Flavor Characterization of
Greening-affected Orange Juice
Elizabeth Baldwin, Anne Plotto, Jinhe Bai, John
Manthey, Randy Cameron, Gary Luzio, Jan
Narciso, Bill Widmer, Smita Raithore, Sharon
Dea, Sophie Deterre and Mike Irey
Asymptomatic vs symptomatic fruit
Asymptomatic
or NonSymptomatic
symptomatic
Healthy
Symptomatic fruit are
reported to impart sour,
bitter taste to resulting
juice (Plotto et al., 2010;
Dagulo et al., 2010) due in
part to decreased sugars,
sometimes increased
acids and increased levels
of bitter limonoids (limonin
and nomilin
Mild
Severe
Size and color of fruit
Average Diameter (cm)
7.2
Average Diameter of Hamlin Oranges (Dec 3,
2012)
7.1
6.9
6.72
6.72
6.9
6.81
6.73
6.53
6
4.8
3.6
2.4
1.2
0
Conv
N1
N2
Fruit Diameter (cm)
Treatments
Midsweet January 2013
HLB (a/b)
Midsweet January 2013
Fruit size (diameter)
0.6
145
140
135
130
125
120
115
0.4
0.2
Healthy
HLB
1
2
3
4
Replicate samples
5
Healthy
0
-0.2
-0.4
-0.6
0
10
20
30
40
50
HLB
Materials and methods
Fruits were commercially processed
Materials and methods
Sensory Analysis: Difference from control test (DFC)
Same or different?
Rate the difference
Smell
0
1
Taste
0
1
2
3
4
5
6
7
8
9
10
2
3
4
5
6
7
8
9
10
Describe the difference
……………………………………………………………..
18-23 panelists
Two replications for statistical analysis (ANOVA)
Sensory analysis – Trained descriptive panel: 10-12
panelists trained to rate descriptors of orange juice
flavor
Methods – Chemical analyses
Soluble solids – refractometer
Titratable Acidity – titrator
Individual sugars – HPLC
Individual acids – HPLC
Limonoids – HPLC-MS
Limonin and nomilin thresholds
• Threshold is the concentration of a compound that can be detected
(in whatever matrix: air, water, model solution, juice)
• Therefore, the lower the threshold, the lower the concentration
necessary for detection
• The threshold of limonin was reported to be 1.0 mg/L in water and
about 6 mg/L in orange juice (Guadagni et al. 1973).
• The threshold of limonin could be modified by adjusting pH in the
testing matrix (model solution or orange juice), and by adding citric
acid or sucrose
• The highest limonin threshold (lowest bitterness) was observed at
pH 3.8 in either the model solution or orange juice (Guadagni et al.
1973)
• Rouseff and Matthews (1984) reported nomilin detection and
recognition thresholds in water of 0.8 mg/L and 2.1 mg/L
respectively
Threshold for limonin and nomilin
Simple matrix: 6 % sucrose and
0.15% citric acid
Limonin
Nomilin
Limonin and nomilin (1:1 ratio)
Limonin varied, nomilin constant
at 2 mg/L
Matrix
Complex matrix
Commercial juice
V09 juicez
H10 juicey
H11 Juicex
Overall OJw
Detection
Threshold
(mg/L)
4.0 ± 0.2
5.4 ± 0.2
1.8 ± 0.30z
2.6 ± 0.3
Limonin Thresholds
Detection
Recognition
(mg/L)
(mg/L)
5.0 ± 0.2
10.2 ± 0.2
2.4 ± 0.3
3.9 ± 0.4
3.0 ± 0.3
6.0 ± 0.3
2.6 ±0.4
4.7 ± 0.4
2.0 ± 0.3
4.3 ± 0.3
2.5 ± 0.3
4.7 ± 0.3
Recognition
Threshold
(mg/L)
7.0 ± 0.3
8.4 ± 0.2
3.9 ± 0.2z
4.6 ± 0.2
Nomilin Thresholds
Detection
Recognition
(mg/L)
(mg/L)
2.5 ± 0.2
5.3 ± 0.2
1.0 ± 0.3
1.7 ± 0.4
1.5 ± 0.3
2.9 ± 0.3
1.2 ± 0.4
2.1 ± 0.4
1.0 ± 0.3
2.1 ± 0.3
1.2 ± 0.3
2.6 ± 0.3
Dea et al.,
2013
Complex matrix: 5.2 % sucrose, 2.15 % glucose, 2.5 % fructose, 0.75 % citric acid
and 0.25 % malic acid
Affect of sugars and acids on detection of
bitterness due to limonin and nomilin
Hamlin
(H10)
Unspiked control
Control- spiked (L&N at
threshold)
Acid-spiked (L&N + 0.3
g/100 mL citric acid)
pH
4.01 ±
0.01
4.00 ±
0.01
3.69 ±
0.02
Soluble
solids
content
(°Brix
corrected)
10.85 ± 0.05
Titratable
acidity
(mg/100 mL
Citric acid)
0.613 ± 0.003
10.81 ± 0.00
0.612 ± 0.004
11.09 ± 0.01
0.899 ± 0.004
SSC/T
A
ratio
17.68
± 0.17
17.65
± 0.12
12.33
± 0.05
Dea et al.,
2013
sensory
Sweet
Sour
Bitter
6.37 b
5.19 bc
3.04 c
5.33 bc
6.37 ab
5.52 a
5.00 c
7.56 a
5.00 ab
8.41 a
4.30 c
4.04 bc
Sugar-spiked (L&N + 3.5
g/100 mL sucrose)
Valencia Unspiked control
(V09)
Control –spiked (L&N at
threshold)
Acid-spiked (L&N + 0.35
g/100 mL citric acid)
4.00 ±
0.01
13.83 ± 0.00
0.593 ± 0.005
23.31
± 0.19
4.00 ±
0.02
4.00 ±
0.02
3.68 ±
0.02
11.90 ± 0.01
0.689 ± 0.002
6.88 b
4.73 b
1.31 c
11.95 ± 0.10
0.690 ± 0.006
5.81 bc
5.31 b
5.96 a
12.30 ± 0.06
1.025 ± 0.013
17.27
± 0.04
17.33
± 0.04
12.00
± 0.10
4.73 c
7.77 a
5.23 ab
Sugar-spiked (L&N + 3.7
g/100 mL sucrose)
4.01 ±
0.02
15.05 ± 0.07
0.666 ± 0.006
22.60
± 0.10
8.31 a
4.08 b
4.15 b
Recap for limonin and nomilin
perception
• Limonin and nomilin are synergistic in
bitterness perception
• Panelists not sensitive to limonin were
sometimes sensitive to nomilin (probably
binds a different receptor on tongue)
• Nomilin descriptors included metallic,
tingly and lingering (aftertaste), while
limonin descriptors were just bitter
• Bitter, metallic, tingly and lingering
aftertaste are typical also for HLB juice
Blending to manage HLB off-flavor
• Symptomatic fruit are small, green and
asymetrical and should be graded out at
the processing plants
• Symptomatic fruit often fall off the tree so
many would not be harvested
• Nevertheless, symptomatic fruit can impart
off-flavor to the juice
• How much symptomatic juice could be
blended with normal (healthy) juice before
flavor is affected?
Detection of symptomatic HLB juice in healthy (normal)
juice
% panelists detecting HLBs
juice in blend (n = 52)
% panelists detecting HLBs
juice in blend (n = 57)
100
90
96.5
A
84.2
80
73.7
70
Hamlin
60
49.1
50
42.1
40
26.3
30
10
0
100
90
Detection
19.3
20
5.3
0.0
0.0
6.25
12.5
Recognition
25
100
90.4
B
80
70
50
100.0
94.2
73.1
Valencia
60
48.1
50
40
26.9
30
19.2
20
10
0
7.7
7.7
1.9
6.25
12.5
g 100 mL
25
-
50
HLBs juice in blend
100
Chemical analyses of healthy and HLBs
juice used in blending study
SSC
pH
TA
Healthy
HLBs
11.4 ± 0.6
11.8 ± 0.3
4.0 ± 0.0
3.9 ± 0.0
0.52 ± 0.00
0.55 ± 0.03
SSC/TA
ratio
21.8 ± 1.1
21.6 ± 1.6
Healthy
12.2 ± 0.2
3.8 ± 0.2a
0.68 ± 0.01 b
17.9 ± 0.1 a
HLBs
11.8 ± 0.3
3.6 ± 0.1 b
1.15 ± 0.03 a
10.2 ± 0.1 b
Cultivar
Hamlin
Valencia
Chemical analyses of healthy and HLBs juice used in
blending study
Sucrose*
Glucose*
Fructose*
Healthy (0 HLBs)
3.63 ± 0.24a
2.70 ± 0.38
2.14 ± 0.88
1.2 ± 0.17
0.40 ± 0.03 a 0.53 ± 0.17 b
0.07 ± 0.008 b
HLBs
50 HLBs
25 HLBs
12.5 HLBs
6.25 HLBs
2.99 ± 0.10 b
3.31
3.47
3.55
3.59
3.31 ± 0.26
3.01
2.86
2.78
2.74
2.57 ± 0.18
2.35
2.24
2.19
2.16
1.3 ± 0.19
1.25
1.22
1.21
1.21
0.31 ± 0.02 b 2.14 ± 0.19 a
0.36
1.34
0.38
0.94
0.39
0.73
0.40
0.64
0.38 ± 0.01 a
0.22
0.15
0.11
0.09
Healthy (0 HLBs)
3.94 ± 0.19 a
2.77 ± 0.64
2.49 ± 0.14
1.34 ± 0.03
0.44 ± 0.02 a 0.53 ± 0.07 b
0.17 ± 0.009 b
HLBs
50 HLBs
25 HLBs
12.5 HLBs
6.25 HLBs
2.35 ± 0.30 b
3.15
3.54
3.74
3.84
1.74 ± 0.40
2.26
2.51
2.64
2.71
3.00 ± 0.94
2.75
2.62
2.55
2.52
1.54 ± 0.09
1.44
1.39
1.36
1.35
0.28 ± 0.06 b 1.23 ± 0.37 a
0.36
0.88
0.40
0.71
0.42
0.62
0.43
0.58
0.51 ± 0.131 a
0.34
0.25
0.21
0.19
Orange Juice
Citric Acid* Malic Acid*
Limonin**
Nomilin**
Hamlin (g/ 100
mL HLBs)
Valencia (g/100
mL HLBs
Materials and methods
Electronic tongue
7 electrochemical
sensors + reference
electrode
Separation of juice blends by e-tongue
1
2
A
Hamlin
B
Valencia
PCA of the blends of Healthy juices with HLB symptomatic juices. Blends were composed of
mL/100 mL HLB Juice: 0%, 6.25%, 12.5%, 25%, 50%, and 100%. A = Hamlin, B =
Valencia
Managing HLB
• Rigorous control, scouting and removal of infected trees is optimal
and proven the best practice to limit the spread of HLB.
• Today most growers have reached their actual or perceived economic
threshold and can no longer afford to or are no longer willing to
remove infected trees.
(Spann, T.M., Schuman, A.W., Rouse, B. , Ebel, B., 2011, Citrus
Industry)
• Therefore, many growers have chosen to maintain the health and
productivity of their trees through an enhanced foliar nutrition
program.
• This policy could conceivably lead to 100% infection
Nutritional programs
• Maury Boyd initiated the original foliar nutrition program.
• HLB symptomatic trees receiving a comprehensive foliar
nutrition treatment have improved visually in some cases
and appear to be maintaining some level of productivity.
• Can a good foliar nutrition program that alleviates tree HLB
symptoms alleviate HLB off-flavor symptoms in the fruit?
Harvests and treatments
Harvests
Hamlin
•
•
•
•
December 2009
December 2010
January 2011
January 2012
Valencia
• April 2011
• April 2012
Typical nutritional treatment ingredients
Oxidate
Renew 14-7-8
Magnesium sulfate
Techmargum
Zinc sulfate
Sodium molybdate
Potassium nitrate 13-0-44 spray grade
435 spray oil
Materials and methods
Fruits were commercially processed
Materials and methods
Sensory Analysis: Difference from control test (DFC)
Same or different?
Rate the difference
Smell
0
1
Taste
0
1
2
3
4
5
6
7
8
9
10
2
3
4
5
6
7
8
9
10
Describe the difference
……………………………………………………………..
18-23 panelists
Two replications for statistical analysis (ANOVA)
Sensory analysis – Trained descriptive panel: 10-12
panelists trained to rate descriptors of orange juice
flavor
Healthy and HLB trees
Maury Boyd
Untreated Healthy
Untreated HLB
HLB N2
Healthy and HLB fruit, different
nutritional programs
H-C
HLB-C
H-N1
H-N2
HLB-N1
HB-N2
Trained panel symptomatic fruit juice
F-Orange
A-Burning
9.0
8.0
A-Astringent
7.0
F-Grapefruit
F-Fruity-noncitrus
6.0
A-Bitter
F-Orange peel
5.0
4.0
3.0
M-Burning
F-Green
2.0
1.0
M-Astringent
F-Stale
0.0
M-Tingling
F-Oxidized oil
M-Body
F-Typical HLB
T-Metallic
T-Sweetness
T-Bitterness
T-Sourness
T-Umami
Ham 12/15/10 HLBs
Hamlin 1/17/2012 U-HLBs
Valencia 4/20/2012 U-HLBs
Trained panel healthy, HLBa and HLBs
F-Orange
A-Burning 8.0
F-Grapefruit
F-Fruity-nonA-Astringent
citrus
6.0
F-Orange
A-Bitter
peel
4.0
M-Burning
F-Green
2.0
M-Astringent
F-Stale
0.0
F-Oxidized
oil
F-Typical
HLB
T-Sweetness
T-Sourness
M-Tingling
M-Body
T-Metallic
T-Bitterness
T-Umami
Valencia 4/20/2012 U-Healthy
Valencia 4/20/2012 U-HLBa
Valencia 4/20/2012 U-HLBs
Trained panel nutritional treatments
Hamlin
N1 Healthy
N1 HLBs
N2 Healthy
N2 HLBa
N2 HLBs
Trained panel nutritional treatments
Valencia
N1 Healthy
N1 HLBa
N1 HLBs
N3 Healthy
N3 HLBa
N3 HLBs
HAMLIN - Conv. Healthy vs. Conv. HLB
symptomatic
2009-2010
(Dec. 2009)
2010-2011
(Jan. 2011)
2011-2012
(Jan. 2012)
Healthy
HLBs
Smell
1.0 b
2.6 a
Taste
1.1 b
6.3 a
Healthy
HLBs
Smell
0.6 b
1.7 a
Taste
0.7 b
3.8 a
Healthy
HLBs
Smell
1.3 a
1.6 a
Taste
1.8 b
2.9 a
Smell: Lime, grapefruit, sulfur, old juice
Taste: Bitter, sour/acid, astringent, less sweet,
medicinal
Smell: Old juice, fermented, overripe
Taste: Bitter, sour, grapefruit, fermented
metallic, less sweet, overripe
Taste: Some bitterness, sweeter, less citrus
flavor
Hamlin differences in aroma or taste
Nutritional DFC – healthy, HLBa HLBs
Month Year/Fruit type
2009
Dec Asymp
Dec Symp
Dec Asymp
Trt
Aroma
Taste
Healthy/HLB Healthy/HLB
Descriptors
SS/TA L+N (PPM)
Healthy/HLB Healthy/HLB
Conv Healthy vs Conv HLB
Conv Healthy vs Conv HLB
Conv Healthy vs HLB N1
NS
1.0/2.6
1.1/2.4
1.4/4.1 Taste - less sweet, bitter, sour/tart, grapefruit, less flavor
1.1/6.3 Smell - Lime, grapefruit, old juice; Taste - Bitter, sour, medicinal
1.3/4.1 Smell - Grapefruit, green, lemon; Taste - Bitter, sour, astringent, metallic, grapefruit, less citrus flavor
27/21
27/20
27/22
3.6/4.7
3.6/7.9
3..6/6.1
Dec
Dec
Dec
Dec
Dec
Dec
Dec
2010
Healthy
Healthy
Asymp
Asymp
Asymp
Asymp
Asymp
Conv Healthy vs N2 Healthy
Conv Healthy vs N3 Healthy
Conv Healthy vs Conv HLB
Conv Healthy vs N2 HLB
Conv Healthy vs N3 HLB
N2 Healthy vs N2 HLB
N3 Healthy vs N3 HLB
ND
0.7/1.1
0.7/2.3
0.5/1.3
0.5/1.4
ND
0.4/1.4
ND
0.9/1.5
1.0/5.7
0.8/5.3
0.9/6.6
0.8/5.3
0.7/6.5
Smell - Nutty, less orange, flat; Taste - bitter, watery, flat, bland, sour
Smell - bitter, rotten, cat, stinky, burnt; Taste - bitter, astingen, tart, grapefruit
Smell - Less citrus aroma; Taste - Bitter, sour, grapefruit, metallic
Smell - less orange; Taste - sour, bitter, grapefruit, acid, less flavor, astringent, tart, less sweet
Taste - Bitter, sour, grapefruit, astringent, metallic
Smell - Less orange aroma, flat; Taste - Bitter, sour, grapefruit, metallic
18/18
18/17
18/17
18/16
18/15
18/16
17/15
2.2/2.5
2.2/1.9
2.2/8.25
2.2/8.1
2.2/11.9
2.5/8.1
1.9/11.9
Jan
Jan
Jan
Jan
2011
Asymp
Symp
Asymp
Symp
Conv Healthy vs Conv HLB
Conv Healthy vs Conv HLB
Conv Healthy vs N1 HLB
Conv Healthy vs N1 HLB
0.4/0.8
0.6/1.7
ND
0.5/1.4
1.1/3.0
0.7/3.8
0.7/2.1
0.8/3.9
Smell - less citrus, week, off-odor; Taste - bitter, sour, less citrus, metallic, less sweet, grapefruit
Smell - Old juice, fermented, overripe; Taste - Bitter, sour, grapefruit, metallic, overripe
Taste: Bitter, metallic, bland
Smell - Old juice, less citrus aroma; Taste - Bitter, less sweet, some metallic, bland/flat
26/25
26/24
26/23
26/24
0.8/2.6
0.8/3.4
0.8/2.7
0.8/2.3
Hamlin continued
Month Year/Fruit type
Trt
Aroma
Taste
Healthy/HLB Healthy/HLB
Descriptors
SS/TA L+N (PPM)
Healthy/HLB Healthy/HLB
2012
Jan
Jan
Jan
Jan
Jan
Jan
Jan
Jan
Jan
Jan
Jan
Jan
Healthy
Healthy
Asymp
Symp
Asymp
Symp
Asymp
Symp
Asymp
Symp
Asymp
Symp
Conv Healthy vs N2 Healthy
Conv Healthy vs N3 Healthy
Conv Healthy vs Conv HLB
Conv Healthy vs Conv HLB
Conv Healthy vs N2 HLB
Conv Healthy vs N2 HLB
Conv Healthy vs N3 HLB
Conv Healthy vs N3 HLB
N2 Healthy vs N2 HLB
N2 Healthy vs N2 HLB
N3 Healthy vs N3 HLB
N3 Healthy vs N3 HLB
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
2.2
ND
1.8/3.1
ND
1.8/2.9
1.7/4.2
1.6/2.1
1.4/3.9
1.4/2.9
2.1/3.5
ND
ND
2.3/3.6
Taste: Less orange flavor, 'flat', watered down
Taste: some bitter, sweeter. Less citrus flavor
Taste: Flat, metallic, off-flavor, sour, bitter
Taste: More sour, bitter and metallic
Taste: Watered down, bad aftertaste, bitter, sour
Taste: Sweeter, bitter, sour
Taste - sweeter, less sweet, bitter, sour
Taste - off-flavor, bitter
27/23
27/24
27/27
27/30
27/27
27/29
27/27
27/23
23/27
23/29
24/27
24/23
?
?
Valencia differences in aroma or taste
Nutritional DFC – Healthy HLBa, HLBs
Month Year/Fruit type
Trt
Aroma
Taste
Healthy/HLB Healthy/HLB
April
April
April
April
April
April
April
April
April
April
April
April
2011
Healthy
Healthy
Asymp
Symp
Asymp
Symp
Asymp
Symp
Asymp
Symp
Asymp
Symp
Conv Healthy vs N1 Healthy
Conv Healthy vs N2 Healthy
Conv Healthy vs Conv HLB
Conv Healthy vs Conv HLB
Conv Healthy vs N1 HLB
Conv Healthy vs N1 HLB
Conv Healthy vs N2 HLB
Conv Healthy vs N2 HLB
N1 Healthy vs N1 HLB
N1 Healthy vs N1 HLB
N2 Healthy vs N2 HLB
N2 Healthy vs N2 HLB
ND
ND
ND
ND
1.0/1.7
0.9/1.2
ND
ND
ND
0.7/1.1
ND
ND
ND
ND
ND
1.2/2.1
1.5/2.5
1.4/3.4
ND
ND
1.4/1.7
1.4/3.9
1.3/2.2
1.3/2.3
April
April
April
April
April
April
April
April
April
April
April
April
2012
Healthy
Healthy
Asymp
Symp
Asymp
Symp
Asymp
Symp
Asymp
Symp
Asymp
Symp
Conv Healthy vs N1 Healthy
Conv Healthy vs N2 Healthy
Conv Healthy vs Conv HLB
Conv Healthy vs Conv HLB
Conv Healthy vs N1 HLB
Conv Healthy vs N1 HLB
Conv Healthy vs N2 HLB
Conv Healthy vs N2 HLB
N1 Healthy vs N1 HLB
N1 Healthy vs N1 HLB
N2 Healthy vs N2 HLB
N2 Healthy vs N2 HLB
ND
ND
ND
ND
ND
ND
ND
0.9/1.4
ND
1.4/2.2
ND
ND
1.9/2.4
2.1/3.1
ND
1.6/2.7
1.4/2.4
1.3/2.3
1.7/3.0
1.5/3.7
ND
3.0/4.1
ND
1.4/2.7
Descriptors
Taste: Sweeter, more sour bitter, off-flavor
Taste: Sweeter, more sour bitter, off-flavor
Taste: Off-flavor, fermented, buitter, musty, vanilla
Taste - more sour, more sweet
Smell - slight off-odor; Taste - bitter, cooked, metallic
Sweeter, more flavorful
Sweeter, more body
Taste: Sweeter, more smooth, more flavor
Taste: sweeter, less sweet, more flavor, less taste
Taste: Sweeter
Taste: Sweeter, some bitterness, unpleasant aftertaste
Taste: Less sweet, more sour, metallic, flat
Taste: Sweeter, slight metallic
Taste: Sour, bitter metallic
Smell - less citrus flavor; Taste - less sweet, less sweet/sour
Taste - less sweet, more sour, less flavor, off-flavor, less sweet/sour
SS/TA L+N (PPM)
Healthy/HLB Healthy/HLB
15/17
15/13
15/14
15/13
15/15
15/14
15/17
15/15
17/15
17/14
13/17
13/15
17/17
17/15
17/22
17/22
17/18
17/16
17/16
17/12
17/18
17/16
15/16
15/12
0.5/2.3
0.5/1.2
0.5/1.9
0.5/1.3
0.5/1.1
0.5/2.1
0.5/0.6
0.5/1.3
1.2/1.1
1.2/2.1
2.3/0.6
2.3/1.3
Conclusions
• Limonin and nomilin are synergistic in terms of
perception of bitterness
• Need about 25-50% HLBs juice in normal juice to detect
a flavor difference (25%) and recognize more bitterness
(50%)
• Generally, HLB effect on flavor is more evident for
Hamlin than Valencia (Valencia has less L&N, is sweeter
and has more volatiles?)
• Generally, HLB has more effect earlier than later in the
season
• Generally flavor of HLBa still similar to healthy, while
HLBs is different
• In some cases (2012), nutritional treatments seemed to
result in higher sugars and perceived sweetness
• Continuing study for 2013 and 2014
Acknowledgements:
Beto Amador and JBT for processing the juices
samples
Mike Irey and Southern Gardens Citrus for
harvesting and supplying fruit for the studies
Thank you!