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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 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 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 Intrinsic effects ◦ Plant varieties ◦ Maturation stage Extrinsic effects ◦ Light ◦ Nutrients (N, P, …) ◦ pH of soil ◦ Irrigation system ◦ Wounding ◦ Pathogen infection ◦ Fungal elicitors Pre Harvest Techniques Variety 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 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 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 Production of anthocyanins studied in Lollo Rosso lettuce ‘Revolution’ Plastics that block progressively at 20-nm intervals across the UV region 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 Carrot cells were cultured Irradiated by white fluorescent lamp at various intensities Cells were collected by centrifuge Extracted with acidified methanol Increased sharply (0- 2000 Ix) 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 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 Color of flowers affected by the pH of soil Grape cell cultures 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 Storage Techniques ◦ Temperature ◦ Light exposure ◦ Environmental Gases Preservation & Maturation Techniques ◦ CA, MAP, SO2, Hexanal/Hexenal, Ozone ◦ Ethylene ◦ Methyl Jasmonate Post Harvest Techniques Storage Temperature ◦ Influences enzymatic activity ◦ High heat denatures enzymes ◦ Freezing slows enzyme activity Abiotic stress increases production of secondary metabolites Minimize PPO activity ◦ Lessen exposure to oxygen Post Harvest Techniques Storage Freezing ◦ Small increase in ANC content with time 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 Light ◦ Required for metabolite production ◦ Effects vary with type: UV or Visible ◦ Production of chromophores to protect against oxidative effects Post Harvest Techniques Storage Exposure of light equivalent of a normal day ◦ >double amount of ANC content in most products than dark storage UV-C ◦ 20% ANC increase in blueberries compared to visible light treatment ◦ Reduction of ripe-rot, shelf life extension Gamma Radiation ◦ Conflicting evidence, generally lowered ANC Post Harvest Techniques Storage 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 CA, MAP, SO2, Hexanal/Hexenal, Ozone ◦ Reduce microbial activity, esp. fungi ◦ Alters respiration and enzyme activity ◦ Control senesce 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 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 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 Temperature pH Ozone Effect of Enzymes High pressure processing Interaction with ascorbic acid Processing Modulations Temperature 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 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 Ozone is a very powerful oxidant Anthocyanins are very sensitive to ozone treatment 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 Polyphenoloxidase (PPO) ◦ Causes browning in fruits When PPO completely inactivated, stability of pelargonidins was intact during storage 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 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 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 Studied extensively May occur by oxidation or condensation of the ascorbic acid with the pigments from the anthocyanins 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 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