Effect of Sucrose Addition on the Sugar and Sorbitol Composition of Frozen Sweet Cherries and Their Derived Concentrates. CHRISTOPHER J. CORNWELL ...
Effect of Sucrose Addition on the Sugar and Sorbitol Composition of Frozen Sweet Cherries and Their Derived Concentrates CHRISTOPHER
J. CORNWELL,
RONALD
ABSTRACTGlucose, fructose, sucrose, and sorbitol contents of Van and Black Republican varieties of sweet cherries were determined by high performance liquid chromatography (HPLC) and enzymic analytical procedures. Samples analyzed included frozen fruit, fruit packed as 3.6 parts fruit plus 1 part added sucrose,and cherry juice concentrates derived from the sugar-packed fruit. Cherry fruit showed invert patterns of glucose and fructose and contained trace amounts of sucrose, and 2.6-3.9g sorbitol/lOOg. Sucrose was either not detected or found in trace amounts in samples to which sucrose had been added. There is evidence that sucrose hydrolysis was caused by presence of invertase. The percent sorbitol content could be used to detect addition of sucrose. Results for sugar and sorbitol content as determined by HPLC or enzymic methods were very similar.
INTRODUCTION A FREQUENT COMMERCIAL practice is to pack sweet cherry fruit in 55 gallon drums with added sucrose and hold
in frozen storage for subsequent use in yogurts, ice creams, sauces, bakery items, etc. The free-run juice produced when the barrels are thawed is rich in color and flavor and yields a high-quality syrup when concentrated. The major objective of this study was to determine whether addition of sucrose to cherry fruit could be detected in the frozen samples and derived juice concentrates through analyses of their fructose, glucose, sucrose, and sorbitol contents. Wrolstad and Shallenberger (198 1) recently published a table listing the free sugar and sorbitol content of sweet cherry fruit as reported in the literature
by various workers. Sweet cherries
have essentially an invert pattern, containing nearly equal quantities of glucose and fructose and either no or trace levels of sucrose. Their sorbitol content is among the highest of the various commercial fruits. Richmond et al. (198 1) analyzed the sugar and sorbitol content of several fruits by high-performance liquid chromatography (HPLC) and reported a similar pattern for sweet cherry fruit. Another objective of this study was to compare the effectiveness of enzymic analytical procedures with HPLC for glucose, fructose, sucrose, and sorbitol measurements. HPLC instrumentation requires considerable investment while enzymic analysesutilize equipment which is more commonly available in industrial laboratories. EXPERIMENTAL Sample Frozen samples were obtained from The Dalles Cherry Growers, Inc., The Dalles, OR 97058, and stored at -15°C until use. Samples included individually quick frozen unpitted sweet cherry fruit (Van and Black Republican varieties, 1978 season) and two lots each of the same fruit packed as 3.6 parts fruit and one part added sucrose. Also provided were samples of cherry juice concentrate which had been manufactured from frozen Black Republican fruit packed with added sucrose.The fruit which had been frozen in 55 gal drums was thawed at ambient temperature and then heated to 60°C. Authors Cornwell, Wrolstad, and Reyes are with the Dept. Of Food Science & Technology, Oregon State Univ., Corvallis, OR 97331. Author Reyes is on sabbatical leave from Universidade Estadual De CampinadF. E.A.A., Campinas, S.P. Brasil.
E. WROLSTAD,
and FELIX
G. R. REYES
The concentrate was made from the freerun juice. Three lots of concentrate were from the 1978 season, one lot was from 1977 and an additional lot was from the 1976 season. All concentrates were 5 1” Brix. Extraction and isolation of sugarsand sorbitol for HPLC analysis Fruit samples were homogenized in a Waring Blendor. Forty ml 95% ethanol were added to lO.Og of homogenate and the mixture was sonicated for 2 min through direct immersion of an ultrasonic probe (Bronwill Biosonik III Ultrasonicator; dial setting of 70). Juice concentrate samples were diluted (25.Og + 75.0 ml distilled
water), and a 10.0-galiquot combinedwith 40 ml ethanol.Samples were held at 2°C for 1 hr to facilitate precipitation of high molecular weight components, and then filtered by suction through a pad of log of hydrated polyvinylpyrrolidone (PVPP) on Whatman No. 1 filter paper; the PVPP had been washed by the procedure of Loomis, 1974. The PVPP pad and sample residue were rinsed with 25 ml 80% ethanol three times; the filtrate and washings were combined
and concentratedto about 5 ml on a rotary evaporator(water bath = 3?C, pressure = 23 torr) to ensure ethanol removal from samples. The samples were passed through two 2 x 7 cm columns in series, the first column containing 6 ml Biorad AG 50 W-X4 cation exchange resin, (200-400 mesh, Na+ form) and the second containing 6 ml Biorad AC 1 X-8 anion exchange resin (200-400 mesh, F- form). The columns were washed with deionized distilled water and the
eluantscollectedin 50 ml volumetric flaskscontaining5 ml of 10% mannitol as an internal standard and 1.0 ml 0.5% CaCl2’2H20. Samples were filtered through 0.45 I.rrn Millipore filters prior to injection. HPLC analysis of extracts The HPLC system was composed of a Varian 5000 liquid chromatograph equipped with a column heater and refractive index detector, a 7.8 X 300 mm Aminex HPX-87 column (Biorad Labora-
tories), and a Hewlett Packard3380 A integrator.Column temperature was maintainedat 85°C. The mobile phasewasdeionized,0.45 urn Millipore filtered water containing 0.1% CaCl,*2H?O: the flow rate was 1 ml/min. Injection volume was 20 ~1. Refractive index range was 0.5 x 1O-5 RI units. Relative detector response (RDR) factors for sucrose, glucose, fructose, and sorbitol were determined to be 1.01, 1.03, 1.04, and 1.07, respectively, using the method described by Akhavan et al. (1980). Percent recoveries (R) for standards were determined by submitting reagent sugar solutions in 80% ethanol through the isolation and separation procedures; percent recoveries for sucrose, glucose, fructose, and sorbitol were 98.7, 98.3, 95.5, and 98.3, respectively. Content of individual sugars was calculated by the following formula: g sugar/lOOg sample = (As)(Wtis)(DF)/(Ais)(R)(RDR) where As = peak area of sugar; Ais = peak area of internal standard; W& = weight of internal standard; DF = dilution factor; R = percent b
.
recovery;and RDR = relativedetectorresponse. Enzymic analysis of sugarsand sorbitol
Sugarand sorbitol contents were determinedenzymically with Glucose/Fructose, D-Sorbitol,
and Sucrose/Glucose kits from
BoehringerMannheim Biochemicals,IndianapoIis,IN, accordingto the manufacturer’s instructions. Samples (2-Sg) were homogenized in a Waring Blendor and diluted with 2L distilled water; a 50-ml aliquot was centrifuged in a Sorvall RCZB centrifuge at 12,061 x g for 10 min and a lo-ml aliquot of the supernatant diluted 25-fold. After combining 2-ml aliquots with reagents absorbance readings at 340 nm were determined on a Perkin Elmer model 550 spectro-
photometerand the concentrationcalculated. -Continued
Volume
47 (1981)--JOURNAL
OF FOOD
on next page
SCIENCE-281
CHERRY
I i I
I
SUGAR
COMPOSITION.
..
Determination of invertase activity in Black Republican fruit Presence of invertase activity in the Black Republican frozen fruit sample was determined from glucose analysis of the following samples: (1) lO.Og cherry fruit; (2) lO.Og cherry fruit plus 5.Og sucrose; (3) 5.Og sucrose. All samples were made up to 100 ml with pH 4.5 acetate buffer and incubated at 50°C for 25 min. Glucose determinations were made using the enzymic procedure.
RESULTS
& DISCUSSION
FIG. 1 SHOWS typical HPLC chromatograms of cherry extract samples and Table 1 lists the free sugar and sorbitol content of all samples aa determined by HPLC. The fruit samples had invert sugar patterns, containing nearly equal quantities of glucose and fructose. Sorbitol was found in
Van Cherries + Sucrose
Van Cherries
man
I-HPLC chromatogram of extracts of Van cherries and Van cherries plus added sucrose. Column * Aminex HPX-87; mobile phase = water containing 0.01% CaC12; flow rate = 1 ml/min; injection volume = 20 pl. Typical retention times: Sucrose (sue), 4.70 min; glucose (glul. 5.98 min; fructose ffru), 8.08 min; mannitol-internal standard (man), 10.65 min; sorbitol (sorb), 13.53 min. Fig.
fru
sorb
lLA
2 sue
1 -Free
sugars and sorbitol
Fructose Sample Van frkt Mean Black
Republican
gll 00s
% C.V.
% T.S.
s/l 00s
6.38 6.80 6.59
5.01 1.32 4.50
46.1
7.32 7.55 7.44
1.78 1.19 2.15
8.86 6.93 6.79
3.15 1.44 2.79
44.9
8.04 8.31 8.18
2.1 1 0.48 2.32
14.99
49.6
15.55 15.26 15.40
1.03 0.46 1.29
49.1
15.04 15.34 15.19
0.66 2.48 1.38
48.6
14.73 14.99 14.86
2.38 2.80 1.21
48.3
13.99 13.74 13.86
0.79 2.69 1.30
49.8
21.22 21.41 21.31
2.59 1.86 0.81
48.9
23.22 23.16 23.19
2.24 0.99 0.17
48.8
22.80 22.12 22.46
1.80 2.03 2.14
49.4
19.64 19.03 19.34
3.56 0.68 2.22
48.7
21.61 21.14 21.37
0.46 1.47 1.54
fruit
Mean Van Fruit (Lot 1) Mean
+ Sucrose
14.93
0.93 0.27 0.50
Van Fruit (Lot 2) Mean
t Sucrose
14.64 14.55 14.75
0.27 2.09 1 .Ol
Black Republican fruit + Sucrose (Lot 1) Mean
14.03 14.55 14.05
2.85 4.64 7.11
Black Republican fruit + sucrose (Lot 2) Mean
12.95 12.97 12.96
1.16 1.54 0.10
Syrup from Republican Mean
Black (Lot 11 (19781
21.02 21.02 21.02
3.28 1.09 0
Syrup from Republican Mean
Black (Lot 2) (1978)
22.26 2
2.92 0.95 0.49
Syrup from Republican Mean
Black (Lot 31 119781
21.89 20.98 2(.44
3.85 2.95 2.98
Syrup from Republican Mean
Black (Lot 4) (1978)
19.38 1m 19.03
2.89 0.58 2.57
Syrup from Republican Mean
Black (Lot 5) 11977)
20.57 20.25 20.41
1.31 1.63 1.12
a % C.V. = pa;cent b fructose ratlo.
282-Volume
14.87
coefficient
47
Of variance;
(1981)-JOURNAL
%T.S.
of cherry
samples
determined
Glucose
= percent
OF
total
FOOD
% C.V.
wgars;
total
SCIENCE
I
I
I
1
0
5
IO
I5
min by HPLP
sucrose % T.S.
52.1
54.1
51.2
50.6
51.4
51.7
50.3
51.1
51.2
50.2
51.0
g/l 009
% C.V.
% T.S.+S
14.29
2.81 2.57 2.59
3.45 3.89 1.16
15.3
15.13
3.94 3.88 3.91
1.01 5.41 1.02
20.5
30.09
1.92 1.99 1.96
7.81 9.64 0.05
6.5
30.02
2.26 2.10 2.18
6.64 8.57 5.04
6.8
28.91
2.85 3.02 2.93
9.82 6.29 4.09
9.2
26.82
3.08 2.76 2.92
2.59 10.14 7.88
9.8
42.33
3.23 2.91 3.07
10.52 10.31 7.49
6.8
45.38
3.18 3.30 3.24
10.69 4.84 2.47
6.7
43.90
3.22 3.28 3.25
7.45 6.40 1.23
6.9
38.50
3.52 u 3.45
9.37 1.77 2.89
8.2
0.2
41.87
3.68 3.54 3.81
5.16 12.99 2.77
7.9
%T.S. + 6 = perc**t
tota,
g/l oog
% C.V.
% T.S.
1.13
0.27 0.26 0.26
3.70 3.64 3.84
1..8
1.20
0.16 0.15 0.18
18.7 13.3 6.25
1.03
0.04 0.08 0.06
1.05
1.07
1.01
1.04
50
0.2 b
&
b
0
0.3
0
0
0
0
0
0
0
1.05
0
1.02
0.15 0.11 0.13
b b 23.1 b
1.05
0.18 0 0.09
suga,s = g1ucoH) + f,“ctcse
1 .l
b b
0.16
0
+ s.“c,osa;
b
Sorbitol
Total Sugars gl1Wg
Glu/Fru
1.03
n
--ii
Time, Table
sorb
.-C
0.3
*ugars + so,b,to,;
g,u,tru
= glucose:
I
large amounts while only small quantities of sucrose were found. This compositional pattern is consistent with the sugar and sorbitol content for sweet cherries as recently compiled from the literature by Wrolstad and Shallenberger (1981). The fructose, glucose, sucrose and sorbitol content, the glucose:fructose ratio, and the total sugar content of the Van and Black Republican samples are within one standard deviation of the mean values reported in the literature compilation. There is a greater difference between the sorbitol contents of Van and Black Republican varieties (15.3 and 20.5% of total sugars plus sorbitol, respectively) than there is between fructose, glucose, and sucrosecontents. The most striking information in Table 1 is that either no or a very low level of sucrose was detected in the samples to which sucrose had been added. Clearly, sucrosehydrolysis is occurring during processing and/or storage. Glinskaya and Shkurina (1969) have previously reported invertase activity in cherry fruit. The results presented in Table 2 demonstrate the presence of invertase activity in the Black Republican fruit sample where 58% inversion of sucrose occurred after 25 min incubation. It is highly likely that invertase activity prior to freezing, during storage, and/or during thawing accounts for there being little or no sucrose in the various samples. The glucose:fructose ratio in Van and Black Republican fruit, 1.13 and 1.20, respectively, is very close to the mean value for sweet cherries, 1.17 (standard deviation = 0.357), compiled from the literature (Wrolstad and Shallenberger, 1981). Sucrose hydrolysis in the samples containing added sucrose lowered the glucose:fructose ratio to values ranging from 1.01-l .07, which is still within the range reported in the literature. The glucose:fructose ratio has very limited value in detecting added sucrose in these samples as the fruit itself has close to an invert pattern. Cherry fruit is very high in sorbitol, but there can be considerable quantitative variation with variety and season. The sorbitol content of Van and Black Republican cherries in this study was 15.3 and 20.5% of total sugarsplus sorbitol, respectively. Neubeller and Stosser (1977) studied the sugar and variation of 20 varieties of sweet cherries over 3 yr and reported the sorbitol content varied from 15.5 - 24.0% Table 24ucrose
Inversion Fruit
Sample
by Black Sucrose
Republication
Fruit
addition
Measure
(cl)
(cl)
1
10
2 3
10
0 5 5
0.76 2.44 0.16
0
% inversion
sample
=
glucose
2 glucose yield
-
from
(sample
100%
Table d-Free
sugars and sorbitol
1 + sample
hydrolysis
XlOO=$$$f=
of cherry
samples determined
Fructose % T.S.
analysesa Sucrose
1 .oo
0.78
% T.S.
GWFru
g/lOOg
%C.V.
%T.S.
Total Sugars
Sorbitol g/lOOg
%C.V.
2.65 3.61 -2.52
2.64 6.46
%TS+S
g/l OOg
% C.V.
48.3
6.62
0.46
51.9
45.9
7.82
0.78 0.85
54.2 51.7
1.08 1.18 1 .OJ
n.d. n.d. n.d.
0 0 0
27.9
13.92
1.97
51.7
1 .OJ
n.d.
0
26.9
3.05
0.76
10.2
21.5
0.28
50.4
1 .Ol
n.d.
0
42.7
3.70
1.57
8.0
6.13 6.61 13.48
0.46
48.3
14.42
13.00
1.89
48.3
21.22
0.58
49.7
coefficient + sorbitol):
by enzymic
Glucose
% C.V.
Van fruit Black Republican fruit Van fruit + Sucrose (Lot 11 Black Republican fruit
%c.V. = Percent fructose + sucrose
sucrose
58%
gllOOg
+ Sucrose (Lot 1) Syrup from Black Republican + Sucrose /Lot 1)
3 glucose)
of added
Sample
a
glucose
M
of total sugars plus sorbitol. Sucrose addition has a dilution effect on sorbitol content; the percent sorbitol content in all samples with added sucrose is reduced from the percent sorbitol of the fruit samples by more than one standard deviation of sorbitol variation as compiled from the literature (Wrolstad and Shallenberger, 1981). The low percent sorbitol content of the samples with added sucrose (6.59.8%) is the best indication in this study that those samples are not derived 100% from fruit. Since the fruit samples with added sucrose were packed as 3.6 parts fruit and one part sucrose, the percent sorbitol would be estimated to be 5.8 and 8.4% for the Van and Black Republican samples, respectively. This is somewhat lower than was actually measured in all four lots of fruit packed with sucrose. Nonuniform distribution of sucrose in the drums and nonrepresentative sampling might account for this discrepancy. The syrup concentrates were produced from the free-run juice of the sugar packed fruit. The percent sorbitol levels of the three lots produced from 1978 seasonBlack Republican fruit is lower than the 8.4% level of sorbitol estimated to be the percent sorbitol content of the sugar packed fruit. These results suggestthat there is not complete diffusion of the sorbitol from the fruit to the juice and the free-run juice from which the concentrate is made contains a somewhat higher proportion of sucrose than the sample as a whole. Duplicate runs were carried out for all samples analyzed by HPLC and the value for each duplicate is shown in Table 1. The percent coefficient of variance (%C.V.) listed for each replicate represents the variation among the three HPLC injections of the sample extract. The %C.V. for individual sample extracts was less than five percent, except for some samples of low sucrose or sorbitol content. The %C.V. listed for the mean value of each sample is the variation between the two duplicates and would represent variation due to sampling, extraction, and isolation. Variation between duplicates is low, being less than five percent for most samples. Table 3 lists the free sugar and sorbitol content for five of the samplesas determined by enzymic methods. Duplicate runs were not made and the %C.V. represents the variation between analytical determinations for the same extract. This variation is low. Sucrose was not detected in any of the five samples by the enzymic procedure. As the enzymic assay for sucrose measuresthe increase in glucose concentration after sucrose hydrolysis, it will not be a sensitive procedure for determining trace amounts of sucrose in presence of large quantities of glucose. Results for fructose, glucose, and sorbitol content as determined by the enzymic (Table 3) and HPLC methods (Table 1) are very similar. The correlation coefficient for fructose content as determined by the two methods for the same five samples is 0.994; for glucose the correlation coefficient is 0.997 and for sorbitol it is 0.847. -Continued on page 290
of variance; % T.S. = Percent total sugars Qlulfru = glucose:fructose ratio; n.d. = not
(glucose detected.
+ fructose
Volume
+ sucrose);
% T.S.+S
= Percent
47 (1981kJOlJRNAL
12.7 14.4
total
sugars
O F FOOD
+ Sorbitol
17.2 20.0 8.3
(glucose
SCIENCE-283
+
