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LWT - Food Science and Technology 43 (2010) 476–481

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LWT - Food Science and Technology

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Extraction and application of antioxidants from black glutinous rice

Kanitha Tananuwong*, Wanida TewaruthDepartment of Food Technology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand

a r t i c l e i n f o

Article history:Received 6 August 2008Received in revised form13 July 2009Accepted 15 September 2009

Keywords:Black glutinous ricePhenolicsAntioxidantExtractionApplication

* Corresponding author. Fax: þ662 254 4314.E-mail address: kanitha.t@chula.ac.th (K. Tananuw

0023-6438/$ – see front matter � 2009 Elsevier Ltd.doi:10.1016/j.lwt.2009.09.014

a b s t r a c t

This research aimed to determine optimum extraction condition of black glutinous rice crude extract andto determine its application as an antioxidant in fish oil enriched mayonnaise. Black glutinous rice flourwas extracted twice with 70:30 acetone-water mixture (v/v) at pH 2 and 6.8 for 2, 4 and 8 h of totalextraction times. Total phenolic content (TPC), total monomeric anthocyanin content (TMA) and anti-oxidant activities as determined by ferric reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity assays of the crude extracts were measured. Theextraction with pH 6.8 solvent for 4 h yielded the crude extract with significantly highest antioxidantactivities analyzed by both FRAP and DPPH tests (p� 0.05) although its TPC and TMA were not greatest.The freeze-dried extract from this condition was then added into fish oil enriched mayonnaise at500 mg/kg and 1000 mg/kg (oil weight basis). Conjugated diene hydroperoxides (CDH), thiobarbituricacid reactive substance (TBARs) and color in CIELAB system of the mayonnaise samples stored at 30 �Cwere determined up to 30 days. The samples contained 1000 mg/kg crude extract had lowest rate of CDHand TBARs increase but had greatest extent of color deterioration, possibly due to anthocyanin degra-dation and Maillard reaction.

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1. Introduction

Pigmented rice (Oryza sativa L.) has been consumed for a longtime in Asia, especially China, Japan, Korea and many countries inSoutheast Asia. Several varieties of pigmented rice, particularly redand black rice, have been cultivated in Thailand. Among thesevarieties, black glutinous rice is the most famous one, generallyused as an ingredient in snacks and desserts. Nowadays, wholegrain pigmented rice has been categorized as one of the potentfunctional foods since it contains high amounts of phenoliccompounds, especially anthocyanins in pericarp (Abdel-Aal, Young,& Rabalski, 2006; Ryu, Park, & Ho, 1998; Yawadio, Tanimori, &Morita, 2007). Many studies have demonstrated antioxidantactivity and radical scavenging ability of the pigmented rice and/orits extract in both in vitro and in vivo models (Hu, Zawistowski, Ling,& Kitts 2003; Ichikawa et al., 2001; Nam et al., 2006; Oki et al.,2002; Toyokuni et al., 2002) as well as other biological effects of theextracts including antimutagenic and anticarcinogenic activities(Hyun & Chung, 2004; Nam, Choi, Kang, Kozukue, & Friedman,2005), reduction of the atherosclerotic plague formation (Xia, Ling,Ma, Kitts, & Zawistowski, 2003), aldose reductase inhibitory activity(Yawadio et al., 2007) and attenuation of some metabolic

ong).

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abnormalities associated with high fructose diets, including glucoseintolerance and hyperlipidemia (Guo et al., 2007). These researchescould assure the health benefits of the pigmented rice.

In the study of phenolic antioxidants from plant materials,solvent extraction has mostly been used to obtain the phenolicfraction due to its simplicity and low cost. Organic solventscommonly used for the extraction include absolute methanol,ethanol and acetone (Sun & Ho, 2005; Yawadio et al., 2007; Yu,Perret, Davy, Wilson, & Melby, 2002). The mixtures of those organicsolvents with water were also widely used (Awika, Rooney, &Waniska, 2004; Nam et al., 2006; Perez-Jimenez & Saura-Calixto,2005). The extractability of phenolic compounds and their antiox-idant activities in the crude extract depends on many factorsincluding polarity and pH of solvents, extraction time andtemperature, as well as chemical structure of phenolic compounds(Awika et al., 2004; Perez-Jimenez & Saura-Calixto, 2006; Sun & Ho,2005). Therefore, it is necessary to find the optimum extractionsystem in order to obtain the extract with high amount of phenoliccompounds and high antioxidant activities. However, most studieson antioxidant activity of pigmented rice extract did not reportdetails on the optimization of the solvent extraction process.Moreover, no published data on the application of pigmented ricecrude extract as an antioxidant in food products is present. Theobjectives of this study were to determine the optimum conditionof solvent extraction of black glutinous rice crude extract and todetermine its application as an antioxidant in food.

K. Tananuwong, W. Tewaruth / LWT - Food Science and Technology 43 (2010) 476–481 477

Our preliminary study indicated that aqueous acetone wassuperior to aqueous ethanol when used to prepare crude extractfrom black glutinous rice. At similar solvent pH and extraction time,the acetone system provided the extract with greater content ofphenolic compounds and antioxidant activities. Moreover, boilingpoint of acetone was approximately 22 �C lower than that ofethanol (Lide, 2008). Lower temperature for solvent evaporationcould be used, which could save energy and reduce thermaldegradation of the antioxidants during evaporation process. Interms of regulatory status, acetone was allowed to use as anextraction solvent for food additives according to the FAO JECFAMonographs (JECFA, 2006) and the directive 2009/32/EC (EC,2009). Aqueous acetone containing 70 ml acetone/100 ml solventwas then selected as a model system for considering the optimumsolvent pH and extraction time for the crude extract preparation.The freeze-dried crude extract from the selected condition, asa natural antioxidant, was then added to fish oil enriched mayon-naise for the application study.

2. Materials and methods

2.1. Materials

Black glutinous rice (Oryza sativa L.) cv. Kam Doi Saked used inthis study was obtained from Chiang Mai province, Thailand. Whiteendosperm of the rice kernel was covered with dark purple-redhull. The hulled rice kernels were milled and passed through the210 mm sieve. The rice flour was then packed in a polypropylene bag(50 mm thickness), sealed, and kept at �18 �C until use. Soybean oilwas purchased from Thai Vegetable Oil Pubic Company Limited,Bangkok, Thailand. Tuna fully refined oil was purchased from T.C.Union Global Public Company Limited, Bangkok, Thailand. Soybeanoil and fish oil contained approximately 100 mg/kg and 1000 mg/kg of a-tocopherol, respectively.

2.2. Chemicals

Acetone was purchased from Ajax Finechem (Taren Point, NSW,Australia). Folin-Ciocalteu reagent was purchased from Carlo ErbaReagenti (Rodano, MI, Italy). Gallic acid, trolox, tripyridyltriazine(TPTZ), 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and 2-thiobarbituricacid were purchased from Fluka Analyticals (Seelze, Germany).

2.3. Preparation of crude extract

The black glutinous rice flour was extracted with acetone:watermixture (70:30 v/v). The pH of the solvent was varied to 2 (acidic)and 6.8 (neutral). For each trial, 10 g of the flour sample was mixedwith 100 ml of the solvent. The extraction was done twice with anequal period of time for each round to obtain the total extractiontime of 2, 4 and 8 h. All extractions were done at 32 �C in a shakingwater bath. For each round of the extraction, the supernatant andpellet was separated by centrifugation at 1250� g for 15 min atroom temperature. The supernatant from first and second round ofextraction was combined and evaporated. The final volume of thecrude extract was adjusted to 40 ml, kept in brown glass bottles andstored at �18 �C until use. The extraction was done in triplicate.

2.4. Determination of total phenolic content

Total phenolic content was determined by the method of Folin-Ciocalteu assay (Waterhouse, 2005). One ml of sample was added

into a 100 ml volumetric flask, followed by 70 ml of distilled waterand 5 ml of Folin-Ciocalteu reagent. The solution was swirled to mixand incubated 7 min at room temperature. Fifteen ml of sodiumcarbonate solution was subsequently added and made up the finalvolume with distilled water. The solution was mixed and incubatedat room temperature for 2 h. The absorbance was measured bya spectrophotometer (Lambda 25 UV–vis Spectrometer, PerkinElmer instrument, USA) at 765 nm. With the calculation from gallicacid standard curve, the results were expressed as mg gallic acid/gflour.

2.5. Determination of total monomeric anthocyanins

Total monomeric anthocyanin content was determined by thepH differential method (Giusti & Wrolstad, 2005). To measure theabsorbance at pH 1.0 and 4.5, the crude extract was diluted 20 timeswith pH 1.0 potassium chloride buffer and pH 4.5 sodium acetatebuffer, respectively. The maximum absorption of the sample in pH 1buffer was found at 513 nm, which indicated that the majoranthocyanin in the extract was likely to be cyanidin-3-glucoside(Giusti & Wrolstad, 2005). Therefore, the total monomeric antho-cyanin content of crude extract was calculated in terms of cyanidin-3-glucoside. The concentration of monomeric anthocyanin pigmentwas calculated by the following equation:

Monomeric anthocyanin pigmentðmg=lÞ

¼hAdiff �MW� DF� 1000

i.3 (1)

where MW represents molecular weight of cyanidin-3-glucoside(449.2), DF is the dilution factor (20), 3 is molar absorptivity ofcyanidin-3-glucoside (26,900 l/mol cm) and Adiff was calculatedfrom the following equation:

Adiff ¼ ðA513 � A700ÞpH1:0�ðA513 � A700ÞpH4:5 (2)

Note that A700 was measured and subtracted off in order toeliminate the effect of haze or sediments in the sample.

2.6. Ferric reducing antioxidant power (FRAP) assay

FRAP assay was performed by a method adapted from Benzieand Strain (1996) Ten ml of sample was added to 990 ml of FRAPsolution (acetate buffer:ferric chloride solution:Tripyridyltriazinesolution of 10:1:1 by volume) in a cuvette. The mixture was held for4 min at room temperature before measuring the absorbance at593 nm. The corrected absorbance was calculated by subtractingthe absorbance of the reagent blank from the absorbance of thesample read after 4 min. The antioxidant activity was calculated asmmol trolox/g flour using trolox standard curve.

2.7. DPPH radical scavenging activity assay

DPPH assay was conducted with a method described by Brand-Williams, Cuvelier, and Berset (1995) with modifications. Differentdilutions of the crude extracts were prepared. Fifty ml of the dilutedcrude extract was mixed with 950 ml of 0.3 mmol/l DPPH solution ina cuvette and held for 30 min at room temperature. The absorbancewas then read at 515 nm. EC50 (g flour/g DPPH), which is theconcentration of the antioxidant that caused the decrease of DPPHradicals to half of its initial concentration, was determined from thegraph of the equivalent amount of the sample in DPPH solution(g flour/g DPPH) and radical scavenging activity (%). The latterparameter was calculated from equation (3).

K. Tananuwong, W. Tewaruth / LWT - Food Science and Technology 43 (2010) 476–481478

Radical scavenging activityð%Þ ¼ 1� Asample=Areagent blank

Table 1Total phenolic content, total monomeric anthocyanin content and antioxidantactivities by FRAP and DPPH assays of black glutinous rice crude extract obtainedfrom different extraction times and pH of solvent (acetone-water mixture,70:30 v/v).a, b, c

SolventpH

Totalextractiontime (h)

Totalphenolics (mggallic acid/gflour)

Totalmonomericanthocyanins(mg/g flour)

FRAP (mmoltrolox/gflour)

EC50 (gflour/gDPPH)

2.0 2 1838� 48 bc 342� 6a 9.31� 0.31 c 77.6� 0.5 a2.0 4 1815� 96 c 352� 35 a 10.10� 0.13 b 59.3� 0.5 b2.0 8 1898� 48 abc 313� 30 ab 11.46� 0.01 a 51.9� 0.5 c6.8 2 1992� 83 a 275� 26 b 9.81� 0.41 bc 46.9� 2.0 d6.8 4 1878� 104 abc 288� 22 b 10.92� 0.54 a 42.9� 2.7 e6.8 8 1968� 75 ab 298� 11 b 11.46� 0.13 a 46.0� 1.8 d

a Mean� standard deviation of triplicate analyses.b Means followed by the same letter within a column do not differ significantly

(p> 0.05).c Crude extract from the optimum condition, solvent pH 6.8 and 4 h extraction

time, were freeze-dried and added in fish oil enriched mayonnaise.

h � �i

� 100 ð3Þ

2.8. Preparation of fish oil enriched mayonnaise

Within 100 g of product, fish oil enriched mayonnaisecomposed of 78.9 g of oil mixture (3:1 weight ratio of soybean oil tofish oil), 9.5 g of distilled vinegar, 8.9 g of egg yolk, 1.3 g of salt, 1.0 gof potassium sorbate and 0.7 g of mustard. Dried crude extract ofblack glutinous rice from the optimum extraction condition wasalso used as an ingredient in the mayonnaise sample. Focusing onthe antioxidant activities of the crude extracts, the extraction withpH 6.8 solvent for 4 h was selected as the optimum condition (seedetails in the Results & Discussion part). Without further purifica-tion step, the crude extract from this condition was freeze-dried at�53 �C under vacuum (�124 psi) for 48 h. The dried powder wasdissolved in vinegar before mixing with other ingredients. The finalconcentration of the crude extract in the oil phase of the mayon-naise was varied to 0 mg/kg (control), 500 mg/kg and 1000 mg/kg(or 395 mg/kg and 790 mg/kg based on the total weight ofmayonnaise). The mayonnaise from each batch was divided, packedin Nylon/DL/LLDPE bags (70 mm thickness) and heat-sealed. Head-space oxygen in each bag was limited. The samples were incubatedin the dark at 30 �C for 30 days for the oxidative stability test andcolor measurement. The samples were prepared in triplicate (3batches per treatment). Note that each bag of the samples was usedonly once for the measurement at a specific storage time.

2.9. Oxidative stability test

2.9.1. Sample preparationThe extraction of the lipid phase from the mayonnaise samples

was done according to the method of Jacobsen, Meyer, and Adler-Nissen (1998). The sample was frozen at �40 �C for at least 22 h todestabilize the emulsion and thawed at 5 �C for 2 h. The thawedmayonnaise was centrifuged at 25,400� g at 4 �C for 10 min. Theseparated oil phase was then used in the measurement of conjugatediene hydroperoxides and thiobarbituric acid reactive substances(TBARs) assay.

2.9.2. Measurement of conjugated diene hydroperoxidesConjugated diene hydroperoxides were evaluated by the

method of Frankel, Huang, Kanner, and German (1994) Fifty mg ofoil phase from mayonnaise sample was added to 5 ml of isooctane.The absorbance was measured at 234 nm. Concentration ofconjugated diene hydroperoxides was calculated with Beer-Lambert law using molar absorptivity of 26,000 l/mol cm. The finalvalue was expressed as mmol hydroperoxides/kg oil.

2.9.3. Thiobarbituric acid reactive substances (TBARs) assayTBARs in the samples were analyzed according to the method

described by Pegg (2005). Fifty mg of oil phase from a mayonnaisesample was transferred to 25 ml volumetric flask and made up thevolume with 1-butanol. The solution was mixed thoroughly. Five mlof the solution was then mixed with 5.0 ml of 0.2 g/100 ml TBA in1-butanol and incubated for 2 h at 95 �C. The absorbance of thesolution was read at 532 nm. TBA value (mg�1) was expressed asthe increasing absorbance due to the reaction of the equivalent1 mg of sample per 1 ml volume with TBA which was calculated bythe following equation:

TBA value ¼h50�

�Asample � Areagent blank

�i.m (4)

where m represents mass of oil sample (mg).

2.10. Color measurement

Color of the mayonnaise samples was measured by MinoltaChroma Meter CR-300, equipped with a liquid tube holder CR-A71(Konica Minolta, Japan). The color was expressed in CIELAB (L*, a*,b*) system (MacDougall, 2002).

2.11. Statistical analysis

Analysis of variance (ANOVA) of the experimental data wasperformed and least significant difference test was used to evaluatethe different between means at the 95% confidence interval.

3. Results and discussion

3.1. Determine the optimum extraction condition of black glutinousrice crude extract

The overall results of the phenolic contents and antioxidantactivities of the crude extracts obtained from different extractionconditions were shown in Table 1. Statistical analysis showed thatextraction time did not significantly affected total phenolic contentand total monomeric anthocyanin content of the crude extract(p> 0.05), while this factor significantly influenced antioxidantactivities of the extract as determined from FRAP and DPPH assay(p� 0.05). The crude extracts obtained from longer extraction timetended to have greater antioxidant activities. Acidity of the solventhad greater effects on total monomeric anthocyanin contents andDPPH assay. At the similar extraction duration, acidic solvent (pH 2)provided the extract with significantly higher amount of totalmonomeric anthocyanin than neutral solvents (pH 6.8) (p� 0.05).This could be due to the higher stability of anthocyanin in acidicsolution. (Abdel-Aal & Hucl, 2003;). However, the acidic solventyielded the crude extract with lower antioxidant activity as deter-mined by DPPH assay. Note that the DPPH assay was expressed asEC50 which was the concentration of antioxidant that caused thedecrease of DPPH radicals to half of its initial concentration.Therefore, lower EC50 value provides higher antioxidant efficiency.Perez-Jimenez and Saura Calixto (2006) showed that DPPH EC50value of gallic acid-catechin mixture (1:1 molar ratio) in acidic (pH2) or neutral solvent was similar but kinetic of the DPPH-scav-enging reaction in both solvent systems was different. In the acidicsystem, the reaction took more time to reach steady state. As for theDPPH test condition used in this study, the 30-min reaction timewas sufficient to provide steady state for the systems containing

K. Tananuwong, W. Tewaruth / LWT - Food Science and Technology 43 (2010) 476–481 479

either neutral or acidic extracts. Hence, the lower antioxidantactivity of the acidic extracts from this research could be due to thealteration of antioxidant composition. At similar extraction dura-tion, acidic solvent might extract a group of compounds with lessDPPH radical-scavenging activities.

In order to determine the optimum extraction condition, theresults from the determination of both phenolic compounds andthe antioxidant activities of the extracts had to be consideredtogether. The overall data from Table 1 depicted that the extractingcondition that provided the highest amount of total phenolics ortotal monomeric anthocyanins did not exactly provide the extractswith the highest antioxidant efficiency for both FRAP assay andDPPH assay. Weak correlation between phenolic content andantioxidant activities was found in earlier researches (Chu, Sun,Wu, & Liu, 2002; Heinonen, Lehtonen, & Hopia, 1998; Sun & Ho,2005). This could be explained as followed. Firstly, althoughphenolic compounds were proposed to be major antioxidants inthe crude extracts, other types of antioxidants, including caroten-oids, tocopherols and minerals, might also be present in the extractand influence the overall antioxidant activities measured. Secondly,the antioxidant activity did not only depend on the amount ofphenolic compounds but it also depended on chemical structure ofphenolic compounds (Heim, Tagliaferro, & Bobilya, 2002; Rice-Evans, Miller, & Paganga, 1996). Thirdly, synergistic effects amongantioxidants in the crude extracts could be available, which wouldfurther weaken the relationship between total phenolic contentand measurable antioxidant activities. Therefore, total phenoliccontent may not always be a good indicator for antioxidant activ-ities of the crude extracts. Moreover, the results from DPPH assaydid not totally agree with the FRAP assay results. The conflictedresults of FRAP and DPPH assay are also found in other reports(Deepa, Kaur, Singh, & Kapoor, 2006; Stratil, Klejdus, & Kuban,2006). This might be because the two systems evaluated thedifferent mechanisms of antioxidants. The FRAP assay was based onthe electron transfer reactions whereas DPPH assay evaluated bothelectron transfer and hydrogen atom transfer reactions (Prior, Wu,& Schaich, 2005). Hence, similar antioxidant compound might reactdifferently in different assays. In addition, iron binding capacity ofantioxidants, especially phenolic compounds, in the crude extractsmight also influence the results from FRAP assay. This assay wasbased on the presumption that the electron transfer reactions werecomplete within 4 min. However, different phenolic compoundsmight have different orders of reactivity with iron. The shortreaction time might not represent the complete reactions for everytype of phenolic compounds presented in the sample (Prior et al.,2005). Composition of phenolic compounds in the crude extractprepared with different extraction conditions might be varied,which might also lead to different iron binding ability and even-tually affect the results from FRAP assays. Nevertheless, FRAP andDPPH assays have usually been used together to evaluate theoverall activities of antioxidants (Deepa et al., 2006; Jiang et al.,2006; Perez-Jimenez & Saura-Calixto, 2005; Stratil et al., 2006).These two assays were less interfered by other substances such asamino acids and glucose comparing to ABTS and ORAC assay(Perez-Jimenez & Saura-Calixto, 2006). Therefore, these methodscould provide more accurate results especially for the test of anti-oxidant activities in non-purified samples.

Since the best extraction conditions for total phenolic content,total monomeric anthocyanin contents and antioxidant activitiesfrom FRAP assay and DPPH assay were not agreeable, the optimumcondition was selected from the shortest total extraction timewhich gave an acceptable amount of total phenolics and totalmonomeric anthocyanins, but yielded the highest antioxidantactivities. That system was the extraction with 70 ml acetone/100 ml acetone-water mixture at pH 6.8 for 4 h total extraction

time. Total phenolic content of the crude extract obtained from thiscondition was 1878 mg gallic acid/g flour, which was within therange of total phenolics in rice, 540–3130 mg gallic acid/g grain,reviewed by Fardet, Rock, and Remesy (2008). Meanwhile, totalmonomeric anthocyanin content of the crude extract, 288 mg cya-nidin equivalent/g flour, was much lower than total anthocyanincontent in black rice, 3276 mg/g grain, as reported by Abdel-Aal andHucl (2003). Genetic variation of the rice samples could result indifferent color intensity of the rice bran. Besides, extraction solventcould influence the measurable amount of anthocyanins. Awikaet al. (2004) reported that the crude extracts of black sorghum grainprepared with acidified methanol (1 g HCl/100 ml solvent) con-tained monomeric anthocyanin nearly twice as much as theextracts from aqueous acetone (70 ml acetone/100 ml solvent).Anthocyanin content of black rice reported in the study of Abdel-Aal and Hucl (2003) was also determined from the acidifiedmethanolic extracts (1.0 mol/l HCl in methanol). Lu and Foo (2001)investigated the reaction between acetone and anthocyanin whichyielded pyrano-anthocyanins. This reaction, which could occur atroom temperature during extraction step, might further reduce themeasurable amount of monomeric anthocyanin in our study.

The crude extract from the optimum condition was further usedto study its application in fish oil enriched mayonnaise as antioxi-dant and colorant. Solid yield of the freeze-dried extract from thisoptimum extraction condition was 2.16 g/100 g rice flour. Extrac-tion of antioxidants directly from the rice bran would help increasethe yield of dried extract. However, the whole grain pigmented riceis regularly consumed. Hulled black glutinous rice is much abun-dant in local market than the isolated bran. Therefore, the flourprepared from the whole grain was selected as a raw material inthis fundamental study.

3.2. Study the application of black glutinous rice crude extract as anantioxidant in fish oil enriched mayonnaise

The addition of dried black glutinous rice crude extract at 500and 1000 mg/kg (oil weight basis) could retard an increase inconjugated diene hydroperoxides and thiobarbituric acid reactivesubstances (TBARs) (Fig. 1) in fish oil enriched mayonnaise. After 18day-storage of the control sample, the conjugated diene hydro-peroxides decreased while the TBA value abruptly increased. Thisoccurrence corresponded to the theory that the increasing ofsecondary oxidative product was the result of hydroperoxidedegradation. However, this change could not be found in thesamples containing the rice extract. Higher concentration of theextract showed greater antioxidant activities. It has been proposedthat the antioxidant activities of phenolic compounds can occurfrom 3 mechanisms as chain-breaking antioxidant, hydroperoxidedestroyer and metal chelator (Frankel, 2005; Heim et al., 2002).Hence, phenolic compounds in black glutinous rice crude extractcould retard the formation of both primary and secondary oxidativeproducts in fish oil enriched mayonnaise by the suggested mech-anisms. Although the major group of antioxidants in black gluti-nous rice crude extract could be considered as hydrophilic phenoliccompounds which mainly dissolved in an aqueous phase inmayonnaise, some of them could diffuse to the oil-water interface.Therefore, the antioxidant activity could still be observed. Theeffect of partitioning of antioxidant on its activities was describedelsewhere (Frankel, 2005).

Although the TBA test showed that the black glutinous ricecrude extract could efficiently increase the oxidative stability of themayonnaise, the oxidative changes in the samples could be influ-enced from other antioxidant. As previously mentioned, thesoybean oil and fish oil contained tocopherols. Moreover, egg yolkalso contained some carotenoids. So there were three categories of

0.0

0.5

1.0

1.5

2.0

2.5

3.0

0 5 10 15 20 25 30Storage time (Days)

edixorepordyH

)lio gk/lom

m(

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0 5 10 15 20 25 30Storage time (day)

gm( eulav

AB

T1-)

Fig. 1. Conjugated diene hydroperoxides and TBA value of oil phase extracted from fishoil enriched mayonnaise during storage. Black glutinous rice crude extract were addedat different concentrations (A 0 mg/kg, control; - 500 mg/kg; C 1000 mg/kg). Errorbars extend one standard deviation above and below the mean of triplicatemeasurements.

40

45

50

55

60

65

70

75

80

85

Storage time (day)

*L

-10

-8

-6

-4

-2

0

2

0 5 10 15 20 25 30

Storage time (day)

*a

10

15

20

25

30

35

40

0 5 10 15 20 25 30

0 5 10 15 20 25 30

Storage time (day)

*b

Fig. 2. L*, a*, b* values of fish oil enriched mayonnaise during storage. Black glutinousrice crude extract were added at different concentrations (A 0 mg/kg, control; -

500 mg/kg; C 1000 mg/kg). Error bars extend one standard deviation above andbelow the mean of triplicate measurements.

K. Tananuwong, W. Tewaruth / LWT - Food Science and Technology 43 (2010) 476–481480

antioxidants in this system, which were phenolic compounds fromblack glutinous rice crude extract, tocopherols and carotenoids. Thesynergistic effect of all antioxidant might improve the antioxidantefficiency of black glutinous rice crude extract in fish oil enrichedmayonnaise.

Since the black glutinous rice crude extract itself has dark purplecolor from anthocyanins, addition of the crude extract into themayonnaise samples also caused a slight change in the mayonnaisecolor. The samples turned to light pink or light purple color,depending on the concentration of the extract added. Therefore, itis necessary to evaluate the color change in the samples duringstorage.

Fig. 2 demonstrated the changes in L*, a*, b* values of mayon-naise samples during storage. Lightness and yellowness of allmayonnaise samples decreased while redness increased. Thisindicated the darkening or browning effect. The color changesmight partly result from Maillard reaction. Since egg yolk containedsmall amounts of reducing sugars and free amino acids, the Mail-lard reaction could slowly occurred at acidic condition in thesample (pH 3–4). The color change occurred greater in the samplecontaining black glutinous rice crude extract especially with1000 mg/kg crude extract addition. This might partly be due toadditional sugar molecules from anthocyanins which couldparticipate in the Maillard reaction. Another reason for the color

change could be due to the oxidative degradation of anthocyanin toundesirable brown-colored products (Delgado-Vargas & Paredes-Lopez, 2002).

In order to evaluate the application of the black glutinous ricecrude extract in fish oil enriched mayonnaise, the results fromoxidative stability test and color measurement should be consid-ered together. Although addition of the extract at high concentra-tion (1000 mg/kg) could better increase the oxidative stability ofthe product, greater color deterioration could be observed.

K. Tananuwong, W. Tewaruth / LWT - Food Science and Technology 43 (2010) 476–481 481

Therefore, the addition of 500 mg/kg crude extract might be moreappropriate since both oxidative stability and color change could becompromised. However, higher concentration of the extract couldbe used with less extent of color change if the extract was frac-tionated, purified, and/or chemically modified. For instance, acyl-ation, which is the attachment of acyl group onto the glucosidicpart of anthocyanin, might be applied to increase the stability ofanthocyanin color at high temperature and high pH (4–7) (Del-gado-Vargas & Paredes-Lopez, 2002). By this way, the rice extractswith anthocyanins could be used as both antioxidant and colorantin food and beverages. Further studies are still needed to provethese suggestions.

Acknowledgement

Financial supports from Grant for Development of New FacultyStaff, Chulalongkorn University and Grant for Graduate Research,Graduate School of Chulalongkorn University are gratefullyacknowledged.

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