Research Article Antioxidant Activity and Functional...

Research ArticleAntioxidant Activity and Functional Properties ofPolymerized Whey Products by Glycation Process

Liliana Ortega1 Anabel Romero1 Claudia Muro1 and Francisco Riera2

1Departamento de Ingenierıa Quımica e Investigacion Instituto Tecnologico de Toluca Apartado Postal 890 52149 MetepecMEX Mexico2Departamento de Ingenierıa Quımica y Tecnologıa de Medio Ambiente Universidad de Oviedo Oviedo 33006 Asturias Spain

Correspondence should be addressed to Claudia Muro cmuroittolucaedumx

Received 14 March 2015 Revised 8 June 2015 Accepted 23 June 2015

Academic Editor Xingxun Liu

Copyright copy 2015 Liliana Ortega et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The antioxidant properties of sweet and acid whey products were incremented by polymerization of their proteins by glycation ofwhey protein concentrates (WPC) and their hydrolyzates (WPCH)with ribose and glucose in individual experiments under similarconcentration Heating at 50∘C during 20 h maximum and pH 7 and pH 9 were used in all tests The higher activity was found inWPC glycosylates products with ribose at pH 7 and heating during 10ndash15 h In comparable form antioxidant activity in WPCHwas incremented by prior hydrolysis to glycation with 25ndash45 of hydrolysis degree Further functional properties of whey proteins(solubility emulsion and foam) were also improved by the polymerization with ribose The color of polymerized products due toMaillard reactions was associated with antioxidant activity of each compound however comparative color in glycosylates productswith glucose and ribose did not show this effect

1 Introduction

Today is known the mediator action of some biopolymers onstructure and lipid oxidation of sensitive foods among themare the functionalwhey proteins [1ndash3]Due to their antioxida-tive effects (which include chelating of prooxidant transitionmetals) lowmolecular weight easy absorption high activityhypoallergenicity and relatively high stability under differentconditions whey proteins may be utilized in formulations ofsubstitutes of synthetic compounds such as butylated hydrox-yanisole (BHA) butylated hydroxytoluene (BHT) and tert-butyl hydroquinone (TBHQ) Mainly proteins as lactoferrin[4] serum albumin [5] and free radical scavenging by aminoacid residues such as cysteine and tyrosine [6 7] are describedas retardants of the fast deterioration of foods and to preventthe autoxidation of their components

However in order to improve their properties and func-tionality currently various researches have been carried outto get better effects of whey products on foods by treatmentsaimed at modifying chemical structure Recent studies showthat antioxidant capacity can be incremented by enzymatic

hydrolysis [8ndash11] chemical methods such as acetylationdeamination succinylation and reductive alkylation andcreating covalent cross-links (polymerization) between foodbiopolymers and carbohydrates [12 13] In particular specialattention has been provided to the effect exerted by reducingsugars on the structure and protein via Maillard reaction(MR)

Additionally polymerization or glycation has also asignificant impact on protein functionality as gelling heatstability emulsifying and foaming capability Thus thesecompounds can exhibit improvement in the antioxidantcapacity and functionality when they are added in emulsions[14 15] MR products can also increment the propertiesof the food such as appearance flavor and texture duringtheir processing and storage [16ndash18] Furthermore they alsomay significantly improve the other biological competencesas antimicrobial ability and antihypertensive properties [19ndash21] However despite these benefits the glycation by MRmay negatively affect the purpose of modification of wheyproteins Products of MR can act as prooxidants dependingon the states or progress of the reaction The thermal and pH

Hindawi Publishing CorporationInternational Journal of Polymer ScienceVolume 2015 Article ID 154262 10 pageshttpdxdoiorg1011552015154262

2 International Journal of Polymer Science

conditions nature and concentration of proteins and sugarscontribute to this drastic and contrary effect

Sugar fragmentation and degradation of amino acids(Strecker degradation) occurring in advanced stages of thereaction may aggravate antioxidant action In addition poly-merized compounds which in turn produce irreversible orintermediates reactions and the interaction between themmay also affect the antioxidant activity of the productsof glycation Besides training of melanoidins characterizedmany of them with typical aroma and flavor [22] are found asproduct of MR in advanced stages Therefore the manipula-tion of whey proteins properties is an important topic in foodindustries and more studies are necessary to determine thepotential glycation and their influence on their functionalityas food additive Due to the complexity of MR it is necessaryto deepen the study of parameters to these reactions and typeof carbohydrate utilized in the glycation

The aim of this work was to evaluate the antioxidantactivity and functional properties of products obtained bycombination of enzymatic hydrolysis and glycation process ofproteins concentrates from milk bovine whey Hydrolyzateswere obtained by contact with Bacillus subtilis Nature of thesugar on functionality of products was analyzed by individualexperiments with ribose and glucose under controlled condi-tions of carbohydrate concentration temperature and pH

2 Materials and Methods

21 Materials Samples of 2 L of acid and sweet bovinewhey milk were provided by a cheese industry localized inMexico The 221015840azino-bis(3-ethylbenzothiazoline-6-sulfonicacid) (ABTS) and reducing sugars glucose ribose and ara-binose were purchased from Sigma Aldrich Other chemicalsagents used were of analytical grade

22 Preparation of Whey Protein Concentrates Samples ofwhey of 1 L were filtered to remove fat and casein particlesby vacuum filtration usingWhatman filter paper number 40

Then whey was concentrated by ultrafiltration process(UF) to obtain whey proteins concentrates (WPC) In thiscase was used cross-flow zirconium-titanium ceramic mem-brane of 15 kDa cut-off and polymeric membrane (hollowfiber) of 6 kDa cut-off (Pall) Tubular housing of tangen-tial flow of membrane was adapted to a peristaltic pumpThe transmembrane pressures were adjusted and controlledto 30 and 03 bar in ceramic and polymeric membranerespectively Whey feeding was performed at 25∘C to bothconcentration processes In eachUF experiment two streamswere obtained the retentate (WPC) and permeate (lactoseand minerals mainly)

Diafiltration was performed by addition of five volumesof deionized water to the retentate to maintain a constantvolume during UF process [23]

UF was stopped after approximately 18 h of filtrationProteins in whey and WPC were determined by Biuretmethod The percent yield (119884) of WPC was calculated using

119884 = 1198812119862211988111198621(100) (1)

where 1198622is protein concentration in the retentate after UF

1198621is protein concentration in the whey sample 119881

1is initial

volume ofwhey sample and1198812is final volume in the retentate

after UFTo perform the next experiments WPC products were

chosen according to the highest proteins concentration

23 Preparation of Hydrolyzed Products from Whey ProteinConcentrates Hydrolyzed productsWPCHwere obtained bydirect contact of substrates WPC with suspended biomass ofBacillus subtilis in relation 1 1 WPC samples were previouslyadjusted to pH 7 B subtilis was previously maintained in thelaboratory on nutrient broth agar slants (5 gL beef extract10 gL peptone 5 gL sodium chloride 18 gL agar and pH 7ndash74) and stored at 4∘C [23]

The hydrolysis was performed in a shaking bath (Shel-Lab) at 100 rpm and 50∘C In this experiment WPCwas usedas control group the sample was treated under the sameexperimental conditions

Upon completion of the hydrolysis experiment (24 h)the B subtilis was inactivated at a temperature of 70∘C in awater bath for 3min then the tubes were centrifuged for 15minutes at 5000 rpm and the supernatant was diluted withdilution factor of 100 to determine the protein concentrationby Biuret method [23] Degree of hydrolysis (DH) of WPCwas determined by measuring the proteins concentrationevery 2 hours during 12 h of uninterrupted hydrolysis andwascalculated by [23]

DH =119862119900minus 119862119891

119862119891

(100) (2)

where 119862119900is the initial protein concentration in WPC and

119862119891is the protein concentration in supernatant hydrolyzed

product for hydrolysis time

24 Preparation of Polymerized Products from Whey byGlycation Process Polymerized products from whey by gly-cation process were obtained by the coupling of the samplesWPC and WPCH with a carbohydrate (ribose and glucose)according to a method previously developed [24]

Carbohydrate solution was previously prepared by bufferdissolution pH 7 of sodium citrate and buffer dissolution pH9 of sodium carbonate each one with 02 g of carbohydrate

Samples of WPC and WPCH were placed in directcontact with carbohydrate (1 4 vv) at pH 7 and pH 9 inindividual experiments The mixtures were stirred at roomtemperature for 1 h to form uniform dispersion Afterwardsthe mixtures were then incubated at 50∘C during 20 h

For the period of glycation process each mixture wassampled in intervals of time of 5 h to 20 h Productswere char-acterized by color change antioxidant activity and functionalproperties

Sweet and acid whey were used as control group thesamples were only heated under the same experimentalconditions

Glycated productswere identified asWWPC-RWPC-Gand WPCH-R WPCH-G for whey whey concentrates and

International Journal of Polymer Science 3

hydrolyzed product with ribose and glucose in different timesof hydrolysis and glycation processes respectively

25 Determination of the Properties of Whey Products

251 Antioxidant Activity The antioxidant activity of wheyproducts was assessed by scavenging of theABTS radical [25]

ABTS∙+ radical was previously prepared as follows00336 g of ammonium persulfate and 00194 g of ABTSwere diluted in 30mL of distilled water This solution wasincubated during 16 h at 25∘C in the dark After this time40mL of ABTS∙+ solution was dissolved in 960mL ofabsolute ethanol reaching an absorbance of 07 plusmn 02 to754 nm

A calibration curve using ascorbic acid 0002019M wasbuilt as standard substance Solutions from 0 to 08mMascorbic acid were prepared in this stage

Aliquots of 20 120583L of these solutions were added to 980 120583Lof ABTS solution (absorbance of 07 plusmn 02 to 754 nm)Each mixture was incubated in the dark for 7min and theabsorbance at 754 nm was measured for all samples

The ABTS radical scavenging activity (AA) was calcu-lated as inhibition percentage according to

AA =119860119888minus 119860119904

119860119888

(100) (3)

where 119860119888is the absorbance of the control (ABTS solution

without samples) and 119860119904is the absorbance of the samples of

ascorbic acidAntioxidant activity of whey products was analyzed in a

similarmanner Briefly 20120583L of each productwasmixedwith980 120583L of the diluted ABTS the mixture was incubated inthe dark for 7min and the absorbance at 734 nm was alsomeasured

The change in absorbance with the percent inhibition ofthe radical ABTS∙+ was calculated for each sample The datawere reported as antioxidant activity equivalent to ascorbicacid (AAEAA) and AA [23]

252 Functional Properties The functional properties ofwhey products were analyzed by measuring their solubilityat pH 45 and 68 emulsions and foaming capacities

Solubility was calculated as the total protein concentra-tion in the supernatant of products after centrifugation at pH46 as a percentage of the initial concentration at pH 68 [26]

Whey products (25 plusmn 5mg) were dispersed in 10mL ofdeionized water and the pH was adjusted with HCl (1 N)or NaOH (1N) when necessary The dispersion was mixedfor 30min and centrifuged continuously (119909g times 2056) for10min (Hettich Zentrifugen 32 R) The amount of protein inthe supernatant was determined by the Biuret method thesolubility was expressed as the percentage of protein in thesupernatant the total in the initial products

Emulsification capacity of whey products and their sta-bility were analyzed using the method developed by Pearceand Kinsella [27] In this case 15mL of corn oil was added to225mg whey products previously dissolved in 45mL 001Mphosphate buffer pH 7 The mixture was homogenized at20000 rpm at room temperature during 1min

The stability of products was determined in qualitativeform when a breakdown of the emulsion was observed thevolume of added oil was registered and used to calculate thepercent CEM by

CEM = 1198811198811

(100) (4)

where 119881 is mL of oil and 1198811is mL of whey products

Regarding foaming capacity (FC) of products it wasdetermined at pH 7 adjusting the samples with NaOH (01 N)at 20∘C

For the test 1mL of sample was mixed with 72mL ofdistilled water with stirring for 3min using a high-speedstirrer of 2000 rpm FC was determined by volume of sampleachieved after this time using

CE =119881119891minus 119881119894

119881119894

(100) (5)

where 119881119891and 119881

119894are the final and the initial volumes of

sample respectivelyThe color change of whey products obtained by MR

during glycation was also determined Spectra of 200 to700 nm using a Perkin Elmer spectrophotometer Lambda 35were obtained for each sample The results were shown asidentification of redness and yellowness color in the productsSample whey was used as standard to obtain the spectra

26 Statistical Analysis Statistica 70 software (Statsoft IncTulsa OK USA) was utilized to analyze the data obtainedfrom whey characteristics antioxidant activity and func-tional properties on glycation products in experiments withthree replicates Results were compared using ANOVA andTukey post hoc test (119875 lt 005)

3 Results and Discussion

31 Products fromWhey Proteins Concentrates Thesweet andacid whey samples showed pH of 65 and 45 respectivelyvalues representing the difference between both whey typeswhich depends on the technological process used to manu-facture the cheese

The average content (789 plusmn 01 and 873 plusmn 01 gL)indicated that both whey types did not have significantdifference in protein content and the values obtained werewithin those reported by Tovar Jimenez et al [23] (6ndash13 gL)Difference inwhey featuresmay be attributed to themilk usedand the cheese elaboration

Concerning UF processes of whey it was found thatperforming the UF with polymeric membrane was possibleto achieve an excellent yield in the protein concentrationamong 96ndash98 for sweet and acid whey The products fromUF were identified asWPC-1 (from sweet whey and retentatefrom polymeric membrane) WPC-2 (from acid whey andretentate from polymeric membrane) WPC-3 (from sweetwhey and retentate from ceramic membrane) WPC-4 (fromacid whey and retentate from ceramic membrane)

Table 1 shows protein composition of WPC productsobtained by UF processes with ceramic membrane of 15 kDa


Table 1 Protein concentration (gL) in crude whey and WPCproducts by UF processes

Membrane Ceramic PolymericSample type Whey WPC WPCAcid 873 plusmn 05 535 plusmn 04 855 plusmn 03Sweet 789 plusmn 03 458 plusmn 05 741 plusmn 03

96 98

45

60

0

20

40

60

80

100

120

1 2 3 4

Yiel

d (

)

PolymericSweet Acid Sweet Acid

Ceramic

Figure 1 Percentage of yield in WPC by UF processes of sweet andacid whey with polymeric and ceramic membrane

and polymeric membrane of 3 kDa while Figure 1 shows thefinal percent yield (119884) of each WPC Difference between theyields in the UF is due to cut-off of membranes Thereforeit is possible that the proteins of great molecular weight asalbumin Immunoglobulins and lactoferrin are found intactin the WPC from ceramic membrane but fewer amountsof 120573-lactoglobulin and 120572-lactalbumin are present in theretentates from 15 kDa membrane since another amount ofthese proteins is found in their permeate streams

Under yield result inUF process in this stagewere chosenthe products WPC-1 and WPC-2 (retentates from polymericmembrane) to continue with their hydrolysis

32 Hydrolyzed Products from Whey Protein ConcentratesDegree of hydrolysis (DH) achieved during 24 h was 45ndash60 from WPC-1 and WPC-2 respectively Data of hydrol-ysis process are showed in Figure 2 This test indicated thatduring 12 h the changes were observed asmore representativein the two types of whey due to enzymatic action from Bsubtilis After this period the DH increased very slowly

Differences of enzymatic action from B subtilis have beenreported in other researches however other substrates andenzymatic extracts or purified enzymes from B subtilis havebeen used in these experiments For example maximumvalues around 70 of hydrolysis were found on rapeseedmeal with enzymatic extract [28] and similar percentagewith commercial proteases such as Alcalase on soybeanand Subtilisin on whey proteins can be seen in [29 30]Particularly in [30] it was found that 26 DH of wheyproteins is obtained after two hours of direct contact withSubtilisin to achieve 70 in 10 h which could be comparedwith DH obtained in this work

0

10

20

30

40

50

60

0 5 10 15 20 25

DH

()

Time (h)

WPI-1WPI-2

Figure 2 DH from WPC-1 and WPC-2 with biomass from Bsubtilis

33 Polymerized Products from Whey Proteins by GlycationProcess Hundred eighty coded polymerized products wereobtained during 20 h Data are reported by conforming sweetor acid whey produced by only heating (W-1 and W-2)glycation of WPC from each whey type and ribose andgalactose (WPC-1 WPC-2 WPC-1-R WPC-1-G WPC-2-R and WPC-2-G) and glycation of hydrolyzed productsobtained with 25 35 and 45 DH of WPC of sweet wheyhydrolysis and 25 45 and 60 DH of WPC of acid wheyduring 4 8 and 12 h respectively (WPCH-1-25-R WPCH-1-35-R WPCH-1-45-R WPCH-2-25-R WPCH-2-45-R andWPCH-2-60-R) for pH 7 and pH 9 and of the same form toglucose

Tables 2 to 6 show a summary of the main results onfeatures of some glycosylated product from sweet and acidwhey with ribose and glucose In particular products withhigher antioxidant activity are shown in Figure 3

Principally data of Table 2 correspond to glycosylatedproducts from sweet and acid whey by heating of samplesAAEAAvalues indicated that a significant enhancement (055to 075) was observed in the products by only acid and sweetwhey heating for 15ndash20 h at pH 7 However no enhancementwas observed in samples at pH 9

In relation toWPC products a higher AAEAA value wasobtained in WPC-R conjugates (056 to 102) at pH 7 withheating time while the increment of AAEAA in WPC-Gconjugates was less (056 to 078) at pH 7 and pH 9 Data canbe observed in Tables 3 and 4

Information from WPCH conjugate products corre-sponding to glycosylated WPC with ribose previouslyhydrolyzed with biomass from B subtilis is shown in Tables5 and 6 AAEAA values of these products indicate thathydrolyzed WPCH can increment their AAEAA value bypolymerization with carbohydrates In general WPCH con-jugates exhibited adequate potency to react with free radicalsDH was sufficient to liberate reactive peptides and producepolymerized products with high AAEAAA values in allexperiments Particularly productswith 25DH fromWPC-acid whey-ribose at pH 7 achieved 155ndash185 of AAEAA and


Table 2 Features of glycosylated product from sweet and acid whey with heating to 50∘C pH 7 and pH 9

Sample Glycosylation time (h) Product Antioxidant activity Functional propertiesAA AAEAA (mML) Solubility Emulsion Foam

Sweet whey at pH 7

0 W7-1 2510 plusmn 02 055 80 plusmn 02 20 plusmn 01 13 plusmn 025 W7-1-30 2905 plusmn 02 057 78 plusmn 02 22 plusmn 01 13 plusmn 0210 W7-1-60 3243 plusmn 01 068 78 plusmn 02 21 plusmn 01 13 plusmn 0115 W7-1-120 3467 plusmn 02 070 75 plusmn 02 22 plusmn 01 12 plusmn 0220 W7-1-180 3531 plusmn 02 075 75 plusmn 02 20 plusmn 01 11 plusmn 01

Sweet whey at pH 9


Acid whey at pH 7


Acid whey at pH 9


Table 3 Features of glycosylated product fromWPC of sweet and acid whey with ribose heating to 50∘C pH 7 and pH 9


WPCsweet whey with ribose at pH 7

0 WPC7-1-R 51 plusmn 06 056 90 plusmn 02 80 plusmn 02 25 plusmn 025 WPC7-1-30-R 53 plusmn 05 058 88 plusmn 02 80 plusmn 02 25 plusmn 0210 WPC7-1-60-R 55 plusmn 05 065 88 plusmn 02 78 plusmn 01 26 plusmn 0215 WPC7-1-120-R 60 plusmn 02 070 85 plusmn 01 78 plusmn 01 26 plusmn 0220 WPC7-1-180-R 77 plusmn 02 075 85 plusmn 01 75 plusmn 01 27 plusmn 02



WPCacid whey with ribose at pH 7





Table 4 Features of glycosylated product fromWPC of sweet whey with glucose and heating to 50∘C pH 7 and pH 9


WPCsweet whey with glucose at pH 7

0 WPC7-1-G 50 plusmn 05 030 70 plusmn 01 62 plusmn 01 25 plusmn 015 WPC7-1-30-G 58 plusmn 06 042 75 plusmn 01 66 plusmn 02 28 plusmn 0110 WPC7-1-60-G 59 plusmn 05 042 75 plusmn 01 65 plusmn 02 30 plusmn 0115 WPC7-1-120-G 60 plusmn 07 052 77 plusmn 02 65 plusmn 01 30 plusmn 0220 WPC7-1-180-G 62 plusmn 05 055 75 plusmn 03 61 plusmn 01 25 plusmn 01



WPCacid whey with glucose at pH 7



0 WPC9-2-G 55 plusmn 03 056 76 plusmn 01 65 plusmn 02 28 plusmn 015 WPC9-2-30-G 58 plusmn 03 068 78 plusmn 01 65 plusmn 01 25 plusmn 0110 WPC9-2-60-G 69 plusmn 03 068 80 plusmn 02 69 plusmn 03 28 plusmn 0215 WPC9-2-120-R 78 plusmn 03 078 75 plusmn 02 65 plusmn 03 25 plusmn 0120 WPC9-2-180-R 75 plusmn 03 075 73 plusmn 02 65 plusmn 01 20 plusmn 01

products with 45 DH at pH 9 achieved only 11 of AAEAAwhereas conjugate products by glycation of hydrolyzed withglucose showed always a lower increase in AAEAA

In terms of reactants nature it is understandable thatWCPH products were more reactive than WPC this is dueto presence of free amino acids However it was found thatmajor contributors to the radical scavenging capacity wereprimarily composed of WCPH although other authors notedthat either the intermediates or the final brown polymer canfunction as hydrogen donors and that sugar caramelizationcan also contribute to the antiradical activity [31]

The most reactive peptides are the ndashNH2groups of lysine

as well as the group ndashNH2located at the amino terminus

Therefore a higher content of residues lysine could lead toa higher degree of protein glycation The imidazole group ofhistidine the indole group of tryptophan and the guanidinogroup of arginine residues are also likely to react withreducing sugars but this possibility is lesser than aminogroups [32]

On the other hand different coloration in conjugatesproducts was also observed during the heating periodfor each carbohydrate However comparative experimentsshowed that glycosylated products with glucose developedan intense yellow color and characteristic odor after 15 h ofheating decreasing at pH 9 whereas glycosylated productswith ribose acquired a faint brownish yellow in similarperiod

Thus it is possible that ribose present as more reactivethan the glucose during the different stages of MR joining

075076075076

102085

0750808

13

w7-1-180w7-2-180

WPC7-1-180-RWPC9-1-180-RWPC7-2-180-R

WPCH7-1-120-35-RWPCH9-2-120-25-G

WPCH9-2-60-25-RWPCH7-2-60-60-R

WPCH9-2-180-45-RAAEAA

Figure 3 AAEAA of representative glycosylated products fromwheymilk in individual experimentswith ribose and glucose at 50∘Cand variations of pH 7 and pH 9

the products substantially to ribose molecules this reactiveproperty could be associated with antioxidant power of wheyproducts

Recent researches have shown that the antioxidant prop-erties of the proteins are enhanced by their conjugation par-ticularly with glucose Conjugate products from ovalbumin-saccharides [33] whey protein isolate (WPI) [34] and specificconjugates 120572-lactalbumin-glucose [35] showed an incre-ment in their antioxidant activity when the proteins werepolymerized with glucose more greatly These results werehighly consistent with the browning intensity of productsAuthors found that particularlyWPI-conjugates with glucoseafter 7 days of dry heating at 60∘C showed an increase in


Table 5 Features of glycosylated product from hydrolyzates fromWPC of sweet whey with ribose heating to 50∘C pH 7 and pH 9


Hydrolyzate (25 HD) fromWPC of sweet whey withribose at pH 7

0 WPCH7-1-25-R 60 plusmn 07 054 90 plusmn 01 80 plusmn 01 10 plusmn 015 WPCH7-1-30-25-R 65 plusmn 05 055 90 plusmn 01 78 plusmn 01 15 plusmn 0110 WPCH7-1-60-25-R 68 plusmn 07 055 90 plusmn 02 75 plusmn 01 15 plusmn 0115 WPCH7-1-120-25-R 70 plusmn 08 058 89 plusmn 02 75 plusmn 02 15 plusmn 0220 WPCH7-1-180-25-R 65 plusmn 06 049 85 plusmn 01 70 plusmn 02 10 plusmn 02



Hydrolyzate (45 HD) fromWPC of sweet whey withribose at pH7








antioxidant activity and an increase in the color It was seeninUV-vis absorption and fluorescence intensity Additionallycomparative data onWPI alone indicated that the conjugatesexhibited a better thermal stability

Similar results were found in the present research how-ever here it is shown that conjugates-ribose enhances antiox-idant action than glucose Difference of antioxidant actioncan also be associated with the presence of complex mix ofcompounds developed during glycation of whey productsGlucose could play an important role in the reductionof antioxidant activity Particularly hydroxyl groups couldinhibit this power [36 37]

Carbohydrate type and pH could explain these resultsHowever reactivity of carbohydrate in glycation process hasnot been described still in clear form According to [37] ithas been shown that reactivity decreases with the molecularweight of carbohydrate which would explain that cross-linking and aggregation protein with ribose is incrementedin comparison with the glucose and this behavior couldbe associated with the increment of antioxidant activity

of conjugate whey products-ribose Reactivity of differentsugars is also given by the availability of its carbonyl groupfor example glucose presents at least two closed forms (cyclicanomers) in which the carbonyl group has disappearedThusa correlation between the reaction rate of glycation and theproportion of the open form of each sugar is also found inthese data

Influence of pH in MR reaction during glycation processmay furthermore be observed on antioxidant action of wheyproducts at basic pH generally the polymerization rateincreases However the results cannot be described thusradically

In an early stage MR generates Amadori products(glycosylamines N-substituted by sugar-amine condensa-tion) which are present due to reactions between aminegroups of proteins and carbonyl groups within the reducingsugar while intermediate stage and advanced stage arecharacterized by formation of reactive intermediates such assugar-derived dicarbonyl compounds as 1-amino-2-deoxy-2-ketoses (eg methylglyoxal 3-deoxyosones or glyoxal)


Table 6 Features of glycosylated product from hydrolyzates fromWPC of acid whey with ribose heating to 50∘C pH 7 and pH 9


Hydrolyzate (25 HD) fromWPC of acid whey with riboseat pH 7

0 WPCH7-2-25-R 60 plusmn 05 060 88 plusmn 03 80 plusmn 01 30 plusmn 025 WPCH7-2-30-25-R 65 plusmn 06 058 90 plusmn 02 80 plusmn 02 35 plusmn 0210 WPCH7-2-60-25-R 68 plusmn 05 060 90 plusmn 01 80 plusmn 02 37 plusmn 0215 WPC7-2-120-25-R 67 plusmn 05 063 85 plusmn 01 78 plusmn 02 35 plusmn 0220 WPC7-2-180-25-R 65 plusmn 05 062 85 plusmn 01 79 plusmn 02 32 plusmn 02











which are produced with time resulting in the formation ofpolymerized compounds that may be interfering substancesaffecting the antioxidant activity In addition alkaline andorheat treatment of proteins may be associated with the pro-duction of several types of cross-linked amino acids such asdehydroalanine lysinoalanine and histidinoalanine whichmay affect also antioxidant activity and the nutritional valueof the treated whey proteins [37] Dehydration of sugarsoccurs by two routes Under acidic conditions are formedfurfurals and at alkaline conditions are formed the reductoneThe enolization of the C

2and C

3carbons and removal of the

amino group in position 1 give rise to the formation of 23-dicarbonyl compounds (via 23-E) which causes further frag-mentation of low molecular weight compounds as ketoalde-hydes reductone and a-dicarbonyl compounds while atacidic pH enolization of C

1andC

2carbon and removal of the

amino group in position 1 and the carboxyl group at position3 are favored resulting in 12-dicarbonyl compounds (via 12-E) as glyoxal and methylglyoxal 1-desoxiglucosulosa and 3-desoxiglucosulosa which may undergo further dehydrationresulting in furfural such as 5-(hydroxymethyl) furfural(HMF) [38]

Concerning functional properties of whey glycosylatedproducts in this work it is shown that conjugates fromWPCand WPCH had an excellent solubility however this valuereduced notably in long heating products Figure 4 showsfunctional properties of glycosylated products from wheymilk in individual experiments with ribose and glucose at50∘C and variations of pH 7 and pH 9

Emulsion property did not change with respect to valuein whey while foam property was incremented in some casesIn general a high volume of foam was obtained in WCP andhydrolyzed with heating in 10 h at pH 7

Under these data the best products found in these testswere those fromWPC-R andWPCH-R at pH 7 with heatingfor 10ndash15 hTherefore conjugates obtained could be excellentantioxidants andor functional additives in formulated foods[39]

Current studies have shown that conjugates from sodiumcaseinate [40] and 120573-lactoglobulin [41] with galactose havethe capacity to form and stabilize food emulsions The bestresults were obtained in these experiments with glycatedproteins at 50∘C and pH 5 during 48 h (corresponding to thelatest steps of MR) This behavior can be attributed to the


0 20 40 60 80 100 120

W7-1-180W7-2-180

WPC7-1-180-RWPC9-1-180-RWPC7-2-180-RWPC7-2-180-G

WPCH7-1-120-35-RWPCH7-2-60-60-RWPCH9-2-60-25-R

WPCH9-2-180-45-RWPCH9-2-120-25-G

Foam ()Emulsion ()

Solubility ()AA ()

Figure 4 Functional properties of glycosylated products fromwheymilk in individual experiments with ribose and glucose at 50∘C andvariations of pH 7 and pH 9

higher adsorption efficiency and degree of interfacial interac-tion exhibited by the glycoconjugates with this carbohydrateand the fact that alkaline pH reduces significantly the foamproperty

4 Conclusions

The antioxidant activity and functional properties of sweetand acid whey products were incremented by polymerizationof their proteins by glycation with heating and addition ofglucose and ribose

Glycation of whey protein concentrates (WPC) and thehydrolyzates (WPCH) with ribose and glucose showed dif-ferent oxidation values

The higher activity was found in WPC glycosylatesproducts with ribose at pH 7 and heating at 50∘C during10ndash15 h In the same way antioxidant activity in WPCH wasincremented by prior hydrolysis to glycation in productswith 25ndash45 hydrolysis degree at pH 9 In these processeswas found an important dependence on pH conditions andcarbohydrate reactivity therefore antioxidant activity wasassociated with carbohydrate type used in glycation of wheyproteins

Functional properties of these products (solubility emul-sion and foam) were also maintained or improved by theglycation with ribose

The color in glycosylates products with glucose was moreintense than in products with ribose however antioxidantactivity was not associated with this change Formation ofhydroxyl groups could probably inhibit this action

Other researchers are required to identify the structureof the compounds in the conjugates that could inhibit thecapacities of conjugates with glucose In addition practicalapplications of these products are necessary to test theirantioxidant capacity as additive in foods

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] J Liu Q Ru and Y Ding ldquoGlycation a promising methodfor food protein modification physicochemical properties andstructure a reviewrdquo Food Research International vol 49 no 1pp 170ndash183 2012

[2] C Sun and S Gunasekaran ldquoEffects of protein concentrationand oil-phase volume fraction on the stability and rheology ofmenhaden oil-in-water emulsions stabilized by whey proteinisolate with xanthan gumrdquo Food Hydrocolloids vol 23 no 1 pp165ndash174 2009

[3] L M Tong S Sasaki D J McClements and E A DeckerldquoMechanisms of the antioxidant activity of a high molecularweight fraction of wheyrdquo Journal of Agricultural and FoodChemistry vol 48 no 5 pp 1473ndash1478 2000

[4] J M C Gutteridge S K Paterson AW Segal and B HalliwellldquoInhibition of lipid peroxidation by the iron-binding proteinlactoferrinrdquo Biochemical Journal vol 199 no 1 pp 259ndash2611981

[5] E Meucci A Mordente and G E Martorana ldquoMetal-catalyzedoxidation of human serum albumin conformational and func-tional changes Implications in protein agingrdquo The Journal ofBiological Chemistry vol 266 no 8 pp 4692ndash4699 1991

[6] A Moure H Domınguez and J C Parajo ldquoAntioxidant prop-erties of ultrafiltration-recovered soy protein fractions fromindustrial effluents and their hydrolysatesrdquo Process Biochem-istry vol 41 no 2 pp 447ndash456 2006

[7] I Arcan and A Yemenicioglu ldquoAntioxidant activity of proteinextracts from heat-treated or thermally processed chickpeasand white beansrdquo Food Chemistry vol 103 no 2 pp 301ndash3122007

[8] G-N Kim H-D Jang and C-I Kim ldquoAntioxidant capacity ofcaseinophosphopeptides prepared from sodium caseinate usingAlcalaserdquo Food Chemistry vol 104 no 4 pp 1359ndash1365 2007

[9] T Bayram M Pekmez N Arda and A S Yalcin ldquoAntioxidantactivity of whey protein fractions isolated by gel exclusionchromatography and protease treatmentrdquo Talanta vol 75 no3 pp 705ndash709 2008

[10] A Dryakova A Pihlanto P Marnila L Curda and HJ T Korhonen ldquoAntioxidant properties of whey proteinhydrolysates as measured by three methodsrdquo European FoodResearch and Technology vol 230 no 6 pp 865ndash874 2010

[11] C Muro Urista R Alvarez Fernandez F Riera Rodrıguez AArana Cuenca and A Tellez Jurado ldquoReview production andfunctionality of active peptides from milkrdquo Food Science andTechnology International vol 17 no 4 pp 293ndash317 2011

[12] J A Gerrard ldquoProtein-protein crosslinking in food methodsconsequences applicationsrdquo Trends in Food Science and Tech-nology vol 13 no 12 pp 391ndash399 2002

[13] N Liu T Zhang Y J Wang Y P Huang J H Ou and PShen ldquoA heat inducible tyrosinase with distinct properties fromBacillus thuringiensisrdquo Letters in Applied Microbiology vol 39no 5 pp 407ndash412 2004

[14] G Liu and Q Zhong ldquoThermal aggregation properties of wheyprotein glycated with various saccharidesrdquo Food Hydrocolloidsvol 32 no 1 pp 87ndash96 2013

[15] S-C Liu D-J Yang S-Y Jin C-H Hsu and S-L ChenldquoKinetics of color development pH decreasing and anti-oxidative activity reduction of Maillard reaction in galac-toseglycine model systemsrdquo Food Chemistry vol 108 no 2 pp533ndash541 2008


[16] M Murakami H Tonouchi R Takahashi et al ldquoStructuralanalysis of a new anti-hypertensive peptide (120573-lactosin B)isolated from a commercial whey productrdquo Journal of DairyScience vol 87 no 7 pp 1967ndash1974 2004

[17] G P Rizzi ldquoFree radicals in theMaillard reactionrdquo Food ReviewsInternational vol 19 no 4 pp 375ndash395 2003

[18] M-N Maillard C Billaud Y-N Chow C Ordonaud and JNicolas ldquoFree radical scavenging inhibition of polyphenoloxi-dase activity and copper chelating properties of model Maillardsystemsrdquo LWTmdashFood Science and Technology vol 40 no 8 pp1434ndash1444 2007

[19] F J Morales and M-B Babbel ldquoAntiradical efficiency ofMaillard reaction mixtures in a hydrophilic mediardquo Journal ofAgricultural and Food Chemistry vol 50 no 10 pp 2788ndash27922002

[20] M S Rao S P Chawla R Chander and A Sharma ldquoAntioxi-dant potential of Maillard reaction products formed by irradia-tion of chitosanmdashglucose solutionrdquoCarbohydrate Polymers vol83 no 2 pp 714ndash719 2011

[21] J A Rufian-Henares and F J Morales ldquoFunctional propertiesof melanoidins in vitro antioxidant antimicrobial and antihy-pertensive activitiesrdquo Food Research International vol 40 no 8pp 995ndash1002 2007

[22] M Carabasa-Giribet and A Ibarz-Ribas ldquoKinetics of colourdevelopment in aqueous glucose systems at high temperaturesrdquoJournal of Food Engineering vol 44 no 3 pp 181ndash189 2000

[23] X Tovar Jimenez A Arana Cuenca A Tellez Jurado A AbreuCorona and C R Muro Urista ldquoTraditional methods for wheyprotein isolation and concentration effects on nutritional prop-erties and biological activityrdquo Journal of the Mexican ChemicalSociety vol 56 no 4 pp 369ndash377 2012

[24] A Fechner A Knoth I Scherze and G Muschiolik ldquoStabil-ity and release properties of double-emulsions stabilised bycaseinate-dextran conjugatesrdquo Food Hydrocolloids vol 21 no5-6 pp 943ndash952 2007

[25] M Ozgen R N Reese A Z Tulio Jr J C Scheerens andA RMiller ldquoModified 22-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) method to measure antioxidant capacityof selected small fruits and comparison to ferric reducingantioxidant power (FRAP) and 221015840-diphenyl-1-picrylhydrazyl(DPPH) methodsrdquo Journal of Agricultural and Food Chemistryvol 54 no 4 pp 1151ndash1157 2006

[26] C V Morr and E Y W Ha ldquoWhey protein concentrates andisolates processing and functional propertiesrdquo Critical Reviewsin Food Science and Nutrition vol 33 no 6 pp 431ndash476 1993

[27] K N Pearce and J E Kinsella ldquoEmulsifying properties ofproteins evaluation of a turbidimetric techniquerdquo Journal ofAgricultural and Food Chemistry vol 26 no 3 pp 716ndash7231978

[28] R He X Ju J Yuan L Wang A T Girgih and R E AlukoldquoAntioxidant activities of rapeseed peptides produced by solidstate fermentationrdquo Food Research International vol 49 no 1pp 432ndash438 2012

[29] L Zhang J Li and K Zhou ldquoChelating and radical scavengingactivities of soy protein hydrolysates prepared from microbialproteases and their effect on meat lipid peroxidationrdquo Biore-source Technology vol 101 no 7 pp 2084ndash2089 2010

[30] S B Kim H S Shin and J W Lim ldquoSeparation of calcium-binding protein derived from enzymatic hydrolysates of cheesewhey proteinrdquo Asian Australasian Journal of Animal Sciencesvol 17 no 5 pp 712ndash718 2004

[31] W-Q Wang Y-H Bao and Y Chen ldquoCharacteristics andantioxidant activity of water-solubleMaillard reaction productsfrom interactions in a whey protein isolate and sugars systemrdquoFood Chemistry vol 139 no 1ndash4 pp 355ndash361 2013

[32] X Q Huang Z C Tu H Xiao et al ldquoCharacteristics andantioxidant activities of ovalbumin glycated with differentsaccharides under heat moisture treatmentrdquo Food ResearchInternational vol 48 no 2 pp 866ndash872 2012

[33] Q Liu B Kong J Han C Sun and P Li ldquoStructure andantioxidant activity of whey protein isolate conjugated withglucose via theMaillard reactionunder dry-heating conditionsrdquoFood Structure vol 1 no 2 pp 145ndash154 2014

[34] M Daglia A Papetti C Aceti B Sordelli C Gregotti andG Gazzani ldquoIsolation of high molecular weight componentsand contribution to the protective activity of coffee againstlipid peroxidation in a rat liver microsome systemrdquo Journal ofAgricultural and Food Chemistry vol 56 no 24 pp 11653ndash11660 2008

[35] M S Rao S P Chawla R Chander and A Sharma ldquoAntioxi-dant potential of Maillard reaction products formed by irradia-tion of chitosanndashglucose solutionrdquo Carbohydrate Polymers vol83 no 2 pp 714ndash719 2011

[36] C M Oliver L D Melton and R A Stanley ldquoFunctionalproperties of caseinate glycoconjugates prepared by controlledheating in the lsquodryrsquo staterdquo Journal of the Science of Food andAgriculture vol 86 no 5 pp 732ndash740 2006

[37] J Buchert D E Cura H Ma et al ldquoCrosslinking food proteinsfor improved functionalityrdquoThe Annual Review of Food Scienceand Technology vol 1 no 1 pp 113ndash138 2010

[38] Y Sun S Hayakawa M Chuamanochan M Fujimoto AInnun and K Izumori ldquoAntioxidant effects of Maillard reac-tion products obtained from ovalbumin and different D-aldohexosesrdquo Bioscience Biotechnology and Biochemistry vol70 no 3 pp 598ndash605 2006

[39] F Nacka J-M Chobert T Burova J Leonil and T HaertleldquoInduction of new physicochemical and functional propertiesby the glycosylation of whey proteinsrdquo Journal of ProteinChemistry vol 17 no 5 pp 495ndash503 1998

[40] M Corzo-Martınez C Carrera-Sanchez M Villamiel J MRodrıguez-Patino and F J Moreno ldquoAssessment of interfacialand foaming properties of bovine sodium caseinate glycatedwith galactoserdquo Journal of Food Engineering vol 113 no 3 pp461ndash470 2012

[41] M Corzo-Martınez C Carrera Sanchez F J Moreno J MRodrıguez Patino and M Villamiel ldquoInterfacial and foamingproperties of bovine 120573-lactoglobulin galactose Maillard conju-gatesrdquo Food Hydrocolloids vol 27 no 2 pp 438ndash447 2012

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of



CeramicsJournal of


CompositesJournal of

NanoparticlesJournal of



International Journal of

Biomaterials


NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research



MetallurgyJournal of


BioMed Research International

MaterialsJournal of


Nano

materials


Journal ofNanomaterials


conditions nature and concentration of proteins and sugarscontribute to this drastic and contrary effect

Sugar fragmentation and degradation of amino acids(Strecker degradation) occurring in advanced stages of thereaction may aggravate antioxidant action In addition poly-merized compounds which in turn produce irreversible orintermediates reactions and the interaction between themmay also affect the antioxidant activity of the productsof glycation Besides training of melanoidins characterizedmany of them with typical aroma and flavor [22] are found asproduct of MR in advanced stages Therefore the manipula-tion of whey proteins properties is an important topic in foodindustries and more studies are necessary to determine thepotential glycation and their influence on their functionalityas food additive Due to the complexity of MR it is necessaryto deepen the study of parameters to these reactions and typeof carbohydrate utilized in the glycation

The aim of this work was to evaluate the antioxidantactivity and functional properties of products obtained bycombination of enzymatic hydrolysis and glycation process ofproteins concentrates from milk bovine whey Hydrolyzateswere obtained by contact with Bacillus subtilis Nature of thesugar on functionality of products was analyzed by individualexperiments with ribose and glucose under controlled condi-tions of carbohydrate concentration temperature and pH

2 Materials and Methods

21 Materials Samples of 2 L of acid and sweet bovinewhey milk were provided by a cheese industry localized inMexico The 221015840azino-bis(3-ethylbenzothiazoline-6-sulfonicacid) (ABTS) and reducing sugars glucose ribose and ara-binose were purchased from Sigma Aldrich Other chemicalsagents used were of analytical grade

22 Preparation of Whey Protein Concentrates Samples ofwhey of 1 L were filtered to remove fat and casein particlesby vacuum filtration usingWhatman filter paper number 40

Then whey was concentrated by ultrafiltration process(UF) to obtain whey proteins concentrates (WPC) In thiscase was used cross-flow zirconium-titanium ceramic mem-brane of 15 kDa cut-off and polymeric membrane (hollowfiber) of 6 kDa cut-off (Pall) Tubular housing of tangen-tial flow of membrane was adapted to a peristaltic pumpThe transmembrane pressures were adjusted and controlledto 30 and 03 bar in ceramic and polymeric membranerespectively Whey feeding was performed at 25∘C to bothconcentration processes In eachUF experiment two streamswere obtained the retentate (WPC) and permeate (lactoseand minerals mainly)

Diafiltration was performed by addition of five volumesof deionized water to the retentate to maintain a constantvolume during UF process [23]

UF was stopped after approximately 18 h of filtrationProteins in whey and WPC were determined by Biuretmethod The percent yield (119884) of WPC was calculated using

119884 = 1198812119862211988111198621(100) (1)

where 1198622is protein concentration in the retentate after UF

1198621is protein concentration in the whey sample 119881

1is initial

volume ofwhey sample and1198812is final volume in the retentate

after UFTo perform the next experiments WPC products were

chosen according to the highest proteins concentration

23 Preparation of Hydrolyzed Products from Whey ProteinConcentrates Hydrolyzed productsWPCHwere obtained bydirect contact of substrates WPC with suspended biomass ofBacillus subtilis in relation 1 1 WPC samples were previouslyadjusted to pH 7 B subtilis was previously maintained in thelaboratory on nutrient broth agar slants (5 gL beef extract10 gL peptone 5 gL sodium chloride 18 gL agar and pH 7ndash74) and stored at 4∘C [23]

The hydrolysis was performed in a shaking bath (Shel-Lab) at 100 rpm and 50∘C In this experiment WPCwas usedas control group the sample was treated under the sameexperimental conditions

Upon completion of the hydrolysis experiment (24 h)the B subtilis was inactivated at a temperature of 70∘C in awater bath for 3min then the tubes were centrifuged for 15minutes at 5000 rpm and the supernatant was diluted withdilution factor of 100 to determine the protein concentrationby Biuret method [23] Degree of hydrolysis (DH) of WPCwas determined by measuring the proteins concentrationevery 2 hours during 12 h of uninterrupted hydrolysis andwascalculated by [23]

DH =119862119900minus 119862119891

119862119891

(100) (2)

where 119862119900is the initial protein concentration in WPC and

119862119891is the protein concentration in supernatant hydrolyzed

product for hydrolysis time

24 Preparation of Polymerized Products from Whey byGlycation Process Polymerized products from whey by gly-cation process were obtained by the coupling of the samplesWPC and WPCH with a carbohydrate (ribose and glucose)according to a method previously developed [24]

Carbohydrate solution was previously prepared by bufferdissolution pH 7 of sodium citrate and buffer dissolution pH9 of sodium carbonate each one with 02 g of carbohydrate

Samples of WPC and WPCH were placed in directcontact with carbohydrate (1 4 vv) at pH 7 and pH 9 inindividual experiments The mixtures were stirred at roomtemperature for 1 h to form uniform dispersion Afterwardsthe mixtures were then incubated at 50∘C during 20 h

For the period of glycation process each mixture wassampled in intervals of time of 5 h to 20 h Productswere char-acterized by color change antioxidant activity and functionalproperties

Sweet and acid whey were used as control group thesamples were only heated under the same experimentalconditions

Glycated productswere identified asWWPC-RWPC-Gand WPCH-R WPCH-G for whey whey concentrates and









AA =119860119888minus 119860119904

119860119888

(100) (3)











CEM = 1198811198811

(100) (4)




CE =119881119891minus 119881119894

119881119894

(100) (5)

where 119881119891and 119881













96 98

45

60

0

20

40

60

80

100

120

1 2 3 4

Yiel

d (

)


Ceramic






0

10

20

30

40

50

60

0 5 10 15 20 25

DH

()

Time (h)

WPI-1WPI-2










Sweet whey at pH 7


Sweet whey at pH 9


Acid whey at pH 7


Acid whey at pH 9






























075076075076

102085

0750808

13

w7-1-180w7-2-180

WPC7-1-180-RWPC9-1-180-RWPC7-2-180-R

WPCH7-1-120-35-RWPCH9-2-120-25-G

WPCH9-2-60-25-RWPCH7-2-60-60-R










































2and C



1andC








0 20 40 60 80 100 120

W7-1-180W7-2-180



WPCH9-2-180-45-RWPCH9-2-120-25-G

Foam ()Emulsion ()

Solubility ()AA ()



4 Conclusions









References


















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials











AA =119860119888minus 119860119904

119860119888

(100) (3)











CEM = 1198811198811

(100) (4)




CE =119881119891minus 119881119894

119881119894

(100) (5)

where 119881119891and 119881













96 98

45

60

0

20

40

60

80

100

120

1 2 3 4

Yiel

d (

)


Ceramic






0

10

20

30

40

50

60

0 5 10 15 20 25

DH

()

Time (h)

WPI-1WPI-2










Sweet whey at pH 7


Sweet whey at pH 9


Acid whey at pH 7


Acid whey at pH 9






























075076075076

102085

0750808

13

w7-1-180w7-2-180

WPC7-1-180-RWPC9-1-180-RWPC7-2-180-R

WPCH7-1-120-35-RWPCH9-2-120-25-G

WPCH9-2-60-25-RWPCH7-2-60-60-R










































2and C



1andC








0 20 40 60 80 100 120

W7-1-180W7-2-180



WPCH9-2-180-45-RWPCH9-2-120-25-G

Foam ()Emulsion ()

Solubility ()AA ()



4 Conclusions









References


















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials






96 98

45

60

0

20

40

60

80

100

120

1 2 3 4

Yiel

d (

)


Ceramic






0

10

20

30

40

50

60

0 5 10 15 20 25

DH

()

Time (h)

WPI-1WPI-2










Sweet whey at pH 7


Sweet whey at pH 9


Acid whey at pH 7


Acid whey at pH 9






























075076075076

102085

0750808

13

w7-1-180w7-2-180

WPC7-1-180-RWPC9-1-180-RWPC7-2-180-R

WPCH7-1-120-35-RWPCH9-2-120-25-G

WPCH9-2-60-25-RWPCH7-2-60-60-R










































2and C



1andC








0 20 40 60 80 100 120

W7-1-180W7-2-180



WPCH9-2-180-45-RWPCH9-2-120-25-G

Foam ()Emulsion ()

Solubility ()AA ()



4 Conclusions









References


















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials






Sweet whey at pH 7


Sweet whey at pH 9


Acid whey at pH 7


Acid whey at pH 9






























075076075076

102085

0750808

13

w7-1-180w7-2-180

WPC7-1-180-RWPC9-1-180-RWPC7-2-180-R

WPCH7-1-120-35-RWPCH9-2-120-25-G

WPCH9-2-60-25-RWPCH7-2-60-60-R










































2and C



1andC








0 20 40 60 80 100 120

W7-1-180W7-2-180



WPCH9-2-180-45-RWPCH9-2-120-25-G

Foam ()Emulsion ()

Solubility ()AA ()



4 Conclusions









References


















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials





















075076075076

102085

0750808

13

w7-1-180w7-2-180

WPC7-1-180-RWPC9-1-180-RWPC7-2-180-R

WPCH7-1-120-35-RWPCH9-2-120-25-G

WPCH9-2-60-25-RWPCH7-2-60-60-R










































2and C



1andC








0 20 40 60 80 100 120

W7-1-180W7-2-180



WPCH9-2-180-45-RWPCH9-2-120-25-G

Foam ()Emulsion ()

Solubility ()AA ()



4 Conclusions









References


















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials








































2and C



1andC








0 20 40 60 80 100 120

W7-1-180W7-2-180



WPCH9-2-180-45-RWPCH9-2-120-25-G

Foam ()Emulsion ()

Solubility ()AA ()



4 Conclusions









References


















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




0 20 40 60 80 100 120

W7-1-180W7-2-180



WPCH9-2-180-45-RWPCH9-2-120-25-G

Foam ()Emulsion ()

Solubility ()AA ()



4 Conclusions









References


















































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials





































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials



Research Article Antioxidant Activity and Functional...

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