Pre and post harvesting
strategies and processing
modulation to enhance
anthocyanin content in food
A Predmore, G Sigurdson, and N Ahmadiania
The Ohio State University
Introduction
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Chemical properties
◦ Flavonoid, C6C3C6 carbon skeleton
◦ Water soluble pigments
◦ Capable of producing red, purple, and
blue colors
◦ Glycosylation at C3 and C5
◦ Acylation of sugar with aliphatic or
aromatic acids
Introduction

Natural antioxidant with possible health benefits
◦ Reduce the risk of
 Cardiovascular disease
 Cancer
 Obesity
◦ Protective properties
 Anti-inflammatory
 Diabetes
(Heins and others 2001; He and Giusti 2011; Pereira and others 2009; Andersen and Markham 2006)
Introduction
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Anthocyanins biosenthesis
Activate the genes necessary to
anthocyanin synthesis
Initiating step involves Phenylalanine
Ammonia Lyase
PAL the rate limiting enzyme
Other in increased activity of the
pentose-phosphate pathway and
decreased protein synthesis
Not always intermediate compounds
carried through to completion
Pre Harvest Techniques
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Intrinsic effects
◦ Plant varieties
◦ Maturation stage
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Extrinsic effects
◦ Light
◦ Nutrients (N, P, …)
◦ pH of soil
◦ Irrigation system
◦ Wounding
◦ Pathogen infection
◦ Fungal elicitors
Pre Harvest Techniques
Variety
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ANC content varies between
different varieties
Solomon A, Golubowicz S, Yablowicz Z, Grossman S, Bergman M, Gottlieb He, Altman A, Kerem Z, Flaishman Ma. 2006. Antioxidant Activities
and Anthocyanin Content of Fresh Fruits of Common Fig (Ficus carica L.). J Agric Food Chem 54: 7717-7723.
Pre Harvest Techniques
Harvest Time
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Two peaks in the
anthocyanins production
during plant maturation
Two varieties of apple ‘Elstar’
and ‘jonagold’
Comparison of the
anthocyanin contents in the
skin
Anthocyanin content high
early and late in the season
Awad MA, Jager AD, Plas LH, Krol AR. 2001. Flavonoid and chlorogenic acid changes in skin of `Elstar' and `Jonagold' apples during development
and ripening. J Hortic Sci 90: 69-83.
Pre Harvest Techniques
Light
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Affect photomorphogenic reactions in plant
Photomorphogenic phenomenon depends on photoreceptors
Photoreceptors helps in the induction of anthocyanins
◦ Phytochrome
◦ Blue/UV light photoreceptor
◦ Cryptochrom
◦ Protochlorophyllide
Photoreceptors alter the expression of the anthocyanin
regulatory and structural genes
Resulted in enhancement of the rate of anthocyanin synthesis
http://www.youtube.com/watch?v=mJBv-vPLJn0&feature=related
Pre Harvest Techniques
Light
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Production of anthocyanins studied in Lollo Rosso
lettuce ‘Revolution’
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Plastics that block progressively at 20-nm intervals
across the UV region
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Total anthocyanins were greatest when both UVA and
UVB were present
Tsormpatsidis E,∗, Henbest R.G.C, Davis F.J, Battey N.H, Hadley P, Wagstaffe A. 2008. UV irradiance as a major influence on growth,
development and secondary products of commercial importance in Lollo Rosso lettuce ‘Revolution’ grown under polyethylene films. Environ
exp bot (63) 232–239
Pre Harvest Techniques
Light
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Carrot cells were cultured
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Irradiated by white fluorescent
lamp at various intensities
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Cells were collected by centrifuge
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Extracted with acidified methanol
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Increased sharply (0- 2000 Ix)
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Slow increase (2000-8000 Ix)
Takeda J. 1988. Light-Induced Synthesis of Anthocyanin in Carrot Cell in Suspension. Exp bot (39) 1065-1077.
Pre Harvest Techniques
Nutrients
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Different level of (N, P, K, Mg and Ca)
One nutrient given at different levels;
remaining four nutrient at normal level
Fruits randomly collected at commercial
harvest time
Nutrient analyzed in the leafs
Negative correlation between N and ANC
concentration
“Carbon-Nutrient Balance” hypothesis
hold true
Low availability of N in organic fertilizers
could increase the ANC concentration
Awad MA, Jager AD. 2002. Relationship between fruit nutrients and concentration of flavonoids and chlorogenic acid in “Elstar” apple skin. J
Hortic Sci 92: 265-276.
Pre Harvest Techniques
pH
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Color of flowers affected by
the pH of soil
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Grape cell cultures
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Low pH caused more
anthocyanin production
Suzuki M. 1995. Enhancement of Anthocyanin Accumulation by High Osmotic Stress and Low pH in Grape Cells (Vitis hybrids). Plant Physiol
147: 152-155.
Post Harvest Techniques
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Storage Techniques
◦ Temperature
◦ Light exposure
◦ Environmental Gases
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Preservation & Maturation Techniques
◦ CA, MAP, SO2, Hexanal/Hexenal, Ozone
◦ Ethylene
◦ Methyl Jasmonate
Post Harvest Techniques
Storage
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Temperature
◦ Influences enzymatic activity
◦ High heat denatures enzymes
◦ Freezing slows enzyme activity
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Abiotic stress increases production of
secondary metabolites
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Minimize PPO activity
◦ Lessen exposure to oxygen
Post Harvest Techniques
Storage
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Freezing
◦ Small increase in ANC content with time
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Cooler Conditions (0-10 °C)
◦ High increases, in may products
◦ 71% increase in pomegranate at 10 °C after 6
wks
◦ 2-6 time increase in strawberry and raspberry
between 10-20 °C after 10 days
Post Harvest Techniques
Storage
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Light
◦ Required for metabolite production
◦ Effects vary with type: UV or Visible
◦ Production of chromophores to protect
against oxidative effects
Post Harvest Techniques
Storage
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Exposure of light equivalent of a normal day
◦ >double amount of ANC content in most
products than dark storage
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UV-C
◦ 20% ANC increase in blueberries compared to
visible light treatment
◦ Reduction of ripe-rot, shelf life extension
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Gamma Radiation
◦ Conflicting evidence, generally lowered ANC
Post Harvest Techniques
Storage
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Environmental Gases: Carbon
Dioxide
◦ Slows microbe growth
◦ Reduces PPO and other enzyme activity
◦ Inverse relationship with PAL
 Increase CO2 leads to decrease of PAL activity
◦ Reversible action
 ANC increases after moving to normal air storage
Post Harvest Techniques
Preservation Treatment
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CA, MAP, SO2, Hexanal/Hexenal, Ozone
◦ Reduce microbial activity, esp. fungi
◦ Alters respiration and enzyme activity
◦ Control senesce
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Generally caused ANC losses
◦ 8 μL hexanal MAP treatment for 38 days: no loss
◦ Intermittent ozone shock treatment: no loss
Post Harvest Techniques
Maturation Treatment
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Ethylene Gas
◦ Produced by fresh produce during ripening
◦ Induces ripening: flavor & secondary
metabolite production
◦ Levels as low as 15 μL/L of air, increase PAL
activity
◦ No significant changes in already ripened fruit
Post Harvest Techniques
Maturation Treatment
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Methyl Jasmonate
◦ Two-fold mechanism
 Stress/Injury Reaction
 Causes increased ethylene production
◦ Linear trend of anthocyanin increases with MJ
concentration
 Increases of 3x-100x, ANC type dependant in
apples
◦ Application of light still required
Processing Modulations
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Temperature
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pH
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Ozone
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Effect of Enzymes
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High pressure processing
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Interaction with ascorbic acid
Processing Modulations
Temperature
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Anthocyanins very sensitive to heating
◦ Anthocyanin content in elderberries was
decreased by 50% after 3h at 90⁰C
◦ Anthocyanin content in blackberry and
strawberry puree significantly decreased after
o
70 C treatment for 2 min
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Logarithmic pattern of destruction as
temperature increases
Sadilova, E., Stintzing, F. C., & Carle, R. (2006). Thermal degradation of acylated and nonacylated anthocyanins. Journal of Food
Science, 71, C504eC512.
Patras, A., Brunton, N. P., Gormely, T. R., & Butler, F. (2009). Impact of high pressure processing on antioxidant activity, ascorbic
acid, anthocyanins and instrumental colour of blackberry and strawberry puree. Innovative Food Science and Emerging
Technologies, 10(3), 308e313.
Processing Modulations
pH
•As the pH increases, typically the stability of
anthocyanins decreases
•High pH in grapes can have effects on the color
and stability of the pigments
Morris, J.R., W.A. Sistrunk, J. Junek and C.A. Sims, (1986). Effects of maturity, juice storage, and juice extraction and
temperature on quality on concord grape juice. J. Am. Sci, 111: 742-746.
Laleh, G. H., Frydoonfar, H., Heidary, R., Jameei, R., & Zare, S. (2006). The effect of light temperature, pH and species on
stability of anthocyanin pigments in four berneris species. Pakistan Journal of Nutrition, 5, 90–92.
Processing Modulations
Ozone
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Ozone is a very powerful oxidant
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Anthocyanins are very sensitive to ozone
treatment
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In a strawberry juice model, treatment
with 7.8% w/w ozone
◦ 92.8% reduction in anthocyanin content
Tiwari, B. K., O’Donnell, C. P., Patras, A., Brunton, N. P., & Cullen, P. J. (2009). Effect of ozone processing on
anthocyanins and ascorbic acid degradation of strawberry juice. FoodChemistry, 113(4),1119e1126.
Processing Modulations
Effect of Enzymes
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Polyphenoloxidase (PPO)
◦ Causes browning in fruits
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When PPO completely inactivated,
stability of pelargonidins was intact during
storage
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β-glucosidase and peroxidase also play
roles in anthocyanin degradation during
storage
Garcia-Palazon, A., Suthanthangjai, W., Kajda, P., & Zabetakis, I. (2004). The effects of high hydrostatic pressure on bglucosidase, peroxidase and polyphenoloxidase in red raspberry (Rubus idaeus) and strawberry (Fragaria x
ananassa). Food Chemistry, 88, 7e10.
Processing Modulations
High Pressure Processing
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Method of preserving and sterilizing food
using very high pressure
Anthocyanins are stable during HPP
Varying stability after storage
◦ Based on MPa of treatment
◦ Amount of PPO inactivation
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Cyanidin 3-glucoside and cyanidin 3sophoroside had higher rates of stability
during storage after 800MPa treatment than
400 or 600MPa in raspberries
Suthanthangjai, W., Kajda, P., & Zabetakis, I. (2005). The effect of high hydrostatic pressure on the anthocyanins of
raspberry (Rubus idaeus). Food Chemistry, 90, 193e197.
Processing Modulations
Interaction with Ascorbic Acid
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Studied extensively
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May occur by oxidation or condensation
of the ascorbic acid with the pigments
from the anthocyanins
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Causes numerous interactions including:
◦ Degradation of both compounds
◦ Decrease in color
◦ Decrease in the quality of the product
Markakis, P., Livingstone, G. E., & Fillers, G. R. (1957). Quantitative aspects of strawberry pigment degradation. Food
Research, 22, 117e130.
Conclusion
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Many factors influence anthocyanin content and
stability including:
◦ Pre-harvest factors
 Varity affects the ANC production
 ANC content varies during maturation
 White light increase ANC concentration
 Higher concentration of Nitrogen could decrease ANC
 pH of soil could affect the ANC production
◦ Post-harvest factors
◦ Processing modulations
 Most processing conditions degrade anthocyanins
 More research is needed for alternative methods