Extraction and application of antioxidants from black glutinous rice

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

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Extraction and application of antioxidants from black glutinous rice Kanitha Tananuwong*, Wanida Tewaruth Department of Food Technology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand

a r t i c l e i n f o

a b s t r a c t

Article history: Received 6 August 2008 Received in revised form 13 July 2009 Accepted 15 September 2009

This research aimed to determine optimum extraction condition of black glutinous rice crude extract and to determine its application as an antioxidant in fish oil enriched mayonnaise. Black glutinous rice flour was extracted twice with 70:30 acetone-water mixture (v/v) at pH 2 and 6.8 for 2, 4 and 8 h of total extraction times. Total phenolic content (TPC), total monomeric anthocyanin content (TMA) and antioxidant activities as determined by ferric reducing antioxidant power (FRAP) and 2,2-diphenyl-1picrylhydrazyl (DPPH) free radical scavenging activity assays of the crude extracts were measured. The extraction with pH 6.8 solvent for 4 h yielded the crude extract with significantly highest antioxidant activities 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 at 500 mg/kg and 1000 mg/kg (oil weight basis). Conjugated diene hydroperoxides (CDH), thiobarbituric acid reactive substance (TBARs) and color in CIELAB system of the mayonnaise samples stored at 30  C were determined up to 30 days. The samples contained 1000 mg/kg crude extract had lowest rate of CDH and TBARs increase but had greatest extent of color deterioration, possibly due to anthocyanin degradation and Maillard reaction. Ó 2009 Elsevier Ltd. All rights reserved.

Keywords: Black glutinous rice Phenolics Antioxidant Extraction Application

1. Introduction Pigmented rice (Oryza sativa L.) has been consumed for a long time in Asia, especially China, Japan, Korea and many countries in Southeast Asia. Several varieties of pigmented rice, particularly red and black rice, have been cultivated in Thailand. Among these varieties, black glutinous rice is the most famous one, generally used as an ingredient in snacks and desserts. Nowadays, whole grain pigmented rice has been categorized as one of the potent functional foods since it contains high amounts of phenolic compounds, especially anthocyanins in pericarp (Abdel-Aal, Young, & Rabalski, 2006; Ryu, Park, & Ho, 1998; Yawadio, Tanimori, & Morita, 2007). Many studies have demonstrated antioxidant activity and radical scavenging ability of the pigmented rice and/or its 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 the extracts 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

* Corresponding author. Fax: þ662 254 4314. E-mail address: [email protected] (K. Tananuwong). 0023-6438/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2009.09.014

abnormalities associated with high fructose diets, including glucose intolerance and hyperlipidemia (Guo et al., 2007). These researches could 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 phenolic fraction due to its simplicity and low cost. Organic solvents commonly 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 organic solvents with water were also widely used (Awika, Rooney, & Waniska, 2004; Nam et al., 2006; Pe´rez-Jime´nez & Saura-Calixto, 2005). The extractability of phenolic compounds and their antioxidant activities in the crude extract depends on many factors including polarity and pH of solvents, extraction time and temperature, as well as chemical structure of phenolic compounds (Awika et al., 2004; Pe´rez-Jime´nez & Saura-Calixto, 2006; Sun & Ho, 2005). Therefore, it is necessary to find the optimum extraction system in order to obtain the extract with high amount of phenolic compounds and high antioxidant activities. However, most studies on antioxidant activity of pigmented rice extract did not report details on the optimization of the solvent extraction process. Moreover, no published data on the application of pigmented rice crude extract as an antioxidant in food products is present. The objectives of this study were to determine the optimum condition of solvent extraction of black glutinous rice crude extract and to determine its application as an antioxidant in food.

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

Our preliminary study indicated that aqueous acetone was superior to aqueous ethanol when used to prepare crude extract from black glutinous rice. At similar solvent pH and extraction time, the acetone system provided the extract with greater content of phenolic compounds and antioxidant activities. Moreover, boiling point of acetone was approximately 22  C lower than that of ethanol (Lide, 2008). Lower temperature for solvent evaporation could be used, which could save energy and reduce thermal degradation of the antioxidants during evaporation process. In terms of regulatory status, acetone was allowed to use as an extraction solvent for food additives according to the FAO JECFA Monographs (JECFA, 2006) and the directive 2009/32/EC (EC, 2009). Aqueous acetone containing 70 ml acetone/100 ml solvent was then selected as a model system for considering the optimum solvent pH and extraction time for the crude extract preparation. The freeze-dried crude extract from the selected condition, as a natural antioxidant, was then added to fish oil enriched mayonnaise for the application study.

2. Materials and methods 2.1. Materials Black glutinous rice (Oryza sativa L.) cv. Kam Doi Saked used in this study was obtained from Chiang Mai province, Thailand. White endosperm of the rice kernel was covered with dark purple-red hull. The hulled rice kernels were milled and passed through the 210 mm sieve. The rice flour was then packed in a polypropylene bag (50 mm thickness), sealed, and kept at 18  C until use. Soybean oil was 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. Soybean oil 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 Erba Reagenti (Rodano, MI, Italy). Gallic acid, trolox, tripyridyltriazine (TPTZ), 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and 2-thiobarbituric acid were purchased from Fluka Analyticals (Seelze, Germany).

2.3. Preparation of crude extract The black glutinous rice flour was extracted with acetone:water mixture (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 mixed with 100 ml of the solvent. The extraction was done twice with an equal period of time for each round to obtain the total extraction time of 2, 4 and 8 h. All extractions were done at 32  C in a shaking water bath. For each round of the extraction, the supernatant and pellet was separated by centrifugation at 1250  g for 15 min at room temperature. The supernatant from first and second round of extraction was combined and evaporated. The final volume of the crude extract was adjusted to 40 ml, kept in brown glass bottles and stored 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 FolinCiocalteu assay (Waterhouse, 2005). One ml of sample was added

477

into a 100 ml volumetric flask, followed by 70 ml of distilled water and 5 ml of Folin-Ciocalteu reagent. The solution was swirled to mix and incubated 7 min at room temperature. Fifteen ml of sodium carbonate solution was subsequently added and made up the final volume with distilled water. The solution was mixed and incubated at room temperature for 2 h. The absorbance was measured by a spectrophotometer (Lambda 25 UV–vis Spectrometer, Perkin Elmer instrument, USA) at 765 nm. With the calculation from gallic acid standard curve, the results were expressed as mg gallic acid/g flour.

2.5. Determination of total monomeric anthocyanins Total monomeric anthocyanin content was determined by the pH differential method (Giusti & Wrolstad, 2005). To measure the absorbance at pH 1.0 and 4.5, the crude extract was diluted 20 times with pH 1.0 potassium chloride buffer and pH 4.5 sodium acetate buffer, respectively. The maximum absorption of the sample in pH 1 buffer was found at 513 nm, which indicated that the major anthocyanin in the extract was likely to be cyanidin-3-glucoside (Giusti & Wrolstad, 2005). Therefore, the total monomeric anthocyanin content of crude extract was calculated in terms of cyanidin3-glucoside. The concentration of monomeric anthocyanin pigment was calculated by the following equation:

Monomeric anthocyanin pigmentðmg=lÞ h i. ¼ Adiff  MW  DF  1000 3

(1)

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

Adiff ¼ ðA513  A700 ÞpH1:0 ðA513  A700 ÞpH4:5

(2)

Note that A700 was measured and subtracted off in order to eliminate 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 Benzie and Strain (1996) Ten ml of sample was added to 990 ml of FRAP solution (acetate buffer:ferric chloride solution:Tripyridyltriazine solution of 10:1:1 by volume) in a cuvette. The mixture was held for 4 min at room temperature before measuring the absorbance at 593 nm. The corrected absorbance was calculated by subtracting the absorbance of the reagent blank from the absorbance of the sample read after 4 min. The antioxidant activity was calculated as mmol trolox/g flour using trolox standard curve.

2.7. DPPH radical scavenging activity assay DPPH assay was conducted with a method described by BrandWilliams, Cuvelier, and Berset (1995) with modifications. Different dilutions of the crude extracts were prepared. Fifty ml of the diluted crude extract was mixed with 950 ml of 0.3 mmol/l DPPH solution in a cuvette and held for 30 min at room temperature. The absorbance was then read at 515 nm. EC50 (g flour/g DPPH), which is the concentration of the antioxidant that caused the decrease of DPPH radicals to half of its initial concentration, was determined from the graph of the equivalent amount of the sample in DPPH solution (g flour/g DPPH) and radical scavenging activity (%). The latter parameter was calculated from equation (3).

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h  i Radical scavenging activityð%Þ ¼ 1  Asample =Areagent blank  100

ð3Þ

2.8. Preparation of fish oil enriched mayonnaise Within 100 g of product, fish oil enriched mayonnaise composed of 78.9 g of oil mixture (3:1 weight ratio of soybean oil to fish oil), 9.5 g of distilled vinegar, 8.9 g of egg yolk, 1.3 g of salt, 1.0 g of potassium sorbate and 0.7 g of mustard. Dried crude extract of black glutinous rice from the optimum extraction condition was also used as an ingredient in the mayonnaise sample. Focusing on the antioxidant activities of the crude extracts, the extraction with pH 6.8 solvent for 4 h was selected as the optimum condition (see details in the Results & Discussion part). Without further purification step, the crude extract from this condition was freeze-dried at 53  C under vacuum (124 psi) for 48 h. The dried powder was dissolved in vinegar before mixing with other ingredients. The final concentration of the crude extract in the oil phase of the mayonnaise 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 of mayonnaise). The mayonnaise from each batch was divided, packed in Nylon/DL/LLDPE bags (70 mm thickness) and heat-sealed. Headspace oxygen in each bag was limited. The samples were incubated in the dark at 30  C for 30 days for the oxidative stability test and color measurement. The samples were prepared in triplicate (3 batches per treatment). Note that each bag of the samples was used only once for the measurement at a specific storage time. 2.9. Oxidative stability test 2.9.1. Sample preparation The extraction of the lipid phase from the mayonnaise samples was done according to the method of Jacobsen, Meyer, and AdlerNissen (1998). The sample was frozen at 40  C for at least 22 h to destabilize the emulsion and thawed at 5  C for 2 h. The thawed mayonnaise was centrifuged at 25,400  g at 4  C for 10 min. The separated oil phase was then used in the measurement of conjugate diene hydroperoxides and thiobarbituric acid reactive substances (TBARs) assay. 2.9.2. Measurement of conjugated diene hydroperoxides Conjugated diene hydroperoxides were evaluated by the method of Frankel, Huang, Kanner, and German (1994) Fifty mg of oil phase from mayonnaise sample was added to 5 ml of isooctane. The absorbance was measured at 234 nm. Concentration of conjugated diene hydroperoxides was calculated with BeerLambert law using molar absorptivity of 26,000 l/mol cm. The final value was expressed as mmol hydroperoxides/kg oil. 2.9.3. Thiobarbituric acid reactive substances (TBARs) assay TBARs in the samples were analyzed according to the method described by Pegg (2005). Fifty mg of oil phase from a mayonnaise sample was transferred to 25 ml volumetric flask and made up the volume with 1-butanol. The solution was mixed thoroughly. Five ml of the solution was then mixed with 5.0 ml of 0.2 g/100 ml TBA in 1-butanol and incubated for 2 h at 95  C. The absorbance of the solution was read at 532 nm. TBA value (mg1) was expressed as the increasing absorbance due to the reaction of the equivalent 1 mg of sample per 1 ml volume with TBA which was calculated by the following equation:

i. h  m TBA value ¼ 50  Asample  Areagent blank where m represents mass of oil sample (mg).

2.10. Color measurement Color of the mayonnaise samples was measured by Minolta Chroma 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 was performed and least significant difference test was used to evaluate the different between means at the 95% confidence interval. 3. Results and discussion 3.1. Determine the optimum extraction condition of black glutinous rice crude extract The overall results of the phenolic contents and antioxidant activities of the crude extracts obtained from different extraction conditions were shown in Table 1. Statistical analysis showed that extraction time did not significantly affected total phenolic content and total monomeric anthocyanin content of the crude extract (p > 0.05), while this factor significantly influenced antioxidant activities of the extract as determined from FRAP and DPPH assay (p  0.05). The crude extracts obtained from longer extraction time tended to have greater antioxidant activities. Acidity of the solvent had greater effects on total monomeric anthocyanin contents and DPPH assay. At the similar extraction duration, acidic solvent (pH 2) provided the extract with significantly higher amount of total monomeric anthocyanin than neutral solvents (pH 6.8) (p  0.05). This could be due to the higher stability of anthocyanin in acidic solution. (Abdel-Aal & Hucl, 2003;). However, the acidic solvent yielded the crude extract with lower antioxidant activity as determined by DPPH assay. Note that the DPPH assay was expressed as EC50 which was the concentration of antioxidant that caused the decrease 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 EC50 value of gallic acid-catechin mixture (1:1 molar ratio) in acidic (pH 2) or neutral solvent was similar but kinetic of the DPPH-scavenging reaction in both solvent systems was different. In the acidic system, the reaction took more time to reach steady state. As for the DPPH test condition used in this study, the 30-min reaction time was sufficient to provide steady state for the systems containing

Table 1 Total phenolic content, total monomeric anthocyanin content and antioxidant activities by FRAP and DPPH assays of black glutinous rice crude extract obtained from different extraction times and pH of solvent (acetone-water mixture, 70:30 v/v).a, b, c Solvent Total Total pH extraction phenolics (mg gallic acid/g time (h) flour)

FRAP (mmol Total trolox/g monomeric anthocyanins flour) (mg/g flour)

EC50 (g flour/g DPPH)

2.0 2.0 2.0 6.8 6.8 6.8

342  6a 352  35 313  30 275  26 288  22 298  11

77.6  0.5 a 59.3  0.5 b 51.9  0.5 c 46.9  2.0 d 42.9  2.7 e 46.0  1.8 d

a

1838  48 bc 1815  96 c 1898  48 abc 1992  83 a 1878  104 abc 1968  75 ab

a ab b b b

9.31  0.31 c 10.10  0.13 b 11.46  0.01 a 9.81  0.41 bc 10.92  0.54 a 11.46  0.13 a

Mean  standard deviation of triplicate analyses. 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. b

(4)

2 4 8 2 4 8

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

either neutral or acidic extracts. Hence, the lower antioxidant activity of the acidic extracts from this research could be due to the alteration of antioxidant composition. At similar extraction duration, acidic solvent might extract a group of compounds with less DPPH radical-scavenging activities. In order to determine the optimum extraction condition, the results from the determination of both phenolic compounds and the antioxidant activities of the extracts had to be considered together. The overall data from Table 1 depicted that the extracting condition that provided the highest amount of total phenolics or total monomeric anthocyanins did not exactly provide the extracts with the highest antioxidant efficiency for both FRAP assay and DPPH assay. Weak correlation between phenolic content and antioxidant 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, although phenolic compounds were proposed to be major antioxidants in the crude extracts, other types of antioxidants, including carotenoids, tocopherols and minerals, might also be present in the extract and influence the overall antioxidant activities measured. Secondly, the antioxidant activity did not only depend on the amount of phenolic compounds but it also depended on chemical structure of phenolic compounds (Heim, Tagliaferro, & Bobilya, 2002; RiceEvans, Miller, & Paganga, 1996). Thirdly, synergistic effects among antioxidants in the crude extracts could be available, which would further weaken the relationship between total phenolic content and measurable antioxidant activities. Therefore, total phenolic content may not always be a good indicator for antioxidant activities of the crude extracts. Moreover, the results from DPPH assay did not totally agree with the FRAP assay results. The conflicted results 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 the different mechanisms of antioxidants. The FRAP assay was based on the electron transfer reactions whereas DPPH assay evaluated both electron transfer and hydrogen atom transfer reactions (Prior, Wu, & Schaich, 2005). Hence, similar antioxidant compound might react differently in different assays. In addition, iron binding capacity of antioxidants, especially phenolic compounds, in the crude extracts might also influence the results from FRAP assay. This assay was based on the presumption that the electron transfer reactions were complete within 4 min. However, different phenolic compounds might have different orders of reactivity with iron. The short reaction time might not represent the complete reactions for every type of phenolic compounds presented in the sample (Prior et al., 2005). Composition of phenolic compounds in the crude extract prepared with different extraction conditions might be varied, which might also lead to different iron binding ability and eventually affect the results from FRAP assays. Nevertheless, FRAP and DPPH assays have usually been used together to evaluate the overall activities of antioxidants (Deepa et al., 2006; Jiang et al., 2006; Pe´rez-Jime´nez & Saura-Calixto, 2005; Stratil et al., 2006). These two assays were less interfered by other substances such as amino acids and glucose comparing to ABTS and ORAC assay (Pe´rez-Jime´nez & Saura-Calixto, 2006). Therefore, these methods could provide more accurate results especially for the test of antioxidant activities in non-purified samples. Since the best extraction conditions for total phenolic content, total monomeric anthocyanin contents and antioxidant activities from FRAP assay and DPPH assay were not agreeable, the optimum condition was selected from the shortest total extraction time which gave an acceptable amount of total phenolics and total monomeric anthocyanins, but yielded the highest antioxidant activities. That system was the extraction with 70 ml acetone/ 100 ml acetone-water mixture at pH 6.8 for 4 h total extraction

479

time. Total phenolic content of the crude extract obtained from this condition was 1878 mg gallic acid/g flour, which was within the range of total phenolics in rice, 540–3130 mg gallic acid/g grain, reviewed by Fardet, Rock, and Re´me´sy (2008). Meanwhile, total monomeric anthocyanin content of the crude extract, 288 mg cyanidin equivalent/g flour, was much lower than total anthocyanin content in black rice, 3276 mg/g grain, as reported by Abdel-Aal and Hucl (2003). Genetic variation of the rice samples could result in different color intensity of the rice bran. Besides, extraction solvent could influence the measurable amount of anthocyanins. Awika et al. (2004) reported that the crude extracts of black sorghum grain prepared with acidified methanol (1 g HCl/100 ml solvent) contained monomeric anthocyanin nearly twice as much as the extracts from aqueous acetone (70 ml acetone/100 ml solvent). Anthocyanin content of black rice reported in the study of AbdelAal and Hucl (2003) was also determined from the acidified methanolic extracts (1.0 mol/l HCl in methanol). Lu and Foo (2001) investigated the reaction between acetone and anthocyanin which yielded pyrano-anthocyanins. This reaction, which could occur at room temperature during extraction step, might further reduce the measurable amount of monomeric anthocyanin in our study. The crude extract from the optimum condition was further used to study its application in fish oil enriched mayonnaise as antioxidant and colorant. Solid yield of the freeze-dried extract from this optimum extraction condition was 2.16 g/100 g rice flour. Extraction of antioxidants directly from the rice bran would help increase the yield of dried extract. However, the whole grain pigmented rice is regularly consumed. Hulled black glutinous rice is much abundant in local market than the isolated bran. Therefore, the flour prepared from the whole grain was selected as a raw material in this fundamental study. 3.2. Study the application of black glutinous rice crude extract as an antioxidant in fish oil enriched mayonnaise The addition of dried black glutinous rice crude extract at 500 and 1000 mg/kg (oil weight basis) could retard an increase in conjugated diene hydroperoxides and thiobarbituric acid reactive substances (TBARs) (Fig. 1) in fish oil enriched mayonnaise. After 18 day-storage of the control sample, the conjugated diene hydroperoxides decreased while the TBA value abruptly increased. This occurrence corresponded to the theory that the increasing of secondary oxidative product was the result of hydroperoxide degradation. However, this change could not be found in the samples containing the rice extract. Higher concentration of the extract showed greater antioxidant activities. It has been proposed that the antioxidant activities of phenolic compounds can occur from 3 mechanisms as chain-breaking antioxidant, hydroperoxide destroyer and metal chelator (Frankel, 2005; Heim et al., 2002). Hence, phenolic compounds in black glutinous rice crude extract could retard the formation of both primary and secondary oxidative products in fish oil enriched mayonnaise by the suggested mechanisms. Although the major group of antioxidants in black glutinous rice crude extract could be considered as hydrophilic phenolic compounds which mainly dissolved in an aqueous phase in mayonnaise, some of them could diffuse to the oil-water interface. Therefore, the antioxidant activity could still be observed. The effect of partitioning of antioxidant on its activities was described elsewhere (Frankel, 2005). Although the TBA test showed that the black glutinous rice crude extract could efficiently increase the oxidative stability of the mayonnaise, the oxidative changes in the samples could be influenced from other antioxidant. As previously mentioned, the soybean oil and fish oil contained tocopherols. Moreover, egg yolk also contained some carotenoids. So there were three categories of

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change could be due to the oxidative degradation of anthocyanin to undesirable brown-colored products (Delgado-Vargas & ParedesLopez, 2002). In order to evaluate the application of the black glutinous rice crude extract in fish oil enriched mayonnaise, the results from oxidative stability test and color measurement should be considered together. Although addition of the extract at high concentration (1000 mg/kg) could better increase the oxidative stability of the product, greater color deterioration could be observed.

3.0

Hy d ro p ero x id e (mmo l/k g o il)

2.5 2.0 1.5 1.0

85

0.5

80 75

0.0 5

10 15 20 Storage time (Days)

25

30

70 65

-1

TBA v alu e (mg )

L*

0

0.50

60

0.45

55

0.40

50

0.35

45

0.30

40 0

0.25

5

10

15

20

25

30

Storage time (day)

0.20 0.15

2

Storage time (day)

0.10 0

0.05 0.00 5

10 15 20 Storage time (day)

25

Fig. 1. Conjugated diene hydroperoxides and TBA value of oil phase extracted from fish oil enriched mayonnaise during storage. Black glutinous rice 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 and below the mean of triplicate measurements.

0

5

10

15

20

25

30

0

5

10

15

20

25

30

-2

30 a*

0

-4

-6 -8

-10

40 35 30

b*

antioxidants in this system, which were phenolic compounds from black glutinous rice crude extract, tocopherols and carotenoids. The synergistic effect of all antioxidant might improve the antioxidant efficiency of black glutinous rice crude extract in fish oil enriched mayonnaise. Since the black glutinous rice crude extract itself has dark purple color from anthocyanins, addition of the crude extract into the mayonnaise samples also caused a slight change in the mayonnaise color. The samples turned to light pink or light purple color, depending on the concentration of the extract added. Therefore, it is necessary to evaluate the color change in the samples during storage. Fig. 2 demonstrated the changes in L*, a*, b* values of mayonnaise samples during storage. Lightness and yellowness of all mayonnaise samples decreased while redness increased. This indicated the darkening or browning effect. The color changes might partly result from Maillard reaction. Since egg yolk contained small amounts of reducing sugars and free amino acids, the Maillard reaction could slowly occurred at acidic condition in the sample (pH 3–4). The color change occurred greater in the sample containing black glutinous rice crude extract especially with 1000 mg/kg crude extract addition. This might partly be due to additional sugar molecules from anthocyanins which could participate in the Maillard reaction. Another reason for the color

25 20 15 10

Storage time (day) Fig. 2. L*, a*, b* values of fish oil enriched mayonnaise during storage. Black glutinous rice 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 and below the mean of triplicate measurements.

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

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