 (240) 0.15 (180) 0.11 (240) 0.35 (240) 8975 0.10 (135) 0.15 (180) 0.09 (225) 0.36 (225) 9280 0.10 (90) 0.22 (145) 0.08 (120) 0.39 (90) 102

% Decrease 70 63.64 28.57 66.00 19.2375 56.52 28.57 70.00 20.0080 51.88 21.43 68.00 11.32

lanes 2e4. In the final lines (3e7), the same sample reveals theappearance of conjugated proteins that have a molecular sizegreater than 120 kDa. Moreover, not all the proteins present thesame degree of conjugation with the time. Proteins of severalmolecular size can be detected in accordance with the differentnumber of conjugated dextranmolecules. This fact is revealed in gellines (3e7) with the appearance of smearing, resulting from theformation of a molecular size gradient of conjugated proteins. InFig. 2B is showed the same molecular size gradient. More gels werecarried out under same conditions and always was detected thissmearing, meaning that sample was formed by proteins of all sizesin the range from 50 to more than 120 kDa. In any case, in finalsample the most part of the proteins can be detected in the upperpart of the gel, meaning a high yield of protein conjugation.

Fig. 3 shows the final size distribution of haemoglobin conju-gates at different temperatures. The detected bands reveal how theprofile of molecular sizes is quite similar at 75 and 80 �C, whereasthe band indicated with an arrow is not produced at the lowertemperatures.

From the point of view of industrial application, a compromisemust be reached between the time employed and the decrease infree amino groups. Thus, 75 �C is found to be the best choice toachieve conjugation with an industrial application. However,bearing in mind the similar patterns obtained in SDS-PAGE and thetime required for conjugation at 80 �C, the latter temperature waschosen in this study to create the samples analyzed.

3.3. Solubility

Considering that this property is one of the most important inproteins that are to be used as food additives in meat products andbearing in mind its influence on the remainder of the functionalproperties measured, solubility was the first parameter to beevaluated. The methodology employed has already been describedin a previous section of the paper.

The solubility values of native and conjugated proteins arecompared at different pH in Fig. 4. Solubility decreased after proteinconjugation in all the protein fractions studied.

Fig. 2. Evolution of the conjugation at 80 �C of Fraction II. Total protein staining (A) and specific glycoprotein staining (B). First lane is the weight marker; lane 2 is the native sample,while lanes 3e7 are the evolution of the conjugation process at different times: 0.5, 1, 2, 6 and 24 h.

C. Álvarez et al. / Food Hydrocolloids 28 (2012) 267e274 271

Most likely, this loss in solubility is caused by the high degree ofcross-linking achieved between proteins. This effect results inprecipitation of proteins, more copious than that observed in nativeproteins. It has been reported (Katayama, Shima, & Saeki, 2002;Saeki & Inoue, 1997) that only certain degrees of conjugation aresuitable for producing an increase in solubility and that this degreeis not necessarily the highest level that may be reached. In ourstudy, the protein was conjugated until the number of free aminogroups remained stable. Hence, if solubility weremeasured at shortconjugation times, better results would be obtained. Nonetheless,the decrease in solubility is always less than 15%, except in the caseof haemoglobin, the solubility of which decreases by around 80%.

3.4. Thermal stability

This property is important to ascertain whether the proteins areable to resist thermal treatment, which is very widely used in thefood industry. Such treatment can damage the protein, which losesits structure and precipitates, as well as causing a decrease in itsnutritional value. This functional property was studied at pH 4 and7, usual conditions in the food industry. The results obtained (Fig. 5)show the stability of the treated proteins before and after conju-gation with dextran.

Thermal stability is enhanced considerably in the conjugatedprotein compared to the native protein for all the fractions ofproteins tested, at both pH. The plasmatic Fractions II and III behavevery similarly. The resistance to thermal treatment of Fraction IV(composed mainly of albumin) increased markedly; the effectobserved at a neutral pH being noteworthy. In this particular case,practically all the native protein became insoluble (close to 90%),whereas a high proportion of the conjugated albumin remained in

the soluble state (almost 40%). On the other hand, Fraction IV wasmore stable at pH¼ 4, although the conjugated fraction showedgreater stability than its native counterpart.

Finally, the isolated haemoglobin presents a very similar profileto the albumin fraction. However, haemoglobin is less resistant atacid pH, though more stable at neutral pH than albumin.

Despite native proteins being more soluble than conjugates atroom temperature, conjugates show better resistance to aggrega-tion and subsequent precipitation when thermal treatment isapplied. Dextran prevents aggregation, because it hindersproteineprotein interactions and protects the native structure ofthe protein. Dextran is thought to decrease the amount of solventavailable for the protein, so the protein is forced to stay ina compact, steady state, thus preventing precipitation (Sasahara,McPhie, & Minton, 2003). This effect is known as the crowdingeffect. These results indicate that proteins linked to dextranincrease their resistance to thermal treatment; which is very usefulto maintain the properties of proteins during the different thermaltreatments employed in the food industry.

3.5. Gelling properties

This functional property was studied under three differentparameters: LGC (lowest gelifying concentration), Tgel (tempera-ture of gelification) and maximum strength of the gel, representedby the G0

max module. The results obtained for these tests are shownin Table 2.

It must be stressed that although the apparent LGC of conju-gated proteins worsened, the total protein in the conjugatedcomplex is 25% of the total amount of the gelling product employed(the complex was prepared in a protein:dextran proportion of 1:3).

Fig. 3. Size evolution of conjugates of haemoglobin and dextran at different temper-atures. Lanes 1, 4 and 7 show native haemoglobin; lanes 2e3, 5e6 and 8e9 areduplicates of the samples obtained at the end of the experiment at 70, 75 and 80 �C,respectively.

Fig. 5. Variation of protein thermal stability after dextran conjugation at pH 4 and 7conjugated at pH 4: ; native at pH4: ; conjugated at pH 7: and native at pH 7: .

C. Álvarez et al. / Food Hydrocolloids 28 (2012) 267e274272

Thus, the LGC remains constant if only the amount of protein isconsidered. Furthermore, the native haemoglobin did not producegels in the concentration ranges studied, although the conjugatedhaemoglobin was able to do so.

In the case of Tgel, variation in this parameter can be observed.In every case, the temperature needed to produce a gel increasedaround 15 �C. This makes it possible to widen the range oftemperatures under which a gel can be formed, with gel productionfalling within the 45e82 �C range. This parameter is related to theimproved thermal resistance achieved after conjugation. Thedextran exerts an effect that protects the proteins from precipita-tion and loss of their tertiary structure (shown as thermal stabilityin previous section), effects that are indispensable to form a gel.

Fig. 4. Solubility of plasmatic fractions and haemoglobin native and conjugated. Solidlines represents native protein, dot lines represents conjugated protein. FII: ; FIII: ;FIV: and haemoglobin: .

Hence, a higher temperature is required to unfold the protein andconsequently form the gel.

The lower strength observed in the gels formedwith conjugatedproteins is due to the spatial position of dextran between theprotein molecules. The values tested in native proteins are in linewith those reported in the literature (Cordobés, Partal, & Guerrero,2004; Selmane, Christophe, & Gholamreza, 2008). The decrease inthis parameter is more noticeable in conjugates made with hae-moglobin (around 85%). The effect of dextran is not so notable inthe rest of the proteins assayed, affording gels only 50% weakerthan those formed with native proteins. The molecular size ofdextran is sufficient to prevent good proteineprotein interaction,which is the response of the strength of the gel formed with nativeproteins. The same effect that decreases the solubility of theconjugates causes the loss in strength in the formed gels.

The storage modulus during heating presents an identicalprofile in all the cases studied. A continuous increase in the value ofG0 is observed above the Tgel point (where the value of G0 is alwaysless than 1 Pa). The slope of the curve depends on the type ofsample used, being more pronounced when native proteins wereanalyzed and resulting in higher strengths being detected.

It may be concluded that the conjugates can be used as ingre-dients in foodstuffs that require thermal treatment, preventingprotein precipitation or degradation. The range of temperatures toobtain a protein gel and the strength of such a gel increases withthe conjugation process.

3.6. DSC analysis

This technique is widely used to study thermal transitions offood proteins, providing information on the modification of nativeprotein to its heat-denatured state. This phenomenon is accom-panied by a significant uptake of heat, which is observed asa negative peak on the DSC thermogram (Cordobés et al., 2004).

The transition temperatures of each conjugated protein areshown in Table 3. It can be seen that glycation of the proteinsproduces a 15% increase in transition temperature in the case of

Table 2Variation of LGC and Tgel between native and conjugated proteins.

LGC % (p/v) Tgel (�C) G0max (Pa)

Native Conjugated Native Conjugated Native Conjugated

FII 2 10 44.8 69.6 40,000 18,500FIII 2 10 56.9 71.8 47,000 25,800FIV 4 12 67.7 82.4 4200 1800Haemoglobin n.d. 14 59.5 77.2 23,500 3750

Table 3Native and conjugated proteins transition temperature. Data showed in Celsiusgrades.

Native Conjugated

FII 71.4 82.4FIII 81.7 87.6FIV 80.1 91.3Haemoglobin 77.7 89.0

C. Álvarez et al. / Food Hydrocolloids 28 (2012) 267e274 273

haemoglobin and of 14% in the case of Fraction IV. These results arein agreement with the data shown previously for the Tgeltemperature and thermal stability. It has been demonstrated thatconjugation produces proteins that are more resistant to thermaltreatments, thus preventing their precipitation and gelification.

The highest temperature observed in the DSC test, comparedwith Tgel measurements, has been reported in other studies in theliterature (Laca et al., 2010; Saeki & Inoue, 1997). The lowesttemperature obtained in the rheological test is related to the firststage of protein precipitation, an increase in proteineproteininteractions and an aggregation that leads to the formation ofa gel network. On the other hand, the highest peak temperaturedetected in the DSC test is related to the denaturation temperature,an effect that mainly occurs just after gel formation.

3.7. Emulsifying capacity

A priori, this property is one of the most enhanced after theMaillard reaction, seeing as the protein is an amphipathic moleculeand the linking to dextran (which is strongly hydrophilic) increasesthis property. The protein hydrophobic groups are able to adsorbthe lipid phase and the dextran can easily solvate the water phase.The emulsifying capacity was measured in order to confirm thiseffect. The results are shown in Fig. 6.

When a final concentration of conjugated protein of 10 mg/mL isused, there is a significant increase in emulsifying capacity. Themodified globulins (Fractions II and III) were able to emulsify the oiladded to the medium completely. Fraction IV possesses a very lowcapacity to produce emulsions, although conjugation with dextranincreases this capacity from 28% to 42% of total oil volume. Finally,haemoglobin shows the best results, when starting out from aninitial emulsification capacity of 55%, 92% emulsification can beachieved after protein modification.

Regarding another aspect, when 4 mg/mL concentration is usedto make emulsions, a negative effect on this property is observed inFractions II and III. There is just 1 mg/mL of protein at this lowconcentration, the rest of the conjugated product being formed bydextran. The proteic part of the conjugated product (which containsthe hydrophobic groups of the glycoprotein) is not sufficient to

Fig. 6. Emulsifying capacity of native and conjugate proteins. Conjugated 10 mg/mL:; native 10 mg/mL: ; conjugated 4 mg/mL: and native 4 mg/mL: .

adsorb the lipid phase and no hydrophobic group is free. Hence,emulsion does not take place. Fraction IV and haemoglobin havea higher concentration of non-polar groups than Fractions II and III,so their emulsifying capacity is not decreased at low concentra-tions. There are sufficient hydrophobic groups to form an emulsionand the addition of hydrophilic groups from dextran improves thisproperty.

3.8. IMC results

The variation in energy in the system during conjugation wasmeasured. Fig. 7 shows a typical curve obtained from the IMCsystem. The detected variation in energy is due initially to theformation of the bonds between the protein and dextran and, ina secondary phase of the process, to the appearance of an Amadoriproduct, Advanced Maillard Products (AMP) (Oliver et al., 2006)and other substances not well characterized. Thus, not all thedetected energy variation can be attributed solely to the conjuga-tion reaction.

In all the experiments carried out, the variation in energy wasdetected after 70 min of exposure of the sample at 60 �C. Theaverage value obtained integrating the curve from six replicates toobtain the variation in enthalpy was DH¼�44.2� 5.4 J/g. If thisvalue is related to the molecular weight of haemoglobin:DH¼�2800 kCal/mol of Hb. The bond energy (E) of C]O and C]Nare 190 and 147 kCal/mol, respectively. During the early stages ofMaillard’s reaction, a C]O bond is broken and a C]N bond isformed, so the energy balance of the process is DE¼�43 kCal/mol.If the entire DE detected were due to conjugation, the total bondsformed (DH/DE) would comprise an average number of 65 for eachhaemoglobin molecule. It was observed (Table 1) that only 10e20amino groups are conjugated. These data confirm that severalchemical reactions take place after conjugation is produced, thusincreasing the variation in enthalpy of the system.

4. Food applications

The possible applications of the modified proteins as foodingredients are summarized in Table 4. Results published ina previous paper (Alvarez et al., 2009) are compared with theresults obtained after conjugation. It can be seen that a highernumber of blood proteins may be used for a variety of purposes.Furthermore, the increase in thermal stability enables the use ofhigher temperatures during food processing.

In other studies, whole plasma was conjugated with glucose orgalactomannan (Benjakul, Lertittikul, & Bauer, 2005; Lertittikul,Benjakul, & Tanaka, 2007; Matsudomi, Inoue, Nakashima, Kato, &Kobayashi, 1995) in order to modify various functional

Fig. 7. Variation of energy detected at 60 �C in an haemoglobin/dextran mixtureduring the formation of conjugates.

Table 4Comparison of blood protein applications in different foods according to functionalproperties.

Food Functionalproperty

Appropriatenative proteina

Appropriatemodified native

Meat products Emulsification,solubility, gelification

Globulins FII, FIII, FIV,haemoglobin

Cakes andpastries

Emulsification Plasma FII, FIII,haemoglobin

Biscuits Solubility Decoloured globin e

Yoghurt Solubility, gelification Plasma, albumin FII, FIII, FIV

a Referred in Alvarez et al. (2009).

C. Álvarez et al. / Food Hydrocolloids 28 (2012) 267e274274

properties, but just one product with defined characteristics wasobtained. The use of fractions is interesting because it allows us toobtain a wide range of proteins with different functional proper-ties from a single protein source. This enables the use of bloodproteins in a higher number of food products than when justanalyzing whole plasma.

Whole plasma can be assumed to have intermediate values.Conjugation of whole plasma was not carried out. Our resultssuggest that, in conjugated plasma, thermal stability will beenhanced, Tgel will be increased, the gels formed will be weaker,and solubility will be worse than in native plasma. Even so, thechanges obtained would be in the same sense as for isolatedproteins.

Thus, we strongly recommend fractionation in order to obtaina pool of proteins with different functional properties that may beused in foodstuffs.

5. Conclusions

Dextran and proteins from porcine blood (haemoglobin, glob-ulins and albumin) were used to produce glycoproteins, assayingdifferent temperature conditions and evaluating the modificationof their functional properties. Temperature was the main factortested to create new glycoproteins in a fast, easy and economicalway. As a result of these experiments, 80 �C was found to be thebest choice to achieve these goals. Only 60 min are needed to obtainglycoproteins that can be used to produce positive modifications inthe functional properties of blood proteins.

Although solubility decreases in all cases, the remaining func-tional properties tested were improved or favourably modified inall the fractions of blood proteins used in this study. The thermalstability of conjugates is more than 40% higher than that of nativeproteins at the usual pH employed in the food industry. Enhancedthermal stability allows the use of these glycoproteins in industrialprocesses that require the application of heat.

The range of temperatures at which a protein forms a gel wasincreased by 15 �C while maintaining the same concentration ofprotein employed. Moreover, it was possible to produce a gel ofhaemoglobin, which is not possible with native haemoglobin at thetested concentration. Moreover, the hardness of the gel can bemodified by reducing its strength.

The improvement in emulsifying capacity was due to increasedamphipathy when adding dextran hydrophilic groups. The bestresults are obtained when comparing concentrations of 10 mg/mL,the conjugated protein being a 20% better emulsifier. This findingshows that a high amount of oil can be adsorbed using the suitableprotein at the appropriate concentration.

IMC reveals that other chemical reactions start immediatelyafter conjugation and increase the enthalpy of the system.

References

Akhtar, M., & Dickinson. (2003). Emulsifying properties of whey proteinedextranconjugates at low pH and different salt concentrations. Colloids and Surfaces B:Biointerfaces, 31, 125e132.

Alvarez, C., Bances, M., Rendueles, M., & Díaz, M. (2009). Functional properties ofisolated porcine blood proteins. International Journal of Food Science & Tech-nology, 44(4), 807e814.

Aoki, T., Hiidome, Y., Sugimoto, Y., Ibrahim, H., & Kato, Y. (2001). Modification ofovalbumin with oligogalacturonic acids through the Maillard reaction. FoodResearch International, 34, 127e132.

Bautista, J., Corpas, R., Cremades, O., Hernandez-Pinzon, I., Romos, R., Villanueva, A.,et al. (2000). Sunflower protein hydrolysates for dietary treatment of patientswith liver failure. Journal of American Oil Chemical Society, 77, 121e126.

Benjakul, S., Lertittikul, W., & Bauer, F. (2005). Antioxidant activity of Maillardreaction products from a porcine plasma proteinesugar model system. FoodChemistry, 93(2), 189e196.

Campbell, L., Raikos, V., & Euston, S. (2003). Modification of functional properties ofegg-white proteins. Molecular Nutrition & Food Research, 47, 369e376.

Coffman, C., & García, V. (1977). Functional properties and amino acid content ofa protein isolate from mung bean flour. Journal of Food Technology, 12(5),473e484.

Cohn, E. J., Strong, L. E., Hughes, W. L., Mulford, D. J., Asworth, J. N., Melin, M., et al.(1946). Preparation and properties of serum and plasma proteins. IV. A systemfor the separation into fractions of the protein and lipoprotein components ofbiological tissues and fluids. Journal of the American Chemical Society, 68,459e463.

Cordobés, F., Partal, P., & Guerrero, A. (2004). Rheology and microstructure of heatinduced egg yolk gels. Rheologica Acta, 43(2), 184e195.

Dávila, E., Parés, D., Cuvelier, G., & Relkin, P. (2006). Heat-induced gelation ofporcine blood plasma proteins as affected by pH. Meat Science, 76, 216e225.

De Vouno, M., Penteado, C., Lajolo Franco, M., & Pereira dos Santos, N. (1975).Functional and nutritional properties of isolated bovine blood proteins. Journalof Science and Food Agriculture, 30(8), 809e815.

Fields, R. (1971). The measurement of amino groups in proteins and peptides.Biochemical Journey, 124, 581e590.

Inklaar, P., & Fortuin, A. (1969). Determining the emulsifying and emulsion stabi-lizing capacity of protein meat additives. Journal of Food Technology, 23,103e107.

Jimenez-Castaño, L., Villamiel, M., & López-Fandiño, R. (2007). Glycosylation ofindividual whey proteins by Maillard reaction using dextran of differentmolecular mass. Food Hydrocolloids, 21, 433e443.

Jung, S. H., Choi, S. J., Kim, H. J., & Moon, T. W. (2006). Molecular characteristics ofbovine serum albuminedextran conjugates. Bioscience, Biotechnology andBiochemistry, 70(9), 2064e2070.

Katayama, S., Shima, J., & Saeki, H. (2002). Solubility improvement of shellfishmuscle proteins by reaction with glucose and its soluble state in low-ionicstrength medium. Journal of Agricultural and Food Chemistry, 50, 4327e4332.

Kato, A., Murata, K., & Kobayashi, K. (1993). Improvement of emulsifying propertiesof egg white proteins by attachment of polysaccharide through Maillard reac-tion in a dry state. Journal of Agriculture and Food Chemical, 41, 540e543.

Laca, A., Paredes, B., & Díaz, M. (2010). A method of egg yolk fractionation. Char-acterization of fractions. Food Hydrocolloids, 24(4), 434e443.

Lamsal, B. P., Jung, S., & Johnson, L. A. (2007). Rheological properties of soy proteinhydrolysates obtained from limited enzymatic hydrolysis. LWT e Food Scienceand Technology, 40, 1215e1223.

Lertittikul, W., Benjakul, S., & Tanaka, M. (2007). Characteristics and antioxidativeactivity of Maillard reaction products from a porcine plasma proteineglucosemodel system as influenced by pH. Food Chemistry, 100(2), 669e677.

Matsudomi, N., Inoue, Y., Nakashima, H., Kato, A., & Kobayashi, K. (1995). Emulsionstabilization by Maillard-type covalent complex of plasma protein with gal-actomannan. Journal of Food Science, 60(2), 265e268.

Oliver, C. M., Melton, L. D., & Stanley, R. A. (2006). Creating proteins with novelfunctionality via the Maillard reaction: a review. Critical Reviews in Food Scienceand Nutrition, 46, 337e350.

Rich, L. M., & Foegeding, E. A. (2000). Effects of sugars on whey protein gelation.Journal of Agricultural and Food Chemistry, 48, 5046e5052.

Saeki, H., & Inoue, K. (1997). Improved solubility of carp myofibrillar proteins in lowionic strength medium by glycosylation. Journal of Agricultural and FoodChemistry, 45, 3419e3422.

Sasahara, K., McPhie, P., & Minton, A. P. (2003). Effect of dextran on protein stabilityand conformation attributed to macromolecular crowding. Journal of MolecularBiology, 326(4), 1227e1237.

Selmane, D., Christophe, V., & Gholamreza, D. (2008). Extraction of proteins fromslaughterhouse by-products: influence of operating conditions on functionalproperties. Meat Science, 79, 640e647.

Sun, Y., Hayakawa, S., & Izumori, K. (2004). Modification of ovalbumin with a rareketohexose through the Maillard reaction: effect on protein structure and gelproperties. Journal of Agriculture Food Chemistry, 52, 1293e1299.