JOURNAL OF FUNCTIONAL FOODS 3 (2011) 79–87

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Determination of the total phenolics in juices and

superfruits by a novel chemical method 5

Marjorie B. Medina*
Residue Chemistry AND Predictive Microbiology RESEARch Unit, EASTERN REGIONAL RESEARch Center, AgriculturAL RESEARch Service,
US DEPARTMEnt of Agriculture, 600 EAST MERMAID LANE, Wyndmoor, PA 19038, USA

ARTICLEINFO
ABSTRACT

Article history: The total phenols in foods and beverages are currently measured with Folin–Ciocalteu,
Received 21 December 2010 through its reducing capacity. A novel method was developed to quantify polyphenols or
Received in revised form phenolic compounds through direct interactions of polyphenols with Fast Blue BB in an
23 February 2011 alkaline medium and the absorbance was measured at 420 nm. The gallic acid calibration
Accepted 28 February 2011 lines (0–500 lg/mL) had a linear correlation (R) greater than 0.99 in all analysis. The limit
Available online 29 March 2011 of detection was <10 lg/mL and the limit of quantification was 10 lg/mL. The total phenolics
in beverage samples had gallic acid equivalents (GAE) of 168–1868 (Fast Blue BB) and
Keywords:
70– 1390 (Folin–Ciocalteu) mg GAE/100 mL in juices and juice mixes. The solid samples
Total phenols
(fresh fruits and dry fruits) were extracted with 70% ethanol and the resulting Fast Blue BB:
Fast Blue BB diazonium dye
Folin-Ciocalteu were 37–1291:21–315 and 337–595:399-1036 GAE (mg/100 g) in fresh
Folin–Ciocalteu
and dry fruits, respectively. The Fast Blue BB:Folin–Ciocalteu ratios ranged from <1 to 4.8.
Superfoods and superfruits
The freeze-dried acai powder had 10,000:3000 mg GAE/100 g (Fast Blue BB:Folin–
Juices
Ciocalteu) while the fresh blueberries had 1291:315 GAE/100 g. The novel method
demonstrated that GAE values were higher with Fast Blue BB method than with the
Folin–Ciocalteu method.
Published by Elsevier Ltd.

1. Introduction dard procedure for measuring total ‘‘phenolics’’. It indirectly

measures the ‘‘total phenolics’’ through the reducing
Polyphenolics contribute to antioxidant properties of food, capacity of components of food or beverage samples. The
juices and beverages, and are essential to the human diet. basic mech- anism is an oxidation/reduction interaction
These phytochemicals have various preventive and detecting the presence of phenols, and the reducing
disease fighting properties. The polyphenolics contribute agents and possibly metal chelators. Phenols and
to antioxi- dant properties and these compounds include polyphenols are detected through the transfer of electrons
flavonoids, flavonols, flavonolols, flavonones, isoflavones, in an alkaline medium from the phenolic compounds to
flavones, anth- ocyanidins, flavanalols, chalcones, phosphomolybdic/phosphotung- stic acid complexes in the
anthocyanins, chlorogenic acid isomers, cinnamic and Folin–Ciocalteu reagent (Singleton, Orthofer, & Lamuela-
benzoic acid families, stilbenes, lignins, lignans, tannins, Raventos, 1999; Singleton & Rossi, 1965). A blue color is
tocopherols and tocotrienols (Shah- idi & Naczk, 2004). formed with the reducing compounds and Fo- lin–Ciocalteu
These compounds have been identified in fruits, reagents, and measured at 725 nm. The concen- tration of
vegetables, grains, nuts, oilseeds, herbs and other plant each sample was calibrated against gallic acid. The
materials. The Folin and Ciocalteu (1927) method is the interferences with the ‘‘total phenols’’ measurement were
stan-

5
Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and
does not imply recommendation or endorsement by the US Department of Agriculture.
* Tel.: +1 215 233 6436; fax: +1 215 233 6559.
E-mail address: marjorie.medina@ars.usda.gov
1756-4646/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.jff.2011.02.007
80 JO
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contributed by non-phenolic antioxidants and reducing Blue BB diazonium salt is based on the coupling of phenolic
sub- stances (ascorbic acid, glucose, fructose, sulphites) compounds with the diazonium salt resulting in the forma-
that are common food additives or are naturally present tion of azo complexes. Aromatic diazonium ions normally
in juices,
fruits and vegetables. Amino acids (tyrosine, tryptophan) couple with active substrates such as phenols (Morrison &
and proteins containing these amino acids also formed a blue Boyd, 1969; Smith & March, 2007). Fast Blue BB salt contains
color with the Folin–Ciocalteu reagent (Peterson, 1979, 1983). a diazonium group, –+N@N–, where the nitrogen is
There is a need for a simple, rapid and direct detection of retained in coupling with the reactive activating group (–
phenolics or polyphenols in foods, beverages, and agricultural OH) of the phenolic group. Coupling mostly occurs PARA to
by-products. The novel total phenolics method utilizing Fast the phenolic activating group, unless the position is already
occupied, then

Fig. 1A – Proposed interactions of Fast Blue BB salt with phenolic acids, caffeic and chlorogenic acids in NaOH buffer
solution. In this figure, the coupling (–N@N–) are shown in the para positions relative to the activating group (–OH) of the
phenolic moeity. It is also possible that coupling can occur at the ortho position(s).
 JOURNAL OF FUNCTIONAL FOODS 3 (2011) 79–87 81

the substitution occurs in the ortho position to the activating

orientin, taxifolin) is shown in Fig. 1B. Medina and Nagdy
group. Phenols are coupled in slightly alkaline solution where
(1993) also showed a proposed interaction between Fast
they can be converted to the more active phenoxide ions.
Cor- inth V diazonium group and the phenolic –OH of
The distribution of naturally occurring phenolics, such as
zeranol, a resorcylic compound. The use of other azo-
flavo- noids and the chemical structure of these phenolics
forming dyes for detection of related phenolic compounds
were de- scribed by Shahidi and Naczk (2004). Quercetin,
were reported for detection of veterinary estrogens
kaempferol, chlorogenic acid, caffeic acid, coumaric acid are
(Gunther, 1978; Medina & Schwartz, 1992; Wortberg,
found in goji beries (Wang, Chang, Stephen Inbaraj, & Chen,
Woller, & Chulamorakot, 1978); hu- man estrogens (Weiss,
2010); cyani- dins, orientin, and taxifolin are present in
Esterbauar, & Scherr, 1975; Weiss, Win- ter, Scherr, &
acai (Schauss et al., 2006). The proposed interaction of
Bayer, 1976); zearalenone mycotoxin (Scott, Panalaks,
gallic acid phenolic groups with the diazonium moiety of Fast
Kanhere, & Miles, 1978); phenolic constituents of plant cell
Blue BB indicated coupling in the para and ortho –OH
walls (Harris, Hartley, & Barton, 1982).
positions (Medina, 2011). Fig. 1A shows the proposed
The interactions of the Fast Blue BB with gallic acid were
interactions of Fast Blue BB with phenolic acids (chlorogenic previously optimized (Medina, 2011). The absorbance mea-
and caffeic acids) where these compounds are major sured at 420, 500 and 520 nm yielded an optimum at 420 nm
constituents of fruits and coffee beans. The azo complex with a Fast Blue BB concentration of 0.1%. Reaction time at
formation with major phenolic con- 60 min showed a linear response. Effects of the alkali
(Na2CO3
stituents (cyanidin, pelargonidin, kaempferol, delphinidin, and NaOH) were also compared and results showed that 5%

Fig. 1B – Proposed interactions of Fast Blue BB salt with flavonoids (cyanidin, pelargonidin, kaempferol, delphinidin,
orientin, taxifolin). In this figure, the coupling (–N@N–) are shown in the para and ortho positions relative to the activating
group (–OH) of the phenolic moeity.
82 JOURNAL OF FUNCTIONAL FOODS 3 (2011) 79–87

NaOH had a faster completion of reaction compared to 20%

and the results were expressed as gallic acid equivalents
Na2CO3. These optimum conditions resulted in a three-step (GAE).
procedure, transferring the samples to tubes, adding the Fast
Blue BB reagent and alkali, and the absorbance was read at 2.3. Fast Blue BB (FBBB) method for gallic acid standard
420 nm. The azo-based assay had higher gallic acid
equivalent (GAE) values than the standard Folin–Ciocalteu Gallic acid calibration standards with concentrations of 0, 10,
for ‘‘total phenolics’’ in tea and coffee preparations with ratios 50, 100, 200 250, 500 ppm were prepared as in Section 2.2
of 2–6 while the packaged juice and juice drink samples had and 1 millilitre (mL) was transferred to borosilicate tubes. A
ratios from <1 to 4. The lower ratios indicate that these 0.1 mL aliquot of 0.1% Fast Blue BB reagent was added to
samples contain high amounts of non-phenolic antioxidant all gallic acid standard tubes, mixed for 1 min and 0.1 mL 5%
particu- larly in fruit juices or ‘juice drinks’ that are typically NaOH was added. The reaction was allowed to complete at
fortified with vitamin C and high fructose corn syrup. Both room temperature for 60 or 90 min. Aliquots (200 lL)
chemicals have reducing properties and are detected by the were trans- ferred to microtitre plates and the optical density
Folin–Cio- calteu but not with the Fast Blue BB method. was mea- sured at 420 nm. The gallic acid concentrations
The objectives of this research were to optimize the reac-
were plotted against the optical density.
tion time period between 60 and 90 min; optimize effects of
ethanol (0–70%) on gallic acid; and measure the total pheno- 2.4. FBBB interaction with gallic acid in water and ethanol
lics of food samples such as super foods (juices of fresh and
dehydrated fruits) with Fast Blue BB method; compare the To determine the effects of water and ethanol on the Fast
gal- lic acid equivalent values with Folin–Ciocalteu; and Blue BB reagent, gallic acid (0–500 ppm) was dissolved in
determine the reproducibility of the Fast Blue BB method. 0%, 10%, 25%, 50% and 70% ethanol and allowed the
reaction to com-
plete at 60 min, then measured the optical densities at
2. Materials and methods 420 nm. This test was to determine the solvent
combination for extraction of solid samples.
2.1. Reagents and equipments
2.5. Analysis of super fruit juices and juice mix
Fast Blue BB (4-benzoylamino-2,5-
dimethoxybenzenediazoni- um chloride hemi-[zinc chloride]) Fast Blue BB method described earlier (Section 2.3) was
salt, Folin–Ciocalteu’s Phe- nol Reagent (2 M), gallic acid followed for the total phenol analysis, except that each
were from Sigma–Aldrich (St. Louis, MO, USA). Borosilicate sample was analyzed with a blank containing only the
culture tubes (12 · 75 mm) and IKA Ultra-Turrax T25
sample and addition of 0.2 mL DIH 2O. Eleven samples of
homogenizer were from Fisher Scientific (Pittsburgh, PA,
‘‘super fruit’’ juices and juice mix were analyzed. Aliquots
USA); Biotek Synergy HT Multi-label Plate Reader from
of these juices were analyzed after opening of the
Biotek (Winooski, VT, USA); Tomy MTX50 centri- fuge
container or stored in -80 °C freezer. Clear juices were
(Peninsula Laboratories Inc., Belmont, CA, USA); and IKA
analyzed with and without filtration but beverages with
Vibrax VXR rotary shaker (IKA Works, Wilmington, NC),
visible solid suspensions were homogenized with Ultra-
Freeze-dried acai powder was a gift from Dr. Alexander
Turrax or sonicator prior to analysis. Superfruit juices and
Schauss, AIBMR Life Sciences (Puyallup, WA, USA). The
juice mixes were typically analyzed at 1:10–1:50 (v/v)
juice samples and fruits were obtained from the local markets
dilutions. Each sample was accompanied by analysis of
in Philadelphia, PA, USA.
blank samples to measure natural non-phenolic
interferences at 420 nm. For total phenol analysis with Fo-
2.2. Folin–Ciocalteu total phenolics assay
lin–Ciocalteu, 50 lL samples were analyzed following the pro-
cedure in Section 2.2. The sample dilution factor can be
The modified Folin–Ciocalteu procedures (Singleton et al.,
determined prior to the quantitative analysis by making
1999) and a procedure published by The Tea Association
sev- eral dilutions, transferring one millilitre of diluted
of the USA (2008) were utilized in this study. Gallic acid
samples to tubes, adding the reagents and measuring the
stock solution, (1 mg/mL) and working standard concentra-
optical density. The dilution that yielded an absorbance
tions of 0, 10, 25, 50, 100, 250 and 500 lg/mL (parts per mil-
within the linear curve is selected for the quantitative
lion, ppm) were prepared in deionized water (DIH 2O). The
assay. Otherwise, several dilutions can be assayed to
modified Folin–Ciocalteu procedure consisted of transferring
obtain results within the calibra- tion standard or the assay
50 lL standard or sample into a 4–5 mL borosilicate
may be repeated for the proper dilutions.
tube, followed by additions of 430 lL DIH2O and 20 lL
Folin–Cio- calteu reagent. After mixing the samples, 50 lL Calculation of the total phenolic content of the fruit juice
20% Na2CO3 and 450 lL DIH2O were added. The sample and beverage samples were derived from the gallic acid cali-
mixtures were allowed to stand for 1 h at room temperature bration standard and the results of samples tested were ex-
and were mixed after every addition of reagents. Aliquots pressed as gallic acid equivalents. The values from the
of 200 lL were transferred to clear microtitre wells in calibration curves were in lg GAE/mL of the diluted extract
duplicate and the absorbance was measured at 725 nm and GAE values from the blank samples were subtracted
using a Biotek Synergy HT. The phenolic content of samples from each sample and the resulting GAE values were
was measured against the gallic acid (GA) calibration multiplied by the dilution factor to obtain lg/mL of the original
standard (0–500 ppm) beverage. The GAE values (lg/mL) in the original samples
 JOURNAL OF FUNCTIONAL FOODS 3 (2011) 79–87 83
were further
84 JOURNAL OF FUNCTIONAL FOODS 3 (2011) 79–87

divided by 10 to directly convert to mg GAE/100 mL 3. Results and discussion

beverage samples.
3.1. Fast Blue BB method

2.6. Fresh fruits, soft dehydrated fruits and dry powder Fig. 2 shows that linearity and the slope of the calibration
lines improved with 90 min reaction time compared to
Solid soft foods like fruits (25–50 g) were cut to small pieces 60 min. The results were higher at concentrations >100 ppm
and homogenized in a mini food processor until the sample when analyzed at 60 min vs 90 min. Therefore, the Fast Blue
was smooth. The homogenates were transferred to plastic BB procedure in this study utilized the 90 min reaction time
storage bags and kept in —80 °C freezer. Samples (2.5 g) for measuring the phenolics in the beverage and fruit sam-
were weighed in 15 mL round bottom tubes, followed by ples. The typical interactions with gallic acid showed
addition of 5 mL 70% ethanol. The samples were
R = 0.9985 (±0.0007 SD) at 90 min vs R = 0.9909 (±0.0047
homogenized with an ultra-turrax (UT), for 1 min (2·)
SD) at
processing, mixed with a rotary shaker for 60 min and
60 min in six calibration lines. The mean calibration
centrifuged at 6000 rpm (2961g) for 10 min. The supernatant
lines were Y = 0.133395 + 0.0048102x (90) min and Y =
was decanted and transferred to calibrated glass tubes and
0.1832466 + 0.00227735x (60 min) which showed slight
the volume was measured. The samples were extracted
changes in slopes and y-intercepts. The limits of detection
again with another 5 mL of 70% eth- anol, homogenized,
(LOD) was <10 lg/mL and limits of quantitation (LOQ) was
mixed for 30 min and centrifuged at 6000 rpm (2961g) for 10
10 lg/mL. The LOD was determined from 3 · SD and the
min. The supernatant was added to the first extract and the
LOQ was 10 · SD of the zero value. R values in this study
total volume was measured. Aliquots of the pooled extracts
were greater than 0.99.
were diluted with DIH2O and analyzed for total phenols by the
Fast Blue BB and Folin–Ciocalteu methods. Typical dilutions
3.2. Fast Blue BB interaction with gallic acid in water and
for analysis were 1:5, 1:10 or 1:20 (v/v). Highly colored
ethanol
extracts were analyzed at higher dilu- tions. These dilutions
can be screened prior to analysis by analyzing the diluted
The effects of Fast Blue BB interaction with gallic acid dis-
extracts with the total phenol methods without the calibration
solved in various concentrations of ethanol were
standards. The excess extracts were stored in —20 °C or —
determined for utilization in sample extraction of
80 °C for longer storage. Total phenols were analyzed with
polyphenols in solid samples. Table 1 shows the linear
Folin–Ciocalteu (Section 2.2) and Fast Blue BB method
correlations of two trials of various concentrations of gallic
(Sections 2.3 and 2.4) The total phenolic content of samples
acid (0, 10–500 lg/mL) dis- solved in 0%, 10%, 25%, 50%
was expressed in GAE values. The calibration curve values
and 70% ethanol. The Fast Blue BB assay showed that
were in lg GAE/mL of the diluted extract and multi- plied
the highest correlations (R) with gallic acid (0–500 lg/mL)
by the dilution factor to obtain lg/mL of the original ex- tract.
were shown in 0%, 50% and 70% ethanol. Since the fresh
This value was then divided by a conversion factor consisting
fruits have high water content, 70% ethanol was used for
of weight of sample divided by total volume of ex- tract and
extraction of the fresh fruit samples and the dehydrated
these resulted to lg GAE/g sample. This GAE value (lg/g)
samples.
was further divided by 10 to directly convert to mg
GAE/100 g samples.
Freeze-dried acai powder (1 g) was extracted utilizing four
extraction procedures. Preparation A: 20 mL 70% ethanol
were added and homogenized with Ultra-Turrax and
3 1-89-60
centrifuged. After decanting the supernatant, 10 mL of 70% 1-89-90
2-92-60
ethanol were added to the pellet and mixed in a shaker for 30
2.5 2-92-90
min. The mixture was centrifuged and the supernatants were 3-95-60
3-95-90
pooled with the first extract. Preparation B1: Another 1 g acai 2 4-103-60
Absorbance, 420

aliquot was extracted with 25 mL of 70% ethanol. Samples 4-103-90

5-123-60
were mixed at room temperature in an IKA vibrax rotary 1.5 5-123-90
shaker for 1 h, the extract was separated by centrifugation 6-129-60
6-129-90
and the supernatants were analyzed. Preparation B2 (1 + 24 1
h): The pellet of the 1 h extract was extracted again with 10
mL of 70% ethanol for 24 h at room temperature in a shaker. 0.5
The mixture was centrifuged and the supernatant was
analyzed. The phenolic values of the 1 h extraction and the 0
pellet ex- tracts were added and reported as ‘1 h + 24 h’ GAE -100 0 100 200 300 400 500 600

values. Preparation B3. A 1 g acai powder was extracted with Gallic Acid (g/mL)
20 mL of 70% ethanol for 24 h at room temperature in a Fig. 2 – Calibration curves of typical interactions of gallic
shaker. The extracts were diluted to fit the gallic acid acid (0, 10–500 lg/mL) and 0.1% FBBB azo dye measured
calibration stan- dard of 0, 10–500 ppm. Duplicate samples at 420 nm after 60 and 90 min reaction times. The top
were analyzed in 3 separate trials with the Folin–Ciocalteu lines are calibration lines with OD measured at 90 min.
and Fast Blue BB methods. There is an improvement in linearity of the calibration
standard compared to the 60 min reaction (bottom lines).
 JOURNAL OF FUNCTIONAL FOODS 3 (2011) 79–87 85

Table 1 – Fast Blue BB interactions with gallic acid in 0–70% ethanol.

a
GA (lg/mL) Mean OD (0– RSD (%) Ethanol (%) b
R R (mean) 0–
70% n = 10) 0–500 lg/mL 500 lg/mL
Gallic Acid Gallic Acid

0 0.1006 11.4 0 0.9942 0.9900

0.1035 7.7 0.9858
50 0.3229 5.7 10 0.9893 0.9609
0.4145 7.6 0.9365
100 0.4308 6.8 25 0.8860 0.9172
0.507 14.5 0.9483
250 0.7348 9.6 50 0.9870 0.9912
0.7305 7.3 0.9954
500 1.185 17.1 70 0.9911 0.9745
1.465 0.9579
17.3
a
Mean OD of Fast Blue BB and gallic acid interaction at 0–500 ppm and effects of water and ethanol (0–70%) as solvents for gallic acid. OD
was read after 60 min reaction time.
b
Linear regression correlation (R) of gallic acid concentrations vs % ethanol. Highest R values are at 0%, 50% and 70% ethanol. Each value is a
mean of duplicate analysis.

3.3. Analysis of superfruit juices and juice mix calteu values had Fast Blue BB:Folin–Ciocalteu ratio of
around 1 or less suggest the presence of high concentration
The United States Food and Drug Administration (FDA)
of non- phenolic compounds. Pomegranate is perceived to be
guide- lines on fruit juices indicate that beverages that
a super- fruit. Analysis of the fresh pomegranate seeds
purport to contain juice (fruit or vegetable juice) must
extracted by mashing in a mortar and pestle resulted in mean
declare the % juice. ‘‘This includes non-carbonated
GAE values of 182 and 152 mg/100 g by Fast Blue BB and
beverages, full strength (100%) juices, concentrated
Folin–Ciocalteu methods with GAE ratio of 1.2.
juices, diluted juices, and bever- ages that purport to
contain juice but contain no juice’’ (Code of Federal
3.4. Fresh fruits, soft dehydrated fruits and dry powder
Regulations, 2009a). ‘‘Beverages that are 100% juice may be
called ‘juice’. However, beverages that are diluted to less Fresh fruits were stored in —80 °C prior to sample
than 100% juice must have the word ‘‘juice’’ qualified with preparation or homogenization. After extraction, the extracts
a term such as ‘beverage’, ‘drink’, or ‘cocktail’’’ (Code of were also stored at —20 or —80 °C for repeat analysis. The
Fed- eral Regulations, 2009b). However, the Food and Drug results from analysis of nine fruit samples are shown in Table
Admin- istration has no guidelines on superfoods but 3. Blueber- ries had the highest GAE values of 1291 mg
instead the agency has issued guidelines on antioxidants GAE/100 g in domestic fruits. The black grapes and black
(Code of Federal Regulations, 2009c). A superfood is plums had the next highest with 572 and 441 GAE/100 g,
perceived to have high nutrient density with preventive or respectively. The peach and pear samples were analyzed
healing properties of many chronic diseases. A superfood with and without skins, and results showed higher GAE
is defined ‘‘as a natural food regarded as especially values in fruits with skin when analyzed with Fast Blue BB
beneficial because of its nutrient profile or its health- and Folin–Ciocalteu methods. Again, the inter-assay
protecting qualities’’ (Oxford Dictionary, 2009). Sloan variability (RSD) was less than 10%. Blueberries and black
(2008) predicted that superfoods was second in the Top plum had the highest phen- olics ratio of 4.1 and 3.4,
10 Food Trends. Sloan reported that this was a result of respectively. The number (n) of Fast Blue BB analysis were
recent scientific validation of the health benefits of super- at times lower than the number of anal- ysis with Folin–
foods convincing the consumers the key benefits of a new Ciocalteu method. Initial analysis of samples containing high
trend in whole food nutrition.’’ phenolics with Fast Blue BB resulted in high GAE values
Our study showed that some fruits and juices show high (>500 lg/mL) where the optical density could not be
total phenolic values and may be considered as superfoods. measured and this replicate analysis was disregarded. At
The juices analyzed in this study were diluted with DIH 2O varying sample dilutions, the GAE values fell outside the reli-
prior to analysis. Analysis of juices from superfruits are able GAE concentration (>50 and <500 lg/mL GAE) of the
shown in Table 2 with GAE values of up to 1800 mg/100 mL Fast Blue BB calibration curve. Fast Blue BB and Folin–
(600 mg per serving) in the ‘Miracle Fruits’ juice mix by the Ciocalteu analyses were repeated with diluted samples.
Fast Blue BB method compared to about 1400 mg GAE/ Table 3 also shows results of analysis of dehydrated
100 mL by the Folin–Ciocalteu method. The acai commercial super- fruits. The goji berry, jujube and prune (dried plum)
drinks, mixes and smoothies had GAE values ranging from had high non-phenolic antioxidant shown by the high total
294 to 1256 mg/100 mL. The Fast Blue BB:Folin–Ciocalteu phenolics by the FC method and a low ratio of <1. The
GAE ratios ranged from 1.9 to 4.0. Juices and juice mixes second goji sam- ple (b) had been exposed to air oxidation
with deep colors had GAE ratios >2 such as in most acai and therefore had lower GAE values. Table 4 shows the
prepara- tions. Fresh squeezed blood orange 295 mg/100 results on the analysis of the freeze-dried acai resulting in
mL GAE value and a ratio of 2.28. Juices with higher Fast mean GAE values of
Blue BB:Folin–Cio-
86 JOURNAL OF FUNCTIONAL FOODS 3 (2011) 79–87

Table 2 – Total phenolics (mg GAE/100 mL) in juices of ‘‘superfruits’’ by Fast Blue BB (FBBB) and Folin–Ciocalteu methods.
Super fruit juices/mixes FBBB method RSD (%) Folin–Ciocalteu RSD (%) Ratio
mg GAE/100 g (n) mg GAE/100 g (n) Folin Ciocalteu:FBBB

Acai + Lime 1227 (3) 5 338 (3) 5 3.6

1256 (3) 0 310 (3) 3 4.0
Acai-I 858 (2) 6 315 (3) 10.8 2.7
Stored (—80) 685 (3) 2 291 (3) 1 2.35
Acai-M 495 (3) 4 276 (3) 3 1.79
Acai-Z 395 (2) 5.1 114 (3) 1.8 3.46
Stored (—80) 284 (2) 4.5 99 (2) 3.0 2.87
Berry boost 172 (3) 1 283 (3) 2 0.61
Black currant 261 (3) 5 115 (3) 3 2.27
Black currant nectar, organic 120 (3) 2 70 (3) 3 1.7
Blood orange, fresh squeeze 295 (3) 5 129 (3) 3 2.28
Miracle fruit juice mix 1 1795 (2) 2 1390 (2) 4 1.07
1868 (4) 4 na
1750 (3) 3 1463 2 1.20
Miracle fruit juice mix 2 1101 (3) 4 816 (3) 11 1.35
Prune juice 243 (3) 1 184 (3) 1 1.32
296 (3) 13 179 (3 1 1.65
Pomegranate juice, fresh extract of seed coat 193 (3) 4 161 (3) 1 1.20
168 (3) 8 142 (3) 6 1.18

Purple corn drink

Chicha 526 (2) 1.1 142 (4) 5 3.7
Stored (—80) 501 (2) 3.6 144 (2) 2.1 3.5
Polyphenols measured as gallic acid equivalents (GAE) per 100 mL. Each data represents GAE values derived from (n) inter-assay trials.

Table 3 – Total phenolics (mg GAE/100 g) in select fresh and dried fruits by Fast Blue BB (FBBB) and Folin–Ciocalteu
methods.

Samples FBBB method FBBB method Folin–Ciocalteu Folin–Ciocalteu FBBB:Folin Ciocalteu

mg GAE/100 g mg
GAE/100 g
Mean (n) RSD (%) Mean (n) RSD (%) Ratio

Fresh fruits
Blueberry 1291 (4) 7 315 (4) 7 4.1
Cherry, sweet bing 205 (6) 5.2 80 (6) 4.0 2.6
81 (6) 4.7 2.5
Grape, black 572 (3) 5 372 (3) 5 1.5
Peach, white with skin 160 (3) 3 98 (3) 4 1.6
Peach, white peeled 91 (3) 5 46 (3) 0 2
Plum, red 161 (3) 1 69 (3) 5 2.3
Plum, Black 441 (6) 5.4 131 (6) 9.2 3.4
Raspberry 393 (4) 2 163 (4) 2 2.4
Strawberry, organic 282 (3) 2 159 (3) 13 1.8

Dry fruits
a
Goji-WF (dry) 562 (6) 1.3 1036 (3) 1.2 0.54
Goji-B (dry) 530 (6) 10.3 895 (3) 10.2 1.70
b
Jujube (dry) 595 (3) 17 944 (3) 3 0.63
Prune 337 (3) 4 399 (3) 1 0.84
Polyphenols measured as gallic acid equivalents (GAE) per 100 mL. Each value is the mean of multiple analyses (n). Each data represents GAE
values derived from (n) inter-assay trials.
a
Goji berry is the common name for wolfberry or Lycium BARBARUM.
b
Jujube or, red date or chinese date, is a species of Ziziphus in the buckthorn family RHAMNACAE. Samples were extracted with a rotary shaker.
Each data represents GAE values derived from (n) inter-assay trials.

9167–10,003 mg/100 g with Fast Blue BB but the mean

tion of the pellet compared to a 24 h extraction on a rotary
Folin– Ciocalteu GAE values were 2380–2984 with Fast Blue
shaker (preparation B). That is, the homogenization
BB:Fo- lin–Ciocalteu ratios of 3.3–3.4. Higher phenolic
extrac- tion yielded higher results than the 24 h shaking
values were obtained in samples extracted (2x) with ultra-
method. With the shaker method, the 1 h extraction had
turrax homoge- nization (preparation A) followed with a 30
higher GAE values than the 24 h extraction. There was
min shaker extrac-
also a color fading
 JOURNAL OF FUNCTIONAL FOODS 3 (2011) 79–87 87

Table 4 – Total Phenolics in freeze-dried acai powder measured as gallic acid equivalent (mg/100 g) by Fast Blue BB (FBBB)
and Folin–Ciocalteu methods.

Samples FBBB Method RSD (%) Folin–Ciocalteu RSD (%) Ratio

mg GAE/100 g (n) 90 min mg GAE/100 g (n) FBBB:Folin–Ciocalteu

ACAI (freeze dried)

Prep A 9698 (3) 3 2938 (3) 2 3.30

Prep B1
Trial 1 1 h 8900 (2) 2 2772 (2) 3.2
Trial 2 9650 (2) 2650 (2) 3.6
Trial 3 8950 (2) 2750 (2) 3.2
Mean (6) 9167 [1 SD = 342] 3.7 2724 1.9 3.4

Prep B2 (1 + 24 h)
Trial 1 9710 (2) 3032 (2) 3.2
Trial 2 10,545 (2) 2875 (2) 3.7
Trial 3 9755 (2) 3045 (2) 3.2
Mean (6) 10,003 [1 SD = 380] 3.8 2984 2.6 3.4

Prep B3 (24 h only)

Trial 1 7725 2425 3.2
Trial 2 8275 2275 3.6
Trial 3 7875 2450 3.2
Mean (6) 7958 [1 SD = 231] 2.9 2380 3.2 3.3
Acai powder (Euterpe OlerACEA) is from the berries of a palm tree in the genus of Euterpe.
Polyphenols were measured as mg gallic acid equivalents per 100 g. Each value is the mean of multiple analyses (n). Each data represents
GAE values derived from (n) inter-assay trials. Prep A: Acai powder was extracted 2· with 70% ethanol using an ultra-turrax homogenizer.
Prep B: Samples were extracted with the rotary shaker. B1 (1 h); B2 (1 + 24 h); B3 (24 h only).

with the 24 h method. However, the double extraction

studies on the Fast Blue BB interactions with pure
(1 h + 24 h extraction of the pellet) yielded higher results
phenolic compounds, miniaturization of the assay protocol,
than the 1 h alone and the homogenization method. The
identifica- tion of the azo complexes and further
phenolic results from all three trials had <5% variability
applications with more food commodities.
(RSD).

Acknowledgments
4. Conclusions
The technical assistance of Sarah Picozzi is greatly appreci-
This novel method is simple and rapid with the use of the ated. The author also thanks Dr. Alexander Schauss of
Fast Blue BB reagent and NaOH added to samples, and the AIBMR Life Sciences for the freeze-dried acai powder.
absor- bance was read at 420 nm after 90 min reaction time.
The Fast Blue BB method quantified the polyphenols in
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